Cu en Resea ch in Food Science 6 (2023) 100425
A ailable online 22 Decembe 2022
2665-9271/© 2022 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Rela ionship be ween he dynamics o ola ile a oma compounds and
mic obial succession du ing he ipening o aw ewe milk-de i ed
Idiazabal cheese
Go ka San ama ina-Ga cía
a
,
*
, Gus a o Amo es
a
, Igo He n´
andez
a
, La a Mo ´
an
b
, Luis Ja ie
R. Ba ´
on
b
, Mailo Vi o
a
,
**
a
Lac ike Resea ch G oup, Depa men o Biochemis y and Molecula Biology, Facul y o Pha macy, Uni e si y he Basque Coun y (UPV/EHU), Paseo de la
Uni e sidad 7, 01006, Vi o ia-Gas eiz, Spain
b
Lac ike Resea ch G oup, Depa men o Pha macy and Food Sciences, Facul y o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU), Paseo de la Uni e sidad 7,
01006, Vi o ia-Gas eiz, Spain
ARTICLE INFO
Keywo ds:
Vola ile composi ion
Odou -ac i e compounds
Mic obio a
Co ela ion analysis
O2PLS
CCo A
ABSTRACT
Cheese mic obio a con ibu es o a ious biochemical p ocesses ha lead o he o ma ion o ola ile compounds
and he de elopmen o la ou du ing ipening. None heless, he ole o hese mic oo ganisms in ola ile a oma
compounds p oduc ion is li le unde s ood. This wo k epo s o he i s ime he dynamics and odou impac o
ola ile compounds, and hei ela ionship o mic obial shi s du ing he ipening o a aw ewe milk-de i ed
cheese (Idiazabal). By means o SPME-GC-MS, 81 ola ile compounds we e iden i ied, among which acids p e-
domina ed, ollowed by es e s, ke ones and alcohols. The ipening ime in luenced he abundance o mos ol-
a ile compounds, hus he momen s o g ea es abundance we e de e mined (such as 30–60 days o acids).
Th ough Odou Impac Ra io (OIR) alues, es e s and acids we e epo ed as he p edominan odou -ac i e
chemical amilies, while indi idually, e hyl hexanoa e, e hyl 3-me hyl bu anoa e, e hyl bu anoa e, bu anoic
acid o 3-me hyl bu anal we e no able odo an s, which would p o ide ui y, ancid, cheesy o mal odou no es.
Using a bidi ec ional o hogonal pa ial leas squa es (O2PLS) app oach wi h Spea man’s co ela ions, 12 bac-
e ial gene a we e epo ed as key bac e ia o he ola ile and a oma ic composi ion o Idiazabal cheese, namely
Psych obac e , En e ococcus, B e ibac e ium, S ep ococcus, Leuconos oc, Ch omohalobac e , Ch yseobac e ium, Ca -
nobac e ium, Lac ococcus, Obesumbac e ium, S eno ophomonas and Fla obac e ium. Non-s a e lac ic acid bac e ia
(NSLAB) we e highly ela ed o he o ma ion o ce ain acids, es e s and alcohols, such as 3-hexenoic acid, e hyl
bu anoa e o 1-bu anol. On he o he hand, he s a e LAB (SLAB) was ela ed o pa icula ke ones p oduc ion,
speci ically 3-hyd oxy-2-bu anone; and en i onmen al and/o non-desi able bac e ia o ce ain ke ones, hy-
d oca bons and sulphu compounds o ma ion, such as 2-p opanone, -3-oc ene and dime hyl sulphone. Addi-
ionally, he SLAB Lac ococcus and Psych obac e , B e ibac e ium and Ch omohalobac e we e desc ibed as ha ing a
nega i e e ec on a oma de elopmen caused by NSLAB and ice e sa. These esul s p o ide no el knowledge o
help unde s and he a oma o ma ion in a aw ewe milk-de i ed cheese.
1. In oduc ion
Idiazabal cheese is a adi ional semi-ha d o ha d cheese om he
Basque Coun y (sou hwes e n Eu ope), which is manu ac u ed om
aw milk o La xa and/o Ca anzana au och honous b eed sheep. I s
p oduc ion is egula ed by i s P o ec ed Designa ion o O igin (PDO)
since 1996 (O icial Jou nal o he Eu opean Communi ies, 1996). The
Idiazabal cheese making p ocess is s ic ly egula ed and es ablishes a
minimum ipening ime o 60 days (Bole ín O icial del Es ado, 1993).
None heless, p oduce s may employ di e en lock managemen and
cheese making p ac ices ha , in u n, a ec he quali y o he milk and
inal cheese. The mos no able di e ences a e ela ed o he lock
* Co esponding au ho .
** Co esponding au ho .
E-mail add esses: [email p o ec ed] (G. San ama ina-Ga cía), [email p o ec ed] (G. Amo es), [email p o ec ed] (I. He n´
andez),
[email p o ec ed] (L. Mo ´
an), [email p o ec ed] (L.J.R. Ba ´
on), [email p o ec ed] (M. Vi o).
Con en s lis s a ailable a ScienceDi ec
Cu en Resea ch in Food Science
jou nal homepage: www.sciencedi ec .com/jou nal/cu en - esea ch-in- ood-science
h ps://doi.o g/10.1016/j.c s.2022.100425
Recei ed 12 Sep embe 2022; Recei ed in e ised o m 10 Decembe 2022; Accep ed 21 Decembe 2022
Cu en Resea ch in Food Science 6 (2023) 100425
2
managemen and g azing p ac ices, such as alley o moun ain g azing
(Abillei a e al., 2010a; Valdi ielso e al., 2016); he use o a isanal o
comme cial enne (Vi o e al., 2003) o he echnological condi ions
used o cheese making and ipening (Aldalu e al., 2021).
Du ing cheese ipening, a ious biochemical p ocesses ake place
ha a e esponsible o he syn hesis o ola ile compounds and,
consequen ly, la ou de elopmen (Fox e al., 2017; Thie y e al.,
2017). Vola ile compounds o igina e p ima ily om h ee g oups o
me abolic pa hways: he me abolism o esidual lac ose, lac a e and
ci a e; he lipolysis and subsequen me abolism o he eleased ee a y
acids (FFAs); and he p o eolysis and me abolism o he esul ing pep-
ides and ee amino acids (FAAs) (Le Qu´
e ´
e and Buchin, 2022). I has
been epo ed ha ipening ime a ec s he ola ile composi ion o
Idiazabal cheese (Ba on e al., 2005a, 2007). Howe e , hese s udies
only ocused on ce ain ime poin s o ipening and did no comp e-
hensi ely cha ac e ize how ola ile chemical amilies and indi idual
compounds e ol e h oughou his p ocess. This aspec is o special in-
e es since he p esence o absence, abundance and p opo ions o each
compound cha ac e ize he ype o cheese and i s a oma (Le Qu´
e ´
e and
Buchin, 2022). In ac , he imbalance o excessi e concen a ions o
se e al compounds ha e been ela ed o o - la ou s (Zabale a e al.,
2016). Mo eo e , i is gene ally ecognized ha no all ola ile com-
pounds con ibu e o cheese a oma (S a owicz, 2021), ha is o say, no
all a e odou -ac i e compounds (Fox e al., 2017; Na ella e al., 2020).
Despi e he la ge wo k done o elucida e he la ou and key a oma ic
compounds o Idiazabal cheese (Ba on e al., 2005a, 2007; Abillei a
e al., 2010b; Valdi ielso e al., 2016), i is unknown how he
odou -impac o ola ile compounds e ol e du ing ipening.
Cheese mic obio a con ibu es o nume ous biochemical eac ions
in ol ed in he o ma ion o la ou compounds (Be uzzi e al., 2018;
Le Qu´
e ´
e and Buchin, 2022) and, indeed, has been desc ibed as
esponsible o he pa icula senso y p ope ies o aw milk cheeses,
such as Manchego o E onka i/Roncal (Balles e os e al., 2006). The
mic obio a o Idiazabal cheese has ecen ly been cha ac e ized by means
o high- h oughpu sequencing (HTS) (San ama ina-Ga cía e al.,
2022a), allowing a be e unde s anding o mic obial communi ies han
cul u e-based me hods (Yelu i Jonnala e al., 2018). O e all, i has been
obse ed ha he ipening ime modula es he bac e ial composi ion.
Speci ically, he s a e LAB (SLAB) (Lac ococcus) p edomina es up o 30
o 60 days and hen, non-s a e LAB (NSLAB) (Lac obacillus, Leuconos-
oc, En e ococcus, S ep ococcus and Ca nobac e ium) p oli e a e, while
he ela i e abundance o non-desi able and/o en i onmen al bac e ia
is educed (such as Pseudomonas, S aphylococcus o Ch omohalobac e ).
Mo eo e , bac e ial composi ion di e s la gely among p oduce s and
se e al bac e ial gene a no epo ed p e iously in any aw ewe milk
and de i ed cheeses ha e been iden i ied (such as Bu iauxella o Obe-
sumbac e ium) (San ama ina-Ga cía e al., 2022a).
In ecen imes, se e al s udies ha a emp o elucida e he ela-
ionship be ween mic obial communi ies and ola ile compounds o -
ma ion in e men ed p oduc s ha e been published (Zhong e al., 2021;
Xia e al., 2022). Howe e , ew s udies ha e ocused on cheese (Zheng
e al., 2018; Chen e al., 2021a) and in o ma ion on aw ewe
milk-de i ed cheeses is sca ce (Ca dinali e al., 2021). In addi ion,
al hough he aim o hese s udies was o unde s and he associa ion
be ween mic obial communi ies and he a oma o ma ion in e men ed
p oduc s, only one wo k has analysed he co ela ion o odou -ac i e
ola ile compounds in e men ed milk (Xia e al., 2021). In o de o
elucida e such co ela ions, complex chemome ic app oaches a e
needed and he mul i a ia e bidi ec ional o hogonal pa ial leas
squa es (O2PLS) (T ygg and Wold, 2003) is one o he mos use ul ap-
p oaches (Bouhaddani e al., 2016). None heless, i s combina ion wi h
o he pa ame e s, such as co ela ion coe icien s, is he mos app o-
p ia e app oach (Galindo-P ie o e al., 2014).
The e o e, his s udy aimed o (1) cha ac e ize how ola ile com-
pounds and hei odou impac e ol e du ing he ipening o aw ewe
milk-de i ed Idiazabal cheese, in o de o (2) in es iga e how hey ela e
o shi s du ing ipening o he unique mic obio a ound in his PDO
cheese and (3) highligh he ele ance o app op ia e ad anced s a is-
ical app oaches o ob ain insigh s and imp o e cheese quali y. Mo e-
o e , he po en ial di e ences among p oduce s p oducing he same
ype o cheese we e also analysed. To he bes o ou knowledge, no such
a comp ehensi e s udy has been conduc ed o da e on any ype o
cheese.
2. Ma e ials and me hods
2.1. Cheese sampling
Fou a isanal Idiazabal PDO cheese p oduce s (iden i ied as A, B, C
and D) we e selec ed o he sampling. Idiazabal cheeses we e p oduced
om aw milk o La xa sheep, each p oduce employing he milk o i s
own lock and ollowing he speci ica ions issued by Idiazabal Desig-
na ion o O igin Regula o y Boa d (Bole ín O icial del Es ado, 1993).
B ie ly, he milk was empe ed o 25 ◦C and he mesophilic lyophilized
s a e cul u e Choozi MM 100 LYO 50 DCU (mix u e o Lac ococcus
lac is subsp. lac is, Lac ococcus lac is subsp. c emo is and Lac ococcus lac is
subsp. lac is bio a . diace ylac is) (DuPon NHIB Ib´
e ica S.L., Ba celona,
Spain) was added. Milk was coagula ed using a isanal enne o com-
me cial enne NATUREN® 195 P emium (Ch . Hansen Holding A/S,
Hø sholm, Denma k). Cheese ipening was ca ied ou in chambe s
main ained a 80–95% ela i e humidi y and 8–14 ◦C empe a u e.
Cheese samples o analysis we e collec ed in duplica e a six ime poin s
du ing ipening (1, 7, 14, 30, 60 and 120 days) (n =48). Samples we e
collec ed and anspo ed o he labo a o y unde e ige a ed condi-
ions (3 ◦C) and hen s o ed in a eeze (−80 ◦C). Be o e analysis, cheese
samples we e de os ed o 24 h a 5 ◦C and kep a oom empe a u e o
1 h.
2.2. Solid-phase mic oex ac ion (SPME) me hodology
The p epa a ion o Idiazabal cheese samples and he SPME p oced-
u e was ca ied ou as p e iously desc ibed (Valdi ielso e al., 2016).
B ie ly, emo ing he ind, 15 g o cheese we e g ound oge he wi h 20
g o anhyd ous Na
2
SO
4
( eagen g ade, Scha lab, Ba celona, Spain).
A e wa ds, 5
μ
L o cyclohexanone (0.5
μ
g/L) (≥99.5%, Sigma-Ald ich,
Mad id, Spain) we e added as in e nal s anda d (IS) solu ion and i was
e-homogenized. The ex ac ion o he ola ile compounds was ca ied
ou by adding 2.5 g o he a o emen ioned mix u e o a 10 mL ambe ial
(Agilen Technologies). The ials we e sealed wi h a PTFE sep a and a
s eel magne ic cap (18 mm PTFE/SIL, Agilen Technologies) p io o
placing hem in he sample ay a oom empe a u e o analysis. SPME
p ocedu e was ca ied ou employing a PAL RSI 85 au osample (CTC
CombiPAL, Zwingen, Swi ze land) equipped wi h a
empe a u e-con olled ai incuba o . A e 15 min o p e-equilib a ion
ime a 60 ◦C, ola ile compounds we e apped on o a 50/30
μ
m
DVB/Ca boxen/PDMS ib e (57298-U, Supelco, Mad id, Spain) a 60 ◦C
o 30 min.
2.3. Gas ch oma og aphy–mass spec ome y (GC-MS) analysis
Vola ile compounds we e analysed in a 7820A Gas Ch oma og aph
sys em, equipped wi h a spli /spli less injec o and coupled o a 5975
se ies MS de ec o (Agilen Technologies), essen ially as p e iously
desc ibed (Ba on e al., 2005b). Vola ile compounds we e deso bed
om he ib e in he on injec ion po o 10 min a 240 ◦C in spli less
mode (spli al e was opened a 200 mL/min a e 10 min o he in-
jec ion). Then, ola ile compounds we e sepa a ed in a Supelcowax-10
used silica capilla y column (59.5 m leng h, 0.25 mm i.d.; 0.25
μ
m
ilm hickness) (Supelco, Mad id, Spain) and helium (99.999% pu i y,
Ai liquid, Mad id, Spain) was employed as he ca ie gas a a cons an
p essu e o 16 psi. O en was held a 40 ◦C o 10 min, hen aised a a
a e o 5 ◦C/min un il 110 ◦C, inc eased again a 10 ◦C/min un il 240 ◦C,
G. San ama ina-Ga cía e al.
Cu en Resea ch in Food Science 6 (2023) 100425
3
and inally held a 240 ◦C o 15 min. Vola ile compounds we e hen
ans e ed o he MS de ec o h ough a ans e line a 280 ◦C and MS
de ec o ope a ed a 150 ◦C in ull scan mode wi h 70 eV as o al ion
cu en .
The ch oma og aphic da a ob ained we e analysed wi h MSD
ChemS a ion Da a Analysis e sion 5.52 (Agilen Technologies). Vola ile
compounds we e en a i ely iden i ied by compa ing hei mass spec a
(ma ch ac o >800) wi h hose o he Na ional Ins i u e o S anda ds
and Technology spec a lib a y (NIST e sion 2.0, Gai he sbu g, USA).
Mean linea e en ion index (LRI) alue o each ch oma og aphic peak
was calcula ed om he analysis o each Idiazabal cheese sample (4
eplica es) and he sa u a ed alkanes s anda d mix u e (3 eplica es ×2
imes h ough he expe imen ) (ce i ied e e ence ma e ial 49452-u,
C7-C40, Sigma-Ald ich). Then, posi i e iden i ica ion o ola ile com-
pounds was ca ied ou by compa ing he LRI and mass spec a wi h
hose o comme cially a ailable high pu i y s anda ds (≥90%, supplied
by Sigma-Ald ich and Honeywell Fluka, Mad id, Spain). The limi o
de ec ion (LOD) was es ablished a wice he noise (a bi a y uni s) o
he ch oma og am. Peak a ea quan i ica ion was ca ied ou by he o al
ion cu en (TIC) and he con en o ola ile compounds was exp essed
as ela i e abundance (peak a ea in a bi a y uni s ela i e o he in-
e nal s anda d (IS)), as exp essed by he ollowing equa ion:
Vola ile ela i e abundace =peak a ea
IS a ea ×2,5 g
sample weigh (g)×100
The mean abundance o he ola ile compounds o each cheese
sample was ob ained om he peak a eas (>LOD) o each compound, as
long as hey we e de ec ed in a leas wo o he ou sample eplica es.
The ela i e abundance o ola ile compounds in cheese samples was
exp essed as mean ±s anda d de ia ion.
2.4. S a is ical analysis o ola ile composi ion
Di e en packages and so wa e we e employed o da a analysis.
The IBM SPSS s a is ical package e sion 26.0 (IBM SPSS Inc., Chicago,
2019) was used o da a p epa a ion and analysis. K uskal-Wallis anal-
ysis o a iance wi h Bon e oni co ec ion was pe o med in IBM SPSS
package o de e mine he e ec o ipening ime and p oduce ac o s on
ola ile chemical amilies and indi idual compounds. Hie a chical
Clus e ing Analysis (HCA) o ola ile compounds was pe o med using
log ans o med, when necessa y, and Uni Va iance (UV) scaled da a
and plo ed in o a hea -map wi h RS udio e sion 1.3.959 and R e sion
3.6.3 (R Co e Team, 2020) wi h “gplo s” package (Wa nes e al., 2020).
The aim was o analyse ola ile compounds g oupings du ing ipening
ime. Then, ends along ipening we e analysed h ough a P incipal
Componen Analysis (PCA) using he SIMCA so wa e e sion
15.0.0.4783 (Ume ics AB, Umeå, Sweden). The numbe o p incipal
componen s (PC) was de e mined by eigen alues (g ea e han 2.5) and
c oss alida ion. O hogonal Pa ial Leas Squa es Disc iminan Analysis
(OPLS-DA) was also pe o med in SIMCA, in o de o analyse whe he
samples di e ed acco ding o he p oduce . Va iable In luence on P o-
jec ion (VIP) alues and loadings weigh s we e used o analyse he
impo ance o each ola ile compound in he model.
2.5. Calcula ion o Odou Impac Ra io
To ob ain a measu e o he odou impac o each ola ile compound
de ec ed in Idiazabal cheese samples, Odou Impac Ra io (OIR) was
calcula ed as Abillei a e al. (2010b) desc ibed, wi h mino modi ica-
ions, and as he ollowing equa ion exp esses:
OIR =Vola ile ela i e abundance
odou h eshold(
μ
g/L o
μ
g/kg)
Compounds wi h OIR alues g ea e han 1 indica e ha he abun-
dance o he ola ile compound was highe han he odou h eshold
(OT) and hus, we e odou -ac i e compounds. OTs a e usually
calcula ed in wa e , howe e , alues could change depending on he
ma ix ( an Geme , 2011). The e o e, a ailable OT alues measu ed in
cheese we e aken o a oid he ma ix e ec (lis ed in Table 2).
2.6. Co ela ion be ween bac e ial communi ies and ola ile compounds
An HTS analysis was pe o med o cha ac e ize he bac e ial commu-
ni ies and he shi s ha occu du ing ipening o he collec ed Idiazabal
cheese samples, as desc ibed by San ama ina-Ga cía e al. (2022a). B ie ly,
10 g o cheese we e suspended in 90 mL o 2% (w/ ) s e ile sodium ci a e
(pH 8.0), and homogenized six imes (each o 20 s ON and 10 s OFF) in a
s omache (Mas ica o Basic 400; IUL Ins umen s, K¨
onigswin e , Ge -
many). Then, 1.5 mL o he esul ing suspension was cen i uged (8000×g
o 10 min a 4 ◦C) and he a -con aining supe na an was disca ded. The
ob ained pelle was esuspended in 600
μ
L o sodium ci a e, and cen i-
uged h ee imes (8000×g o 10 min a 4 ◦C). DNA was ex ac ed wi h he
DNeasy Blood & Tissue Ki (Qiagen, Valencia, CA, USA) and he 16S RNA
gene lib a y was p epa ed using Nex e a XT DNA Lib a y P epa a ion Ki
(Illumina Inc., San Diego, CA, USA). The V3–V4 egions o he
16S RNA gene we e ampli ied by PCR ( o wa d p ime :
5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCW-
GCAG-3′; e e se p ime : 5′-GTCTCGTGGGCTCGGAGATGTGTA
TAAGAGACAGGACTACHVGGGTATCTAATCC-3′), as desc ibed by Klind-
wo h e al. (2013). Then, 16S RNA gene sequencing was pe o med on
he Illumina MiSeq pla o m using he MiSeq Reagen Ki 3 (2 ×300 bp)
(Illumina Inc.). MiSeq Repo e so wa e was used o quali y il e ing and
imming o aw eads and axonomic classi ica ion was pe o med using
he MG-RAST web da a analysis ool (Meye e al., 2008), based on he
Sil a SSU da abase (P uesse e al., 2007). Bac e ial abundance was p e-
sen ed as ela i e abundance (%) based on he iden i ied sequences.
To s udy he ela ionship be ween he iden i ied bac e ial gene a and
he dynamics o ola ile a oma compounds, an O2PLS app oach was
applied o log ans o med, when necessa y, and UV scaled da a in
SIMCA. Main bac e ial gene a we e selec ed as X- a iables and ola ile
compounds as Y- a iables. The model was alida ed, among o he s, by
R2 and Q2 alues, Pe mu a ion es o Inne Rela ion plo . The key
bac e ial gene a o he ola ile composi ion o Idiazabal cheese we e
iden i ied based on VIP alues and loading weigh s, oge he wi h
Spea man’s Rank Co ela ions calcula ed in SPSS and in e p e ed in a
hea map wi h an HCA analysis pe o med in R wi h “phea map”
package (Kolde, 2019). The esul ed co ela ions we e e i ied by a
Canonical Co ela ion Analysis (CCo A) mul i a ia e s a is ical
app oach pe o med in R wi h “ egan” package (Oksanen e al., 2020).
3. Resul s and discussion
3.1. Changes in ola ile composi ion
Table 1 summa izes he a e age ela i e abundance o he indi idual
ola ile compounds iden i ied du ing he ipening ime o he collec ed
Idiazabal cheese samples om he ou p oduce s (A, B, C and D). The
a e age ela i e abundance pe cen age o he iden i ied ola ile chem-
ical amilies is epo ed in Supplemen a y Table 1. A o al o 81 ola ile
compounds we e iden i ied, which could be g ouped in o se en amilies
acco ding o hei chemical s uc u e, namely acids (9 indi idual acids),
alcohols (21), aldehydes (7), ke ones (12), es e s (24), hyd oca bons (5),
sulphu compounds (2) and e penes (1). Es e s and alcohols cons i u ed
he highes numbe o indi idual compounds, as p e iously epo ed o
Idiazabal cheese (Ba on e al., 2005a, 2007; Abillei a e al., 2010b;
Valdi ielso e al., 2016), al hough a highe numbe o alcohols we e
iden i ied in he p esen s udy. O e all, 18 indi idual compounds no
epo ed p e iously in Idiazabal cheese we e de ec ed. Compa ed o he
la es wo ks on aw ewe milk-de i ed cheeses, a g ea e numbe o in-
di idual compounds ha e been iden i ied (Gaglio e al., 2019a; Ca di-
nali e al., 2021).
Acids we e he p edominan chemical amily (79.5–88.9% o he
G. San ama ina-Ga cía e al.
Cu en Resea ch in Food Science 6 (2023) 100425
4
Table 1
A e age ela i e abundance ±s anda d de ia ion o indi idual ola ile compounds iden i ied h oughou ipening (1, 7, 14, 30, 60 and 120 days) o Idiazabal cheese samples om 4 p oduce s (n =48).
ID LRI
a
Vola ile compounds Ripening ime (days)
b
P- alue
c
1 7 14 30 60 120 RT P
Acids
C1 1647 Ace ic acid
d
8.06 ±22.8 353 ±542 ND 24.3 ±36.4 61.6 ±101 120 ±243 NS NS
C2 1698 n- Bu anoic acid
d
1681 ±1594 6884 ±5928 9289 ±7950 10674 ±6878 12053 ±7633 6624 ±8670 * **
C3 1880 n- Pen anoic acid
d
10.5 ±14.4 55.1 ±50.4 1135 ±3050 4166 ±11566 48.1 ±57.5 428 ±942 NS *
C4 1921 n- Hexanoic acid
d
3168 ±2911 12617 ±9968 16782 ±13507 24546 ±15744 25632 ±19485 15567 ±22362 NS ***
C5 2067 (E)-3-Hexenoic acid
d,
ND ND ND 682 ±1692 196 ±365 2682 ±5030 NS ***
C6 2105 n- Hep anoic acid
d
ND 102 ±113 435 ±817 183 ±199 192 ±210 151 ±237 NS **
C7 2192 n- Oc anoic acid
d
1174 ±1231 3727 ±2899 5186 ±4099 7982 ±5158 9623 ±8291 6362 ±7524 * ***
C8 2395 n- Nonanoic acid
d
ND ND ND ND ND 307 ±617
C9 2396 n- Decanoic acid
d
490 ±502 1439 ±1119 2185 ±1695 4073 ±2525 4441 ±3872 2959 ±3862 * **
To al s aigh -chain acids 6532 ±6275 25177 ±20619 35012 ±31119 52331 ±43801 52247 ±40014 35200 ±49486 * ***
Alcohols
C10 936 E hanol
d
171 ±58.5 696 ±931 743 ±1065 1288 ±2102 443 ±505 145 ±154 NS NS
C11 1040 1-P opanol
d
ND ND ND ND ND 287 ±723
C12 1145 1-Bu anol
e
ND 4.76 ±8.93 10.6 ±19.8 10.6 ±19.6 20.3 ±31.4 22.4 ±24.9 * ***
C13 1205 3-Me hyl-1-bu anol
e
7.46 ±15.1 34.1 ±29.8 58.2 ±87.8 52.3 ±55.9 29.5 ±16.7 6.51 ±7.76 * ***
C14 1249 1-Pen anol
e
7.76 ±6.20 4.60 ±8.55 ND 19.4 ±52.6 ND ND ** NS
C15 1351 1-Hexanol
e
ND ND 8.12 ±15.5 8.69 ±16.1 17.4 ±20.8 26.0 ±20.6 *** NS
C16 1482 2-E hyl-1-hexanol
d
9.66 ±7.22 18.2 ±22.2 5.41 ±5.99 62.9 ±147 15.8 ±8.09 6.71 ±9.78 NS *
C17 1552 1-Oc anol
e
ND ND ND ND ND 3.15 ±4.24
To al p ima y alcohols 196 ±87.1 757 ±1001 825 ±1194 1442 ±2393 526 ±582 497 ±945 NS NS
C18 1025 2-Bu anol
e
ND ND ND 88.5 ±124 812 ±1102 424 ±334 *** NS
C19 1120 2-Pen anol
e
ND ND ND 1.60 ±2.97 3.41 ±8.25 27.0 ±18.2 *** NS
C20 1122 3-Me hyl-2-bu anol
d,
ND ND ND ND 4.41 ±8.43 7.14 ±13.3 NS NS
C21 1218 2-Hexanol
e
ND ND ND ND ND 1.44 ±2.75
C22 1316 2-Hep anol
d
ND ND ND 30.7 ±82.2 3.73 ±6.99 12.2 ±19.4 * NS
C23 1509 2-Nonanol
e
ND ND ND ND ND 7.29 ±11.4
C24 1640 2,3-Bu anediol
d
ND 27.1 ±76.7 ND 32.1 ±59.6 ND ND NS *
C25 1646 Men hol
d,
6.65 ±6.63 13.0 ±10.4 27.5 ±41.2 11.2 ±12.6 ND ND ** **
To al seconda y alcohols 6.65 ±6.63 40.1 ±87.1 27.5 ±41.2 164 ±281 823 ±1126 479 ±399 *** NS
C26 1352 2-Me hyl-3-pen anol
d
9.41 ±17.4 ND ND ND ND ND
To al e ia y alcohols 9.41 ±17.4 ND ND ND ND ND
C27 1115 2-P open-1-ol
d,
ND ND 10.7 ±20.9 26.8 ±49.7 31.8 ±81.4 1.79 ±5.06 NS ***
C28 1396 2-Nonen-1-ol
d,
0.497 ±0.921 ND ND ND ND ND
C29 1407 (E)-4-Hexen-1-ol
d,
ND ND ND ND ND 2.09 ±3.94
C30 1320 6-Hep ene-2,4-diol
d,
8.00 ±10.0 8.23 ±18.7 ND ND ND ND ** *
To al allyl alcohols 8.50 ±11.0 805 ±18.7 10.7 ±20.9 26.8 ±49.7 31.8 ±81.4 3.88 ±9.01 NS ***
To al alcohols 221 ±122 1602 ±1107 863 ±1256 1633 ±2724 1381 ±1789 980 ±1353 * NS
Aldehydes
C31 1083 Hexanal
e
ND ND 1.55 ±2.88 ND ND ND
C32 1187 Hep anal
d
3.62 ±6.94 ND ND ND ND ND
C33 1287 Oc anal
e
0.0911 ±0.258 ND ND ND ND ND
C34 1399 Nonanal
e
7.93 ±5.03 14.2 ±4.22 15.2 ±4.82 24.2 ±35.2 14.4 ±8.11 9.73 ±12.1 NS NS
To al s aigh -chain aldehydes 11.6 ±12.2 14.2 ±4.22 16.7 ±7.70 24.2 ±35.2 14.4 ±8.11 9.73 ±12.1 NS NS
C35 919 3-Me hyl-bu anal
d
2.99 ±5.54 3.96 ±4.83 1.29 ±2.44 2.44 ±4.63 8.40 ±10.8 43.5 ±31.5 *** NS
To al b anched-chain aldehydes 2.99 ±5.54 3.96 ±4.83 1.29 ±2.44 2.44 ±4.63 8.40 ±10.8 43.5 ±31.5 *** NS
C36 1541 Benzaldehyde
e
ND ND ND 3.90 ±11.0 12.4 ±23.0 1.80 ±2.24 * **
C37 1667 Benzeneace aldehyde
d,
ND ND 7.98 ±15.6 ND ND ND
To al a oma ic aldehydes ND ND 7.98 ±15.6 3.90 ±11.0 12.4 ±23.0 1.80 ±2.24 NS NS
To al aldehydes 14.6 ±17.7 18.2 ±9.05 26.0 ±25.7 30.5 ±50.8 35.2 ±41.9 55.0 ±45.8 * NS
Ke ones
C38 817 2-P opanone
d
13.8 ±16.0 13.1 ±16.9 10.6 ±12.6 56.9 ±142 ND ND * ***
C39 904 2-Bu anone
e
2.84 ±5.25 ND 25.2 ±21.3 1962 ±2305 1465 ±1572 243 ±335 *** NS
C40 978 2-Pen anone
e
ND ND ND 107 ±129 57.2 ±77.8 98.9 ±73.9 *** NS
(con inued on nex page)
G. San ama ina-Ga cía e al.
Cu en Resea ch in Food Science 6 (2023) 100425
5
Table 1 (con inued)
ID LRI
a
Vola ile compounds Ripening ime (days)
b
P- alue
c
1 7 14 30 60 120 RT P
C41 1184 2-Hep anone
e
17.5 ±9.45 34.6 ±12.7 39.7 ±17.2 163 ±226 82.1 ±60.3 170 ±211 ** *
C42 1288 2-Oc anone
e
1.82 ±2.83 28.4 ±74.6 1.57 ±2.90 22.5 ±57.0 1.92 ±3.82 3.28 ±6.14 NS ***
C43 1392 2-Nonanone
e
12.4 ±8.40 28.7 ±13.1 34.5 ±20.8 91.0 ±69.7 64.7 ±56.1 99.2 ±111 * *
C44 1447 8-Nonen-2-one
d
ND ND ND ND ND 8.91 ±11.2
C45 1604 2-Undecanone
e
ND ND 2.72 ±5.07 7.09 ±10.8 6.59 ±7.77 0.716 ±1.35 * NS
To al me hyl ke ones 48.4 ±42.0 105 ±117 114 ±79.8 2409 ±2940 1678 ±1778 624 ±749 *** NS
C46 918 (E,E)-6,10-Dime hyl-5,9- dodecadien-2-one
d,
ND ND ND ND 29.2 ±64.9 ND
C47 981 2,3-Bu anedione
e
60.3 ±72.1 76.9 ±79.1 86.2 ±76.7 24.9 ±46.4 22.7 ±46.6 ND ** **
C48 1292 3-Hyd oxy-2-bu anone
d
349 ±564 477 ±401 533 ±451 380 ±377 179 ±245 66.8 ±110 * ***
C49 1367 2-Hyd oxy-3-pen anone
d,
ND ND 4.69 ±8.77 1.45 ±2.85 ND ND NS **
To al dike ones and o he ke ones 409 ±636 554 ±480 624 ±536 406 ±427 230 ±357 66.8 ±110 * ***
To al ke ones 457 ±678 659 ±597 738 ±616 2815 ±3367 1908 ±2135 691 ±859 ** *
Es e s
C50 1186 Me hyl hexanoa e
e
ND 3.81 ±7.83 3.33 ±6.17 ND ND ND NS **
C51 1187 Me hyl 4-me hyl pen anoa e
d,
2.87 ±5.34 7.40 ±8.64 3.33 ±6.17 ND 3.84 ±7.20 4.53 ±8.66 NS **
To al me hyl es e s 2.87 ±5.34 11.2 ±16.5 6.66 ±12.3 ND 3.84 ±7.20 4.53 ±8.66 NS **
C52 1038 E hyl bu anoa e
d
4.32 ±3.12 27.0 ±45.3 53.8 ±97.8 78.3 ±116 62.8 ±81.3 288 ±362 ** **
C53 1055 E hyl 2-me hyl bu anoa e
d
ND ND ND ND 1.77 ±3.28 3.11 ±5.75 NS **
C54 1069 E hyl 3-me hyl bu anoa e
d
ND ND ND ND 44.2 ±112 33.9 ±65.4 NS **
C55 1134 E hyl pen anoa e
e
ND ND ND ND 2.12 ±4.92 2.42 ±4.54 NS **
C56 1234 E hyl hexanoa e
e
18.7 ±12.0 183 ±267 343 ±554 453 ±584 480 ±647 216 ±270 * *
C57 1292 E hyl hex-4-enoa e
d,
ND ND ND 7.24 ±14.1 7.70 ±15.1 0.292 ±0.559 NS ***
C58 1335 E hyl hep anoa e
e
ND ND 4.78 ±8.89 5.77 ±10.7 3.86 ±9.01 33.8 ±53.2 NS ***
C59 1434 E hyl oc anoa e
e
5.27 ±3.72 31.7 ±45.9 71.5 ±117 120 ±139 101 ±171 58.3 ±95.9 * **
C60 1537 E hyl nonanoa e
d,
ND ND ND ND ND 1.18 ±2.20
C61 1641 E hyl decanoa e
d
4.47 ±3.82 20.3 ±24.1 458 ±1275 1002 ±2560 87.9 ±140 320 ±559 ** NS
C62 1855 E hyl dodecanoa e
d
ND ND ND ND 0.640 ±1.80 1291 ±2894 NS *
To al e hyl es e s 32.7 ±22.7 262 ±383 930 ±2051 1667 ±3424 791 ±1185 2248 ±4312 *** **
C63 1122 P opyl bu anoa e
e
ND ND ND ND 3.35 ±8.57 6.74 ±7.88 ** ***
C64 1320 P opyl hexanoa e
e
ND ND ND ND 14.6 ±19.6 27.1 ±28.0 *** NS
C65 1521 P opyl oc anoa e
e
ND ND ND ND ND 5.68 ±8.53
To al p opyl es e s ND ND ND ND 18.0 ±28.2 39.5 ±44.4 *** NS
C66 1218 Bu yl bu anoa e
d
ND ND ND ND 2.20 ±5.05 2.98 ±3.31 ** *
C67 1412 Bu yl hexanoa e
d
ND ND ND ND 3.34 ±9.45 7.81 ±7.31 *** NS
To al bu yl es e s ND ND ND ND 5.54 ±14.5 10.8 ±10.6 *** NS
C68 1267 Pen yl bu anoa e
d,
ND ND 2.11 ±3.90 3.62 ±6.81 2.45 ±5.45 127 ±237 NS ***
To al pen yl es e s ND ND 2.11 ±3.90 3.62 ±6.81 2.45 ±5.45 127 ±237 NS ***
C69 1414 Hexyl hexanoa e
d
ND ND ND ND ND 3.08 ±6.05
To al hexyl es e s ND ND ND ND ND 3.08 ±6.05
C70 1131 1-Me hylp opyl bu anoa e
d,
ND ND ND ND ND 5.56 ±7.07
C71 1323 2-Me hylp opyl hexanoa e
d
ND ND ND ND 11.4 ±22.1 35.5 ±32.4 *** NS
C72 1372 2-P openyl hexanoa e
d
ND ND ND ND 2.54 ±5.47 0.485 ±0.900 NS **
C73 1461 3-Me hylbu yl hexanoa e
d,
ND ND ND ND 1.55 ±3.49 40.2 ±74.8 NS **
To al b anched-alkyl es e s ND ND ND ND 15.5 ±31.0 81.7 ±115 *** NS
To al es e s 35.6 ±28.0 273 ±400 939 ±2067 1671 ±3431 836 ±1271 2515 ±4734 *** **
Hyd oca bons
C74 700 Hep ane
e
7.46 ±17.2 13.0 ±17.6 7.10 ±17.2 11.9 ±14.2 5.45 ±15.4 ND NS NS
To al sa u a ed hyd oca bons 7.46 ±17.2 13.0 ±17.6 7.10 ±17.2 11.9 ±14.2 5.45 ±15.4 ND NS NS
C75 688 1,3-Pen adiene
d
ND ND ND ND ND 2.42 ±4.55
C76 837 -3-Oc ene
d
35.6 ±54.4 35.7 ±44.8 36.9 ±47.0 239 ±621 16.3 ±33.0 17.5 ±33.5 NS ***
C77 956 1,3-Oc adiene
d,
5.93 ±11.3 7.08 ±13.9 7.50 ±14.7 98.5 ±268 6.71 ±15.3 6.22 ±11.7 NS ***
C78 1041 Toluene
e
20.2 ±12.2 42.9 ±28.4 24.9 ±19.4 112 ±261 19.9 ±23.5 68.0 ±112 NS NS
To al unsa u a ed hyd oca bons 61.7 ±77.9 85.7 ±87.1 69.3 ±81.2 449 ±1150 42.9 ±71.8 94.1 ±162 NS ***
To al hyd oca bons 69.2 ±95.1 98.7 ±105 76.4 ±98.4 461 ±1164 48.4 ±87.2 94.1 ±162 NS ***
Sulphu compounds
(con inued on nex page)
G. San ama ina-Ga cía e al.
Cu en Resea ch in Food Science 6 (2023) 100425
6
o al abundance) (Supplemen a y Table 1), as obse ed p e iously
(Ba on e al., 2005a, 2007; Abillei a e al., 2010b; Valdi ielso e al.,
2016), and he highes abundances we e ound be ween 30 and 60 days
o ipening (P ≤0.05) (Table 1). All he iden i ied acids we e
s aigh -chain a y acids, p edomina ing hexanoic (44.2–51.1% o o al
acids), bu anoic (18.8–27.3%) and oc anoic acids (14.8–18.4%). These
esul s ag ee wi h p e ious s udies (Ba on e al., 2005b, 2007; Abillei a
e al., 2010b; Valdi ielso e al., 2016). Howe e , 3-hexenoic acid was
iden i ied o he i s ime and no b anched-chain acids we e de ec ed in
his s udy. Vola ile acids also p edomina e in o he aw ewe
milk-de i ed cheeses, such as Vas edda della alle del Belìce cheese, and
simila changes du ing ipening ha e been epo ed (Delgado-Ma ínez
e al., 2019; Gaglio e al., 2019a). S aigh -chain a y acids a e also
abundan in o he aw ewe milk-de i ed cheeses (Gaglio e al., 2019a;
Ca dinali e al., 2021), al hough, in some cases, such as in To a del
Casa , ace ic acid, b anched-chain o long-chain FFAs p edomina e
(Delgado-Ma ínez e al., 2019). Mo eo e , clea di e ences we e
obse ed among Idiazabal cheese p oduce s (P ≤0.001). Samples om
p oduce B, who used comme cial enne , showed lowe abundance o
acids compa ed o p oduce s A, C and D ha used a isanal enne . This
was expec ed due o he highe p egas ic lipase ac i i y o a isanal
enne compa ed o comme cial enne (Vi o e al., 2003) and i s sn-3
s e eospeci ici y (Thie y e al., 2017; Amo es e al., 2021). Lipop o ein
lipase (LPL) om aw milk and mic obial lipases and es e ases a e also
impo an lipoly ic agen s in aw milk cheeses (Thie y e al., 2017; Le
Qu´
e ´
e and Buchin, 2022). Howe e , LPL ac i i y is e y low in Idiazabal
cheese (Ch´
a a i e al., 1998).
Es e s we e he second amily in o de o impo ance h oughou
ipening (0.859–11.3%) (Supplemen a y Table 1), whose abundance
inc eased un il 120 days (P ≤0.001) (Table 1). E hyl es e s p edomi-
na ed (89.4–99.8% o o al es e s h oughou ipening), speci ically,
decanoic, hexanoic, dodecanoic and bu anoic e hyl es e s. Mo eo e ,
se e al es e s we e iden i ied o he i s ime in Idiazabal cheese, such
as me hyl 4-me hyl pen anoa e o pen yl bu anoa e. Among mino es-
e s, a signi ican inc ease was also obse ed o p opyl, bu yl and
b anched-alkyl es e s du ing ipening (P ≤0.01). These esul s pa ially
ag ee wi h p e ious wo ks in Idiazabal cheese, as a as iden i ied
compounds and hei abundance a e conce ned (Ba on e al., 2007;
Abillei a e al., 2010b; Valdi ielso e al., 2016). Compa ed o o he aw
ewe milk-de i ed cheeses, he e a e no able di e ences, al hough e hyl
es e s ha e also been desc ibed as p edominan (Delgado-Ma ínez
e al., 2019; Gaglio e al., 2019a; Ca dinali e al., 2021). Signi ican
di e ences among p oduce s we e only obse ed o me hyl es e s (P ≤
0.01), wi h p oduce s B and C showing he highes abundance; and o
e hyl and pen yl es e s (P ≤0.01 and P ≤0.001, espec i ely), o which
p oduce A p esen ed he highes abundance. In gene al, he p esence o
es e s has been associa ed o LAB es e ase ac i i y (Liu e al., 2004;
Be uzzi e al., 2018) and consequen ly, he obse ed di e en ia ion
among p oduce s could be ela ed o he di e en LAB composi ion.
Ke ones abundance was ema kable (3.68–7.16%) (Supplemen a y
Table 1), which also changed du ing ipening ime (P ≤0.01) (Table 1).
Me hyl ke ones abundance inc eased un il 30 days and dec eased a -
e wa ds bu emaining p edominan , while dike ones and o he ke ones
showed he g ea es abundance a he beginning o he ipening
(Table 1). These esul s would explain why me hyl ke ones ha e been
desc ibed as p edominan in Idiazabal cheese be o e (Ba on e al.,
2005a, 2007; Valdi ielso e al., 2016). Indi idually 2-bu anone p e-
domina ed, as obse ed in p e ious s udies (Ba on e al., 2007; Abil-
lei a e al., 2010b; Valdi ielso e al., 2016), oge he wi h
3-hyd oxy-2-bu anone ha has p e iously been ela ed o moun ain
g azing (Valdi ielso e al., 2016). Some ke ones, such as 2-hyd oxy-3--
pen anone o 6,10-dime hyl-5,9-dodecadien-2-one, ha e no been
iden i ied in Idiazabal cheese o da e (Table 1). Di e ences among
p oduce s we e only obse ed o dike ones and o he ke ones (P ≤
0.001), wi h p oduce s C and D showing he highes abundance.
Compa ed o o he aw ewe milk-de i ed cheeses, he e a e g ea
Table 1 (con inued)
ID LRI
a
Vola ile compounds Ripening ime (days)
b
P- alue
c
1 7 14 30 60 120 RT P
C79 746 Dime hyl sulphide
e
1.55 ±2.86 ND ND 17.2 ±46.3 ND ND NS **
C80 1934 Dime hyl sulphone
d
25.0 ±34.0 8.05 ±15.7 7.41 ±14.0 61.4 ±163 7.76 ±14.5 6.77 ±12.5 NS ***
To al sulphu compounds 26.6 ±36.8 8.05 ±15.7 7.41 ±14.0 78.6 ±209 7.76 ±14.5 6.77 ±12.5 NS ***
Te penes
C81 1195 D-Limonene
e
0.151 ±0.430 1.35 ±3.83 ND 1.02 ±2.89 ND ND NS NS
To al e penes 0.151 ±0.430 1.35 ±3.83 ND 1.02 ±2.89 ND ND NS NS
a
LRI: linea e en ion index.
b
ND: no de ec ed.
c
RT: ipening ime ac o e ec ; P: p oduce ac o e ec ; NS: P >0.05, *P ≤0.05, **P ≤0.01, ***P ≤0.001.
d
Ten a i ely iden i ied ola ile compounds.
e
Posi i ely iden i ied ola ile compounds.
Vola ile compounds no p e iously desc ibed in Idiazabal cheese.
G. San ama ina-Ga cía e al.
Cu en Resea ch in Food Science 6 (2023) 100425
7
di e ences in he iden i ied ke ones and hei abundance du ing
ipening (Delgado-Ma ínez e al., 2019; Gaglio e al., 2019a; Ca dinali
e al., 2021). Gezginc e al. (2021) ha e also epo ed 2-bu anone as he
mos common ke one in Tulum cheese, whe eas 2-hep anone, 2-non-
anone and hyd oxyace one p edomina e in G an O ino cheese (Gaglio
e al., 2019b). Ke ones a e o med by ca abolism o FFAs, in ol ing he
oxida ion o β-ke oacids and deca boxyla ion (Fox e al., 2017; Be uzzi
e al., 2018), mainly by mould and ungi (Fox e al., 2017; Le Qu´
e ´
e and
Buchin, 2022).
Alcohols we e ano he abundan chemical amily (3.40–5.46%)
(Supplemen a y Table 1), which showed he highes abundance a 30
days o ipening (P ≤0.05) (Table 1). P ima y alcohols we e ema kable
du ing ipening, al hough a dec ease was obse ed om 30 o 120 days,
when he abundance o seconda y alcohols no ably inc eased (Table 1).
Howe e , he ipening ime e ec was only signi ican o seconda y
alcohols (P ≤0.001). These esul s would explain he g ea abundance o
seconda y alcohols epo ed be o e (Ba on e al., 2005a, 2007; Valdi-
ielso e al., 2016). E hanol and 2-bu anol p edomina ed, which
pa ially ag ees wi h p e ious s udies (Ba on e al., 2007; Abillei a
e al., 2010b) and se e al alcohols we e iden i ied o he i s ime in
Idiazabal cheese, such as 3-me hyl-2-bu anol o 2-p open-1-ol. Di e -
ences among p oduce s we e only obse ed o allyl alcohols (P ≤
0.001), wi h p oduce A p esen ing he highes abundance. Compa ed o
o he aw ewe milk-de i ed cheeses, he e olu ion o alcohols and he
p edominan compounds di e (Fe n´
andez-Ga cía e al., 2004; Delga-
do-Ma ínez e al., 2019; Gaglio e al., 2019a; Ca dinali e al., 2021).
Gezginc e al. (2021) ha e also obse ed e hanol and 2-bu anol as p e-
dominan in Tulum cheese, while Gaglio e al. (2019b) ha e epo ed
1-hexanol in G an O ino cheese. Alcohols biosyn hesis has been ela ed
o lac ose me abolism, educ ion o ca bonyl compounds (Le Qu´
e ´
e and
Buchin, 2022) o se e al enzyma ic and non-enzyma ic eac ions om
amino acids o me hyl ke ones (Y on and Rijnen, 2001; Be uzzi e al.,
2018; Le Qu´
e ´
e and Buchin, 2022).
Hyd oca bons, sulphu compounds, aldehydes and e penes we e
mino compounds (<4%) (Supplemen a y Table 1), as obse ed be o e
in Idiazabal cheese (Ba on e al., 2005a, 2007; Abillei a e al., 2010b;
Valdi ielso e al., 2016). Howe e , he e a e di e ences compa ed o
o he aw ewe milk-de i ed cheeses (Delgado-Ma ínez e al., 2019;
Gaglio e al., 2019a; Ca dinali e al., 2021), such as he high abundance
o a oma ic hyd oca bons epo ed o Vas edda della alle del Belìce
cheese (Gaglio e al., 2019a). The ipening ime only had a signi ican
e ec on aldehydes (P ≤0.05) (Table 1), wi h a p edominance o
s aigh -chain aldehydes a 30 days o ipening, a oma ic aldehydes a
60 days and b anched-chain aldehydes a 120 days. Ne e heless, he
ipening ime e ec was only signi ican o he ela i e abundance o
b anched-chain aldehydes (P ≤0.001) (Table 1). Indi idually, nonanal
and 3-me hyl-bu anal we e abundan , unlike in p e ious s udies in
Idiazabal cheese (Ba on e al., 2007; Abillei a e al., 2010b; Valdi ielso
e al., 2016), and benzeneace aldehyde was iden i ied o he i s ime.
The e olu ion o aldehydes and he p edominan compounds di e o
o he aw ewe milk-de i ed cheeses (Fe n´
andez-Ga cía e al., 2004;
Delgado-Ma ínez e al., 2019; Gaglio e al., 2019a; Ca dinali e al.,
2021). The p esence o s aigh -chain aldehydes in cheese is ela ed o
he au oxida ion o unsa u a ed a y acids, bo h ee and es e i ied
(Be uzzi e al., 2018). The p esence o b anched-chain and a oma ic
aldehydes, on he o he hand, is ela ed o he deg ada ion o amino
acids by non-enzyma ic o enzyma ic eac ions by yeas o LAB (Y on
and Rijnen, 2001; Be uzzi e al., 2018; Le Qu´
e ´
e and Buchin, 2022).
Anyway, aldehydes a e ansi o y compounds because o hei apid
educ ion o alcohols o oxida ion o acids (Le Qu´
e ´
e and Buchin, 2022),
which explains he low abundance obse ed. In addi ion, i should be
no ed ha among mino compounds, signi ican di e ences among
p oduce s we e obse ed only o unsa u a ed hyd oca bons and
sulphu compounds (P ≤0.001), wi h p oduce B showing he highes
abundance in bo h cases. O e all, he o ma ion o sulphu compounds
is ela ed o enzyma ic eac ions o sulphu -con aining amino acids,
Fig. 1. “Analysis o ola ile composi ion e olu ion du ing ipening (1, 7, 14, 30, 60 and 120 days) o Idiazabal cheese by means o HCA (A) and PCA (sco es and
loadings plo s, B and C espec i ely) and sco es plo o he OPLS-DA model based on he p oduce (D). Vola ile compounds a e labeled acco ding o he ID (Table 1).
The scale alues o he HCA co espond o log ans o med and UV scaled da a.”
G. San ama ina-Ga cía e al.
Cu en Resea ch in Food Science 6 (2023) 100425
8
Table 2
Es ima ed OIR alues h oughou ipening ime (1, 7, 14, 30, 60 and 120 days) and senso y desc ip ion o each ola ile compound de ec ed in Idiazabal cheese samples
(n =48).
Vola ile compounds OT
a
OIR
b
alues du ing ipening (days) Desc ibed odou no es Re e ences
c
1 7 14 30 60 120
Acids
Ace ic acid 22000 <1 Sou , inega , pungen , acid 1, 2, 3
n- Bu anoic acid 50 33.6 138 186 213 241 132 Rancid, cheesy, pu id, sha p, sou , swea 1, 2, 3
n- Pen anoic acid 137 <1 <1 8.28 30.4 <1 3.13 Swea , pu id, sha p, sou , cheesy, bu ned 1, 3
n- Hexanoic acid 290 10.9 43.5 57.9 84.6 88.4 53.7 Swea , sou , pungen , goa , ancid, cheesy, oo , aecal 1, 2, 3
(E)-3-Hexenoic acid – – Pungen , swea , inega , cheesy, g een 4, 5
n- Hep anoic acid 3000 <1 Swea , ancid, aecal 2, 3
n- Oc anoic acid 450 2.61 8.28 11.5 17.7 21.4 14.1 Swea , goa , soapy, waxy, mus y, aecal, dus , cleane 1, 2, 3
n- Nonanoic acid 4000 <1 Faecal, bu ned, ui y 3
n- Decanoic acid 10000 <1 Fa y, soapy, dus , waxy, bu ned 2, 3
Alcohols
E hanol 8 <1 Alcohol, winey, swee , e he eal 1
1-P opanol 5700 <1 Alcohol, winey, swee 1
1-Bu anol 800 <1 Winey, swee , ui y, usel oil 1
3-Me hyl-1-bu anol 250 <1 Alcohol, winey, ui y, bu ned, he bal 1, 3
1-Pen anol 4000 <1 Alcohol, sha p, ha sh 1
1-Hexanol 50 <1 Winey, oily, lowe , ui y, 1, 2
2-E hyl-1-hexanol 830 <1 G een, ci us, lo al, oily, swee 5, 6
1-Oc anol 42 <1 Fa y, waxy, ci us, oily, walnu , moss, chemical,
me al, bu ned
7, 8
2-Bu anol 59 <1 <1 <1 1.50 13.8 7.19 Winey, alcohol, swee , ui y, usel oil 1, 2
2-Pen anol 41 <1 Alcohol, sligh ly g een, winey, ui y 1, 2
3-Me hyl-2-bu anol 420 <1 F ui y 5
2-Hexanol 82 <1 He bal, g een, chemical, winey, ui y, a y, e penic,
cauli lowe
5, 9
2-Hep anol 70 <1 Ea hy, swee , ui y, oily, g een, he bal 1, 2, 3
2-Nonanol 75 <1 Fa y, mild, g een, melon, coconu 7, 9, 10
2,3-Bu anediol 11000 <1 F ui y 3
Men hol 920 <1 Min y, cooling 11
2-Me hyl-3-pen anol 420 <1 G illed, b ead 3
2-P open-1-ol 5000 <1 Pungen , mus a d 5
2-Nonen-1-ol 130 <1 G een, a y, melon 5
(E)-4-Hexen-1-ol 100 <1 G een, he bal, mus y, oma o 5
6-Hep ene-2,4-diol – – –
Aldehydes
Hexanal 5 <1 G een, he bal, sha p 1, 3
Hep anal 3 1.21 <1 <1 <1 <1 <1 Fa y, ui y, soapy, g een, waxy, he bal 10, 12, 13, 14, 15
Oc anal 1.5 <1 F ui y, g een, ci us, a y, a y- ui y, lemon 10, 12, 13, 14, 15, 16
Nonanal 10 <1 1.42 1.52 2.42 1.44 <1 Swee , a y- lo al, lo al-waxy, osy, ci us, peas,
plas ic
1, 2, 3
3-Me hyl-bu anal 0.200 15.0 19.8 6.46 12.2 42.0 217 Mal , chocola e, o ee, g een 1
Benzaldehyde 325 <1 Almond, che y s one, bu ned suga 7, 14, 17
Benzeneace aldehyde 5.50 <1 <1 1.45 <1 <1 <1 Flo al, honey, daisy, g een, iole -like, hyacin h,
s y ene, osy, d y ui , swee
6, 10, 14, 16, 18, 19
Ke ones
2-P opanone 840 <1 Swee , ui y, e he eal, nausea ing 1
2-Bu anone 30 <1 <1 <1 65.4 48.8 8.09 Swee , e he eal, sligh ly nausea ing 1
2-Pen anone 70000 <1 Swee , ui y, e he eal 1, 2
2-Hep anone 5 3.51 6.92 7.93 32.5 16.4 34.0 Mus y, blue cheese, pungen , soapy, lowe 1, 2, 3
2-Oc anone 41 <1 F ui y, mus y, lo al, g een, he bal, mouldy, humidi y,
soapy, mus y, blue-cheese
6, 8, 9, 10, 18
2-Nonanone 5 2.48 5.75 6.91 18.2 12.9 19.8 Mus y, lo al, ui y, soapy 1, 2
8-Nonen-2-one – – Blue cheese, ui y baked 6, 8, 10
2-Undecanone 6.2 <1 <1 <1 1.14 1.06 <1 F ui y, he bal 2
(E,E)-6,10-Dime hyl-5,9-
dodecadien-2-one
– – –
2,3-Bu anedione 3 20.1 25.6 28.7 8.29 7.56 <1 Bu e y, swee , c eam, ca amel 7, 10, 12, 13, 14, 15,
16, 20
3-Hyd oxy-2-bu anone 850 <1 Bu e y, lowe 1, 3
2-Hyd oxy-3-pen anone 2500 <1 Fa y, u le, ea hy, nu y 5, 6, 10
Es e s
Me hyl hexanoa e 390 <1 Ci us, pineapple, e he eal 9, 16
Me hyl 4-me hyl pen anoa e – – S awbe y, oas ed cocoa 21, 22
E hyl bu anoa e 1 4.32 27.0 53.8 78.3 62.8 288 F ui y, apple, pineapple, banana, swee , lowe 1, 2, 3
E hyl 2-me hyl bu anoa e – – Swee , ui y 10, 13
E hyl 3-me hyl bu anoa e 0.1 <1 <1 <1 <1 442 339 F ui y, oli e, swee 2, 3
E hyl pen anoa e 8.7 <1 F ui y, swee , acid, apple, pineapple, g een, be y,
opical
5, 15, 18
E hyl hexanoa e 1 18.7 183 343 453 480 216 F ui y, apple, pineapple, banana, mouldy, lowe 1, 2, 3
E hyl hex-4-enoa e – – –
E hyl hep anoa e 2.2 <1 <1 2.17 2.62 1.75 15.4 F ui y, pineapple, swee , banana, be y, cognac and
sligh ly g een wi h a seedy nuance
5, 9, 18
(con inued on nex page)
G. San ama ina-Ga cía e al.
Cu en Resea ch in Food Science 6 (2023) 100425
9
mainly me hionine, by mic oo ganisms (Fox e al., 2017; Le Qu´
e ´
e and
Buchin, 2022), while hyd oca bons mainly o igina e om deg ada ion
o ca o ene (Po olo e al., 2007).
Subsequen ly, he dynamics o ola ile compounds du ing ipening
we e analysed by mul i a ia e analysis. Th ough an HCA, he ola ile
composi ion was di ided in o h ee s ages: ini ial s age (1–14 days),
middle s age (30–60 days) and ad anced s age (120 days) (Fig. 1A).
Ace ic and hep anoic acids, me hyl es e s, mos dike ones, mos s aigh -
chain aldehydes and benzeneace aldehyde, some alcohols (such as 2-
me hyl-3-pen anol o men hol), sa u a ed hyd oca bons and e penes
cha ac e ized he ini ial s age. Mos abundan acids, ew e hyl es e s,
mos me hyl ke ones and he es o dike ones, some p ima y and sec-
onda y alcohols, nonanal and benzaldehyde, unsa u a ed hyd oca bons
and sulphu compounds cha ac e ized he middle s age. Finally, 3-hex-
enoic and nonanoic acids, mos es e s, he es o p ima y and seconda y
alcohols, 3-me hyl bu anal and 8-nonen-2-one cha ac e ized he
ad anced s age. The cha ac e is ic p esence o some ola ile compounds
a he ini ial and middle s age o ipening would explain why hey ha e
ei he no been iden i ied o ha e been de ec ed in smalle abundance in
p e ious s udies (Ba on e al., 2005a, 2007; Abillei a e al., 2010b;
Valdi ielso e al., 2016). PCA app oach con i med he HCA esul s
(Fig. 1B–C).
To s udy di e ences in he ola ile composi ion among p oduce s o
he Idiazabal cheeses analysed, an OPLS-DA app oach was applied.
Despi e i s limi a ions, he model epo ed a clea dis inc ion be ween
p oduce s, speci ically, be ween A, B and C-D (Fig. 1D). Oc anoic acid,
e hyl oc anoa e o 2-oc anone we e some o he mos impo an ola ile
compounds o such di e en ia ion. The obse ed di e en ia ion could
be due o se e al easons, such as he ype o enne used (Vi o e al.,
2003), as men ioned abo e; o he le el o enne lipase employed, since
he highe he le el, he g ea e FFAs elease and senso y sco es in
Idiazabal cheese (Amo es e al., 2021). Mo eo e , he sheep g azing
sys em could also ha e a ec ed he ola ile composi ion o Idiazabal
cheese, since clea di e ences ha e been epo ed in cheeses made om
milk om ex ensi e moun ain g azing, compa ed o indoo eeding and
pa - ime g azing sheep (Valdi ielso e al., 2016). Finally, cheese mak-
ing and ipening condi ions (De Filippis e al., 2016) o mic obial
composi ion (Fox e al., 2017), ha e also been epo ed o a ec he
ola ile composi ion o cheese, al hough i has no been s udied in
Idiazabal cheese.
3.2. OIR alues
I is well known ha no all ola ile compounds con ibu e o cheese
a oma, mainly due o hei OTs (S a owicz, 2021). The e o e, OIR alues
we e calcula ed o elucida e he key a oma ic compounds o Idiazabal
cheese (Table 2). O e all, es e s and acids showed he highes OIR
alues du ing ipening and, consequen ly, we e he mos odou -ac i e
compounds. Es e s OIR alues inc eased as ipening p og essed and all
he iden i ied odou -ac i e es e s we e e hyl es e s, p edomina ing e hyl
3-me hyl bu anoa e, e hyl bu anoa e and e hyl hexanoa e (Table 2).
The e o e, odou -ac i e es e s co esponded o p edominan es e s
Table 2 (con inued)
Vola ile compounds OT
a
OIR
b
alues du ing ipening (days) Desc ibed odou no es Re e ences
c
1 7 14 30 60 120
E hyl oc anoa e 65 <1 <1 1.10 1.85 1.55 <1 F ui y, winey, pineapple, ap ico , bu ned, ea hy,
lowe
1, 2, 3
E hyl nonanoa e 377 <1 Cheesy, ui y 3
E hyl decanoa e 23 <1 <1 19.9 43.6 3.82 13.9 F ui y, winey, a y, lowe , humidi y 2, 3
E hyl dodecanoa e 400 <1 <1 <1 <1 <1 3.23 Flowe , anilla 3
P opyl bu anoa e 124 <1 F ui y, swee , pineapple, banana 1, 2
P opyl hexanoa e – – F ui y, pineapple, blackbe y, a y 1, 2
P opyl oc anoa e – – Coconu 5
Bu yl bu anoa e 100 <1 F ui y, pineapple, banana, swee , a y 1, 2
Bu yl hexanoa e 700 <1 Flowe , ui y, pineapple, mouldy 2, 3
Pen yl bu anoa e 210 <1 Swee , ui y, banana, pineapple, che y, opical 5
Hexyl hexanoa e 6400 <1 G een, swee , waxy, ui y wi h opical and be y
no es
5
1-Me hylp opyl bu anoa e – – Swee , ui y, pineapple, um, che y, apple, o e ipe
ui
5
2-Me hylp opyl hexanoa e – – Apple 1
2-P openyl hexanoa e 200 <1 Pineapple, a y- ui y 1
3-Me hylbu yl hexanoa e 320 <1 F ui y, swee , pineapple wi h a sligh ly pungen sou
cheesy no e
5
Hyd oca bons
Hep ane 950 <1 Sol en , swee -e he eal, di usi e 1
1,3-Pen adiene 2500 <1 Plas ic, pain , ke osene 23
-3-Oc ene – – Sha p, he bal, lea he -like 2
1,3-Oc adiene 5600 <1 Woody-moss 13
Toluene 1 20.2 42.9 24.9 112 19.9 68.0 F ui y, swee -gassy, hyd oca bon 1
Sulphu compounds
Dime hyl sulphide 1.2 1.29 <1 <1 14.3 <1 <1 Unpleasan wild adish, cabbage, sulphu ous,
pomeg ana e, co n, ea hy, ancid
10, 12, 15, 20
Dime hyl sulphone 2.5 10.0 3.22 2.96 24.5 3.10 2.71 Swee , lowe , sulphu ous, ho milk, bu ned 9, 10, 14
Te penes
D-Limonene 70 <1 G ass 3
a
OT exp essed as
μ
g/L o
μ
g/kg. Da a a e aken om he ollowing: Abillei a e al. (2010b); Wang e al. (2021); Na ella e al. (2020); Sa hi e al. (2021); Majche
and Jele´
n (2011); Kubícko ´
a and G osch (1998); A aie (2009); an Geme (2011).
b
OIR alues calcula ed as mean ela i e abundance om 4 p oduce s (A, B, C and D) a each ipening ime (1, 7, 14, 30, 60, 120 days)/odou h eshold. OIR wi h
alues highe han 1 a e bold colou ed.
c
Desc ibed odou no es aken om: (1) Ba on e al. (2005a); (2) Abillei a e al. (2010b); (3) Zabale a e al. (2016); (4) Cˆ
ama a e al. (2020); (5) The Good Scen s
Company In o ma ion Sys em (2021); (6) Po eda e al. (2008); (7) Ju ic e al. (2003); (8) Jung e al. (2013); (9) Moio e al. (2000); (10) Cu ioni and Bosse (2002); (11)
Zhang e al. (2022); (12) Na ella e al. (2020); (13) Sympou a e al. (2009); (14) Wang e al. (2020); (15) Ka agul Yucee e al. (2009); (16) Whe s ine e al. (2005);
(17) Chen e al. (2021b); (18) Qian and Reineccius (2002); (19) Fox e al. (2017); (20) Boscaini e al. (2003); (21) Campo e al. (2006); (22) Takeoka e al. (1995); (23)
Ho wood e al. (1981).
G. San ama ina-Ga cía e al.
