Ci a ion: Ne ini, M.; Russo, A.;
Deco osi, F.; Me iggi, N.; Vi i, C.;
Ca alie i, D.; Ma asi, M. A Mic obial
Phenomics App oach o De e mine
Me abolic Signa u es o Enhance
Seab eam Spa us au a a T aceabili y,
Di e en ia ing be ween Wild-Caugh
and Fa med. Foods 2024,13, 2726.
h ps://doi.o g/10.3390/
oods13172726
Academic Edi o : E and o Lei e de
Souza
Recei ed: 3 July 2024
Re ised: 13 Augus 2024
Accep ed: 21 Augus 2024
Published: 28 Augus 2024
Copy igh : © 2024 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/).
oods
A icle
A Mic obial Phenomics App oach o De e mine Me abolic
Signa u es o Enhance Seab eam Spa us au a a T aceabili y,
Di e en ia ing be ween Wild-Caugh and Fa med
Ma a Ne ini 1, Alessand o Russo 1, F ancesca Deco osi 2, NiccolòMe iggi 3, Ca lo Vi i 2,
Duccio Ca alie i 1and Massimiliano Ma asi 1,*
1Depa men o Biology, Uni e si y o Flo ence, Via Madonna del Piano, 50019 Fi enze, I aly;
[email p o ec ed] (M.N.); alessand o. [email p o ec ed] (A.R.); [email p o ec ed] (D.C.)
2Depa men o Ag icul u e, Food, En i onmen and Fo es y (DAGRI), Uni e si y o Flo ence,
50019 Flo ence, I aly; ancesca.deco [email p o ec ed] (F.D.); [email p o ec ed] (C.V.)
3Ins i u e o Ag icul u al Biology and Bio echnology (IBBA), Na ional Resea ch Council (CNR),
56124 Pisa, I aly; niccolo.me iggi@cn .i
*Co espondence: [email p o ec ed]
Abs ac : Backg ound: The need o e icien and simpli ied echniques o sea ood aceabili y is
g owing. This s udy p oposes he Biolog EcoPla e assay as an inno a i e me hod o assessing wild
and a med Spa us au a a aceabili y, o e ing ad an ages o e o he molecula echniques in e ms o
echnical simplici y. Me hods: The Biolog EcoPla e assay, known o i s high- h oughpu capabili ies
in mic obial ecology, was u ilized o e alua e he unc ional di e si y o mic obial communi ies om
a ious o gans o S. au a a (seab eam) om he Medi e anean a ea. Samples we e aken om he
an e io and pos e io gu , cloaca swabs and gills o dis inguish be ween a med and wild-caugh
indi iduals. The analysis ocused on colo de elopmen in OmniLog Uni s o speci ic ca bon sou ces
a 48 h. Resul s: Gills p o ided he mos accu a e clus e iza ion o sample o igin. The assay moni o ed
he de elopmen o colo o ca bon sou ces such as
α
-cyclodex in, D-cellobiose, glycogen,
α
-D-
lac ose, L- h eonine and L-phenylalanine. A mock expe imen using p incipal componen analysis
(PCA) success ully iden i ied he o igin o a blind sample. Shannon and Simpson indexes we e used o
s a is ically assess he di e si y, e lec ing he clus e iza ion o di e en o gan samples; Conclusions:
The Biolog EcoPla e assay p o es o be a quick, cos -e ec i e me hod o disc imina e S. au a a
aceabili y (wild s. a med), demons a ing eliable ep oducibili y and e ec i e di e en ia ion
be ween a med and wild-caugh seab eam.
Keywo ds: aceabili y; ish; ood mic obiology; ood quali y; EcoPla e
1. In oduc ion
Au hen ica ion and ood aud de ec ion is pa o he complex ne wo k o ac ions
ha include ood aceabili y. T aceabili y is a p ocess which in ol es se e al di e en
disciplines, including in o ma ion, logis ics, isk managemen , quali y and sa e y [
1
]. The
de e mina ion o ood o igin holds signi ican impo ance, no jus o ensu ing sa e y and
quali y, bu also o impac ing cus ome sa is ac ion and he p ice consume s a e willing o
pay. Ex ensi e su eys conduc ed among Eu opean ci izens ha e consis en ly shown ha
pu chasing decisions ega ding sea ood p oduc s a e in luenced by ac o s such as p oduc
o igin and he me hods employed in ishing o a ming p ac ices [
2
]. Thus, he in e play be-
ween au hen ici y, b and ecogni ion and consume s’ us plays a i al ole in suppo ing
hei pu chasing choices. In many cases, legal equi emen s a e in oduced o ce i y he spe-
ci ic o igin o ood [
3
] and EU Regula ion 2018/274, including o p e en alle genic o oxic
molecules. To ha end, go e nmen s, in e na ional s anda diza ion and non-go e nmen al
ce i ica ion o ganiza ions play majo oles in p o ec ing ood au hen ici y.
Foods 2024,13, 2726. h ps://doi.o g/10.3390/ oods13172726 h ps://www.mdpi.com/jou nal/ oods
Foods 2024,13, 2726 2 o 12
The globaliza ion o he sea ood ade and he lack o s anda ds o in o ma ion
exchange ha e made acking and acing sea ood e y challenging [
4
]. In he supply
chain and he sea ood indus y, he e is a need o imp o ed aceabili y, sus ainabili y and
ood sa e y.
F om echnical pe spec i es, ood au hen ici y is p o ec ed by employing a numbe
o echniques, anging om chemical analysis, iso ope, and biogeochemical a ge s, o
me agenomics [
5
,
6
]. The use o mic obial me agenomics has a ac ed a en ion o ace-
abili y. The o ensic aspec o he use o mic obial communi ies as a signa u e o a speci ic
en i onmen has been shown in di e en habi a s, showing ha he gene a ion o a ypical
mic obial communi y is no s ochas ic, bu inely egula ed and ep oducible [
7
]. Mic o-
bial communi ies in he en i onmen ha e speci ic signa u es ha a e impossible o e y
di icul o ep oduce
in i o
leading o possible aud. This is ue no only wi h e e -
ence o axonomical popula ions, including e enness and ichness, bu also o me abolic
ai s, which include pa icula o unique genes and me abolic pa hways (bioma ke s) [
8
].
Based on his e idence, mic obial me agenomics has been p oposed as a ool o associa e
speci ic signa u es.
Mic obial communi ies can he e o e con ibu e o ood aud de ec ion [
9
] by (1) au-
hen ica ing he o igin o a p oduc in e ms o geog aphical o bo anical/animal p o e-
nance o o gans; (2) p o ing he absence o adul e a ion. In his scena io, he use o
phenomics (Biolog EcoPla e) as a possible al e na i e o help in iden i ying whe e ish
came om is es ed in his wo k. EcoPla e Biolog is a ype o high- h oughpu mic obial
ecology assay ha is used o measu e he unc ional di e si y o mic obial communi ies and
moni o a ious en i onmen al samples [
10
–
13
]. A pa icula biological sys em, whe he
ecosys em o mic ocosm, depends on he in e play o h ee ac o s—en i onmen , biologi-
cal communi y s uc u e (di e si y) and biological ac i i y ( unc ion) [
14
]. The e o e, he
inal ou come in he pheno ype mic oa ay esul s om his unique in e ac ion. Typically,
he EcoPla e assay in ol es he use o a 96-well mic opla e ha con ains a se o 31 ca bon
sou ces (and one nega i e con ol) epea ed h ee imes, which a e used as sole ca bon and
ene gy sou ces by di e en mic obial unc ional g oups. By measu ing he u iliza ion o
hese ca bon sou ces by he mic obial communi y, we aim o un a el di e ences in he
mic obial communi ies acco dingly, no only wi h he mic obiome associa ed wi h wild
o a med ish, bu also wi h he o gan om which he mic obial communi y is isola ed.
EcoPla es a e pa icula ly use ul o iden i ying he unc ional capabili ies o mic obial
communi ies, which can in o m ou unde s anding o ecosys em p ocesses and help us o
de elop s a egies o managing aceabili y.
2. Ma e ials and Me hods
2.1. Fish Ma e ial and Dissec ions
The s udy a ea was chosen o be he Cos a degli E uschi, ex ending om Li o no o Pi-
ombino (I aly). This egion is no able o i s p oduc ion o seab eam, bo h wild and a med,
which is o subs an ial economic alue. This ocus aligns wi h he a ea’s p ominence in he
sea ood indus y [
15
]. Wild-caugh seab eam (Spa us au a a) and a med seab eams we e
pu chased by a local high-quali y and ISO ce i ied sea ood e aile (ce i ied wi h UNI EN
ISO 9001:2015 and ISO 25012:2014) wi h ce i ied and labeled o igin o ishing, a ming and
da e o deli e y. All seab eam used in his s udy, bo h wild-caugh and sea- a med, came
om a e y limi ed a ea ensu ing ha samples we e ob ained in close p oximi y o each
o he , speci ically in a ew squa e kilome e s om he coas o Tuscany, I aly, wi hin he
FAO ishing a ea 37.1.3 [
16
]. Due o he di icul y o ob aining ma ched ish in he same a ea,
in pa icula wi h e e ence o he wild-caugh indi iduals, expe imen s we e conduc ed on
di e en days. A o al o 46 specimens we e analyzed. The specimens comp ehended gills,
cloaca, an e io and pos e io gu ob ained om 23 indi iduals pu chased a wo di e en
imes, in Ap il 2023 (n= 9) and Feb ua y 2024 (n= 14) o a e age season di e ences:
10 o wild-caugh seab eams, 10 o a med seab eams and 3 o he mock expe imen .
P elimina y expe imen s showed ha gu samples we e unsui able o he analyses. Fo
Foods 2024,13, 2726 3 o 12
his eason, an e io and pos e io gu we e no sampled o he es o he indi iduals. The
a e age ish weigh was 671
±
46 g o wild-caugh seab eams and 411
±
22 g o a med
seab eams. The age o he ish was unknown.
2.2. Sample P epa a ion o EcoPla e Assay
The main o e iew o he p ocess is desc ibed in Figu e 1. Fish we e p ocessed wi hin
30 min o pu chase and wi hin 6 h o ishing. All ish we e deli e ed on ice in e ige a ed
ucks. F om each ish, ou di e en specimens we e collec ed: a cloacal swab, a agmen
o gill, he an e io gu and he pos e io gu . A agmen o 1 g o he le gill was collec ed
using a pai o scisso s p e iously s e ilized on lame (Figu e 1A). The cloaca was sampled
inse ing he collec ion ip o a s e ile swab ( o a dep h o abou 1.5 cm) inside and slowly
o a ing he swab agains he gu wall o 4 o a ions. The swab (co on) po ion was
he e o e cu wi h a scisso p e iously s e ilized on he lame each ime. A e wa ds, he
ish was dissec ed o eco e he en i e gu using a s e ile scalpel; he gu was hen di ided
in o wo po ions, iden i ied as he an e io and pos e io gu . F om hose sec ions 1 g
o an e io and pos e io gu was cu . All samples we e esuspended in 10 mL o s e ile
demine alized wa e and o exed wi h a Vo ex-Genie 2T (Scien i ic Indus ies, New Yo k,
NY, USA) o 10 min o help esuspend he mic oo ganisms in he wa e . A e o exing,
100
µ
L o he swab was di ec ly pla ed in he EcoPla e. Gill suspensions we e dilu ed o 1:10,
hen 100
µ
L we e pla ed in he EcoPla e [
10
–
13
]. Fo gu samples, di e en dilu ions we e
es ed (1:2, 1:10, 1:100) due o he di e ence in u bidi y caused by he s ochas ic p esence
o he gu con en s, hen 100
µ
L o each dilu ion was pla ed in he EcoPla e. EcoPla es we e
incuba ed a 25
◦
C inside an OmniLog Reade (Biolog) and moni o ed au oma ically e e y
15 min o colo changes in he wells o 48 h. Raw kine ic da a (A bi a y OmiLog Uni s)
we e e ie ed using he OmniLog, OL_PM_FM/Kin 1.30, File Managemen /Kine ic Plo
so wa e (Ve sion 1.7) (Biolog, Haywa d, CA, USA). A e 2 days o incuba ion, EcoPla es
we e collec ed and s o ed a −20 ◦C.
Figu e 1. O e iew o expe imen s designed o use 96-well EcoPla e o assess aceabili y (see he
Sec ion 2 o de ails). (A) B ie ly, wild-caugh and a med samples we e ob ained and 1 g o gills
was esuspended in wa e . Simila ly, a swab was used o emo e ecal ma e ial om he cloaca. The
suspensions we e aliquo ed in he 96-well EcoPla e, which was di ided in o h ee echnical eplicas
( ep esen ed by lowe case le e and di e en shadows o he 96-well pla e). (B) Example o ex ac ion
o gills and sampling in he cloaca.
Foods 2024,13, 2726 4 o 12
2.3. S a is ical Analysis
All s a is ical analyses we e ca ied ou using he G aphPad P ism 9.2.0 package.
Di e ences in A bi a y OmniLog Uni s (AOU) o selec ed me aboli es de eloped a 0 h,
24 h and 48 h be ween a med and wild-caugh samples. We employed he K uskall–Wallis
es s o compa ing g oups. P incipal Componen Analysis (PCA) was pe o med using
he p comp unc ion o depic sample dis ibu ion acco ding o he di e en subs a e
consump ion p o iles (D,L-
α
-Glyce ol Phospha e, D-Cellobiose, Glucose-1- Phospha e,
Glycogen, i-E y h i ol, L-Phenylalanine, L-Th eonine, Tween 80,
α
-Cyclodex in,
α
-D-
Lac ose,
β
-Me hyl-D-Glucoside). The e ec o o igin and season was es ed by pe mu a-
ional mul i a ia e analysis based on Euclidean dis ance using (adonis2 unc ion “ egan”)
package e . 2.6-4 [
17
], also e e ed o as adonis PERMANOVA. Bo h PCA and adonis
PERMANOVA analyses we e ca ied ou in R en i onmen e . 4.3.3 [
18
] (R Co e Team,
Vienna, Aus ia, 2024).
Shannon index (H) [19] and Simpson (D) index we e calcula ed wi h he equa ions:
H=−∑pi ×ln(pi)(1)
pi =ai
∑ai (2)
D=∑ai(ai −1)
A(A−1)(3)
Equa ions (1) and (2) a e he Shannon index, whe e pi is he p opo ional colo de el-
opmen o he well (ai) o e he o al colo de elopmen o all wells o a echnical eplica.
Equa ion (3) is he Simpson index whe e (A) is he o al colo de elopmen o all wells.
3. Resul s
3.1. Selec ed Ca bon Sou ces by Mic obial Communi ies om Bo h Wild-Caugh and
Fa med Specimens
Pheno ypic di e ences be ween wild-caugh and a med seab eam om a ious
o gans we e ini ially obse ed. Du ing he 48 h incuba ion pe iod, all selec ed me aboli es
exhibi ed inc eased dye de elopmen . Ou o he ou o gan sec ions examined (an e io
and pos e io gu , cloaca and gills), he an e io and pos e io gu samples we e excluded
om he analysis. This decision was based on he high a iabili y obse ed wi hin he
echnical eplica es due o challenges in achie ing he co ec dilu ions. Fu he mo e, he
p esence o in es inal con en s could no be eliably p edic ed, as bo h ull and emp y gu s
we e sampled du ing he expe imen . F om a echnical s andpoin , sampling he an e io
and pos e io gu also in ol ed complex dissec ion p ocedu es, while accessing he gills
and cloaca was compa a i ely easie , especially o indus ial pu poses. Consequen ly, he
analysis ocused on he gills and cloaca samples, which p o ed o be eliable sou ces.
Mic obio a om gills and cloaca we e he e o e deeply analyzed. To ha end, we
chose h ee ime poin s ha showed he mos signi ican di e ences, which we e a
0 h, 24 h and 48 h o incuba ion. Se e al me aboli es, including,
β
-me hyl-D-Glucoside,
glycogen, D-cellobiose, glucose-1-phospha e and Tween 80, we e selec ed as he mos
p omising me aboli es o be moni o ed. The esul s o ou expe imen s showed lim-
i ed di e ences in A bi a y OmniLog Uni s (AOU) be ween wild-caugh and a med
seab eam when he cloaca was es ed
(Figu e 2A)
. Howe e , when he expe imen was
epea ed wi h he gills, se e al signi ican di e ences we e obse ed o all selec ed me abo-
li es a bo h 24 and 48 h (Figu e 2B). Fu he mo e, when we compa ed he mic obial
communi ies ob ained om he gills in e ms o A bi a y OmniLog Uni s a 48 h, we
ound signi ican di e ences be ween he a med and wild-caugh so s o he ollowing
me aboli es:
β
-me hyl-D-Glucoside (
4.4 ±1.6 AOU
and
107.1 ±18.9 AOU
),
α
-ciclodex in
(
16.6 ±6.7 AOU
and
131 ±18.9 AOU
), glycogen (
21.6 ±5.9 AOU
and
150.2 ±13.5 AOU
),
D-cellobiose
(34 ±10.9 AOU
and
186.6 ±11.8 AOU
)andGlucose-1-phospha e(
41.8 ±15.4 AOU
and 179.4
±
16.3 AOU) (Figu e 2). We selec ed hese me aboli es as hey can disc imina e
Foods 2024,13, 2726 5 o 12
be ween he mic obial communi ies o wild-caugh and a med seab eam on he gills.
Tween 80 (85.7
±
18.9 AOU and 206.2
±
8.7 AOU) was he only me aboli e able o show
disc imina ion be ween wild-caugh and a med ish in samples de i ed om cloaca, e en
a e 24 h.
Figu e 2. The u iliza ion o selec ed ca bon sou ces by mic obial communi ies om bo h wild-caugh
and a med specimens was assessed in cloaca (A) and gills (B), ac oss h ee dis inc ime ames.
Ho izon al ba s ep esen K uskal–Wallis es , e o ba s ep esen s anda d e o s. I e o ba s a e
no isible, i is because he size o he symbol (o box) is bigge han he e o ba s. **** p< 0.001;
*** 0.0002; ** 0.0021; * 0.0332; ns, no signi ican .
3.2. Compa ison o he Gills and he Cloaca
An analysis was also p oposed o disc imina e he sampling si e o he o gan, by
compa ing gills and cloaca si es. The da a a e p esen ed o bo h a med and wild-caugh
ish (Figu e 3). In a med seab eam, no di e ences in e m o AOU we e shown among
Foods 2024,13, 2726 6 o 12
bo h o gans a he ime poin o 47 h. On he o he hand, in wild-caugh seab eam we
selec ed six me aboli es showing he highes di e ences be ween gills and cloaca: D-
Cellobiose (
186.6 ±11.8 AOU
and 48.6
±
12.4 AOU), L-Phenylalanine (
173.4 ±16.4 AOU
and
32.3 ±6.8 AOU),
L-Aspa agine (102.7
±
19.9 AOU and 219.7
±
14.7 AOU), L-Th eonine
(185.1
±
12.3 AOU and 56.7
±
10 AOU), Phenyle hylamine (134.4
±
16.5 AOU and
27.8 ±7.1 AOU) and Tween 80 (78.9 ±18.8 AOU and 206.2 ±8.7 AOU).
Figu e 3. Di e en ca bon sou ce u iliza ion om mic obial communi ies sampled om he gills and
cloaca compa ed a he sampling poin o 48 h. Fa med (A) and wild-caugh (B) sou ces. Ho izon al
ba s ep esen K uskal–Wallis es , while e o ba s ep esen s anda d e o s. I e o ba s a e no
isible, i is because he size o he symbol (o box) is bigge han he e o ba s. **** p< 0.001;
*** 0.0002; ** 0.0021; * 0.0332; ns, no signi ican .
3.3. Mock Expe imen
To e alua e he hypo hesis ha EcoPla e phenomics can aid in iden i ying aceabili y,
a mock (blind) expe imen was conduc ed. The ope a o was gi en uniden i ied esuspen-
sions o gills and cloaca, which could ha e o igina ed om ei he wild-caugh o a med
sou ces. A PCA analysis was pe o med o educe he numbe o a iables associa ed
wi h di e en sampling imes, whe e a ia ions in OmniLog Uni s we e obse ed. Sub-
sequen ly, a PCA analysis was conduc ed using he imepoin s desc ibed in he Sec ion 2
Foods 2024,13, 2726 7 o 12
and da a om he mos di e se me aboli es in gills and cloaca (Figu e 4). PCA based
on Euclidean dis ance showed he samples’ dis ibu ion and he exp ession o he o al
amoun o consumed subs a es, g ouped acco ding o ish o igin bu depending on he
ime poin conside ed (Figu e 4). In de ail, he dis ibu ion o he samples om di e en
ish o igins and he mock expe imen showed a clea o e lap a ime T0 in bo h ish issue
da ase s; his e idence was con i med by adonis PERMANOVA (Gills-T0 and Cloaca-T0 in
Figu e 4). A e 24 and 48 h, a clea sepa a ion among samples om di e en ish o igins
was e iden o bo h he gill and cloaca da ase s including he mock es (Gills-T24 and
48; Cloaca-T24 and T 48 in Figu e 4). In pa icula , he wild-caugh g oup om he gill
da ase showed a highe deg ee o sepa a ion compa ed o he a med g oup. O e all,
he g oup sepa a ion a e 24 and 48 h was mo e e ec i e in he gills da ase and his was
co obo a ed by he R-squa ed alues (also e e ed o as explained a iance) om adonis
PERMANOVA (Figu e 4). The mul i a ia e analyses (adonis PERMANOVA) we e ca ied
ou using a wo- ac o model o mula, adding a seasonal e ec (sampling in Ap il 2023 and
Feb ua y 2024, see Sec ion 2) oge he wi h he o igin a iable. As expec ed, o some ex en ,
he analysis also highligh ed a season e ec , e iden in he cloaca da ase bu negligible
in he gill da ase . The analysis highligh ed a g ea e in luence o season on he mic obial
communi ies esiden in he cloaca and hei e ec on he consump ion o he di e en
subs a es, bu his was no he case o hose o he gills, co obo a ing he e idence ha
he gills ep esen he ideal issue o he de elopmen o his me hodical app oach in
ish con ol.
Figu e 4. Di e ences in subs a e consump ion p o iles in samples om di e en ish o igins.
P incipal Componen Analysis (PCA) based on Euclidean dis ance epo ing samples om di e en
ish o igins (colo scheme). Fish issue (gills o cloaca) and ela ed ime poin (T0, T24 and T48) a e
epo ed a he op o each panel. R-squa ed (R2) alues and signi ican e ec o o igin a iable,
es ed wi h adonis PERMANOVA, a e gi en in squa e b acke s a he op o each panel. A signi ican
e ec is indica ed wi h as e isks (*** p< 0.001).
Foods 2024,13, 2726 8 o 12
O igin was he main a iable able o explain he main di e ences highligh ed in he
p o iles o he subs a es consumed a e 24 and 48 h. This was e iden o he gills as
desc ibed abo e and highligh ed by he R-squa ed alues om he mul i a ia e analysis
(R-squa ed om adonis PERMANOVA in Table S1).
In addi ion, he Shannon and Simpson indexes we e used o signi ican ly assess
simila i ies and di e ences among he gills and cloaca o a med, wild-caugh and unknown
samples by including all 31 wells o each eplica om gills a 48 h (Figu e 5). In e es ingly,
bo h indexes showed signi ican di e ences o he gills in bo h a med and wild-caugh
so s, while he mock sample (wild-caugh seab eams) was he same wi h i s homolog
(Figu e 5A,B). Unexpec edly, in he cloaca sample, bo h indexes also showed signi ican
di e ences be ween a med and wild-caugh seab eams, along wi h he mock samples
(Figu e 5C,D).
Figu e 5. Shannon and Simpson indexes o gills (A,B) and cloaca (C,D) om a med, wild-caugh
and unknown samples (mock expe imen ). Ho izon al ba s ep esen K uskal–Wallis es , while e o
ba s ep esen s anda d e o s; **** p< 0.001; *** 0.0002; ** 0.0021; ns, no signi ican .
In summa y, bo h he PCA analysis and he Shannon and Simpson indexes in his
expe imen demons a ed ha he EcoPla e app oach ob ained om he mic obial commu-
ni y om gills success ully dis inguished be ween wild-caugh and a med samples. The
unknown sample consis en ly aligned wi h he wild-caugh g oup, suppo ing he acing
capabili ies o his sys em.
4. Discussion
The s udy o mic obio a as a acing ool has gained signi ican a en ion in ecen
yea s due o i s c ucial ole in a ious aspec s o ood cha ac e iza ion. Mic obio a has been
used o ace dai y p oduc s [
20
,
21
], ui s [
22
], honey [
23
], wa e [
24
,
25
], mea [
26
–
28
] and
sea ood [
29
]. These examples in ol e mainly wo echniques: Dena u ing G adien Gel
Elec opho esis (DGGE) and Nex Gene a ion Sequencing (NGS).
The composi ion and unc ioning o mic obial communi ies can also be cha ac e ized
by using a phenomics app oach, de eloped o cha ac e ize and analyze he obse able
cha ac e is ics o pheno ypes o a speci ic mic obial communi y [30].
In his s udy, we p esen Biolog EcoPla e as a iable me hod o assessing aceabili y,
o e ing addi ional echniques o e he exis ing DGGE and NGS. The EcoPla e es is no
only easy o pe o m bu is also cos -e ec i e, elimina ing he need o complex equipmen .
E en i we used an OmniLog machine o ini ial high- h oughpu analysis, his app oach
needs a simple 96-well pla e spec opho ome e . Th ough ou mock expe imen , we
success ully demons a ed he e ec i eness o ou me hod in iden i ying he o igin o a
Foods 2024,13, 2726 9 o 12
blind sample. By u ilizing speci ic condi ions, including he use o gill-de i ed ma e ials
and moni o ing OmniLog Uni s (colo de elopmen ) a 48 h o ca bon sou ces such as
D,L-
α
-Glyce ol Phospha e, D-Cellobiose, Glucose-1- Phospha e, Glycogen, i-E y h i ol,
L-Phenylalanine, L-Th eonine, Tween 80,
α
-Cyclodex in,
α
-D-Lac ose and
β
-Me hyl-D-
Glucoside, we achie ed accu a e clus e iza ion o he sample’s o igin.
The Biolog EcoPla e was de eloped wi h a speci ic ocus on communi y analysis and
mic obial ecological s udies. I was ini ially designed in esponse o he needs exp essed by
a g oup o mic obial ecologis s who we e u ilizing he Biolog GN Mic oPla e bu desi ed a
panel ha o e ed eplica e es s [
31
]. Fu he ecen analyses ha e s udied ep oducibili y
by sequencing he bac e ial communi ies en iched wi hin each well. Compa isons o alpha
and be a di e si y in hese sys ems ia NGS showed ha , while he composi ion o he
communi ies ha g ow o inhabi he wells in each subs a e a ay di e ges sha ply om
ha o he o iginal communi y in he inoculum, he inal en ichmen is domina ed by one
o se e al OTUs [
32
]. Each domina ing mic obial communi y is well es ablished and he
en ichmen is ep oducible and s able. The ep oducibili y o EcoPla e has been shown by
o he s udies [
32
–
34
]. Wi h e e ence o he s anda diza ion o indus ial pu poses, we
iden i ied speci ic ime poin s o comple e he analysis; howe e , he obse a ion o a ull
ime-cou se p o ile (kine ics o de elopmen o he pigmen ) may gi e mo e in o ma ion
han measu emen s a one o wo ime poin s [35].
Se e al s udies ha e explo ed he use o mic obio a o aceabili y pu poses in di -
e en ia ing be ween a med o wild-caugh sea ood, including ishing si es. Ou pa allel
esea ch has demons a ed ha by analyzing he bac e ial V3-V4 egion o he 16S RNA
genes, we can e ec i ely p o ile he di e si y o gill bac e ial communi ies in seabass and
seab eam. This p o iling allowed esea che s o iden i y dis inc i e bac e ial signa u es
co ela ed wi h h ee p oxima e ishing zones along he Tuscan coas [
29
]. The use o NGS
has e ealed ha ce ain bac e ial axa a e uniquely ied o hei espec i e ishing a ea,
independen o he ish species. Fu he mo e, his s udy ea i ms he sui abili y o gill
issues o ish- aceabili y esea ch, showing ha he gills’ mic obial communi ies a e
highly sensi i e o en i onmen al changes, hus p o iding a eliable measu e o habi a
a ia ions [29,36].
Ano he s udy co ec ly labeled ish samples iden i ied bo h wi h axonomical name
and “ a med” o “wild-caugh ”; he s udy was conduc ed using 16S DNA mic obial
p o iling and nex -gene a ion sequencing (NGS) [
28
]. The s udy included a med ish,
speci ically ilapia and wild-caugh ish specimens, such as wild salmon. To al DNA
was ex ac ed om he skin mucus o he ish samples ia swabbing. The esea che s
used Illumina MiSeq o sequence he V3-V4 egions o he 16S DNA ha we e ampli ied.
The s udy aimed o assess mislabeling (which also includes “ a med” o “wild-caugh ”)
by analyzing he mic obial p o iles. The esul s showed ha mislabeling was e iden
when assessing Fai h’s phylogene ic di e si y bu no wi h Pielou’s e enness index [
28
].
Speci ically, An a c ic oo h ish and Pa agonian oo h ish we e equen ly mislabeled [
28
].
The au ho s sugges ed he need o iden i y speci ic indica o mic oo ganisms ha a e mo e
abundan in wild condi ions. Fo example, Jan hinobac e ium was ound in highe quan i ies
in he gu o a med A lan ic salmon in p e ious s udies [
28
,
37
], indica ing ha ce ain
mic oo ganisms may se e as indica o s o wild condi ions.
Ou esea ch demons a ed ha ce ain ca bon sou ces such as D,L-
α
-Glyce ol Phos-
pha e, D-Cellobiose, Glucose-1- Phospha e, Glycogen, i-E y h i ol, L-Phenylalanine, L-
Th eonine, Tween 80,
α
-Cyclodex in,
α
-D-Lac ose and
β
-Me hyl-D-Glucoside showed he
abili y o di e si y mic obial communi ies be ween a med and wild-caugh o ganisms,
speci ically in he gills a he 48 h ime poin . In e es ingly, o he ca bon sou ces p esen
in he Biolog EcoPla e, such as amines, some amino acids, some ca boxylic acids and
phenolic compounds, did no exhibi signi ican di e ences. The eason behind he highe
di e en ia ion obse ed wi h hese ca bon sou ces emains in iguing. While D-cellobiose,
α
-cyclodex in and glycogen a e all glucose polyme s o dime s, indica ing a po en ial
en ichmen o chemo gano ophs, we did no in es iga e he speci ic mic obial species
