Ci a ion: Nunes, A.R.;
Sánchez-Juanes, F.; Gonçal es, A.C.;
Al es, G.; Sil a, L.R.; Flo es-Félix, J.D.
E alua ion o Raw Cheese as a No el
Sou ce o Bio e ilize wi h a High
Le el o Biosecu i y o Bluebe y.
Ag onomy 2022,12, 1150. h ps://
doi.o g/10.3390/ag onomy12051150
Academic Edi o : Alain Deloi e
Recei ed: 11 Ap il 2022
Accep ed: 6 May 2022
Published: 10 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/).
ag onomy
A icle
E alua ion o Raw Cheese as a No el Sou ce o Bio e ilize
wi h a High Le el o Biosecu i y o Bluebe y
Ana R. Nunes 1,2, Fe nando Sánchez-Juanes 3,4, Ana C. Gonçal es 1,5 , Gilbe o Al es 1, Luís R. Sil a 1,6 and
JoséDa id Flo es-Félix 1,*
1CICS-UBI—Heal h Sciences Resea ch Cen e, Uni e si y o Bei a In e io , 6201-506 Co ilha, Po ugal;
[email p o ec ed] (A.R.N.); [email p o ec ed] (A.C.G.); [email p o ec ed] (G.A.);
[email p o ec ed] (L.R.S.)
2CNC—Cen e o Neu oscience and Cell Biology, Uni e si y o Coimb a, 3004-504 Coimb a, Po ugal
3Ins i u o de In es igación Biomédica de Salamanca (IBSAL), Complejo Asis encial Uni e si a io de
Salamanca, Uni e sidad de Salamanca, CSIC, 37007 Salamanca, Spain; [email p o ec ed]
4Depa amen o de Bioquímica y Biología Molecula , Uni e sidad de Salamanca, 37007 Salamanca, Spain
5Labo a o y o Pha macology, Facul y o Pha macy, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal
6CPIRN-UDI/IPG—Cen e o Po en ial and Inno a ion o Na u al Resou ces, Resea ch Uni o Inland
De elopmen (UDI), Poly echnic Ins i u e o Gua da, 6300-559 Gua da, Po ugal
*Co espondence: jd lo [email p o ec ed]
Abs ac :
Today’s ag icul u e equi es he sea ch o new and mode n ools in o de o imp o e and
expand he use o i s c ops and o inc ease hei sus ainabili y. The use o plan g ow h-p omo ing PGP
bac e ia (PGPB) is he a ge o much esea ch and seems o be an ideal s a egy as long as he s ains
a e p ope ly selec ed o his pu pose. Among he bac e ia, lac ic acid bac e ia (LAB) a e conside ed
a sui able al e na i e due o hei high biosa e y and mechanisms o p omo ing plan g ow h. In
iew o his, in his wo k we decided o isola e LAB wi h PGP capaci y om aw milk cheese o
he PDO “Se a da Es ela”. A o al o 88 s ains wi h a high di e si y and ema kable capaci y
o con ol ood-bo ne and pa hogenic mic oo ganisms we e isola ed. In addi ion, mos o hem
showed excellen capaci ies o phospha e solubiliza ion and he p oduc ion o indole-3-ace ic acid
and side opho es. Subsequen ly, we also s udied hei inocula ion in bluebe y seedlings. Among
he isola es, s ains QSE20, QSE62 and QSE79 showed he mos ema kable abili y o e icien ly
colonize he hizosphe e o his plan , imp o ing oo de elopmen and inc easing he numbe o
seconda y oo s.
Keywo ds: lac ic bac e ia; PGPB; bluebe y cul u es; oo coloniza ion; aw milk cheese
1. In oduc ion
The s udy o lac ic acid bac e ia (LAB) associa ed wi h he e men a ion p ocesses o
dai y p oduc s, such as cheese, has shown ha hese popula ions play a c ucial ole in he
o ganolep ic p ope ies o e men ed dai y p oduc s [
1
]. Indus ial cheese manu ac u ing
p ocesses a e based on he elimina ion o maximum educ ion o he na i e popula ions
o mic oo ganisms p esen in milk h ough he pas eu iza ion p ocess and he addi ion
o s a e s o con ol he e men a ion p ocess [
2
]. Ne e heless, he e a e a la ge numbe
o cheeses ha belong o adi ional a ie ies and hei p oduc ion equi es he use o
aw milk [
3
]. The e o e, hese cheeses a e p oduced acco ding o adi ional me hods [
3
]
and in compliance wi h P o ec ed Designa ion o O igin (PDO) egula ions [
4
,
5
]. As a
as we know, hey con ain a high di e si y o LAB, bo h a he speci ic and in aspeci ic
le els [
6
–
8
], he o igin o which is ela ed o ho izon al and e ical con aminan s in he
en i onmen o he milk p oduc ion and ex ac ion [
9
]. On he Ibe ian Peninsula, especially
in he inne -wes e n egion, he e is a pa icula a ie y made om aw milk and cu dled
wi h ege able enne om Cyna a ca dunculus [
10
], esul ing in so cheeses wi h nume ous
Ag onomy 2022,12, 1150. h ps://doi.o g/10.3390/ag onomy12051150 h ps://www.mdpi.com/jou nal/ag onomy
Ag onomy 2022,12, 1150 2 o 20
egional a ian s in which e ical ansmission is enhanced, among he well-known a e
To a del Casa cheese, Azei ão cheese and Se a da Es ela cheese [4,8,11,12].
Conside ing ha he use o bac e ial gene a poses some biological isk, se e al e o s
ha e been made in ecen yea s o sea ch o new mic oo ganisms ha ha e he po en ial
o be used as bio e ilize s and bios imulan s wi h no, o ewe , oxic e ec s. Among he
mos s udied mic oo ganisms in he las ew yea s, Se a ia o Pan oea s and ou , bu se e al
species and s ains om hese gene a ha e heal h implica ions due o hei pa hogen po en-
ial [
13
,
14
]. Since LAB a e able o p oduce bac e iocins and con ol pa hogenic bac e ia in
ood o p e en ou b eaks associa ed wi h he consump ion o esh ui s and ege ables, i
is no su p ising ha hey a e also a a ge o many s udies [
15
]. As a as we know, he use
o LAB as plan g ow h-p omo ing bac e ia (PGPB), especially om he plan en i onmen ,
is suppo ed by hei abili y o exe plan g ow h-p omo ing mechanisms, highligh ing
he p oduc ion o side opho es, phy oho mones and/o phospha e solubiliza ion [
16
]. In
addi ion, hey can p oduce seconda y me aboli es ha ha e he abili y o con ol bac e ial
and ungal phy opa hogens by eleasing ly ic enzymes, compe ing o space and/o p o-
ducing bac e iocins [
17
,
18
]. In his way, LAB ha e op imal p ope ies o posi i ely in e ac
wi h plan s and humans and can be conside ed p obio ics ha play a dual ole as p obio ic
agen s [19].
Howe e , LAB a e no he mos abundan mic oo ganisms in ag icul u al mic o-
biomes [
1
,
17
,
20
–
22
], and hei isola ion om he hizosphe e, phyllosphe e and endophy ic
en i onmen s o a ious plan s can be di icul due o hei me abolic needs and biological
p ope ies, as well as hei g ow h equi emen s, which may be limi ed by he p esence o
o he , mo e abundan axa in hese en i onmen s [23].
Fo his eason, he p esen wo k p oposes o use cheeses made om aw milk om
he PDO “Se a da Es ela”, he di e si y o LAB o which is p ac ically unknown, ocusing
on he po en ial abili y o in e ac wi h plan s. Second, we also p opose he applica ion
o his bac e ia o p omo e plan g ow h o he imp o emen o bluebe y cul i a ion, a
booming c op in he Bei a in e io egion, whe e he PDO “Se a da Es ela” is embedded.
Since his c op mus be de eloped unde acidic condi ions, he use o LAB as bio e ilize
could p esen ad an ages o e o he bac e ial bios imulan s due o he acidophilic na u e
o hese bac e ia, combined wi h i s PGP mechanism and high biosecu i y cha ac e is ics.
2. Ma e ials and Me hods
2.1. S ains Isola ion
Pu a i e bio e ilize s ains we e isola ed om aw cheeses om “Se a da Es ela”
PDO due o he absence o a pas eu iza ion p ocess in milk employed in hei p epa a ion.
A o al o h ee di e en comme cial cheeses om Lac ise L d. Limi ed (Seia, Po ugal)
we e selec ed o he s ain isola ion. Th ee eplica es o each cheese we e analyzed in a
o al o 9 samples. The me hodology used o s ains isola ion was desc ibed by Sanchez-
Juanes e al. [
8
], using he MRS aga (Sigma Co., S . Louis, MO, USA), and he inocula ed
pla es we e incuba ed a 28 ◦C o 48 h.
2.2. MALDI-TOF MS Pe o ming and Da a Analysis
The sample p epa a ion and he MALDI-TOF MS analysis we e ca ied ou as p e-
iously published [
8
], using a sa u a ed ma ix solu ion o
α
-HCCA (B uke Dal onics,
B emen, Ge many) dissol ed in 50% ace oni ile and 2.5% i luo oace ic acid. Amoun s o
biomass be ween 5 and 100 mg we e used o ob ain he spec a indica ed by he manu ac-
u e . The calib a ion mass was done based on he B uke Bac e ial Tes S anda ds (BTS),
using masses as a e ages: RL36, 4365.3 Da; RS22, 5096.8 Da; RL34, 5381.4 Da; RL33me h,
6255.4 Da; RL29, 7274.5 Da; RS19, 10,300.1 Da; RNase A, 13,683.2 Da and myoglobin,
16,952.3 Da.
The sco e alues p oposed by he manu ac u e a e he ollowing: a sco e alue
be ween 2.3 and 3.00 indica es highly p obable species iden i ica ion. A sco e alue be ween
2.0 and 2.299 indica es secu e genus iden i ica ion and p obable species iden i ica ion. A
Ag onomy 2022,12, 1150 3 o 20
sco e alue be ween 1.7 and 1.999 indica es p obable genus iden i ica ion, and a sco e
alue < 1.7 indica es no eliable iden i ica ion.
Clus e analysis was pe o med based on a compa ison o s ain-speci ic main spec a
c ea ed as desc ibed abo e. The dend og am was cons uc ed by he s a is ical oolbox
o Ma lab 7.1 (Ma hWo ks Inc., Na ick, MA, USA) in eg a ed wi h he MALDI Bio ype
3.0 so wa e. The pa ame e se ings we e: ‘Dis ance Measu e = Co ela ion a e age’ and
‘Linkage = Comple e’. The linkage unc ion is no malized acco ding o he dis ance be ween
0 (pe ec ma ch) and 1000 (no ma ch).
2.3. Phylogene ic Analysis o pheS Gene
The ampli ica ion and sequencing o he pheS gene we e ca ied ou as indica ed
by Doan e al. [
24
] using he p ime s pheS-21-F (5
0
-CAYCCNGCHCGYGAYATGC-3
0
) and
pheS-23-R (5
0
-GGRTGRACCATVCCNGCHCC-3
0
). The sequences ob ained we e compa ed
wi h hose om GenBank using he BLASTN p og am [
25
]. The ob ained sequences
and hose o ela ed bac e ia e ie ed om GenBank we e aligned using he Clus al W
p og am [
26
]. The phylogene ic dis ances we e calcula ed acco ding o Kimu a’s wo-
pa ame e model [
27
]. The phylogene ic ees we e in e ed using he Neighbo -joining
model [28], and MEGA 7.09 so wa e [29] was used o all he phylogene ic analyses.
2.4. E alua ion o An imic obial Ac i i y
The e alua ion o he an imic obial ac i i y was pe o med based on he me hod o
aga solid di usion by pe o a ion o he cylind ical ca i ies [
30
]. B ie ly, pa hogenic/
oodbo ne bac e ia we e u he cul i a ed on Muelle –Hin on (MH) a 37
◦
C o 24 h.
The echnique was conduc ed using a suspension wi h a s anda d McFa land u bidi y
o 0.5. Each LAB s ain was g own on MRS b o h o 72 h a 28
◦
C and 125 pm. Nex ,
he medium was cen i uged a 6.000
×
g o 4 min, and each well (4 mm high
×
5 mm
diame e ), was dug in o he Muelle –Hil on (MH) aga pla es (55 mm) and illed wi h
10
µ
L o each sample. The supe na an s we e p epa ed by g owing LAB bac e ia on MRS
b o h a 28
◦
C o 120 h a 125 pm. Subsequen ly, hey we e i s ly cen i uged o 2 min
a 12.000 pm and hen il e ed (0.22
µ
m). Nega i e con ol (MRS b o h) and wo posi i e
con ols. penicillin (0.05 mg/mL) and gen amicin A (10 mg/mL), we e also es ed. The
Pe i dishes we e incuba ed a 37
◦
C o 24 h. The an imic obial e ec was measu ed in
iplica e and de e mined in e ms o inhibi o y zone diame e (IZD) in mm.
2.5. Analysis o Plan G ow h P omo ion Po en ial o Isola ed S ains
The solubiliza ion o insoluble phospha e was analysed on Piko skaya pla es con ain-
ing 2% CaHPO
4
, Ca
3
(HPO
4
)
2,
o hyd oxyapa i e and incuba ed o 15 days a 28
◦
C [
31
].
S anda dized measu emen s we e conduc ed employing a phospha e solubiliza ion index
(PSI), which was calcula ed as he a io be ween he halos a ound he colony and he colony
size. Side opho e p oduc ion was e alua ed in M9-CAS-AGAR [
32
] modi ied wi h he
addi ion o a ca ionic sol en , HDTMA, which s abilizes he Fe–CAS complex, and hence
allows he de ec ion o side opho e p oduc ion due o he appea ance o an o ange halo
a ound colonies. On he o he hand, indole ace ic acid p oduc ion was e alua ed in JMM
medium [
32
] supplemen ed wi h 167 mg/L o yp ophan. A e 7 days o incuba ion,
he supe na an s we e eco e ed by cen i uga ion a 5000
×
gand il e ed using 0.22
µ
m
Millipo e il e s (Millipo e Co., Bu ling on, MA, USA). A e wa ds, 1 mL o Salkowsky
eagen was added o 2 mL o supe na an , and he ed colo o med was measu ed by
spec opho ome y a 550 nm using an ATI Unicam 8625 Spec ome e (Ma son, Madison,
WI, USA).
2.6. In-Plan E alua ion o Plan G ow h P omo ion Abili y
Fo plan coloniza ion assays, 30 seeds o each Vaccinium my illus plan we e su ace
s e ilized by imme sion in 70% e hanol o 30 s ollowed by soaking in an aqueous 5%
sodium hypochlo i e solu ion o 2 min. The seeds we e hen washed six imes wi h
Ag onomy 2022,12, 1150 4 o 20
s e ile wa e and ge mina ed in wa e –aga pla es o e laid wi h Wha man numbe 1 s e ile
pape we ed wi h s e ile wa e . Subsequen ly, seeds we e ans e ed o pla es wi h
Rigaud & Puppo Aga pla es o e laid wi h Wha man numbe 1 s e ile pape . Each seed
was inocula ed wi h 300
µ
L o a suspension o selec ed s ains wi h a concen a ion o
10
7
UFC/mL. The pla es we e hen placed in da kness in a g ow h chambe a 24
◦
C un il
he seedling oo s eme ged. Da a we e collec ed on days 7 and 14 a e inocula ion.
2.7. Coloniza ion Abili y
Plan coloniza ion assays was conduc ed employing seedlings g own in pa allel o
PGP abili y assays unde he same condi ions. The e alua ion o coloniza ion abili ies
o isola ed s ains was de eloped by immuno luo escence loca ion o he cells o he
inocula ed s ains. Inmunoloca ion was pe o med ollowing whole-moun p epa a ion.
Roo s we e excised om he plan and ixed in o maldehyde (4%, / ) o e nigh . Then,
oo s we e blocked on a solu ion o powde ed milk (3%, w/ ) and T i on-X (0.03%, / )
in PBS (pH 7.4) o 1 h a oom empe a u e. A e wa ds, oo s we e washed h ee imes
wi h PBS and incuba ed wi h a p ima y an ibody (G am-Posi i e Ma ke An ibody, San a
C uz Bio echnology, Dallas, TX, USA) o e nigh a 4
◦
C. Nex , oo s we e washed h ee
imes wi h PBS and incuba ed wi h a seconda y an ibody (m-IgGkappa BP-CFL 488, San a
C uz Bio echnology, Dallas, TX, USA). Finally, hey we e washed wi h PBS and s o ed
subme ged in PBS a 4 ◦C and p o ec ed om ligh un il hei use.
Uninocula ed oo s o bluebe ies we e included in he expe imen as nega i e con ols.
Fluo escence mic oscopy was ca ied ou wi h a Zeiss Axio Image A1, and he exci a ion
o g een luo escen p o eins and Alexa-488 luo opho e linked o seconda y an ibody was
accomplished using a me cu y lamp. Roo cells we e s ained wi h 10
µ
M o calco luo
whi e (Sigma, S . Louis, MO, USA).
Coloniza ion densi y was pe o med by qPCR analysis. To al DNA was ex ac ed em-
ploying NZY Plan /Fungi gDNA Isola ion ki (NZYTech, Lisbon, Po ugal) om inocula ed
oo s, measu ing oo sec ion leng h o e e ence he inal alues. The qPCR condi ion was pe -
o med ollowing Pon onio e al. [
33
]. P ime s SK w (5-GGGGATAACAYYTGGAAACAG-3)
and SK w (5-CTCGGCTACGTATCATTGTCTTG-3) we e employed o de ec he concen a-
ion o lac ic bac e ia. Each qPCR eac ion was pe o med in iplica e, using NZYSup eme
qPCR G een Mas e Mix (NZYTech, Lisbon, Po ugal) on a CFX96 Bio ad eal- ime The -
mocycle (Bio-Rad, He cules, CA, USA). The PCR co e p og am was as ollows: 95
◦
C o
10 min, ollowed by 35 cycles o 95
◦
C o 10 s, 55
◦
C o 30 s, 72
◦
C o 30 s and 72
◦
C
o 7 min. Mel cu e analysis o PCR amplicons was ini ia ed a 60
◦
C, inc easing by
1
◦
C un il he inal empe a u e o 95
◦
C was eached. A calib a ion cu e o qPCR was
gene a ed using lac ic bac e ia suspension wi h known cell densi ies ( om 3.2
±
0.2 o
7.5 ±0.1 Log CFU)
o a pu e cul u e o s ain Lac iplan ibacillus plan a um QSE79 isola ed
in he p esen s udy.
3. Resul s
3.1. Iden i ica ion o Isola ed S ains
A o al o 88 s ains o LAB we e isola ed om he cheeses o he PDO “Se a da
Es ela”. These cheeses a e p oduced in he adi ional way om aw milk and coagula ed
wi h his le enne and display a e men a ion pe iod o wo mon hs. Fo his pu pose, h ee
di e en cheeses we e used, and h ee samples we e aken om each cheese, yielding coun s
o 11.6 ±2.3 ×108, 9.4 ±0.4 ×108and 12.7 ±1.4 ×108CFU/g o cheese, espec i ely.
The esul s o s ains iden i ica ion using MALDI-TOF showed ha he isola es we e
dis ibu ed among 6 gene a and 8 di e en species, as ollows: Lac iplan ibacillus (L.) plan-
a um,Lac icaseibacillus (La.) pa acasei,La. hamnosus,Lac ococcus (Lc.) lac is,Le ilac obacillus
(L .) b e is,La ilac obacillus (L .) cu a us,Leuconos oc (Le.) mesen e oides,Le. ci eum. O he
88 s ains, 77 ma ched and had sco e alues g ea e han 2.0 as hei species we e a ailable
in he Bio ype 3.0 da abase. On he o he hand, 11 s ains ha ma ched showed a sco e
o less han 2.0 bu g ea e han 1.5 (Table 1). Since hese s ains we e classi ied using
Ag onomy 2022,12, 1150 5 o 20
a g ouping based on he co ela ion mean, a o al o 15 di e en g oups wi h ma ching
dis ibu ion be ween he ob ained species and simila i y sco es was ob ained. G oup I
included s ains wi h a simila i y sco e g ea e han 2.0–2.5; in pa icula , wo subg oups
we e obse ed o L. plan a um and one which ep esen ed he majo i y and showed g ea
homogenei y (Figu e 1).
Table 1.
Resul s o iden i ica ion o isola ed s ains by MALDI-TOF MS and pheS gene sequencing. In
bold, selec ed s ain o each g oup o sequence he pheS gene.
S ains G oup
MALDI TOF Iden i y pheS Iden i y
Species Sco es Close Type S ain Iden i y % Accession
Numbe
QSE21, QSE41,
QSE43, QSE45,
QSE49, QSE50,
QSE52, QSE56,
QSE58, QSE59,
QSE60, QSE66,
QSE71, QSE73,
QSE75, QSE76,
QSE77, QSE78,
QSE81, QSE84,
QSE86, QSE87,
QSE92
I
Lac iplan ibacillus
plan a um 2566–2046
Lac iplan ibacillus
plan a um subsp.
plan a um ATCC 14917T
99.75 OM802174
QSE44, QSE48,
QSE54, QSE61,
QSE79, QSE83
II
Lac iplan ibacillus
plan a um 2464–2076
Lac iplan ibacillus
plan a um subsp.
plan a um ATCC 14917T
99.85 OM802180
QSE64 III
Lac iplan ibacillus
plan a um 1969
Lac iplan ibacillus
plan a um subsp.
plan a um ATCC 14917T
100 OM802177
QSE20, QSE51,
QSE67A, QSE74
IV
Lac icaseibacillus
pa acasei 2426–2085
Lac icaseibacillus pa acasei
subsp. ole ans DSM
20258T
98.80 OM802167
QSE62 V
Lac icaseibacillus
pa acasei 2343
Lac icaseibacillus pa acasei
subsp. pa acasei ATCC
25302T
98.59 OM802175
QSE67B VI
Lac icaseibacillus
pa acasei 2388
Lac icaseibacillus pa acasei
subsp. pa acasei ATCC
25302T
98.60 OM802178
QSE01, QSE04,
QSE14, QSE23,
QSE33, QSE36,
QSE38, QSE40,
QSE47, QSE55,
QSE57, QSE69,
QSE72, QSE82
VII Lac ococcus
lac is 2540–2319 Lac ococcus lac is subsp.
lac is LMG 6890T99.24 OM802173
QSE68 VIII Le ilac obacillus
b e is 2301 Le ilac obacillus b e is
LMG 6906 T99.76 OM802179
QSE02, QSE03,
QSE05, QSE06,
QSE12, QSE13,
QSE15, QSE16,
QSE19, QSE24,
QSE27, QSE30,
QSE32, QSE39
IX La ilac obacillus
cu a us 2447–2001 La ilac obacillus cu a us
JCM 1096 T99.75 OM802171

Ag onomy 2022,12, 1150 6 o 20
Table 1. Con .
S ains G oup
MALDI TOF Iden i y pheS Iden i y
Species Sco es Close Type S ain Iden i y % Accession
Numbe
QSE18, QSE29,
QSE31, QSE35,
QSE42, QSE46,
QSE63, QSE65,
QSE88, QSE89,
QSE91
XLeuconos oc
mesen e oides 2332–1800
Leuconos oc mesen e oides
subsp. c emo is LMG
6909T
99.15 OM802176
QSE11, QSE22,
QSE34, QSE53,
QSE70, QSE80,
QSE90
XI Leuconos oc
mesen e oides 1924–1579
Leuconos oc mesen e oides
subsp. c emo is LMG
6909T
99.44 OM802165
QSE17A,
QSE17B XII Leuconos oc
mesen e oides 1825–1665
Leuconos oc mesen e oides
subsp. c emo is LMG
6909T
99.74 OM802166
QSE37 XIII Leuconos oc
ci eum 1753 Leuconos oc ci eum LMG
9849T98.89 OM802172
QSE26 XIV
Lac icaseibacillus
hamnosus 2447 Lac icaseibacillus
hamnosus ATCC 7469 T98.30 OM802169
QSE28 XV Leuconos oc
mesen e oides 1970
Leuconos oc mesen e oides
subsp. c emo is LMG
6909T
99.74 OM802170
G oup II consis ed o s ains wi h a simila simila i y sco e o g oup I (be ween 2.0
and 2.4) wi h L. plan a um. G oup III also showed iden i ica ion wi h L. plan a um bu had a
simila i y sco e o 1.925. S ains belonging o hese g oups showed simila simila i y sco es
o L. plan a um subspecies ( a iables anging om 2.2 and 2.0), wi h g oup I mos closely
ma ching L. plan a um ssp. a gen o a ensis DSM 16365
T
. In addi ion, g oups I, II and III had
highe sco es compa ed o L. plan a um ssp. plan a um DSM 20174T.
G oup IV included s ains wi h a simila i y sco e be ween 2.0 and 2.5 o L. pa acasei
(Table 1) and a highe sco e compa ed o L. pa acasei ssp. ole ans DSM 20258
T
han L.
pa acasei ssp. pa acasei DSM 5622
T
(sco e alue o 2.005). G oups V and VI consis ed o
s ains wi h sco e alues anging om 2.388 o 2.344 in L. pa acasei and had lowe sco e
alues o L. pa acasei ssp. pa acasi DSM 5622
T
han g oup IV. In hese h ee g oups, he
sco e alue in ela ion o he ype s ain o he nea es subspecies, L. pa acasei ssp. pa acasei
DSM 5622T, was always below 2.0.
S ains o Lc. lac is we e included in g oup VII and had a sco e alue be ween 2.540 and
2.319 (Table 1). These s ains showed high homogenei y in hei dend og am dis ibu ion,
and all o hem had a simila i y sco e o abou 2.200 o Lc. lac is ssp. lac is DSM 20481T.
G oup VIII included only a single s ain ha is consis en wi h L . b e is, showing a
sco e alue o 2.301 and g ea e simila i y o he ype s ain L . b e is DSM 20054T.
G oup IX included 14 s ains wi h sco e alues anging om 2.447 o 2.001 in e ms o
cu a u e simila i y o La ilac ibacillus cu a us, wi h he highe simila i y sco e o La ilac o-
bacillus cu a us DSM 20019
T
(sco es om 1.900 o 1.700) s anding ou . G oup X, consis ing
o 11 s ains, had sco es anging om 2.332 o 1.800 in ela ion o di e en s ains o he
species Leuconos oc mesen e oides, especially o he subspecies mesen e oides o dex anicum.
G oup XI included s ains wi h sco e alues om 1.924 o 1.579, gi ing an iden i ica ion a
he genus le el wi hin Leuconos oc bu likely o he species Le. mesen e oides. G oup XII
included wo di e en s ains ha we e also iden i ied in he genus Leuconos oc, bu wi h
sco e alues (1.825 and 1.665) ha did no assign o a species. Finally, in ag eemen wi h he
sco e alues ob ained, g oups XIII, XIV and XV had only one s ain in each g oup and we e
assigned o he genus Leuconos oc (g oups XIII and XV) and he species Lac icaseibacillus
hamnosus (Table 1).
Ag onomy 2022,12, 1150 7 o 20
Ag onomy 2022, 12, x FOR PEER REVIEW 8 o 22
Figu e 1. Clus e analysis o MALDI-TOF MS spec a o s ains isola ed in his s udy. Dis ance is
displayed in ela i e uni s. Rep esen a i e s ains o each g oup selec ed o pheS gene analysis a e
ma ked in bold.
G oup II consis ed o s ains wi h a simila simila i y sco e o g oup I (be ween 2.0
and 2.4) wi h L. plan a um. G oup III also showed iden i ica ion wi h L. plan a um bu had
a simila i y sco e o 1.925. S ains belonging o hese g oups showed simila simila i y
sco es o L. plan a um subspecies ( a iables anging om 2.2 and 2.0), wi h g oup I mos
closely ma ching L. plan a um ssp. a gen o a ensis DSM 16365T. In addi ion, g oups I, II and
III had highe sco es compa ed o L. plan a um ssp. plan a um DSM 20174T.
G oup IV included s ains wi h a simila i y sco e be ween 2.0 and 2.5 o L. pa acasei
(Table 1) and a highe sco e compa ed o L. pa acasei ssp. ole ans DSM 20258T han L. pa -
acasei ssp. pa acasei DSM 5622T (sco e alue o 2.005). G oups V and VI consis ed o s ains
wi h sco e alues anging om 2.388 o 2.344 in L. pa acasei and had lowe sco e alues
o L. pa acasei ssp. pa acasi DSM 5622T han g oup IV. In hese h ee g oups, he sco e
alue in ela ion o he ype s ain o he nea es subspecies, L. pa acasei ssp. pa acasei DSM
5622T, was always below 2.0.
Figu e 1.
Clus e analysis o MALDI-TOF MS spec a o s ains isola ed in his s udy. Dis ance is
displayed in ela i e uni s. Rep esen a i e s ains o each g oup selec ed o pheS gene analysis a e
ma ked in bold.
In his way, and based on he esul s o some g oups o he dend og am, sequencing
o he housekeeping gene pheS was pe o med o comple e he iden i ica ion o each s ain,
mainly om g oups XIII, XIV and XV, and o cla i y he posi ion o he emaining ones
ela i e o he di e en subspecies. The ob ained da a a e p esen ed in Table 1and Figu e 2.
Thus, QSE60, QSE79 and QSE64, ep esen a i es o g oups I, II and III, showed pe cen age
simila i ies in he pheS gene o 99.75%, 99.85% and 100%, espec i ely, o L. plan a um
subsp. plan a um ATCC 14917
T
, he ype s ain o his subspecies. Mo eo e , he e was
no doub abou i s iden i ica ion because he nex subspecies, a gen o a ensis, appea ed
wi h pe cen ages o simila i y o 90% in he pheS gene (Figu e 2). The ep esen a i e s ain
o g oup IV, QSE20, showed a pe cen age simila i y in he pheS gene o 98.80% o he
ype s ain La.pa acasei subsp. ole ans DSM 20258
T
, while s ains QSE62 and QSE67B,
ep esen a i es o g oups V and VI, showed a simila i ies in he pheS gene o 98.60% and
98.59%, espec i ely, wi h espec o he ype s ain La. pa acasei subsp. pa acasei ATCC
25302
T
. Thus, we can conclude ha hese h ee g oups belong o La. pa acasei, while he
i s g oup belongs o he subsp. ole ance and he o he wo g oups belong o he subsp.
pa acasei, con i ming hei phylogene ic posi ion (Figu e 2). The g oup XIV belonging o
his genus, which consis s o s ain QSE26, was iden i ied as La. hamnosus because i has
Ag onomy 2022,12, 1150 8 o 20
a pe cen age simila i y o 98.30% o he ype s ain o his species and shows a clea ly
de ined phylogene ic posi ion (Figu e 2).
Ag onomy 2022, 12, x FOR PEER REVIEW 10 o 22
assign hese g oups hemsel es o a pa icula subspecies based on he pe cen age simi-
la i y achie ed (Table 1 and Figu e 2). G oup XIII, consis ing only o s ain QSE37, showed
a pe cen age simila i y o he pheS gene o 98.89% o he sequence o he ype s ain o Le.
ci eum, which occupies a unique posi ion in he phylogene ic ee (Figu e 2).
Figu e 2. Neighbo -joining phylogene ic un oo ed ee based on pheS gene pa ial sequences (400
n ) showing he axonomic loca ion o 15 ep esen a i e s ains om di e en g oups o MALDI-
TOF MS wi hin he closely ela ed ype s ains.
3.2. Sc eening o An imic obial Ac i i y
Analysis o he capaci y o LAB o p oduce an imic obial subs ances is a p o en ac
and can be a conside able ad an age in hei compe i ion o space, e.g., in he hizo-
sphe e. The s udy o he capaci y o he ep esen a i e s ains o each MALDI-TOF MS
g oup was ca ied ou by he me hod o aga solid di usion by pe o a ion o he cylin-
d ical ca i ies. S ains QSE20, QSE62, QSE63 and QSE79 showed he highes ac i i ies and
he highes numbe o inhibi ed mic oo ganisms (Figu e 3). O hese, s ain QSE20 s ands
ou as he only s ain ha showed ac i i y agains Salmonella yphimu inum and, oge he
wi h QSE79, agains En e ococcus aecalis. The la e s ain also showed ac i i y agains
eigh s ains s udied, some o which we e o no able impo ance, such as Bacillus ce eus,
E. aecalis, Acine obac e baumanii and S aphylococcus au eus. Al hough mos s ains showed
Figu e 2.
Neighbo -joining phylogene ic un oo ed ee based on pheS gene pa ial sequences (400 n )
showing he axonomic loca ion o 15 ep esen a i e s ains om di e en g oups o MALDI-TOF
MS wi hin he closely ela ed ype s ains.
The iden i ica ion o g oup VII was con i med by he iden i ica ion o s ain QSE38,
whose pheS gene had a pe cen age o 99.24% wi h espec o he sequence o Lc. lac is
subsp. lac is LMG 6890
T
, which is he ype s ain o his species. The s ain QSE68, which
belongs o g oup VIII, con i med he iden i ica ion ob ained ia MALDI-TOF, and showed
a pe cen age simila i y in he pheS gene o 99.76% o L . b e is LMG 6906
T
. G oup IX was
ep esen ed by s ain QSE32, which was consis en wi h he esul s ob ained in MALDI-
TOF, and had a pe cen age simila i y in he pheS gene o 99.75% o he sequence o he
ype s ain L . cu a us JCM 1096T(Figu e 2).
G oups X, XI, XII and XV, ep esen ed by s ains QSE63, QSE11, QSE17A and QSE28,
espec i ely, we e iden i ied as Le. mesen e oides by MALDI-TOF, and hei speci ic a ilia ion
was con i med by sequencing o he pheS gene (Table 1and Figu e 2). Howe e , he da a
om MALDI-TOF showed unce ain a ilia ion wi h espec o he subspecies, while he
pheS gene o hese s ains showed pe cen age simila i ies o 99.15%, 99.44%, 99.74% and
Ag onomy 2022,12, 1150 9 o 20
99.74%, espec i ely, wi h espec o Le mesen e oides subsp. c emo is LMG 6909
T
. The s ains
QSE63, QSE17A and QSE28 showed 98.90% simila i y and s ain QSE11 showed 99.14% in
ela ion o Leuconos oc mesen e oides subsp. dex anicum DSM 20484
T
. As such, i is di icul
o assign he ep esen a i e s ains o g oups X, XI, XII and XV, and hus o assign hese
g oups hemsel es o a pa icula subspecies based on he pe cen age simila i y achie ed
(Table 1and Figu e 2). G oup XIII, consis ing only o s ain QSE37, showed a pe cen age
simila i y o he pheS gene o 98.89% o he sequence o he ype s ain o Le. ci eum, which
occupies a unique posi ion in he phylogene ic ee (Figu e 2).
3.2. Sc eening o An imic obial Ac i i y
Analysis o he capaci y o LAB o p oduce an imic obial subs ances is a p o en ac
and can be a conside able ad an age in hei compe i ion o space, e.g., in he hizosphe e.
The s udy o he capaci y o he ep esen a i e s ains o each MALDI-TOF MS g oup
was ca ied ou by he me hod o aga solid di usion by pe o a ion o he cylind ical
ca i ies. S ains QSE20, QSE62, QSE63 and QSE79 showed he highes ac i i ies and he
highes numbe o inhibi ed mic oo ganisms (Figu e 3). O hese, s ain QSE20 s ands
ou as he only s ain ha showed ac i i y agains Salmonella yphimu inum and, oge he
wi h QSE79, agains En e ococcus aecalis. The la e s ain also showed ac i i y agains
eigh s ains s udied, some o which we e o no able impo ance, such as Bacillus ce eus,E.
aecalis,Acine obac e baumanii and S aphylococcus au eus. Al hough mos s ains showed
ac i i y agains P o eus mi abilis and S. au eus,Se a ia ma censcens also p o ed o be sligh ly
sensi i e o many o he selec ed isola es. Isola es QSE11, QSE17A and QSE38 showed no
ac i i y agains almos all s udied mic oo ganisms (Figu e 3).
Ag onomy 2022, 12, x FOR PEER REVIEW 11 o 22
ac i i y agains P o eus mi abilis and S. au eus, Se a ia ma censcens also p o ed o be
sligh ly sensi i e o many o he selec ed isola es. Isola es QSE11, QSE17A and QSE38
showed no ac i i y agains almos all s udied mic oo ganisms (Figu e 3).
Figu e 3. Inhibi ion ac i i y o each selec ed s ain agains di e en ood-bo ne bac e ial pa hogens.
Uni s a e in cen ime e s o adius om he ma gin o he ca i y.
3.3. Plan G ow h-P omo ion Mechanisms
E alua ion o he capaci y o plan g ow h p omo ion o he selec ed bac e ia
showed ha , in gene al, all g oup-selec ed s ains p esen ed some PGP mechanisms (Fig-
u e 4). In ac , all isola es, excep s ain QSE37, showed he abili y o p oduce lowe le els
o indoleace ic acid ( alues anging om 9 o 68 µg/mL) (Table 2). The second mos com-
mon mechanism was he p oduc ion o side opho es, which was obse ed in all isola es,
excep o s ains QSE28 and QSE37. A his poin , i is necessa y o highligh he ac i i y
o some s ains, such as QSE63, QSE64, QSE11 and QSE79, he PSI p oduc ion o which
anged om 1 o 0.85. On he o he hand, he solubiliza ion o a phospha e sou ce was
obse ed in all isola es, excep s ains QSE28 and QSE38. Howe e , he s ains showed a
g ea e abili y o solubilize icalcium phospha e han dicalcium phospha e, while solu-
biliza ion o hyd oxyapa i e was he leas common mechanism, occu ing only in s ains
al eady able o solubilize o he ino ganic phospha e sou ces. Acco ding o hese da a,
mos o he selec ed s ains seem o be able o p omo e plan g ow h h ough he p esence
o se e al associa ed mechanisms ( he solubiliza ion o phospha e om se e al sou ces,
p oduc ion o side opho es and indole-ace ic acid).
Figu e 3.
Inhibi ion ac i i y o each selec ed s ain agains di e en ood-bo ne bac e ial pa hogens.
Uni s a e in cen ime e s o adius om he ma gin o he ca i y.
Ag onomy 2022,12, 1150 16 o 20
concen a ion in plan –mic oo ganism in e ac ions depends on he expo and/o di usion
mechanisms. The p esence o hese mechanisms u ns LAB in o an in e es ing ool since
hey can coun e ac desi able cha ac e is ics and be conside ed an ideal bio e ilize [66].
Un il now, ew wo ks ha e analyzed he g ow h-p omo ing capaci y o LAB in plan s.
T e dokhlib e al. [
67
] obse ed an inc ease o 8% in he size o whea oo s a e hei
incuba ion wi h L. plan a um UN12. These da a a e much lowe han hose obse ed in he
p esen wo k, whe e he inc ease was g ea e han 50% in mos o he expe imen s. In he
same way, he inocula ion wi h Lac obacillus sp. KLF01, an indole-ace ic acid-p oducing and
phospha e-solubilizing s ain, p oduces an inc ease in oo and ae ial leng hs and chlo o-
phyll con en in oma o and peppe [
68
]. I has also been desc ibed ha he inocula ion o
IAA-p oducing Lac obacillus is capable o imp o ing ge mina ion and oo de elopmen
unde no mal condi ions, such as saline condi ions, in le uce and adish [
69
]. The use o
he LCP-1 s ain belonging o he genus Lac ococcus showed an inc ease in he ge mina ion
a e o oma oes and an imp o emen in hei de elopmen [
70
]. In ou case, he s ains
belonging o he genus Lac ococcus e ealed g ea uni o mi y (Figu e 1) and only showed
an inc ease in he numbe o seconda y oo s. Tha is p obably ela ed mo e o he dai y
indus y i sel han o e ical ansmission om he en i onmen . Al hough he use o
Leuconos oc has no been s udied un il now, in he p esen s udy, ema kable esul s, such
as hose obse ed in Lac iplan ibacillus and Lac icaseibacillus, ha e no been obse ed.
A undamen al aspec o he plan –mic oo ganism in e ac ion in ol es he coloniza-
ion o he oo plan . In his s udy, i was clea which s ains p oduced a g ea e inc ease
in he s udied pa ame e s and mo e e icien coloniza ion o he oo su ace (Figu e 6).
As a as we know, hese oo coloniza ion pa e ns had al eady been obse ed in Rhi-
zobium [
71
], Bacillus [
72
] and Phyllobac e ium [
73
] gene a, and hus hey we e conside ed
e icien bio e ilize s. This po en ial is conside ed a undamen al equi emen in he selec-
ion o a compe en bio e ilize , ega dless o he genus s udied he ein because, al hough
he hizosphe ic o endophy ic loca ion is no necessa y, i s a angemen ensu es a mo e e-
ma kable in e ac ion [
72
]. In u n, he s udied s ains showed simila coloniza ion pa e ns
as o he compe en s ains, such as Bacillus amylolique aciens FZB42, whe e a mo e in ense
coloniza ion is epo ed in he uppe oo zones o di e en plan species [
74
]. This ac
indica es simila beha io be ween di e en species, such as ha o Phylum Fi micu es.
5. Conclusions
The PDO “Se a da Es ela” cheeses p esen a high di e si y o LAB, wi h isola ion
o six gene a and eigh di e en species, namely Lac iplan ibacillus (L.) plan a um, Lac icas-
eibacillus (La.) pa acasei, La. hamnosus, Lac ococcus (Lc.) lac is, Le ilac obacillus (L .) b e is,
La ilac obacillus (L .) cu a us, Leuconos oc (Le.) mesen e oides and Le. ci eum. Some species,
such as Lc. lac is, showed a low in aspeci ic di e si y, while he emaining ones displayed
a no o ious deg ee o he e ogenei y among hem. In addi ion, he MALDI-TOF MS analysis
p o ed, one mo e ime, ha i is a e sa ile and use ul ool o he cha ac e iza ion o ood
mic obial popula ions. In a gene al way, he main selec ed isola es exhibi ed di e en
mechanisms able o p omo e plan g ow h and con ol undesi able bac e ia in ood in
a iable ways. Among he iden i ied isola es, he QSE20, QSE62 and QSE79 s ains exe ed
he mos ema kable oo coloniza ion and also imp o emen o he oo de elopmen
and he numbe o seconda y oo s. The ob ained da a indica e ha aw milk cheeses
can be conside ed a eliable sou ce o he isola ion o highly e icien LAB, which can be
applied o he de elopmen o bio e ilize s gi en hei high biosa e y le el. The use o
hese bac e ia can open a new ho izon in he design o bio e ilize s o he applica ion o
bac e ial bios imulan s ha can be ansmi ed o he consume by e ical ans e and can
ac as p obio ics.
Au ho Con ibu ions:
Concep ualiza ion, G.A. and J.D.F.-F.; me hodology, F.S.-J. and J.D.F.-F.;
alida ion, G.A., L.R.S. and J.D.F.-F.; o mal analysis, J.D.F.-F.; in es iga ion, A.C.G. and A.R.N.; da a
cu a ion, J.D.F.-F.; w i ing—o iginal d a p epa a ion, A.C.G. and A.R.N.; w i ing— e iew and

Ag onomy 2022,12, 1150 17 o 20
edi ing, F.S.-J., G.A., L.R.S. and J.D.F.-F.; supe ision, J.D.F.-F.; p ojec adminis a ion, G.A.; unding
acquisi ion, G.A. 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 Eu opean Union’s Ho izon 2020 P og amme o esea ch
and inno a ion unde he Ma ie Skłodowska-Cu ie g an ag eemen No 101003373. The au ho s a e
g a e ul o he Founda ion o Science and Technology (FCT), he Minis y o Science, Technology and
Highe Educa ion (MCTES), he Eu opean Social Fund (EFS) and he Eu opean Union (EU) o he
PhD ellowship o Ana R. Nunes (SFRH/BD/139137/2018) and Ana C. Gonçal es (2020.04947.BD).
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 :
Sequence da a a e a ailable in he GenBank eposi o y unde accession
numbe s OM802165 o OM802180.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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