Ci a ion: San ama ina-Ga cía, G.;
He nández, I.; Amo es, G.; Vi o, M.
Cha ac e iza ion o Mic obial Shi s
du ing he P oduc ion and Ripening
o Raw Ewe Milk-De i ed Idiazabal
Cheese by High-Th oughpu
Sequencing. Biology 2022,11, 769.
h ps://doi.o g/10.3390/
biology11050769
Academic Edi o : Huizhong Chen
Recei ed: 13 Ap il 2022
Accep ed: 11 May 2022
Published: 18 May 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
biology
A icle
Cha ac e iza ion o Mic obial Shi s du ing he P oduc ion and
Ripening o Raw Ewe Milk-De i ed Idiazabal Cheese by
High-Th oughpu Sequencing
Go ka San ama ina-Ga cía * , Igo He nández , Gus a o Amo es and Mailo Vi o *
Lac ike Resea ch G oup, Depa men o Biochemis y and Molecula Biology, 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;
igo [email p o ec ed] (I.H.); gus a o.amo [email p o ec ed] (G.A.)
*Co espondence: [email p o ec ed] (G.S.-G.); [email p o ec ed] (M.V.);
Tel.: +34-945013799 (G.S.-G.); +34-945013099 (M.V.)
Simple Summa y:
Idiazabal is a adi ional cheese p oduced om aw ewe milk in he Basque
Coun y (Sou hwes e n Eu ope). The senso y p ope ies o aw milk cheeses ha e been a ibu ed,
among o he ac o s, o mic obial shi s ha occu du ing he p oduc ion and ipening p ocesses.
In his s udy, we used high- h oughpu sequencing echnologies o in es iga e he mic obio a o
La xa ewe aw milk and he dynamics du ing cheese p oduc ion and ipening p ocesses. The
mic obio a o aw milk was composed o lac ic acid bac e ia (LAB), en i onmen al bac e ia and
non-desi able bac e ia. Th oughou he cheese making and ipening p ocesses, he g ow h o LAB
was p omo ed, whe eas ha o non-desi able and en i onmen al bac e ia was inhibi ed. Mo eo e ,
some gene a no epo ed p e iously in aw ewe milk we e de ec ed and clea di e ences we e
obse ed in he bac e ial composi ion o aw milk and cheese among p oduce s, in ela ion o LAB
and en i onmen al o non-desi able bac e ia, some o which could be a ibu ed o he p oduc ion o
la ou ela ed compounds.
Abs ac :
In his s udy, we used high- h oughpu sequencing echnologies (sequencing o V3–V4
hype a iable egions o 16S RNA gene) o in es iga e o he i s ime he mic obio a o La xa ewe
aw milk and he bac e ial shi s ha occu du ing he p oduc ion and ipening o Idiazabal cheese.
Resul s e ealed se e al bac e ial gene a no epo ed p e iously in aw ewe milk and cheese, such
as Bu iauxella and Obesumbac e ium. Bo h he cheese making and ipening p ocesses had a signi ican
impac on bac e ial communi ies. O e all, he g ow h o lac ic acid bac e ia (LAB) (Lac ococcus,
Lac obacillus,Leuconos oc,En e ococcus,S ep ococcus and Ca nobac e ium) was p omo ed, whe eas ha
o non-desi able and en i onmen al bac e ia was inhibi ed (such as Pseudomonas and Clos idium).
Howe e , conside able di e ences we e obse ed among p oduce s. I is no ewo hy ha he s a e
LAB (Lac ococcus) p edomina ed up o 30 o 60 days o ipening and hen, he g ow h o non-s a e
LAB (Lac obacillus,Leuconos oc,En e ococcus and S ep ococcus) was p omo ed. Mo eo e , in some
cases, bac e ia ela ed o he p oduc ion o ola ile compounds (such as Ha nia,B e ibac e ium and
Psych obac e ) also showed no able abundance du ing he i s ew weeks o ipening. O e all, he
esul s o his s udy enhance ou unde s anding o mic obial shi s ha occu du ing he p oduc ion
and ipening o a aw ewe milk-de i ed cheese (Idiazabal), and could indica e ha he p ac ices
adop ed by p oduce s ha e a g ea impac on he mic obio a and inal quali y o his cheese.
Keywo ds: cheese quali y; ipening; mic obio a; bac e ial di e si y; 16S RNA sequencing; PCoA
1. In oduc ion
Idiazabal cheese is a semi-ha d o ha d cheese made exclusi ely om he aw milk o
La xa and/o Ca anzana sheep, wi h a minimum ipening ime o 60 days. I s p oduc ion
is loca ed in he Basque Coun y (Sou hwes e n Eu ope) and has a P o ec ed Designa ion
Biology 2022,11, 769. h ps://doi.o g/10.3390/biology11050769 h ps://www.mdpi.com/jou nal/biology
Biology 2022,11, 769 2 o 21
o O igin (PDO) [
1
]. Mos o he p oduce s a ached o he Idiazabal PDO a e small amily
dai ies ha lead he whole p ocess, om li es ock managemen o cheese making and
inal sales. Al hough Idiazabal cheese p oduc ion is a s ic ly egula ed p ocess, p oduce s
may use di e en p ac ices ha may a ec he cha ac e is ics o he inal p oduc . The
mos conside able di e ences in p oduc ion p ac ices a e no iced in he managemen and
eeding o he he d, leading o di e ences in milk quali y [
2
]; in he use o a isanal o
comme cial enne , o in he pa ame e s selec ed du ing cheese making and ipening, since
he Idiazabal PDO speci ica ions es ablish anges [3].
Idiazabal cheese, as o he cheeses p epa ed om aw milk, has a iche and mo e
in ense a oma ic p o ile compa ed wi h hose p oduced om pas eu ized milk [
4
,
5
]. Such
in e es ing senso y p ope ies o aw milk cheeses ha e p e iously been a ibu ed, among
o he ac o s, o he complex dynamics o mic obial composi ion du ing cheese making
and ipening [
5
,
6
]. The quali y o aw milk, use o s a e s and hei in insic cha ac e -
is ics, ype o enne used and ipening ime a e some ac o s ha de e mine he cheese
mic obio a [5,7–9]
. The mic obio a o milk has a di e se and complex composi ion, bu i is
mainly composed o lac ic acid bac e ia (LAB) [
5
,
10
]. In Idiazabal cheese, he mos common
LAB a e Lac ococcus,Lac obacillus and Leuconos oc [
11
,
12
]. These bac e ia me abolize he
lac ose p esen in milk, gene a ing lac ic acid and o he compounds, such as ace ic acid,
e hanol and diace yl. These compounds, along wi h o he s p oduced du ing ipening,
de e mine he senso y p ope ies o cheese [
13
]. Al hough LAB a e p edominan , o he low-
abundance mic oo ganisms a e also pa o he mic obial ecosys em o cheese [
10
,
14
,
15
],
and consequen ly con ibu e o he quali y o he inal p oduc [16,17].
The cha ac e is ics o LAB and o he mic oo ganisms p esen in Idiazabal cheese
ha e been desc ibed and ela ed o i s senso y p ope ies in se e al s udies [
11
,
12
,
18
,
19
].
Howe e , hese s udies we e pe o med 20 yea s ago using cul u e-dependen me hods.
Nowadays, high- h oughpu sequencing (HTS) echnologies a e used o moni o mic obial
communi ies in di e en e men ed p oduc s [
20
–
22
], including cheese [
23
–
25
]. The HTS
echniques allow he de ec ion o a la ge numbe o bac e ia, including hose p esen in ela-
i ely small numbe s [
26
–
29
], hose p esen in a iable bu non-cul i able s a e (VBNC) [
30
]
and hose no de ec ed by o he cul u e-dependen o independen me hods [30–34].
The as majo i y o s udies on cheese ocus on cheese p oduced om cow
milk [24,35,36]
,
and only a ew s udies ha e been ca ied ou on cheese p oduced om he aw milk o
ewe [37–39]
. Mo eo e , li le is known abou he bac e ial composi ion o aw ewe milk [
40
–
42
]
and how i changes du ing cheese making and ipening p ocesses [26,43,44].
The e o e, his s udy aimed o (1) cha ac e ize he bac e ial communi ies o he aw
milk o La xa ewe; (2) analyse he e ec o cheese making and ipening p ocesses on
bac e ial popula ions; and (3) s udy he po en ial di e ences among p oduce s p oducing
he same ype o cheese. To he bes o ou knowledge, no comp ehensi e me agenomic
s udy has been conduc ed o da e on aw ewe milk-de i ed cheeses. Mo eo e , al hough
Idiazabal cheese has an in e na ionally ecognized PDO [
1
], no HTS s udies ha e been
pe o med o cha ac e ize i s bac e ial popula ions.
2. Ma e ials and Me hods
2.1. Milk and Cheese Sampling
To analyse he mic obio a o La xa ewe aw milk and Idiazabal cheeses, samples we e
collec ed om ou a isanal Idiazabal PDO cheese p oduce s (iden i ied as A, B, C and
D), whose dai ies we e si ua ed in di e en geog aphic loca ions h oughou he Basque
Coun y. Milk was kep in e ige a ion anks be o e cheese making. Cheeses we e p o-
duced om he collec ed milk samples, acco ding o speci ica ions issued by he Idiazabal
Designa ion o O igin Regula o y Boa d [
3
], using 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 Ibé ica S.L., Ba celona, Spain) as he s a e .
Milk was coagula ed using a isanal enne p epa ed om he s omachs o La xa lambs (ex-
ac ed du ing he i s mon h o lac a ion, cleaned, d ied, sal ed and g ound, as desc ibed
Biology 2022,11, 769 3 o 21
p e iously [
45
]) o comme 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
8–14
◦
C empe a u e and 80–95% ela i e humidi y. Cheeses we e collec ed in duplica e
a six ime poin s du ing ipening (1, 7, 14, 30, 60 and 120 days). The e o e, a o al o
4 aw milk samples and 48 cheese samples we e analysed. Samples we e collec ed and
anspo ed o he labo a o y unde e ige a ed condi ions (3 ◦C) o analysis.
2.2. DNA Ex ac ion
DNA ex ac ion was pe o med immedia ely a e sample a i al, ollowing he
me hod desc ibed by E kus e al. [
46
], wi h some modi ica ions. To ex ac DNA om
cheese samples, 10 g o each sample was suspended in 90 mL o 2% (w/ ) s e ile sodium
ci a e (pH 8.0), and homogenized in a s omache (Mas ica o Basic 400; IUL Ins umen s,
Königswin e , Ge many) six imes, each o 20 s ON and 10 s OFF. Then, 1.5 mL o he
esul ing suspension was cen i uged a 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 a 8000
×
g o 10 min a 4
◦
C. DNA was ex ac ed
wi h DNeasy Blood & Tissue Ki (Qiagen, Valencia, CA, USA), acco ding o he manu-
ac u e ’s p o ocol. To ex ac DNA om milk samples, 10 mL o aw milk om each
sample was p ocessed as desc ibed abo e, howe e , wi hou he need o homogenisa ion
in he s omache .
2.3. Lib a y P epa a ion and Sequencing
HTS analysis was pe o med in he Sequencing and Geno yping Uni o he Genomic
Facili y/SGIke (suppo ed by UPV/EHU, MICINN, GV/EJ, FSE) o he Uni e si y o he
Basque Coun y. The 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), acco ding o he 16S RNA gene
me agenomics wo k low o Illumina. The V3–V4 egions o he 16S RNA gene we e ampli-
ied by PCR ( o wa d p ime : 5
0
-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCC
TACGGGNGGCWGCAG-3
0
; e e se p ime : 5
0
-GTCTCGTGGGCTCGGAGATGTGTA
TAAGAGACAGGACTACHVGGGTATCTAATCC-3
0
) as desc ibed by Klindwo h e al. [
47
].
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.).
2.4. Bioin o ma ic Analysis
Quali y il e ing and imming o aw eads we e pe o med using he MiSeq Repo e
so wa e (Illumina), and axonomic classi ica ion was pe o med using he MG-RAST web
da a analysis ool [
48
], based on he Sil a SSU da abase [
49
]. Since he sequencing o
mos a iable egions o he 16S RNA gene is e ec i e up o he genus le el, and seldom
disc imina es among species adequa ely [
50
], he axonomic classi ica ion was pe o med
up o he genus ank. Ra e ac ion cu es we e also gene a ed using MG-RAST.
2.5. S a is ical Analysis
Rela i e bac e ial abundance (%) was calcula ed based on he iden i ied sequences,
and h ee signi ican igu es we e used o exp ess he esul s. The IBM SPSS s a is ical
package e sion 26.0 (IBM SPSS Inc., Chicago, IL, USA, 2019) was used o da a p epa a ion
and analysis. Mann–Whi ney U es and K uskal-Wallis analysis o a iance wi h Bon e -
oni co ec ion we e pe o med using he SPSS package. The objec i e was o es ima e
di e ences in eads and ope a ional axonomic uni s (OTUs) be ween milk and cheese
samples, and o analyse he in luence o p oduce , cheese making and ipening ime ac o s
on bac e ial phyla and gene a abundance. To de e mine he di ec ion and s eng h o co -
ela ions among he main bac e ial gene a, Spea man’s ank co ela ion coe icien s we e
calcula ed using SPSS, and displayed as a hea map in RS udio e sion 1.3.959 and R e sion
3.6.3 [
51
] using he “gplo s” package [
52
]. To analyse he e ec o p oduce and ipening
ime ac o s on he abundance o he main bac e ial gene a, Pe mu a ional Mul i a ia e
Biology 2022,11, 769 4 o 21
Analysis o Va iance (PERMANOVA) was compu ed in R using he “ egan” package [
53
].
P incipal Componen Analysis (PCA) o he main bac e ial gene a was pe o med using
hei log- ans o med, when necessa y, and Uni Va iance scaled abundance da a, and
plo ed using he SIMCA so wa e ( e sion 15.0.0.4783; Ume ics AB, Umeå, Sweden). The
numbe o p incipal componen s (PCs) was de e mined by eigen alues (g ea e han 1.5)
and c oss alida ion. The aim was o s udy mic obial dynamics in cheeses acco ding o
p oduce and ipening ime ac o s. An O hogonal Pa ial Leas Squa es Disc iminan
Analysis (OPLS-DA) was pe o med in SIMCA o con i m whe he mic obial communi ies
o samples di e ed acco ding o he p oduce .
Alpha and be a di e si y indices we e calcula ed by aking in o accoun he sequence
abundance o all bac e ial gene a p esen in milk and cheese samples. Alpha di e si y was
assessed in R using di e en packages, depending on he objec i e: “ idy e se” package
o da a cleaning and p epa a ion o analysis [
54
]; “Biodi e si yR” package o calcula -
ing Shannon, Simpson, In e se Simpson, Be ge and Shannon e enness (Je enness and
Ee enness) di e si y indices [
55
]; and “ egan” package o calcula ing Chao1 and ACE
di e si y indices. Signi ican di e ences among p oduce s o each di e si y index we e
analysed in SPSS using K uskal-Wallis es . Be a di e si y indices (B ay–Cu is and Jacca d
dissimila i ies) we e calcula ed using he “ egan” package o R, and plo ed in o a P incipal
Coo dina e Analysis (PCoA) model using he “APE” package o R [56].
3. Resul s and Discussion
3.1. Cha ac e is ics o 16S RNA Gene Sequencing Da a
A o al o 10,798,992 16S RNA gene sequences we e ob ained om La xa ewe aw milk
and Idiazabal cheese samples (n= 52), wi h an a e age sequence leng h o
348 ±101 bp
,
mean GC con en o 53
±
5% and 10,388 OTUs. Al oge he , 24 bac e ial phyla, 209 amilies
and 645 gene a we e iden i ied. Fu he de ails o he eads, OTUs and numbe o iden i ied
phyla, amilies and/o gene a a e summa ised in Table 1. The numbe o sequences
ob ained om cheese samples was signi ican ly g ea e han hose ob ained om milk
samples (p
≤
0.001), al hough no signi ican di e ences we e obse ed in he numbe
o iden i ied OTUs be ween he wo sample ypes (p > 0.05). Mo eo e , bo h milk and
cheese samples ob ained om di e en p oduce s showed signi ican dissimila i ies in he
numbe o eads (p
≤
0.01) and iden i ied OTUs (p
≤
0.001), wi h p oduce A being clea ly
dis inc om he o he h ee p oduce s. In gene al, he a e ac ion cu es showed a clea
and s ong s abilizing endency (Figu e S1), indica ing su icien sampling o mic obial
communi ies. O e all, his s udy epo s a g ea e numbe o sequence eads, OTUs and
axonomic iden i ica ions in aw ewe milk and cheese han p e ious s udies [14,38,39,44].
3.2. In-Dep h Analysis o Mic obial Shi s
3.2.1. Bac e ial Composi ion o he Raw Milk o Ewe
Milk is an impo an sou ce o mic oo ganisms in cheese [
5
,
57
]. A o al o 21 bac e ial
phyla, 165 amilies and 455 gene a we e iden i ied in aw milk samples. A he phylum
le el (Figu e 1A, Table S1), Fi micu es (10.5–54.1%) and P o eobac e ia (16.9–40.7%) we e
he mos dominan , ollowed by Bac e oide es (5.44–19.6%). O he phyla, wi h abundances
highe han 1%, we e de ec ed only in milk samples ob ained om some p oduce s: Ac i-
nobac e ia and Ve ucomic obia in samples ob ained om p oduce C (3.75% and 1.33%,
espec i ely) and D (2.97% and 2.75%, espec i ely), and Planc omyce es in samples om
p oduce D (1.28%). In gene al, he p edominance o Fi micu es and P o eobac e ia in aw
ewe milk is consis en wi h p e ious s udies [
26
,
40
,
42
], al hough di e ences in he abun-
dance o each phylum ha e been epo ed among milk samples collec ed om di e en
b eeds [
26
,
40
–
42
]. Howe e , aw milk samples o La xa ewe we e cha ac e ized by he
high-le el abundance o Bac e oide es and a no able p esence o Ve ucomic obia and Planc-
omyce es in compa ison wi h milk collec ed om o he b eeds [
26
,
40
,
42
]. This indica es a
di e en ial cha ac e is ic o La xa ewe aw milk used o Idiazabal cheese p oduc ion.
Biology 2022,11, 769 5 o 21
Table 1.
Me a axonomic da a o La xa ewe aw milk and Idiazabal cheese samples a 6 ipening imes
(1, 7, 14, 30, 60 and 120 days) om 4 p oduce s (A, B, C and D) (n= 52).
P oduce
Milk/Cheese
Ripening Time
(Day)
Sample ID
Bac e ial Di e si y
Sequences OTUs Mbp
Coun Phyla Families Gene a
A
Milk MA 66,989 8450 2.70 8 103 221
1 1 321,148 21,102 6.84 10 102 244
2 331,816 17,948 5.96 10 107 242
7 9 238,804 12,412 4.21 10 82 182
10 251,225 13,472 4.55 10 92 203
14 17 263,231 13,305 4.51 10 81 176
18 334,305 16,504 5.95 11 90 209
30 25 269,207 14,266 5.18 8 89 195
26 280,285 13,701 4.65 8 84 191
60 33 330,729 15,810 5.50 10 79 179
34 341,419 16,324 5.92 10 79 183
120 41 370,181 22,557 7.61 14 83 187
42 332,471 16,882 6.15 12 78 170
B
Milk MB 6092 2504 0.769 10 66 136
13 257,407 12,967 4.39 9 90 178
4 184,670 10,745 3.66 7 80 158
711 160,436 9174 3.15 7 70 124
12 148,700 8429 3.09 9 67 117
14 19 193,164 8672 2.94 9 57 107
20 134,695 7270 2.48 7 55 107
30 27 140,484 2919 1.31 7 40 60
28 129,491 6422 2.33 6 43 88
60 35 295,377 13,046 4.38 10 64 125
36 157,505 7103 2.46 6 47 91
120 43 294,909 12,781 4.61 10 54 107
44 175,689 8629 3.13 10 53 95
C
Milk MC 10,632 4889 1.41 10 66 135
15 237,563 11,167 3.76 10 83 165
6 200,211 9565 3.24 10 73 156
713 172,573 9232 3.16 9 72 136
14 105,377 2947 1.32 5 39 65
14 21 281,503 11,879 4.03 10 76 152
22 162,633 7438 2.54 8 57 112
30 29 374,652 14,534 5.20 8 75 164
30 178,935 7641 2.65 9 66 125
60 37 390,610 14,658 5.06 12 83 170
38 218,602 8265 2.84 8 57 105
120 45 116,160 5909 2.05 5 47 76
46 188,779 8633 2.99 7 51 83
D
Milk MD 52,040 11,547 3.80 21 151 378
17 210,147 11,128 3.71 10 97 217
8 175,444 8825 3.03 11 91 197
715 136,796 6512 2.38 7 65 120
16 126,129 6062 2.11 8 56 113
14 23 194,767 8284 2.99 11 66 131
24 201,259 9138 3.09 14 73 139
30 31 161,519 7050 2.39 13 68 119
32 187,889 8164 2.78 10 66 136
60 39 86,466 4525 1.68 8 45 84
40 202,656 8933 3.28 7 65 127
120 47 205,505 9667 3.36 11 67 119
48 209,716 10,185 3.51 15 63 123
Biology 2022,11, 769 6 o 21
Biology 2022, 11, x FOR PEER REVIEW 6 o 22
3.2. In-Dep h Analysis o Mic obial Shi s
3.2.1. Bac e ial Composi ion o he Raw Milk o Ewe
Milk is an impo an sou ce o mic oo ganisms in cheese [5,57]. A o al o 21 bac e ial
phyla, 165 amilies and 455 gene a we e iden i ied in aw milk samples. A he phylum
le el (Figu e 1A, Table S1), Fi micu es (10.5–54.1%) and P o eobac e ia (16.9–40.7%) we e
he mos dominan , ollowed by Bac e oide es (5.44–19.6%). O he phyla, wi h abun-
dances highe han 1%, we e de ec ed only in milk samples ob ained om some p oduc-
e s: Ac inobac e ia and Ve ucomic obia in samples ob ained om p oduce C (3.75% and
1.33%, espec i ely) and D (2.97% and 2.75%, espec i ely), and Planc omyce es in sam-
ples om p oduce D (1.28%). In gene al, he p edominance o Fi micu es and P o eobac-
e ia in aw ewe milk is consis en wi h p e ious s udies [26,40,42], al hough di e ences
in he abundance o each phylum ha e been epo ed among milk samples collec ed om
di e en b eeds [26,40–42]. Howe e , aw milk samples o La xa ewe we e cha ac e ized
by he high-le el abundance o Bac e oide es and a no able p esence o Ve ucomic obia
and Planc omyce es in compa ison wi h milk collec ed om o he b eeds [26,40,42]. This
indica es a di e en ial cha ac e is ic o La xa ewe aw milk used o Idiazabal cheese p o-
duc ion.
Figu e 1. Rela i e abundance (%) o bac e ial phyla and gene a o La xa ewe aw milk ((A,B), e-
spec i ely) and 1-day-old ipened Idiazabal cheese samples ((C,D), espec i ely) p oduced by ou
p oduce s (A, B, C, D).
Figu e 1.
Rela i e abundance (%) o bac e ial phyla and gene a o La xa ewe aw milk
(
(A,B), espec i ely
) and 1-day-old ipened Idiazabal cheese samples ((
C
,
D
), espec i ely) p oduced
by ou p oduce s (A, B, C, D).
A o al o 24 gene a wi h abundance g ea e han 1% we e iden i ied, and 10 o hese
gene a showed abundance highe han 5% (Figu e 1B, Table S2). Lac ococcus (
1.64–14.5%
),
Eubac e ium (0.0766–9.18%), Clos idium (0.183–6.09%), Leuconos oc (0–6.10%) and
S aphylococcus
(0.291–5.48%) we e he mos abundan gene a wi hin Fi micu es. Simila ly,
Pseudomonas (7.36–18.5%), Bu iauxella (0–14.1%), Se a ia (0.0245–12.6%) and Raoul ella
(0–6.86%) showed he highes abundance wi hin P o eobac e ia, and Ch yseobac e ium
(
0–11.7%
) wi hin Bac e oide es. Di e ences we e obse ed among milk samples ob ained
om di e en p oduce s (Table S2). While Pseudomonas and Lac ococcus we e iden i ied as
he main gene a common o all analysed aw milk samples, he emaining gene a we e
cha ac e is ic o each p oduce . The abundance o he es o gene a classi ied as “o he s”
and unclassi ied sequences was ema kable (6.25–22.4% and 15.6–50.8%, espec i ely).
Di e ences obse ed in he mic obial composi ion o milk samples a he phylum and
genus le els among p oduce s (Tables S1 and S2) could be caused by a ious ac o s such as
di e ences in lac a ion s age, lock managemen and eeding, o sou ces o mic oo ganisms,
o ins ance, mamma y gland diseases o mic oo ganisms con amina ing he ea su ace,
p ac ices and ma e ials employed du ing milking o dai y en i onmen [
5
,
10
,
15
,
41
]. Mo e-
o e , hese ac o s could explain he di e ences obse ed in bac e ial communi ies be ween
he aw milk o La xa ewe and ha o o he ewe b eeds [40–42].
Biology 2022,11, 769 7 o 21
The iden i ied bac e ial gene a we e di ided in o h ee g oups: LAB, comp ising
gene a p e iously classi ied as LAB [
57
]; en i onmen al bac e ia, including bac e ia de i ed
om he na u al en i onmen [
58
]; and non-desi able bac e ia, con aining gene a exhibi ing
a pa hogenic po en ial [
59
] o ela ed o spoilage [
60
]. The LAB iden i ied in his s udy
included he gene a Lac ococcus,Leuconos oc,En e ococcus,Lac obacillus,Ca nobac e ium
and S ep ococcus. These g am-posi i e bac e ia ha e equen ly been iden i ied in dai y
p oduc s [
57
,
61
], and hei p esence in he aw milk o ewe b eeds, o he han La xa, has
been con i med by HTS, albei a di e en abundances [42–44].
The en i onmen al bac e ial gene a iden i ied in his s udy included Obesumbac e ium,
Rosebu ia and P os hecobac e . These gene a ha e been isola ed om di e en na u al
sou ces, such as soil, esh and sal wa e as well as animal and human gu [
62
–
64
]; howe e ,
o he bes o ou knowledge, no s udy has epo ed hei p esence in aw ewe milk.
The non-desi able bac e ial gene a iden i ied in his s udy included Pseudomonas,
Clos idium,S aphylococcus and Bacillus, which a e widely known pa hogens [
59
,
65
]. Fo
ins ance, Pseudomonas is he mos impo an psych o ophic bac e ia in aw milk, which may
e en p edomina e in e ige a ed milk [
10
]. I comes om na u al en i onmen [
66
] and has
been ela ed o hygiene condi ions [
67
,
68
]. Some species belonging o he gene a Bu iauxella,
Se a ia,Ch yseobac e ium,Eubac e ium,Raoul ella,Ruminococcus,Pan oea,S eno ophomonas,
Bac e oides,Fla obac e ium and Acine obac e ha e also been desc ibed as oppo unis ic o
as eme ging pa hogens [
69
–
79
]. Mo eo e , some o hese gene a, such as Se a ia and
Clos idium, a e also ela ed o milk spoilage, esul ing in o - la ou s [
80
] and o he cheese
blowing de ec because o CO
2
p oduc ion [
81
]. The p esence o hese bac e ia in aw ewe
milk has been epo ed only in a ew s udies [
42
,
59
,
82
,
83
]. To he bes o ou knowledge,
he gene a Bu iauxella,Se a ia,Eubac e ium,Raoul ella,Ruminococcus and Bac e oides ha e
no been iden i ied in aw ewe milk so a .
3.2.2. Bac e ial Shi s du ing he Cheese Making P ocess
Nex , he e ec o he cheese making p ocess, which encompasses all he p oduc-
ion s ages om milk o 1-day-old ipened cheeses, on mic obio a was analysed. In his
way, he bac e ial composi ion o La xa ewe aw milk and 1-day-old ipened Idiazabal
cheese was compa ed. In 1-day-old ipened cheese samples, bac e ia belonging o 19 phyla,
160 amilies
and 450 gene a we e de ec ed; hus, he numbe o iden i ied bac e ial amilies
and gene a we e simila be ween 1-day-old ipened cheese and aw milk samples, bu he
numbe o bac e ial phyla iden i ied in cheese was less han ha iden i ied in aw milk.
Howe e , he cheese making p ocess had a g ea impac on he abundance o bac e ial
communi ies (Figu e 1C,D, Tables S1 and S2). A he phylum ank (Figu e 1C, Table S1),
he ela i e abundance o Fi micu es inc eased ema kably in 1-day-old ipened Idiazabal
cheese samples (63.7–94.7%), while ha o P o eobac e ia dec eased (2.61–22.4%), al hough
emaining as he second mos impo an phyla. In gene al, he abundances o he es o
phyla dec eased, al hough he e ec o he cheese making p ocess was no s a is ically
signi ican in all cases. The abundance o sequences classi ied as “o he s” was conside -
ably educed (<0.01%), and uniden i ied sequences accoun ed o lowe , ye ema kable,
abundance (1.21–18.6%). To da e, e y ew HTS s udies ha e analysed he e ec o he
cheese making p ocess on bac e ial communi ies in aw ewe milk
cheeses [43,44]
, and e en
ewe a he phylum ank [
26
]. In compa ison o aw ewe milk-de i ed cheeses, mo e HTS
s udies ha e been conduc ed on cow milk-de i ed cheeses [
29
,
84
]. De Pasquale e al. [
26
]
ha e epo ed an inc ease in Fi micu es abundance and a dec ease in P o eobac e ia abun-
dance in Canes a o Pugliese aw ewe milk-de i ed cheese, bu he changes we e mo e
d as ic han hose obse ed in his s udy. No in o ma ion could be ound in he li e a u e
conce ning he e ec o he manu ac u ing p ocess on he emaining phyla.
The e ec o he cheese making p ocess on he main bac e ial gene a is shown in
Figu e 1D and Table S2. Wi hin LAB, Lac ococcus was he mos abundan genus in 1-day-old
ipened cheese samples collec ed om all p oduce s (52.5–93.2%), al hough a no ably
lowe abundance was obse ed o p oduce A. The e ec o he cheese making p ocess
Biology 2022,11, 769 8 o 21
on he emaining gene a in he LAB g oup a ied wi h he p oduce . The abundance o
Lac obacillus dec eased in cheese samples collec ed om all p oduce s, excep p oduce
A (<0.200% in all p oduce s); abundances o Leuconos oc and Ca nobac e ium we e highe
in p oduce A samples (4.48% and 4.40%, espec i ely); abundances o S ep ococcus and
En e ococcus we e sligh ly highe in he cheese samples o p oduce A (0.507% and 0.458%,
espec i ely) and p oduce D (0.993% and 0.892%, espec i ely). The genus Lac ococcus was
p edominan du ing he cheese making p ocess because o i s p esence in he s a e cul u e,
con i ming ha bac e ia comp ising he s a e cul u e g ow and p edomina e, as has been
p e iously obse ed o Peco ino Siciliano cheese [
39
]. The p oli e a ion o non-s a e
LAB (NSLAB) has been epo ed p e iously, al hough he e a e clea di e ences acco ding
o he ype o cheese. Fo ins ance, Lac ococcus and Lac obacillus ha e been epo ed as
p edominan in Cacio io e della Sibilla cheese [
43
], whe eas Lac ococcus,Ca nobac e ium and
En e ococcus p edomina e in Canes a o Pugliese cheese [26].
In gene al, he abundance o non-desi able bac e ia was less han 1% a e cheese
making, al hough he abundance o some bac e ial gene a, namely Bu iauxella (0–5.79%),
Se a ia (0.00179–2.16%) and Raoul ella (0.0151–1.43%), was main ained a a ema kable
le el o e en inc eased in cheese ob ained om some p oduce s (Figu e 1D, Table S2).
The oppo unis ic bac e ia Ha nia,B e ibac e ium and Psych obac e [
85
–
87
], which had low
abundance in milk (<1%), also inc eased hei abundances in he cheese o some p oduce s
(0.00282–9.62%, 0.0210–2.43% and 0.00168–2.28%, espec i ely). No ably, hese bac e ial
gene a exhibi lipase and/o p o ease ac i i ies [
88
–
90
], and p oduce in e es ing ola ile
compounds (such as 1-hexanol, 1-p opanol, p opyl bu anoa e o bu yl bu anoa e), a ec ing
cheese quali y [
91
–
93
]. O e all, he abundance o en i onmen al bac e ia dec eased du ing
cheese making, al hough Obesumbac e ium main ained a ema kable abundance in samples
om p oduce A (1.89%). Mo eo e , he en i onmen al genus Ch omohalobac e [
94
], which
showed low abundance in milk, exhibi ed highe abundance in cheese, especially ha
ob ained om p oduce C (1.78%). The abundance o bac e ia classi ied as “o he s” and o
uniden i ied bac e ia in cheese (1.31–2.74% and 1.26–18.6%, espec i ely) was lowe han
ha in milk (Figu e 1D, Table S2). Supp ession o he g ow h o en i onmen al and non-
desi able bac e ia, such as Pseudomonas o S aphylococcus, du ing cheese making has been
epo ed p e iously [
26
,
43
]. None heless, li le has been epo ed abou he p e alence o
oppo unis ic o eme ging pa hogens and en i onmen al bac e ia a e he cheese making
p ocess using aw ewe milk. De Pasquale e al. [26] ha e de ec ed Raoul ella in Canes a o
Pugliese cheese bu no in aw milk and Aleg ía e al. [
32
] ha e epo ed a p e alence o
Ch omohalobac e in esh Oscypek cheese. Ha nia and Psych obac e ha e been iden i ied in
o he cheeses p epa ed om aw ewe milk [
38
,
95
], al hough he e ec o he cheese making
p ocess on he abundance o hese bac e ia is unknown.
The cheese making p ocess adds o he ac o s ha can in luence he bac e ial commu-
ni ies [
9
], in addi ion o ac o s ha de e mine he milk mic obio a (Sec ion 3.2.1). B ie ly,
he con e sion o milk o cheese dec eases he pH o 4.5–5.3, which in e e es wi h he
g ow h o mos bac e ia, excep LAB [
9
,
57
]. The NaCl concen a ion o he b ine and
low sal ole ance o mos bac e ia only acili a e he g ow h o LAB [
57
] and halophiles,
such as Psych obac e [
96
] and Ch omohalobac e [
94
]. The dec ease in mois u e con en
and wa e ac i i y (a
w
) also supp esses he p oli e a ion o mos bac e ia, excep LAB,
because o hei esis ance o educed a
w
alues [
9
,
57
]. Mo eo e , a ia ion ha occu s
in he edox po en ial du ing he con e sion o milk o cheese only allows he g ow h
o acul a i e o obliga e anae obic bac e ia [
57
]. I is wo h men ioning ha a isanal
enne employed o he p oduc ion o some aw ewe milk cheeses could be an impo an
sou ce o mic oo ganisms, o example LAB [
8
]. The use o lamb enne pas e con aining
p egas ic lipase esul s in highe lipolysis and he de elopmen o he cha ac e is ic la ou
o Idiazabal cheese [
97
]. Al hough a isanal enne con ains high le els o a wide ange
o mic oo ganisms, including ae obic mesophilic bac e ia [
98
], no signi ican di e ences
ha e been de ec ed in mic obial coun s in Idiazabal cheeses p epa ed using a isanal o
comme cial enne [
99
]; howe e , i would be in e es ing o elucida e his aspec using
Biology 2022,11, 769 9 o 21
cul u e-independen me hods, such as HTS. Finally, i has been obse ed ha small di -
e ences in he en i onmen o dai y acili ies p oducing a isanal cheeses can lead o he
de elopmen o si e-speci ic “household” mic obio a [
100
]. The e o e, hese ac o s could
explain he di e ences in bac e ial composi ion obse ed among di e en aw ewe milk
cheeses and among p oduce s p oducing he same ype o cheese.
3.2.3. Bac e ial Shi s du ing he Cheese Ripening P ocess
Finally, he e ec o he ipening p ocess on he bac e ial composi ion o cheese was
s udied. A o al o 23 phyla, 197 amilies and 583 gene a we e iden i ied h oughou he
cheese ipening p ocess; hus, he numbe o bac e ial amilies and gene a was highe
du ing cheese ipening han in aw milk and in cheese a e he cheese making p ocess.
A he phylum le el (Table S1), he abundance o Fi micu es inc eased ( om a mean o
79.4% a 1 day o ipening o 97.7% a 120 days o ipening), while ha o P o eobac e ia
dec eased sha ply ( om 8.56% o 0.116%). In gene al, abundance o he emaining phyla
no p edominan du ing he cheese making p ocess and ha o “o he s” and uniden i ied
bac e ia we e educed, excep Ac inobac e ia in he cheese o some p oduce s; none heless,
he change in abundance le els was no signi ican o all phyla. O e all, he p edominance
o Fi micu es and educ ion in he abundance o P o eobac e ia and emaining phyla ha e
p e iously been epo ed in o he aw ewe milk cheeses such as Liq an cheese [38,44].
The cheese ipening ime had a conside able impac on bac e ial abundance a he
genus le el, esul ing in la ge di e ences among p oduce s (Figu e 2, Table S2). Wi hin
LAB, Lac ococcus emained he mos dominan genus in Idiazabal cheese du ing ipening a
all imes and o all p oduce s (mean abundance: 74.9% a 1 day o ipening, and 74.5%
a 120 days o ipening), excep p oduce A, which showed no ably lowe p opo ions
o Lac ococcus. The e ec o ipening ime on bac e ial abundance was signi ican only
o Lac obacillus, wi h an inc ease in i s abundance o all p oduce s ( om 0.0949% o
8.96%), while he e olu ion o he abundance o he emaining gene a a ied wi h he
p oduce . In cheeses om p oduce A, he abundance o Leuconos oc, which inc eased a e
cheese making, was unques ionably p omo ed by ipening ime ( om 4.48% o 31.0%),
whe eas ha o Ca nobac e ium dec eased ( om 4.40% o 0.330%). In cheeses ob ained om
p oduce s A and D, he abundances o S ep ococcus and En e ococcus, which inc eased
du ing he cheese making p ocess, also inc eased du ing ipening ( om 0.750% o 4.52% and
om 0.675% o 2.12%, espec i ely). O e all, aking in o accoun LAB dynamics (
Table S2
)
and hei co ela ions du ing ipening ime (Figu e 3), a clea pa e n was obse ed. The
abundance o Lac ococcus dec eased o e 30 o 60 days o ipening, depending on he
p oduce , when NSLAB (Leuconos oc,Lac obacillus,S ep ococcus and En e ococcus) began
o p oli e a e. In o he wo ds, om he i s ipening mon h on hese NSLAB begin o
p oli e a e and become an impo an pa o he inal mic obio a o he cheese.
The p edominance o bac e ia added as pa o he s a e cul u e has also been p e i-
ously epo ed du ing he ipening o o he aw ewe milk cheeses, such as Peco ino Siciliano
cheese [
38
,
39
]. Howe e , lac ose deple ion, sal concen a ion, and low pH and empe a u e
dec ease he iabili y o s a e LAB, and depending on lysis a es, he NSLAB gain im-
po ance [
101
]. The p oli e a ion o Lac obacillus,Leuconos oc,S ep ococcus o
En e ococcus
has also been obse ed in o he aw ewe milk cheeses [
26
,
38
,
39
,
95
]; howe e , he NSLAB
composi ion o o he ypes o aw ewe milk cheeses is di e en om ha o Idiazabal
cheese [
38
,
39
,
43
,
44
]. These di e ences a e impo an , since NSLAB a ec , among o he s,
he p o eolysis and lipolysis o cheese, and consequen ly, i s inal p ope ies, including
la ou and ex u e [102–104].
Biology 2022,11, 769 16 o 21
Biology 2022, 11, x FOR PEER REVIEW 16 o 22
Figu e 6. PCoA o bac e ial be a di e si y a genus ank based on B ay-Cu is (A) and Jacca d (B)
dissimila i ies.
Taking oge he , alpha and be a di e si y indices con i med he esul s o he in-
dep h analysis o mic obial shi s, uni a ia e analysis (K uskal-Wallis es ) and mul i a -
ia e analyses (PERMANOVA, PCA and OPLS-DA). The cheese making and ipening p o-
cesses had an undoub ed impac on he bac e ial communi ies. O e all, bac e ia om he
s a e cul u e p edomina ed a he beginning o ipening, bu a e 30 o 60 days o ip-
ening, he bac e ia om aw milk, especially NSLAB, began o p oli e a e and become
no iceable. None heless, clea di e ences in he mic obial composi ion o aw ewe milk
and cheese samples we e obse ed among p oduce s, which could indica e ha di e -
ences in p ac ices, such as lock managemen and milking, as well as pa ame e s selec ed
du ing cheese making and ipening p ocesses would de e mine he inal mic obio a.
4. Conclusions
This is he i s HTS s udy ca ied ou wi h he objec i e o cha ac e izing he mic o-
bio a o La xa ewe aw milk and examining he bac e ial shi s ha occu du ing he p o-
duc ion and ipening o Idiazabal cheese. This esea ch con i ms ha HTS echniques al-
low a be e unde s anding o he mic obial communi ies, which could no be achie ed
p e iously using cul u e-dependen echniques. Se e al bac e ial gene a we e de ec ed
o he i s ime in aw ewe milk and cheese. Bo h he cheese making p ocess and ipening
ime had a ema kable impac on bac e ial communi ies, al hough conside able di e -
ences we e obse ed among p oduce s. Thus, he use o aw milk and he p ac ices and
condi ions employed by each p oduce o lock managemen , milking and cheese making
and ipening could de e mine he mic obio a. The g ow h o LAB was p omo ed h ough-
ou he cheese making and ipening p ocesses, whe eas ha o non-desi able and en i-
onmen al bac e ia was inhibi ed. Howe e , LAB composi ion di e ed among p oduce s,
and he g ow h o NSLAB was p omo ed a e 30 o 60 days o ipening. In addi ion, in
some cases, bac e ia ela ed o he p oduc ion o ola ile compounds (such as Ha nia,
B e ibac e ium and Psych obac e ) showed no able abundance du ing he i s ew weeks
o ipening.
Figu e 6.
PCoA o bac e ial be a di e si y a genus ank based on B ay-Cu is (
A
) and Jacca d
(B) dissimila i ies.
Taking oge he , alpha and be a di e si y indices con i med he esul s o he in-dep h
analysis o mic obial shi s, uni a ia e analysis (K uskal-Wallis es ) and mul i a ia e
analyses (PERMANOVA, PCA and OPLS-DA). The cheese making and ipening p ocesses
had an undoub ed impac on he bac e ial communi ies. O e all, bac e ia om he s a e
cul u e p edomina ed a he beginning o ipening, bu a e 30 o 60 days o ipening, he
bac e ia om aw milk, especially NSLAB, began o p oli e a e and become no iceable.
None heless, clea di e ences in he mic obial composi ion o aw ewe milk and cheese
samples we e obse ed among p oduce s, which could indica e ha di e ences in p ac ices,
such as lock managemen and milking, as well as pa ame e s selec ed du ing cheese
making and ipening p ocesses would de e mine he inal mic obio a.
4. Conclusions
This is he i s HTS s udy ca ied ou wi h he objec i e o cha ac e izing he mi-
c obio a o La xa ewe aw milk and examining he bac e ial shi s ha occu du ing he
p oduc ion and ipening o Idiazabal cheese. This esea ch con i ms ha HTS echniques
allow a be e unde s anding o he mic obial communi ies, which could no be achie ed
p e iously using cul u e-dependen echniques. Se e al bac e ial gene a we e de ec ed o
he i s ime in aw ewe milk and cheese. Bo h he cheese making p ocess and ipening
ime had a ema kable impac on bac e ial communi ies, al hough conside able di e ences
we e obse ed among p oduce s. Thus, he use o aw milk and he p ac ices and condi-
ions employed by each p oduce o lock managemen , milking and cheese making and
ipening could de e mine he mic obio a. The g ow h o LAB was p omo ed h oughou he
cheese making and ipening p ocesses, whe eas ha o non-desi able and en i onmen al
bac e ia was inhibi ed. Howe e , LAB composi ion di e ed among p oduce s, and he
g ow h o NSLAB was p omo ed a e 30 o 60 days o ipening. In addi ion, in some cases,
bac e ia ela ed o he p oduc ion o ola ile compounds (such as Ha nia,B e ibac e ium
and Psych obac e ) showed no able abundance du ing he i s ew weeks o ipening.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/biology11050769/s1, Figu e S1: Ra e ac ion cu es o mic obial
popula ions o he s udied samples om each p oduce . Each g aph ep esen s a p oduce (A, B, C
and D) and each line is colou ed acco ding o he Sample ID; Figu e S2: Sco es and loadings plo s o
Biology 2022,11, 769 17 o 21
PCA based on main bac e ial gene a o Idiazabal cheeses om 4 p oduce s (A, B, C and D). Samples
a e colou ed acco ding o he p oduce and labels indica e samples iden i ica ion; Table S1: Mean
and s anda d de ia ion o bac e ial phyla o La xa ewe aw milk and Idiazabal cheese samples a
6 ipening imes (1, 7, 14, 30, 60 and 120 days) om 4 p oduce s (A, B, C and D) (n= 52).; Table S2:
Mean and s anda d de ia ion o bac e ial gene a o La xa ewe aw milk and Idiazabal cheese samples
a 6 ipening imes (1, 7, 14, 30, 60 and 120 days) om 4 p oduce s (A, B, C and D) (n= 52): Table S3:
α
-di e si y indices o La xa ewe aw milk and Idiazabal cheese samples a 6 ipening imes (1, 7, 14,
30, 60 and 120 days) om 4 p oduce s (A, B, C and D) (n= 52).
Au ho Con ibu ions:
Concep ualiza ion, me hodology, alida ion, in es iga ion and esou ces,
G.S.-G., I.H., G.A. and M.V; o mal analysis and da a cu a ion, G.S.-G., I.H. and G.A. w i ing—o iginal
d a p epa a ion, G.S.-G.; w i ing— e iew and edi ing, G.S.-G., I.H., G.A. and M.V.; isualiza ion,
G.S.-G.; supe ision, p ojec adminis a ion and unding acquisi ion, M.V. All au ho s ha e ead and
ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by he Basque Go e nmen , g an o Resea ch G oups numbe
IT944-16. G. San ama ina-Ga cía ecei ed a p edoc o al g an om he Uni e si y o he Basque
Coun y (UPV/EHU).
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen :
The da a a e no publicly a ailable ye as some da a se s a e being used
o addi ional publica ions.
Acknowledgmen s:
The au ho s hank he Idiazabal PDO cheesemake s o collabo a ing wi h his
s udy and he echnical suppo p o ided by SGIke (UPV/EHU, ERDF, EU).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Re e ences
1.
Dia io O icial de las Comunidades Eu opeas. Reglamen o (CE) N
◦
1107/96 de La Comisión de 12 de Junio de 1996 Rela i o Al
Regis o de Las Indicaciones Geog á icas y de Las Denominaciones de O igen Con A eglo Al P ocedimien o Es ablecido En El
A ículo 17 Del Reglamen o (CEE) No 2081/92 Del Consejo. DOCE 1996,148, 1–10.
2.
Valdi ielso, I.; Bus aman e, M.A.; Aldezabal, A.; Amo es, G.; Vi o, M.; Ruiz De Go doa, J.C.; De Renobales, M.; Ba on, L.J.R.
Case S udy o a Comme cial Sheep Flock unde Ex ensi e Moun ain G azing: Pas u e De i ed Lipid Compounds in Milk and
Cheese. Food Chem. 2016,197, 622–633. [C ossRe ] [PubMed]
3.
Bole ín O icial del Es ado. O den del 30 de no iemb e po la que se ap ueba el Reglamen o de la Denominación de O igen
«Idiazabal» y su Consejo Regulado . BOE 1993,289, 34591–34596.
4.
Ba on, L.J.R.; Redondo, Y.; A ambu u, M.; Gil, P.; Pé ez-Elo ondo, F.J.; Albisu, M.; Náje a, A.I.; de Renobales, M.;
Fe nández-Ga cía, E.
Vola ile Composi ion and Senso y P ope ies o Indus ially P oduced Idiazabal Cheese. In . Dai y J.
2007
,
17, 1401–1414. [C ossRe ]
5.
O’Sulli an, O.; Co e , P.D. Mic obio a o Raw Milk and Raw Milk Cheeses. In Cheese: Chemis y, Physics and Mic obiology, 4 h ed.;
McSweeney, P.L.H., Fox, P.F., Co e , P.D., E e e , D.W., Eds.; Academic P ess: San Diego, CA, USA, 2017; Volume 1, pp. 301–316.
[C ossRe ]
6. Peláez, C.; Requena, T. Exploi ing he Po en ial o Bac e ia in he Cheese Ecosys em. In . Dai y J. 2005,15, 831–844. [C ossRe ]
7.
Cosen ino, S.; Palmas, F. Hygienic Condi ions and Mic obial Con amina ion in Six Ewe’s-Milk-P ocessing Plan s in Sa dinia, I aly.
J. Food P o . 1997,60, 283–287. [C ossRe ]
8.
C ucia a, M.; Sannino, C.; E colini, D.; Sca assa, M.L.; De Filippis, F.; Mancuso, I.; La S o ia, A.; Mosche i, G.; Se anni, L. Animal
Renne s as Sou ces o Dai y Lac ic Acid Bac e ia. Appl. En i on. Mic obiol. 2014,80, 2050–2061. [C ossRe ]
9.
Possas, A.; Bonilla-Luque, O.M.; Vale o, A. F om Cheese-Making o Consump ion: Explo ing he Mic obial Sa e y o Cheeses
h ough P edic i e Mic obiology Models. Foods 2021,10, 355. [C ossRe ]
10.
Quigley, L.; O’Sulli an, O.; S an on, C.; Be es o d, T.P.; Ross, R.P.; Fi zge ald, G.F.; Co e , P.D. The Complex Mic obio a o Raw
Milk. FEMS Mic obiol. Re . 2013,37, 664–698. [C ossRe ]
11.
Pé ez-Elo ondo, F.J.; Albisu, M.; Ba cina, Y. Changes in he Mic o lo a o Idiazabal Cheese wi h he Addi ion o Comme cial
Lac ic S a e s. Aus . J. Dai y Technol. 1993,48, 10–14.
12.
Pé ez-Elo ondo, F.J.; Aldámiz Echoba ia, P.; Albisu, M.; Ba cina, Y. Indigenous Lac ic Acid Bac e ia in Idiazabal Ewes’ Milk
Cheese. In . Dai y J. 1998,8, 725–732. [C ossRe ]
13.
Thie y, A.; Pogacic, T.; Webe , M.; Lo al, S. P oduc ion o Fla o Compounds by Lac ic Acid Bac e ia in Fe men ed Foods. In
Bio echnology o Lac ic Acid Bac e ia: No el Applica ions, 2nd ed.; Mozzi, F., Raya, R.R., Vignolo, G.M., Eds.; John Wiley & Sons, L d.:
Hoboken, NJ, USA, 2015; pp. 314–340. ISBN 9781118868386.
Biology 2022,11, 769 18 o 21
14.
Kamila i, E.; Anagnos opoulos, D.A.; Papademas, P.; Kamila is, A.; Tsal as, D. Cha ac e izing Halloumi Cheese’s Bac e ial
Communi ies h ough Me agenomic Analysis. LWT 2020,126, 109298. [C ossRe ]
15.
Zhao, Z.; Ning, C.; Chen, L.; Zhao, Y.; Yang, G.; Wang, C.; Chen, N.; Zhang, Z.; Li, S. Impac s o Manu ac u e P ocesses and
Geog aphical Regions on he Mic obial P o ile o T adi ional Chinese Cheeses. Food Res. In .
2021
,148, 110600. [C ossRe ]
[PubMed]
16.
Ni o, S.; F a ianni, A.; T emon e, P.; So en ino, E.; Tipaldi, L.; Pan ili, G.; Coppola, R. Inno a i e Cacioca allo Cheeses Made
om a Mix u e o Cow Milk wi h Ewe o Goa Milk. J. Dai y Sci. 2014,97, 1296–1304. [C ossRe ] [PubMed]
17.
Zheng, X.; Liu, F.; Shi, X.; Wang, B.; Li, K.; Li, B.; Zhuge, B. Dynamic Co ela ions be ween Mic obio a Succession and Fla o
De elopmen In ol ed in he Ripening o Kazak A isanal Cheese. Food Res. In . 2018,105, 733–742. [C ossRe ]
18.
Mendia, C.; Ibañez, F.C.; To e, P.; Ba cina, Y. In luence o he Season on P o eolysis and Senso y Cha ac e is ics o Idiazabal
Cheese. J. Dai y Sci. 2000,83, 1899–1904. [C ossRe ]
19.
Pé ez-Elo ondo, F.J.; Albisu, M.; Ba cina, Y. Physicochemical P ope ies and Seconda y Mic o lo a Va iabili y in he Manu ac u e
and Ripening o Idiazabal Cheese. Lai 1999,79, 281–290. [C ossRe ]
20.
Hu, Y.; Huang, X.; Yang, B.; Zhang, X.; Han, Y.; Chen, X.-X.; Han, B.-Z. Con as ing he Mic obial Communi y and Me abolic
P o ile o Th ee Types o Ligh -Fla o Daqu. Food Biosci. 2021,144, 101395. [C ossRe ]
21.
Liang, T.; Xie, X.; Zhang, J.; Ding, Y.; Wu, Q. Bac e ial Communi y and Composi ion o Di e en T adi ional Fe men ed Dai y
P oduc s in China, Sou h A ica, and S i Lanka by High-Th oughpu Sequencing o 16S RRNA Genes. LWT
2021
,144, 111209.
[C ossRe ]
22.
Zhang, Q.Q.; Li, D.; Zhang, W.; Jiang, M.; Chen, X.H.; Dong, M.S. Compa a i e Analysis o he Bac e ial Di e si y o Chinese
Fe men ed Sausages Using High-Th oughpu Sequencing. LWT 2021,150, 111975. [C ossRe ]
23.
Biolca i, F.; Fe ocino, I.; Bo e o, M.T.; Dalmasso, A. Sho Communica ion: High-Th oughpu Sequencing App oach o
In es iga e I alian A isanal Cheese P oduc ion. J. Dai y Sci. 2020,103, 10015–10021. [C ossRe ] [PubMed]
24.
Ne o, L.A.; And e a, M.; Almeida, T.T.; Fe ei a, L.R.; Cama go, A.C.; Yama ogi, R.S.; Ca alho, A.F.; Call, D.R. Lac ic Mic obio a
o he Minas A isanal Cheese P oduced in he Se o Region, Minas Ge ais, B azil. LWT 2021,148, 111698. [C ossRe ]
25.
Salaza , J.K.; Ca s ens, C.K.; Ramachand an, P.; Shaze , A.G.; Na ula, S.S.; Reed, E.; O esen, A.; Schill, K.M. Me agenomics o
Pas eu ized and Unpas eu ized Gouda Cheese Using Ta ge ed 16S RDNA Sequencing. BMC Mic obiol.
2018
,18, 189. [C ossRe ]
[PubMed]
26.
De Pasquale, I.; Calasso, M.; Mancini, L.; E colini, D.; La S o ia, A.; De Angelis, M.; Di Cagno, R.; Gobbe i, M. Causal Rela ionship
be ween Mic obial Ecology Dynamics and P o eolysis du ing Manu ac u e and Ripening o P o ec ed Designa ion o O igin
(PDO) Cheese Canes a o Pugliese. Appl. En i on. Mic obiol. 2014,80, 4085–4094. [C ossRe ]
27.
E colini, D.; De Filippis, F.; La S o ia, A.; Iacono, M. “Remake” by High-Th oughpu Sequencing o he Mic obio a In ol ed in
he P oduc ion o Wa e Bu alo Mozza ella Cheese. Appl. En i on. Mic obiol. 2012,78, 8142–8145. [C ossRe ]
28.
Michailidou, S.; Pa lou, E.; Pasen sis, K.; Rhoades, J.; Liko a i i, E.; A gi iou, A. Mic obial P o iles o G eek PDO Cheeses
Assessed wi h Amplicon Me aba coding. Food Mic obiol. 2021,99, 103836. [C ossRe ]
29.
Jiang, Y.; Yu, P.; Liu, X.; Zhao, J.; Zhang, H.; Chen, W. Shi s in Di e si y and Func ion o Bac e ial Communi y du ing Manu ac u e
o Rushan. J. Dai y Sci. 2021,104, 12375–12393. [C ossRe ]
30.
Zhang, F.; Wang, Z.; Lei, F.; Wang, B.; Jiang, S.; Peng, Q.; Zhang, J.; Shao, Y. Bac e ial Di e si y in Goa Milk om he Guanzhong
A ea o China. J. Dai y Sci. 2017,100, 7812–7824. [C ossRe ]
31.
Ab iouel, H.; Ma ín-Pla e o, A.; Maqueda, M.; Valdi ia, E.; Ma ínez-Bueno, M. Biodi e si y o he Mic obial Communi y in a
Spanish Fa mhouse Cheese as Re ealed by Cul u e-Dependen and Cul u e-Independen Me hods. In . J. Food Mic obiol.
2008
,
127, 200–208. [C ossRe ]
32.
Aleg ía, Á.; Szczesny, P.; Mayo, B.; Ba dowski, J.; Kowalczyk, M. Biodi e si y in Oscypek, a T adi ional Polish Cheese, De e mined
by Cul u e-Dependen and -Independen App oaches. Appl. En i on. Mic obiol. 2012,78, 1890–1898. [C ossRe ]
33.
Masoud, W.; Vogensen, F.K.; Lille ang, S.; Abu Al-Soud, W.; Sø ensen, S.J.; Jakobsen, M. The Fa e o Indigenous Mic obio a,
S a e Cul u es, Esche ichia Coli,Lis e ia Innocua and S aphylococcus Au eus in Danish Raw Milk and Cheeses De e mined by
Py osequencing and Quan i a i e Real Time (QRT)-PCR. In . J. Food Mic obiol. 2012,153, 192–202. [C ossRe ] [PubMed]
34.
Ya za, P.; Yilmaz, P.; P uesse, E.; Glöckne , F.O.; Ludwig, W.; Schlei e , K.H.; Whi man, W.B.; Euzéby, J.; Amann, R.; Rosselló-Mó a,
R. Uni ing he Classi ica ion o Cul u ed and Uncul u ed Bac e ia and A chaea Using 16S RRNA Gene Sequences. Na . Re .
Mic obiol. 2014,12, 635–645. [C ossRe ] [PubMed]
35.
Be ani, G.; Le an e, A.; Lazzi, C.; Bo a i, B.; Ga i, M.; Ne iani, E. Dynamics o a Na u al Bac e ial Communi y unde
Technological and En i onmen al P essu es: The Case o Na u al Whey S a e o Pa migiano Reggiano Cheese. Food Res. In .
2020,129, 108860. [C ossRe ] [PubMed]
36.
De Paula, A.C.L.; Medei os, J.D.; Fe nandes, G.R.; da Sil a, V.L.; Diniz, C.G. Mic obiome o Indus ialized Minas F escal Cheese
Re eals High P e alence o Pu a i e Bac e ia: A Conce n in he One Heal h Con ex . LWT 2021,139, 110791. [C ossRe ]
37.
Ca dinali, F.; Fe ocino, I.; Milano i´c, V.; Belleggia, L.; Co aglia, M.R.; Ga o alo, C.; Foligni, R.; Mannozzi, C.; Mozzon, M.;
Cocolin, L.; e al. Mic obial Communi ies and Vola ile P o ile o Queijo de Azei ão PDO Cheese, a T adi ional Medi e anean
This le-Cu dled Cheese om Po ugal. Food Res. In . 2021,147, 110537. [C ossRe ]
38.
Dimo , S.G.; Gyu o a, A.; Zago che , L.; Dimi o , T.; Geo gie a-Mi e a, D.; Peyko , S. NGS-Based Me agenomic S udy o Fou
T adi ional Bulga ian G een Cheeses om Tche ni Vi . LWT 2021,152, 112278. [C ossRe ]
Biology 2022,11, 769 19 o 21
39.
Gaglio, R.; F anciosi, E.; Toda o, A.; Gua cello, R.; Al eo, V.; Randazzo, C.L.; Se anni, L.; Toda o, M. Addi ion o Selec ed
S a e /Non-S a e Lac ic Acid Bac e ial Inoculums o S abilise PDO Peco ino Siciliano Cheese P oduc ion. Food Res. In .
2020
,
136, 109335. [C ossRe ]
40.
End es, C.M.; Cas o, Í.M.S.; T e isol, L.D.; Se e o, J.M.; Mann, M.B.; Va ela, A.P.M.; F azzon, A.P.G.; Maye , F.Q.; F azzon, J.
Molecula Cha ac e iza ion o he Bac e ial Communi ies P esen in Sheep’s Milk and Cheese P oduced in Sou h B azilian Region
ia 16S RRNA Gene Me aba coding Sequencing. LWT 2021,147, 111579. [C ossRe ]
41.
Es eban-Blanco, C.; Gu ié ez-Gil, B.; Puen e-Sánchez, F.; Ma ina, H.; Tamames, J.; Acedo, A.; A anz, J.J. Mic obio a Cha ac e iza-
ion o Sheep Milk and I s Associa ion wi h Soma ic Cell Coun Using 16s RRNA Gene Sequencing. J. Anim. B eed. Gene .
2020
,
137, 73–83. [C ossRe ]
42.
Biçe , Y.; Telli, A.E.; Sönmez, G.; Telli, N.; Uça , G. Compa ison o Mic obio a and Vola ile O ganic Compounds in Milk om
Di e en Sheep B eeds. J. Dai y Sci. 2021,104, 12303–12311. [C ossRe ]
43.
Ca dinali, F.; Osimani, A.; Tacca i, M.; Milano i´c, V.; Ga o alo, C.; Clemen i, F.; Pol e igiani, S.; Zi i, S.; Ra aelli, N.; Mozzon, M.;
e al. Impac o This le Renne om Ca lina Acan hi olia All. Subsp. Acan hi olia on Bac e ial Di e si y and Dynamics o a Special y
I alian Raw Ewes’ Milk Cheese. In . J. Food Mic obiol. 2017,255, 7–16. [C ossRe ] [PubMed]
44.
Ramezani, M.; Hosseini, S.M.; Fe ocino, I.; Amoozega , M.A.; Cocolin, L. Molecula In es iga ion o Bac e ial Communi ies
du ing he Manu ac u ing and Ripening o Semi-Ha d I anian Liq an Cheese. Food Mic obiol.
2017
,66, 64–71. [C ossRe ]
[PubMed]
45.
Bus aman e, M.; Chá a i, F.; San is eban, A.; Ceballos, G.; He nández, I.; JoséMiguélez, M.; A anbu u, I.; Ba ón, L.J.R.; Vi o,
M.; De Renobales, M. Coagula ing and Lipoly ic Ac i i ies o A isanal Lamb Renne Pas es. J. Dai y Res.
2000
,67, 393–402.
[C ossRe ] [PubMed]
46.
E kus, O.; de Jage , V.C.L.; Geene, R.T.C.M.; an Alen-Boe ig e , I.; Hazelwood, L.; an Hijum, S.A.F.T.; Klee ebezem, M.; Smid,
E.J. Use o P opidium Monoazide o Selec i e P o iling o Viable Mic obial Cells du ing Gouda Cheese Ripening. In . J. Food
Mic obiol. 2016,228, 1–9. [C ossRe ] [PubMed]
47.
Klindwo h, A.; P uesse, E.; Schwee , T.; Peplies, J.; Quas , C.; Ho n, M.; Glöckne , F.O. E alua ion o Gene al 16S Ribosomal
RNA Gene PCR P ime s o Classical and Nex -Gene a ion Sequencing-Based Di e si y S udies. Nucleic Acids Res.
2013
,41, e1.
[C ossRe ]
48.
Meye , F.; Paa mann, D.; D’Souza, M.; Olson, R.; Glass, E.M.; Kubal, M.; Paczian, T.; Rod iguez, A.; S e ens, R.; Wilke, A.; e al.
The Me agenomics RAST Se e —A Public Resou ce o he Au oma ic Phylogene ic and Func ional Analysis o Me agenomes.
BMC Bioin o m. 2008,9, 386. [C ossRe ]
49.
P uesse, E.; Quas , C.; Kni el, K.; Fuchs, B.M.; Ludwig, W.; Peplies, J.; Glöckne , F.O. SILVA: A Comp ehensi e Online Resou ce
o Quali y Checked and Aligned Ribosomal RNA Sequence Da a Compa ible wi h ARB. Nucleic Acids Res. 2007,35, 7188–7196.
[C ossRe ]
50.
Johnson, J.S.; Spakowicz, D.J.; Hong, B.Y.; Pe e sen, L.M.; Demkowicz, P.; Chen, L.; Leopold, S.R.; Hanson, B.M.; Ag es a, H.O.;
Ge s ein, M.; e al. E alua ion o 16S RRNA Gene Sequencing o Species and S ain-Le el Mic obiome Analysis. Na . Commun.
2019,10, 5029. [C ossRe ]
51.
R Co e Team. The R P ojec o S a is ical Compu ing. A ailable online: h ps://www. -p ojec .o g/ (accessed on 15 Ma ch 2020).
52.
Wa nes, G.R.; Bolke , B.; Bonebakke , L.; Gen leman, R.; Hube , W.; Liaw, A.; Lumley, T.; Maechle , M.; Magnusson, A.; Moelle , S.;
e al. Package “Gplo s” Ti le Va ious R P og amming Tools o Plo ing Da a [R Package Gplo s Ve sion 3.1.1]. A ailable online:
h ps://c an. -p ojec .o g/package=gplo s (accessed on 15 Janua y 2021).
53.
Oksanen, J.; Blanche , F.G.; F iendly, M.; Kind , R.; Legend e, P.; Mcglinn, D.; Minchin, P.R.; O’ha a, R.B.; Simpson, G.L.; Solymos, P.;
e al. Package “Vegan”. A ailable online: h ps://c an. -p ojec .o g/web/packages/ egan/ egan.pd (accessed on 15 Janua y 2021).
54.
Wickham, H.; A e ick, M.; B yan, J.; Chang, W.; McGowan, L.; F ançois, R.; G olemund, G.; Hayes, A.; Hen y, L.; Hes e , J.; e al.
Welcome o he Tidy e se. J. Open Sou ce So w. 2019,4, 1686. [C ossRe ]
55.
Kind , R.; Coe, R. T ee Di e si y Analysis: A Manual and So wa e o Common S a is ical Me hods o Ecological and Biodi e si y S udies;
Wo ld Ag o o es y Cen e (ICRAF): Nai obi, Kenya, 2005; ISBN 92 9059 179 X.
56.
Pa adis, E.; Claude, J.; S imme , K. APE: Analyses o Phylogene ics and E olu ion in R Language. Bioin o ma ics
2004
,20, 289–290.
[C ossRe ]
57.
Fox, P.F.; Guinee, T.P.; Cogan, T.M.; McSweeney, P.L.H. Fundamen als o Cheese Science, 2nd ed.; Fox, P.F., Guinee, T.P., Cogan, T.M.,
McSweeney, P.L.H., Eds.; Sp inge : New Yo k, NY, USA, 2017; ISBN 9781489976819.
58.
Quin ana, Á.R.; Seseña, S.; Ga zón, A.; A ias, R. Fac o s A ec ing Le els o Ai bo ne Bac e ia in Dai y Fa ms: A Re iew. Animals
2020,10, 526. [C ossRe ] [PubMed]
59.
Van den B om, R.; de Jong, A.; an Engelen, E.; Heu elink, A.; Vellema, P. Zoono ic Risks o Pa hogens om Sheep and Thei
Milk Bo ne T ansmission. Small Rumin. Res. 2020,189, 106123. [C ossRe ] [PubMed]
60.
Lu, M.; Wang, N.S. Spoilage o Milk and Dai y P oduc s. In The Mic obiological Quali y o Food: Foodbo ne Spoile s; Be ilacqua, A.,
Co bo, M.R., Sinigaglia, M., Eds.; Woodhead Publishing: Dux o d, UK, 2017; pp. 151–178. ISBN 9780081005033.
61.
Wang, Y.; Wu, J.; L , M.; Shao, Z.; Hungwe, M.; Wang, J.; Bai, X.; Xie, J.; Wang, Y.; Geng, W. Me abolism Cha ac e is ics o Lac ic
Acid Bac e ia and he Expanding Applica ions in Food Indus y. F on . Bioeng. Bio echnol. 2021,9, 612285. [C ossRe ] [PubMed]
62.
Pa adh, A.D. G am-Nega i e Spoilage Bac e ia in B ewing. In B ewing Mic obiology: Managing Mic obes, Ensu ing Quali y and
Valo ising Was e; Hill, A.E., Ed.; Else ie L d.: Ams e dam, The Ne he lands, 2015; pp. 175–194. ISBN 9781782423492.
Biology 2022,11, 769 20 o 21
63.
S aley, J.T. The Genus P os hecobac e . In The P oka yo es; S a , M.P., S olp, H., T üpe , H.G., Balows, A., Schlegel, H.G., Eds.;
Sp inge : New Yo k, NY, USA, 1981; pp. 477–479. ISBN 9783662131879.
64.
Suchodolski, J.S. Gas oin es inal Mic obio a. In Canine and Feline Gas oen e ology; Washabau, R.J., Day, M.J., Eds.; W.B. Saunde s:
S . Louis, MO, USA, 2012; pp. 32–41. ISBN 9781416036616.
65.
Va ašˇcino á, T.; Pipo á, M.; F aqueza, M.J.R.; Mal’a, P.; Dud iko á, E.; D ážo ská, M.; Lauko á, A. Sho Communica ion:
An imic obial Po en ial o Lac obacillus plan a um S ains Isola ed om Slo ak Raw Sheep Milk Cheeses. J. Dai y Sci.
2020
,103,
6900–6903. [C ossRe ]
66.
Fala deau, J.; Keeney, K.; T mˇci´c, A.; Ki s, D.; Wang, S. Fa m- o-Fo k P o iling o Bac e ial Communi ies Associa ed wi h an
A isan Cheese P oduc ion Facili y. Food Mic obiol. 2019,83, 48–58. [C ossRe ]
67.
Bellassi, P.; Rocche i, G.; Noce i, M.; Lucini, L.; Masoe o, F.; Mo elli, L. A Combined Me abolomic and Me agenomic App oach
o Disc imina e Raw Milk o he P oduc ion o Ha d Cheese. Foods 2021,10, 109. [C ossRe ]
68.
Yelu i Jonnala, B.R.; McSweeney, P.L.H.; Sheehan, J.J.; Co e , P.D. Sequencing o he Cheese Mic obiome and I s Rele ance o
Indus y. F on . Mic obiol. 2018,9, 1020. [C ossRe ]
69.
Ac o , J.K. Clinical Bac e iology. In Else ie ’s In eg a ed Re iew Immunology and Mic obiology; Ac o , J.K., Ed.; W.B. Saunde s:
Philadelphia, PA, USA, 2012; pp. 105–120. ISBN 978-0-323-07447-6.
70.
An onello, V.S.; Dallé, J.; Domingues, G.C.; Fe ei a, J.A.S.; Fon ou a, M.d.C.Q.; Knapp, F.B. Pos -Cesa ean Su gical Si e In ec ion
Due o Bu iauxella ag es is.In . J. In ec . Dis. 2014,22, 65–66. [C ossRe ]
71.
Be gogne-Bé ézin, E. Bac e ia: Acine obac e . In Encyclopedia o Food Sa e y; Mo a jemi, Y., Ed.; Academic P ess: San Diego, CA,
USA, 2014; Volume 1, pp. 337–341. ISBN 9780123786128.
72.
Fusco, V.; Ab iouel, H.; Benoma , N.; Kabisch, J.; Chie i, D.; Cho, G.-S.; F anz, C.M.A.P. Oppo unis ic Food-Bo ne Pa hogens. In
Food Sa e y and P ese a ion; G umezescu, A.M., Holban, A.M., Eds.; Academic P ess: San Diego, CA, USA, 2018; pp. 269–306.
ISBN 9780128149560.
73.
Guiu, A.; Buendía, B.; Llo ca, L.; Gómez Pun e , R.M.; Gi ón, R. Ch yseobac e ium spp., a New Oppo unis ic Pa hogen Associa ed
wi h Cys ic Fib osis? En e m. In ecc. Mic obiol. Clin. 2014,32, 497–501. [C ossRe ]
74.
Li, N.; Wang, Y.; You, C.; Ren, J.; Chen, W.; Zheng, H.; Liu, Z. Va ia ion in Raw Milk Mic obio a h oughou 12 Mon hs and he
Impac o Wea he Condi ions. Sci. Rep. 2018,8, 2371. [C ossRe ]
75.
Mo in, A. Pan oea. In Encyclopedia o Food Mic obiology, 2nd ed.; Ba , C.A., To o ello, M.L., Eds.; Academic P ess: San Diego, CA,
USA, 2014; pp. 1028–1032. ISBN 9780123847331.
76.
Mu phy, E.C.; Mö gelin, M.; Cooney, J.C.; F ick, I.M. In e ac ion o Bac e oides agilis and Bac e oides he aio aomic on wi h he
Kallik ein-Kinin Sys em. Mic obiology 2011,157, 2094–2105. [C ossRe ] [PubMed]
77.
Ti éca , M.; Walle , F.; Vieilla d, M.H.; Cou col, R.J.; Loïez, C. Ruminococcus gna us: An Unusual Pa hogen in Sep ic A h i is.
Anae obe 2014,30, 159–160. [C ossRe ] [PubMed]
78.
Wa´skiewicz, A.; I zykowska, L. Fla obac e ium spp.—Cha ac e is ics, Occu ence, and Toxici y. In Encyclopedia o Food Mic obiol-
ogy, 2nd ed.; Ba , C.A., To o ello, M.L., Eds.; Academic P ess: San Diego, CA, USA, 2014; pp. 938–942. ISBN 9780123847331.
79.
Appel, T.M.; Quijano-Ma ínez, N.; De La Cadena, E.; Mojica, M.F.; Villegas, M.V. Mic obiological and Clinical Aspec s o
Raoul ella spp. F on . Public Heal h 2021,9, 686789. [C ossRe ] [PubMed]
80.
Lo, R.; Xue, T.; Weeks, M.; Tu ne , M.S.; Bansal, N. Inhibi ion o Bac e ial G ow h in Swee Cheese Whey by Ca bon Dioxide as
De e mined by Cul u e-Independen Communi y P o iling. In . J. Food Mic obiol. 2016,217, 20–28. [C ossRe ]
81.
Mo andi, S.; Ba elli, G.; Sil e i, T.; T ingali, S.; Nunzia a, L.; Villa, A.; Acquis apace, A.; B asca, M. Impac o Sal ing and Ripening
Tempe a u es on La e Blowing De ec in Val ellina Case a PDO Cheese. Food Con ol 2021,120, 107508. [C ossRe ]
82.
Fo ou, K.; Tzo a, A.; Voida ou, C.; Alexopoulos, A.; Plessas, S.; A ge is, I.; Bezi zoglou, E.; Ak ida-Deme zi, K.; Deme zis, P.G.
Isola ion o Mic obial Pa hogens o Subclinical Mas i is om Raw Sheep’s Milk o Epi us (G eece) and Thei Role in I s Hygiene.
Anae obe 2011,17, 315–319. [C ossRe ]
83.
Fuka, M.M.; Engel, M.; Skelin, A.; Redžepo i´c, S.; Schlo e , M. Bac e ial Communi ies Associa ed wi h he P oduc ion o A isanal
Is ian Cheese. In . J. Food Mic obiol. 2010,142, 19–24. [C ossRe ]
84.
Ru alcaba-Gómez, J.M.; Delgado-Macuil, R.J.; Zelaya-Molina, L.X.; Maya-Lucas, O.; Ruesga-Gu ié ez, E.; Anaya-Espa za,
L.M.; de la Mo a, Z.V.; López-De la Mo a, D.A.; A eaga-Ga ibay, R.I. Bac e ial Succession h ough he A isanal P ocess and
Seasonal E ec s De ining Bac e ial Communi ies o Raw-Milk Adobe a Cheese Re ealed by High Th oughpu Dna Sequencing.
Mic oo ganisms 2021,9, 24. [C ossRe ]
85.
Bowman, J.P. The Genus Psych obac e . In The P oka yo es; Dwo kin, M., Falkow, S., Rosenbe g, E., Schlei e , K., S ackeb and , E.,
Eds.; Sp inge : New Yo k, NY, USA, 2006; pp. 920–930. ISBN 9780387307466.
86.
Shwe a, F.N.U.; Gu am, P.R.; O’Ho o, J.C.; Khalil, S. B e ibac e ium Species: An Eme ging Oppo unis ic Cause o Bloods eam
In ec ions. Mayo Clin. P oc. 2021,96, 1093–1094. [C ossRe ]
87.
Yin, Z.; Yuan, C.; Du, Y.; Yang, P.; Qian, C.; Wei, Y.; Zhang, S.; Huang, D.; Liu, B. Compa a i e Genomic Analysis o he
Ha nia Genus Re eals an Explici E olu iona y Rela ionship be ween he Species Al ei and Pa al ei and P o ides Insigh s in o
Pa hogenici y. BMC Genom. 2019,20, 768. [C ossRe ]
88.
Anas , J.M.; Dzieciol, M.; Schul z, D.L.; Wagne , M.; Mann, E.; Schmi z-Esse , S. B e ibac e ium om Aus ian Ha d Cheese Ha bo
a Pu a i e His amine Ca abolism Pa hway and a Plasmid o Adap a ion o he Cheese En i onmen . Sci. Rep.
2019
,9, 6164.
[C ossRe ] [PubMed]
Biology 2022,11, 769 21 o 21
89.
Oz u koglu-Budak, S.; Wiebenga, A.; B on, P.A.; de V ies, R.P. P o ease and Lipase Ac i i ies o Fungal and Bac e ial S ains
De i ed om an A isanal Raw Ewe’s Milk Cheese. In . J. Food Mic obiol. 2016,237, 17–27. [C ossRe ] [PubMed]
90.
Zhang, D.; Palme , J.; Teh, K.H.; Flin , S. Iden i ica ion and Selec ion o Hea -S able P o ease and Lipase-P oducing Psych o ophic
Bac e ia om F esh and Chilled Raw Milk du ing up o Fi e Days S o age. LWT 2020,134, 110165. [C ossRe ]
91.
De Pasquale, I.; Di Cagno, R.; Buchin, S.; De Angelis, M.; Gobbe i, M. Spa ial Dis ibu ion o he Me abolically Ac i e Mic obio a
wi hin I alian PDO Ewes’ Milk Cheeses. PLoS ONE 2016,11, e0153213. [C ossRe ]
92.
Dee ae, P.; Bonna me, P.; Spinnle , H.E.; Helinck, S. P oduc ion o Vola ile A oma Compounds by Bac e ial S ains Isola ed om
Di e en Su ace-Ripened F ench Cheeses. Appl. Mic obiol. Bio echnol. 2007,76, 1161–1171. [C ossRe ]
93.
I linge , F.; In Yung, S.A.Y.; Sa hou, A.S.; Delbès-Paus, C.; Mon el, M.C.; Co on, E.; Co on, M.; Helinck, S. Ecological and A oma ic
Impac o Two G am-Nega i e Bac e ia (Psych obac e cele and Ha nia al ei) Inocula ed as Pa o he Whole Mic obial Communi y
o an Expe imen al Smea So Cheese. In . J. Food Mic obiol. 2012,153, 332–338. [C ossRe ]
94.
Quillaguamán, J.; Delgado, O.; Ma iasson, B.; Ha i-Kaul, R. Ch omohalobac e sa ecensis sp. No ., a Psych o ole an Mode a e
Halophile Isola ed om he Saline Andean Region o Boli ia. In . J. Sys . E ol. Mic obiol. 2004,54, 1921–1926. [C ossRe ]
95.
Fuka, M.M.; Wallisch, S.; Engel, M.; Welzl, G.; Ha anek, J.; Schlo e , M. Dynamics o Bac e ial Communi ies du ing he Ripening
P ocess o Di e en C oa ian Cheese Types De i ed om Raw Ewe’s Milk Cheeses. PLoS ONE. 2013,8, e80734. [C ossRe ]
96.
Ga cía-López, M.L.; San os, J.A.; O e o, A.; Rod íguez-Calleja, J.M. Psych obac e . In Encyclopedia o Food Mic obiology, 2nd ed.;
Ba , C.A., To o ello, M.L., Eds.; Else ie : Ams e dam, The Ne he lands, 2014; pp. 261–268. ISBN 9780123847331.
97.
He nández, I.; Ba ón, L.J.R.; Vi o, M.; Pé ez-Elo ondo, F.J.; Flanagan, C.; Rozas, U.; Náje a, A.I.; Albisu, M.; Vicen e, M.S.; de
Renobales, M. Lipolysis, P o eolysis and Senso y P ope ies o Ewe’s Raw Milk Cheese (Idiazabal) Made wi h Lipase Addi ion.
Food Chem. 2009,116, 158–166. [C ossRe ]
98.
Gil, P.F.; Conde, S.; Albisu, M.; Pé ez-Elo ondo, F.J.; E ayo, I.; Vi o, M.; De Renobales, M. Hygienic Quali y o Ewes’ Milk
Cheeses Manu ac u ed wi h A isan-P oduced Lamb Renne Pas es. J. Dai y Res. 2007,74, 329–335. [C ossRe ]
99.
E ayo, I.; Pé ez Elo ondo, F.J.; Gil, P.F.; Albisu, M.; Vi o, M.; Conde, S.; Rod iguez Ba on, L.J.; Náje a, A.I.; Gómez-Hidalgo,
M.E.; Delgado, C.; e al. Hygienic Quali y, Lipolysis and Senso y P ope ies o Spanish P o ec ed Designa ion o O igin Ewe’s
Milk Cheeses Manu ac u ed wi h Lamb Renne Pas e. Lai 2006,86, 415–434. [C ossRe ]
100.
Bokulich, N.A.; Mills, D.A. Facili y-Speci ic “House” Mic obiome D i es Mic obial Landscapes o A isan Cheesemaking Plan s.
Appl. En i on. Mic obiol. 2013,79, 5214–5223. [C ossRe ] [PubMed]
101.
Blaya, J.; Ba zideh, Z.; LaPoin e, G. Symposium Re iew: In e ac ion o S a e Cul u es and Nons a e Lac ic Acid Bac e ia in he
Cheese En i onmen . J. Dai y Sci. 2018,101, 3611–3629. [C ossRe ] [PubMed]
102.
F an zen, C.A.; Ko , W.; Pede sen, T.B.; A dö, Y.M.; B oadben , J.R.; Ne e, H.; Hansen, L.H.; Dal Bello, F.; Øs lie, H.M.; Kleppen,
H.P.; e al. Genomic Cha ac e iza ion o Dai y Associa ed Leuconos oc Species and Di e si y o Leuconos ocs in Unde ined Mixed
Mesophilic S a e Cul u es. F on . Mic obiol. 2017,8, 132. [C ossRe ]
103.
Gobbe i, M.; De Angelis, M.; Di Cagno, R.; Mancini, L.; Fox, P.F. P os and Cons o Using Non-S a e Lac ic Acid Bac e ia
(NSLAB) as Seconda y/Adjunc S a e s o Cheese Ripening. T ends Food Sci. Technol. 2015,45, 167–178. [C ossRe ]
104.
Li, S.; Li, Y.; Du, Z.; Li, B.; Liu, Y.; Gao, Y.; Zhang, Y.; Zhang, K.; Wang, Q.; Lu, S.; e al. Impac o NSLAB on Kazakh Cheese Fla o .
Food Res. In . 2021,144, 110315. [C ossRe ]
105.
Mohamaden, W.I.; Zhen- en, Z.; Hegab, I.M.; Shang-li, S. Expe imen al In ec ion in Mice wi h E winia pe sicina.Mic ob. Pa hog.
2019,130, 38–43. [C ossRe ]
106.
Quigley, L.; O’Sulli an, O.; Be es o d, T.P.; Ross, R.P.; Fi zge ald, G.F.; Co e , P.D. High-Th oughpu Sequencing o De ec ion
o Subpopula ions o Bac e ia No P e iously Associa ed wi h A isanal Cheeses. Appl. En i on. Mic obiol.
2012
,78, 5717–5723.
[C ossRe ]
107.
Sádecká, J.; Šako á, N.; Pangallo, D.; Ko eˇno á, J.; Kolek, E.; Pušká o á, A.; Buˇcko á, M.; Valík, L.; Kuch a, T. Mic obial Di e si y
and Vola ile Odou -Ac i e Compounds o Ba elled Ewes’ Cheese as an In e media e P oduc Tha De e mines he Quali y o
Win e B yndza Cheese. LWT 2016,70, 237–244. [C ossRe ]
108.
D’Amico, D.J.; Donnelly, C.W. G ow h and Su i al o Mic obial Pa hogens in Cheese. In Cheese: Chemis y, Physics and
Mic obiology, 4 h ed.; McSweeney, P.L.H., Fox, P.F., Co e , P.D., E e e , D.W., Eds.; Academic P ess: San Diego, CA, USA, 2017;
Volume 1, pp. 573–594. ISBN 9780122636530.
109.
Gon ijo, M.T.P.; Sil a, J.d.S.; Vidigal, P.M.P.; Ma in, J.G.P. Phylogene ic Dis ibu ion o he Bac e iocin Repe oi e o Lac ic Acid
Bac e ia Species Associa ed wi h A isanal Cheese. Food Res. In . 2020,128, 108783. [C ossRe ]
110.
Kaya, H.I.; Simsek, O. Cha ac e iza ion o Pa hogen-Speci ic Bac e iocins om Lac ic Acid Bac e ia and Thei Applica ion wi hin
Cock ail agains Pa hogens in Milk. LWT 2019,115, 108464. [C ossRe ]
111.
Macedo, A.C.; Malca a, F.X.; Oli ei a, J.C. E ec o P oduc ion Fac o s and Ripening Condi ions on he Cha ac e is ics o Se a
Cheese. In . J. Food Sci. Technol. 1997,32, 501–511. [C ossRe ]
