Ci a ion: Sinis e a-Loaiza, L.;
Alonso-Lo e a, P.; Ca delle-Cobas,
A.; Mi anda, J.M.; Vázquez, B.I.;
Cepeda, A. Compliance wi h
Nu i ional Recommenda ions and
Gu Mic obio a P o ile in Galician
O e weigh /Obese and
No mal-Weigh Indi iduals.
Nu ien s 2023,15, 3418. h ps://
doi.o g/10.3390/nu15153418
Academic Edi o : Yoshi aka
Hashimo o
Recei ed: 30 June 2023
Re ised: 28 July 2023
Accep ed: 28 July 2023
Published: 1 Augus 2023
Copy igh : © 2023 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/).
nu ien s
A icle
Compliance wi h Nu i ional Recommenda ions and Gu
Mic obio a P o ile in Galician O e weigh /Obese and
No mal-Weigh Indi iduals
Lau a Sinis e a-Loaiza, Pa icia Alonso-Lo e a, Alejand a Ca delle-Cobas * , Jose Manuel Mi anda ,
Bea iz I. Vázquez and Albe o Cepeda
Labo a o io de Higiene, Inspección y Con ol de Alimen os, Depa amen o de Química Analí ica,
Nu ición y B oma ología, Campus Te a, Uni e sidade da San iago de Compos ela, 27002 Lugo, Spain;
[email p o ec ed] (L.S.-L.); [email p o ec ed] (P.A.-L.); [email p o ec ed] (J.M.M.);
[email p o ec ed] (B.I.V.); [email p o ec ed] (A.C.)
*Co espondence: alejand a.ca [email p o ec ed]
Abs ac :
Di e en esea ch s udies ha e iden i ied speci ic g oups o ce ain die a y compounds
as he onse and p og ession o obesi y and sugges ed ha gu mic obio a is a media o be ween
hese compounds and he in lamma ion associa ed wi h pa hology. In his s udy, he objec i e was o
e alua e he die a y in ake o 108 o e weigh (OW), obese (OB), and no mal-weigh (NW) indi iduals
and o analyze hei gu mic obio a p o ile o de e mine changes and associa ions wi h Body Mass
Index (BMI) and die . When indi iduals we e compa ed by BMI, signi ican di e ences in ibe and
monounsa u a ed a y acids (MUFAs) in ake we e obse ed, showing highe adequacy o he NW
g oup. The analysis o gu mic obio a showed s a is ical di e ences o 18 ASVs; Anae os ipes and
Faecalibac e ium dec eased in he OW/OB g oup, whe eas he genus Oscillospi a inc eased; he genus
was also ound in he LEFSe analysis as a bioma ke o OW/OB. Rosebu ia aecis was ound in a
signi ican ly highe p opo ion o NW indi iduals and iden i ied as a bioma ke o he NW g oup.
Co ela ion analysis showed ha adequa ion o nu i ional ecommenda ion o ibe indica ed a
highe abundance o P e o ella cop i, linea ly co ela ed wi h F. p ausni zii,Bac e oides caccae, and
R. aecis. The same co ela ion was ound o he adequa ion o MUFAs, wi h hese bac e ia being
mo e abundan when he in ake was adjus ed o o below he ecommenda ions.
Keywo ds: gu mic obio a; obesi y; ibe ; monounsa u a ed a y acids; BMI
1. In oduc ion
Wes e n socie ies ha e unde gone a p ocess ha in ol es majo quali a i e and quan-
i a i e changes in die a y habi s. Thus, adi ional die s ha e been eplaced by die s
cha ac e ized by a highe ene gy load and a dec ease in ibe and complex ca bohyd a es.
These changes, in addi ion o beha io al changes such as less physical ac i i y, ha e esul ed
in an inc ease in wo ldwide o e weigh and obesi y a es [
1
]. Obesi y is he abno mal
o excessi e accumula ion o a ha can be de imen al o heal h. I is o mul i ac o ial
o igin, esul ing om pa hological p ocesses and de i ing om an in e ela ion be ween
nume ous ac o s [2,3].
Acco ding o he Eu opean Heal h Su ey in Spain 2020, abou 16% o he Spanish
popula ion su e s om obesi y, while 37.60% o he popula ion is o e weigh [
4
]. O he
ecen moni o ing wo k e ealed ha Galicia is one o he egions o Spain in which he e
a e highe a es o obesi y in adul s, eaching 26.7% [
5
]. Addi ionally, a s udy epo ed ha
du ing he COVID-19 con inemen , 44% o pa icipan s indica ed an inc ease in hei body
weigh , wi h an a e age inc ease o 2.8 kg [
6
]. Thus, i is p obably ue ha he cu en
o e weigh a e o Galician people could now be highe han hose in he ci ed epo s [
4
,
5
].
Nu ien s 2023,15, 3418. h ps://doi.o g/10.3390/nu15153418 h ps://www.mdpi.com/jou nal/nu ien s
Nu ien s 2023,15, 3418 2 o 19
Se e al au ho s ha e poin ed ou ha he die a y habi s o Galician people do no ol-
low he same pa e ns as in he es o Spain. Howe e , oge he wi h he no h o Po ugal,
hey a e included in he die a y model known as he A lan ic Die [
7
,
8
]. Knowledge o he
die a y pa e ns o he popula ion is essen ial o unde s anding he po en ial impac o he
s a egies implemen ed o p e en he inc ease in obesi y a es [9].
One app oach o ea ing obesi y in ecen yea s in ol es he s udy o he gu mi-
c obio a, he esul s o which in bo h mice and humans desc ibe ema kable di e ences
be ween he gu mic obio a o obese (OB) and no mal-weigh (NW) subjec s. When he e
is an excess o body a , colonic concen a ions o Fi micu es inc ease by mo e han 50%,
while hose o Bac e oides dec ease co ela i ely compa ed o NW subjec s [
10
]. The com-
posi ion o he gu mic obio a o obese indi iduals is cha ac e ized by a dec ease in gene a
such as Akke mansia,Alis ipes,Faecalibac e ium, and Oscillibac e [
11
,
12
] and an inc ease in
S aphylococcus and Clos idium [13–16].
In addi ion, die a y pa e ns a e conside ed modula o s o he gu mic obio a; o
example, excessi e a consump ion induces an imbalance in he gu mic obio a, leading
o gu ba ie dys unc ion, inc eased hos body weigh , and low-g ade in lamma ion
o adipose issue [
17
]. A high-p o ein die , on he o he hand, inc eases he g ow h o
bile- ole an bac e ial species (Alis ipes,Bilophila, and Bac e oides) and dec eases bac e ia
ha hyd olyze disaccha ides o polysaccha ides in o simple ca bohyd a es (Rosebu ia,
Eubac e ium ec ale, and Ruminococcus b omii) [18].
Al hough he e is a ailable e idence abou he di e ences in gu mic obio a composi-
ion in bo h o e weigh o obese and no mal-weigh indi iduals, he e is sca ce in o ma ion
abou po en ial bioma ke s associa ed wi h ce ain die a y compounds. Thus, he hypo h-
esis o he p esen s udy is ha die a y in ake, especially speci ic die a y componen s, is
associa ed wi h di e en gu mic obio a composi ions in bo h o e weigh o obese and
no mal-weigh subjec s. The e o e, he aim o his s udy was o di e en ia e he gu mic o-
bio a o o e weigh (OW) and OB subjec s om ha o NW subjec s, in a sample o Galician
people, compa e he esul s wi h p e ious s udies o es ablish po en ial bioma ke s and
de e mine i s associa ion wi h die a y componen s po en ially diminished o inc eased in
each g oup unde s udy.
2. Ma e ials and Me hods
2.1. Popula ion and Sample Size
This c oss-sec ional s udy is pa o an in e na ional p ojec abou he sea ch o no el
bioma ke s in diabe es and obesi y in Ibe ian-Ame ica (CyTED p ojec 918PTE0540). The
s udy popula ion o he p esen analysis included 108 indi iduals, anging in age om
40 o 70 yea s
, wi h habi ual esidence in Galicia (a no hwes egion o Spain). The sample
size was es ima ed based on p e ious s udies [
19
–
21
]. The pa icipan s we e in o med o
he objec i es o he s udy, and in o med consen was ob ained om each olun ee o
pa icipa e in he s udy, and all da a ob ained we e handled acco ding o Spanish Law
3/2018 on pe sonal da a p o ec ion. This consen de ailed he condi ions o he esea ch,
highligh ing he olun a y na u e o pa icipa ion, he possibili y o wi hd awing om he
s udy e en wi h he accep ance o he consen , and he anonymous ea men o he da a
o esea ch pu poses only.
The ollowing inclusion c i e ia we e used: age be ween 40 and 70 yea s; absence o
diagnosed pa hologies, no ha ing unde gone medical ea men wi h ho mones, co icoids,
o ha ing ecen ly consumed any o he ollowing subs ances: p o on pump inhibi o s,
amphe amines, alpha-ad ene gic d ugs, alpha-blocke s, be a-blocke s, opia es, calcium-
an agonis s, neu olep ics, icyclic an idep essan s, pheno hiazines, cen al ne ous sys em
s imulan s (cocaine, e c.); no ha ing consumed any supplemen con aining p obio ics o
p ebio ics du ing he p e ious 2 mon hs; in he case o women, no being p egnan
E hical app o al o his s udy was ob ained om The Regional E hics Commi ee o
Clinical Resea ch (Galician Heal h Se ice, SERGAS, n
◦
2018/270) in compliance wi h he
Nu ien s 2023,15, 3418 3 o 19
Decla a ion o Helsinki o 1964 ega ding p i acy, con iden iali y, and in o med consen .
All expe imen s we e ca ied ou in acco dance wi h app o ed guidelines and egula ions.
2.2. An h opome ic Measu es
An h opome ic measu emen s we e ob ained. Weigh was de e mined using an InBody
127 digi al scale (InBody, Tokyo, Japan). Heigh was measu ed wi h a po able s adiome e
(ADE MZ10042; Hambu g, Ge many), wi h he subjec up igh and in balance, wi hou
bending he knees. Subsequen ly, he Body Mass Index (BMI) was calcula ed using he
Que ele o mula: BMI = weigh (kg)/[heigh (m)]
2
. Acco ding o he classi ica ion anges
p oposed by he Spanish Obesi y Socie y [
22
], h ee olun ee s we e g ouped in g oups
acco ding o hei BMI: 18–24.9 kg/m
2
o NW, 25–29.9 kg/m
2
o OW, and
≥
30 OB kg/m
2
.
2.3. Die a y In o ma ion
A 72 h die a y eco d was comple ed by he olun ee s o 3 days (2 wo kdays and
1 weekend day). The olun ee s we e gi en ins uc ions on how o eco d hei die a y in ake.
They we e also asked o p o ide in o ma ion on he po ions o ood consumed, he ing edien s
and echniques used in cooking, and he ype and quan i y o be e ages consumed.
The mean daily ene gy and nu ien in ake o each olun ee we e calcula ed using he
open so wa e “die calcula o ”, a ailable on he websi e o he Endoc inology and Clinical
Nu i ion Resea ch Cen e [
23
], which is based on Spanish oods. Da a ob ained om he
so wa e we e compa ed o he nu i ional objec i es o he Spanish adul popula ion
published by he Spanish Socie y o Communi y Nu i ion (SENC, 24) o de e mine hei
nu i ional adequacy. The da a ob ained we e compa ed by sex and by BMI o olun ee s.
Da a abou mic onu ien (mine als and i amins) in ake we e compa ed o Re e ence
Die a y In akes (RDI) es ablished by he Spanish Fede a ion o Nu i ion, Food and Die e ics
Socie ies [24]. The da a ob ained we e compa ed by sex and BMI o he olun ee s.
2.4. Fecal Sample Collec ion and DNA Ex ac ion
Volun ee s ecei ed de ailed ins uc ions o collec ecal samples and we e p o ided
wi h a s e ile con aine ha should deli e o he labo a o y along wi h he 72 h die a y
eco d. Samples should ha e been deli e ed wi hin wo hou s a e de eca ion o , in he
case o no being able o do i , hey should ha e been immedia ely ozen a
−
20
◦
C a e
deposi ion and deli e y o he labo a o y whe e hey we e conse ed ozen un il ea ed
o analysis.
DNA om ecal samples was ex ac ed using he Dneasy Powe soil ki (Qiagen
®
,
Hilden, Ge many) ollowing he manu ac u e ’s ins uc ions. Ex ac ed DNA was hen
quan i ied using a Qubi
™
4 luo ome e (In i ogen, The mo Fishe Scien i ic, Ca lsbad,
CA, USA) and he Qubi ki 1X dsDNA High Sensi i i y (The mo Fishe Scien i ic, Inc.,
Ka ls uhe, Ge many). A e quan i ica ion, DNA samples we e ozen and s o ed a
−
20
◦C un il u he analysis.
2.5. 16S RNA Amplicon Sequencing
Fo 16S RNA amplicon sequencing, 2
µ
L o DNA ex ac ed om each sample was used
o cons uc he lib a ies, and he Ion GeneS udio
TM
S5 Sys em (Li e Technologies, Ca lsbad,
CA, USA) was used. Fo his pu pose, he 16S hype a iable egions we e ampli ied wi h
wo se s o p ime s, 2–4–8 and 3–6, 7–9, and he lib a ies we e p epa ed by using he Ion
16S
TM
Me agenomics Ki (Li e Technologies) and he Ion Xp ess
TM
Plus F agmen Lib a y
Ki (Li e Technologies). Lib a ies con aining equal amoun s o PCR p oduc s pooled wi h a
ba code we e p epa ed by using he Ion Xp ess
TM
Ba code Adap e s Ki (Li e Technologies).
Then, hese lib a ies we e quan i ied by using he Ion Uni e sal Lib a y Quan i a ion Ki
(Li e Technologies). Nex , 10 pM o each lib a y was pooled and loaded on an Ion One-
Touch
™
2 Sys em (Li e Technologies), which au oma ically pe o ms empla e p epa a ion
and en ichmen . Templa e-posi i e ion sphe e pa icles we e en iched wi h Dynabeads
™
MyOne
™
S ep a idin C1 magne ic beads (In i ogen, Ca lsbad, CA, USA) by using an Ion
Nu ien s 2023,15, 3418 4 o 19
One Touch ES ins umen . Finally, an Ion 520 TM chip (Li e Technologies) was loaded wi h
he samples on an Ion GeneS udioTM S5 Sys em sequence using he Ion 520™ & Ion 530™
Loading Reagen s supplied in he OT2-Ki (Li e Technologies).
2.6. S a is ical and Bioin o ma ic Analysis
S uden ’s - es o independen samples was used o compa e quali a i e a iables
be ween di e en g oups (sex o BMI). The X2 es wi h Ya es’s co ec ion was used o
compa e equencies. In all cases, he ob ained di e ences we e conside ed s a is ically
signi ican i he p alue was less han 0.05.
A wo-way ANOVA was used o de e mine signi ica i e di e ences o ime and
subs a es as he wo co a ia es in he gene al linea model using Tukey’s analysis. Fo
signi ican di e ences (p< 0.05) a one-way ANOVA was conduc ed o each subs a e
compa ing 0, 5, 10, and 24 h. Simila ly, each subs a e was compa ed using wo-way
ANOVA; esul s we e signi ican when p< 0.05. The so wa e SPSS
®
.27 o Windows
(SPSS Inc., Chicago, IL, USA) was used o hese analyses.
Fo he analysis o 16S RNA amplicon sequencing, he aw sequencing eads we e
ob ained om he To en Sui e so wa e ( . 5.12.2.) as as q iles. The as q iles we e
p ocessed wi h QIIME 2 so wa e . 2022.11 [
25
]. To p oduce amplicon sequence a ian s
(ASVs), he DADA2 me hod was used o quali y il a ion (Q sco e
≥
30), imming,
denoising, and de eplica ion. Samples wi h ea u es ( axa) wi h a o al abundance (summed
ac oss all samples) o <10 we e emo ed. Taxonomy was assigned o ASVs by using he
q2- ea u e-classi ie classi y-sklea n naï e Bayes axonomy classi ie , which was compa ed
o he G eengenes 13_8 99% ope a ional axonomic uni (OTU) e e ence sequences.
STAMP so wa e ( 2.1.3) o he “S a is ical Analysis o Taxonomic and Func ional
P o iles” [
26
] was used o de e mine s a is ical di e ences in he ob ained ASVs a he
species le el. K uskal–Wallis H es wi h pos hoc Tukey–K ame es was employed.
The OTUs wi h axonomic in o ma ion, ob ained in QIIME2, we e used oge he wi h
a me ada a ile, in ex o ma , o hei analyses in he ee pla o m Mic obiome Analys ,
“a comp ehensi e s a is ical, unc ional and in eg a i e analysis o mic obiome da a” [
27
].
alpha- and be a- di e si y, co ela ion, and LE Se analysis we e ca ied ou .
3. Resul s
3.1. Nu i ional Analysis and Adequa ion o he Objec i es and he Recommended Daily In akes o
he Spanish Popula ion
A o al o 108 subjec s comple ed a 72 h die a y ecall, and hei an h opome ic
measu emen s we e ob ained. O hese, 67 (62%) we e women and 41 (38%) we e men. The
a e age age o he subjec s was 51.0
±
8.0 yea s, and he a e age kilocalo ie in ake was
2344.0
±
556.9 kcal/day. Compa ing his ene gy in ake by sex, a signi ican ly highe in ake
(p= 0.04) was obse ed o men (2502.6
±
538.3) han o women (2247.0
±
537.0). Da a
ob ained o mac onu ien s and o he pa ame e s a e shown in Table 1.
As i can be obse ed, he a e age o al ca bohyd a e in ake in e ms o % o ene gy is
de icien , 88.1% o he indi iduals do no achie e he ecommenda ions. The same occu s
o ibe in ake (84.3% o indi iduals a e in de ici ). Rega ding he lipid p o ile, 89.6%
o he pa icipan s a e abo e he objec i es, which indica es a die ich in a , especially
in sa u a ed a since 88% o he olun ee s showed o possess a consump ion abo e he
ecommenda ions. Fo p o ein, i is necessa y o indica e ha his mac onu ien does
no appea in he ecommenda ions o he Spanish popula ion, since i s de e mina ion is
depending on he o he wo mac onu ien s. The pe cen o p o ein usually ecommended
is 10–15% o o al ene gy. The da a ob ained o he pa icipan s o his s udy indica ed
an adequa ion o 38.9% and an excess o 61.1%.
Fo simple suga , only 13 indi iduals showed o ha e a consump ion below a 10%,
being an a e age in ake o 18.1
±
7.4%. Rega ding ui and ege able consump ion, he
a e age in ake o ui and ege ables was 1.8
±
1.5 se ings, which is <50% o he ecom-
mended i e daily se ings; only 4.6% o he popula ion su eyed had he ecommended
Nu ien s 2023,15, 3418 5 o 19
in ake, while 94.4% did no mee he ecommenda ions. Rega ding o he pa ame e s such
as alcohol consump ion, mos o he olun ee s showed o be below he ecommenda ion
o 1 and 2 S anda d D ink Uni s (SDUs) o alcoholic be e ages, o women and men,
espec i ely. Rega ding wa e consump ion, 45.4% o he pa icipan s did no each he
ecommenda ions o wa e consump ion.
Table 1.
Pa icipan s’ die quali y in e ms o a calo ic lipid p o ile: adequacy o nu i ional objec i es
o he Spanish popula ion (n= 108). Resul s a e exp essed as he a e age
±
s anda d de ia ion. Die
adequacy is exp essed as he numbe o indi iduals mee ing he nu i ional goals and in b acke s is
exp essed as pe cen .
DIET ADEQUACY
CALORIC PROFILE NUTRITIONAL
OBJECTIVES
VALUES OF
PARTICIPANTS’ DIET DÉFICIT ADEQUATE EXCESS
Ca bohyd a es (% Ene gy) 50–55 38.9 ±7.8 96 (88.1%) 11 (10.1%) 1 (0.9%)
Fibe (g/1000 kcal) >14 11.7 ±7.7 91 (84.3%) 15 (13.9%) 2 (1.9%)
Lipids (% Ene gy) 30–35 41.8 ±8.0 7 (6.4%) 15 (13.9%) 86 (79.6%)
LIPID PROFILE
SFA (% Ene gy) ≤7–8 12.0 ±3.2 - 13 (0.0%) 95 (88%)
MUFA (% Ene gy) 20 20.8 ±16.1 36 (33.3%) 45 (41.7%) 27 (25.0%)
PUFA (% Ene gy) 5 5.9 ±3.6 39 (38.0%) 23 (21.2%) 62 (57.4%)
Choles e ol (mg) <300 347.0 ±138.6 0 (0.0%) 46 (42.6%) 62 (57.4%)
OTHERS
Wa e (mL) 2000 2063.3 ±627.9 49 (45.4%) 59 (54.6%) 0 (0.0%)
F ui s and ege ables 5 po ions 1.8 ±1.5 102 (94.4%) 5 (4.6%) 1 (0.9%)
Suga (% Ene gy) 6–10 18.1 ±7.4 - 13 (12.0%) 95 (88.0%)
Alcohol (g) ≤1 SDU women
≤2 SDU men 10.84 ±44.43 - 100 (92.6%) 8 (7.4%)
SFA: Sa u a ed Fa y Acids; MFA: Monounsa u a ed Fa y Acids; PUFA: Polyunsa u a ed Fa y Acids; SDU:
S anda d D ink Uni .
When he da a ob ained we e compa ed by sex (Table S1), he e we e some signi ican
di e ences in die a y pa e ns be ween men and women.
Thus, in gene al, women accomplish mo e nu i ional objec i es han men. On a e age,
men ha e a highe in ake o p o ein, a lowe consump ion o ca bohyd a es, and a highe
consump ion o a han women. Rega ding he lipid p o ile, s a is ical di e ences we e
ound o SFA, whe e women, once again, showed o be e accomplish he nu i ional
objec i es o he Spanish popula ion.
Compa ing in ake da a acco ding o BMI (Table 2), no s a is ical di e ences we e
ound among NW, OW, and OB subjec s o all pa ame e s in es iga ed wi h he excep ion
o o al ene gy, ibe , and MUFAs, o which s a is ical di e ences we e ound. In he case
o ibe , a be e adequa ion (32.5%) was obse ed o NW indi iduals han o OW/OB
(15.2 and 14.3, espec i ely). Fo MUFAs, only 14.3% o he OB olun ee s accomplish he
objec i es, whe eas o he OW and NW g oups, he accomplishmen was highe , 36.4 and
25%, espec i ely. Fo o al ene gy, 2284.0
±
658.6 kcal/day we e ob ained o he NW
g oup, whe eas o he OW and OB g oup, he o al ene gy ob ained was 2221.3
±
497.2 and
2498.4
±
459.1 kcal/day, espec i ely. A signi ican ly highe calo ic in ake was ob ained o
he OB g oup (p= 0.0356).
Rega ding wa e , ui , ege ables, and suga , no s a is ical di e ences we e obse ed
acco ding o BMI. Howe e , in gene al, he NW and he OW g oups showed highe
adequacy o wa e and alcohol consump ion. Fo suga consump ion, he wo s adequacy
was ob ained o he NW g oup, bu hese alues included all he simple suga consumed,
including ha p o ided o ui s and no only he added simple suga , and his is he
g oup consuming mo e ui s and ege ables.
Nu ien s 2023,15, 3418 6 o 19
Table 2.
Mac onu ien in ake o no mal weigh (NW), o e weigh (OW), o obese (OB) subjec s,
and i s adequa ion o he nu i ional objec i es o he Spanish popula ion (n= 108). Calo ic p o ile,
lipidic p o ile, and o he s a e shown. Resul s a e exp essed wi h mean
±
s anda d de ia ion. Die
adequacy is exp essed as he numbe o indi iduals mee ing he nu i ional goals and in b acke s is
exp essed as pe cen .
NW (n= 40) OW (n= 35) OB (n= 33)
CALORIC PROFILE Nu i ional
Objec i es Mean ±SD
In ake
Adequacy:
n(%)
Mean ±SD
In ake
Adequacy:
n(%)
Mean ±SD
In ake
Adequacy:
n(%)
pValue
Ca bohyd a es
(% Ene gy) 50–55% 40.3 ±7.1 5 (12.5%) 37.4 ±7.7 1 (3.0%) 38.8 ±8.6 4(11.4%) 0.274
Lipids
(% Ene gy) 30–35% 40.6 ±6.3 6 (15.0%) 43.6 ±9.6 5 (15.2%) 41.9 ±7.5 5 (14.3%) 0.280
Fibe
(g/1000 Kcal) >14 14.2 ±9.5 13 (32.5%) 12.5 ±10.14 4 (12.0%) 9.1 ±2.9 5 (14.3%) 0.038
LIPID PROFILE
SFA
(% Ene gy) ≤7–8 11.8 ±2.8 3 (7.5%) 11.9 ±3.0 4 (12.1%) 15.5 ±8.1 0 (0.0%) 0.559
MUFA
(% Ene gy) 20 20.3 ±5.7 10 (25.0%) 19.4 ±5.7 12 (36.4%) 6.5 ±5.3 5 (14.3%) <0.001
PUFA
(% Ene gy) 5 5.8 ±2.1 12 (30.0%) 6.5 ±5.3 19 (57.6%) 5.4 ±2.7 6 (17.4%) 0.874
Choles e ol (mg) <300 mg 340.3 ±137.7 19 (47.5%) 348.0 ±129.8 11 (33.0%) 354.1 ±152.1 10 (28.6%) 0.915
OTHERS
Wa e (mL) 2000 mL 2305.36 ±487.6 23 (57.5%) 2128.0 ±682.2 20 (57.4%) 2098.1 ±736.4 16 (48.5%) 0.751
F ui s and ege ables 5 po ions 1.9 ±1.5 3 (7.5%) 1.3 ±1.4 1 (2.8%) 2.2 ±1.5 1 (3.0%) 0.785
Suga 6–10 20.0 ±9.0 1 (2.5%) 19.0 ±7.1 5 (14.3%) 16.0 ±5.1 7 (21.2%) 0.632
Alcohol (g)
≤1 SDU
women
≤2 SDU men
3.8 ±9.8 39 (97.5%) 4.0 ±8.7 32 (97.0%) 3.9 ±8.8 31 (88.6%) 0.298
SFA: Sa u a ed Fa y Acids; MFA: Monounsa u a ed Fa y Acids; PUFA: Polyunsa u a ed Fa y Acids; SDU: S anda d
D ink Uni .
Rega ding he in ake o mic onu ien s, a compa ison be ween women and men can
be seen in Table S2. Signi ican di e ences we e ound be ween sexes o he in ake o
hiamine, ibo la in, niacin, i amin B6, i amin E, phospho us, and i on, wi h he a e age
daily in ake being highe in men han in women o all he mic onu ien s. Rega ding
adequacy, howe e , women possess a majo pe cen age o adequacy han men.
When compa ing mic onu ien in ake by BMI (Table 3), i was ound ha NW subjec s
consumed highe amoun s o some mic onu ien s such as i on, iodine, and asco bic acid
han OW and OB pa ien s, whe eas ocophe ol in ake was signi ican ly highe o OW
subjec s and OB pa ien s, and a highe in ake o calci e ol wi h espec o bo h NW and
OW subjec s was obse ed. Compliance wi h in ake wi h cu en ecommenda ions was
highe in NW subjec s han in OW subjec s o calcium, i on, iodine, zinc, asco bic acid,
and calci e ol. Only in he case o magnesium we e he highes compliance a es ound in
OB subjec s wi h espec o NW and OW pa ien s.
Table 3.
Mic onu ien in ake o no mal weigh (NW), o e weigh (OW), o obese (OB) subjec s, and i s
adequa ion o he Die a y Re e ence In akes (DRI) o he Spanish popula ion (n= 108). Die adequacy is
exp essed as he numbe o indi iduals mee ing he DRI and in b acke s is exp essed as pe cen .
NW (n= 40) OW (n= 35) OB (n= 33)
Vi amins A e age ±SD
In ake
Adequacy
n(%)
A e age ±SD
In ake
Adequacy
n(%)
A e age ±SD
In ake
Adequacy
n(%)
pValue
Thiamine (mg) 1.5 ±0.8 32 (80.0%) 1.9 ±0.9 31 (88.6%) 1.4 ±0.3 29 (87.9%) 0.009
Ribo la in (mg) 1.7 ±0.5 30 (75.0%) 2.1 ±0.6 31 (88.6%) 1.7 ±0.4 27 (81.9%) 0.007
Nu ien s 2023,15, 3418 7 o 19
Table 3. Con .
NW (n= 40) OW (n= 35) OB (n= 33)
Vi amins A e age ±SD
In ake
Adequacy
n(%)
A e age ±SD
In ake
Adequacy
n(%)
A e age ±SD
In ake
Adequacy
n(%)
pValue
Niacin (mg) 30.5 ±10.7 30 (75.0%) 40.4 ±14.5 16 (45.7%) 31.6 ±7.7 25 (75.8%) 0.0005
Vi amin B6(µg) 2.0 ±0.8 35 (87.5%) 2.3 ±1.1 33 (94.3%) 2.0 ±0.6 30 (90.9%) 0.159
Folic acid (µg) 281.3 ±126.3 13 (32.5%) 278.3 ±105.2 10 (28.6%) 255.3 ±74.6 8 (24.2%) 0.537
Vi amin B12 (µg) 6.1 ±6.0 40 (100%) 6.4 ±2.5 35 (100%) 5.6 ±2.8 33 (100%) 0.697
Vi amin C (mg) 151.2 ±87.7 35 (87.5%) 166.2 ±79.6 33 (94.3%) 160.7 ±68.5 33 (100%) 0.738
Vi amin A (µg) 798.4 ±603.1 24 (60.0%) 750.5 ±452.7 19 (54.3%) 745.0 ±406.8 17 (51.5%) 0.868
Vi amin D (µg) 2.9 ±3.2 8 (20.0%) 4.6 ±6.0 11 (31.4%) 4.5 ±6.5 11 (33.3%) 0.321
Vi amin E (mg) 8.3 ±8.1 5 (12.5%) 7.8 ±9.9 4 (11.4%) 5.9 ±5.6 2 (6.1%) 0.416
Mine als
Calcium (mg) 719.8 ±297.1 7 (17.5%) 841.2 ±415.0 12 (34.3%) 792.9 ±348.4 10 (30.3%) 0.331
Magnesium (mg) 325.7 ±134.9 23 (57.5%) 368.9 ±95.8 23 (65.7%) 308.8 ±81.0 15 (45.5%) 0.063
Po assium (mg) 3086.7 ±904.1 22 (55.0%)
4044.5
±
1135.9
26 (74.3%) 3511.8 ±745.6 24 (72.7%) 0.0001
Phospo us (mg) 1415.5 ±500.5 38 (95.0%) 1716.9 ±472.9 34 (97.1%) 1436.8 ±380.6 32 (97.0%) 0.009
I on (mg) 16.7 ±9.0 23 (57.5%) 18.3 ±5.8 29 (82.9%) 14.0 ±2.8 21 (63.6%) 0.035
Iodine (µg) 184.4 ±150.3 16 (40.0%) 347.5 ±197.4 25 (71.4%) 292.5 ±153.6 25 (75.8%) 0.0002
Zinc (mg) 24.4 ±78.7 29 (72.5%) 14.1 ±3.5 34 (97.1%) 10.8 ±2.9 27 (81.8%) 0.0445
Sodium (mg) 2610.9 ±677.5 6 (15.0%)
5609.5
±
1219.7
0 (0.0%) 3877.1 ±346.0 0 (0.0%) <0.0001
3.2. Analysis o he Gu Mic obio a Composi ion
3.2.1. Alpha- and Be a-Di e si y
A o al o 95 ecal samples we e collec ed om he olun ee s. A i s analysis be ween
g oups (NW, OW, and OB) was ca ied ou ; howe e , no di e ences we e ob ained be ween
he OW and OB g oups. The e o e, a second analysis conduc ed by g ouping OW and
OB was de eloped. Al hough he BMI is he mos ex ended me hod, in clinical p ac ice,
o classi y o e weigh and obesi y in adul s, i is no he mos adequa e o de e mine
he amoun o body a . In addi ion, he nu i ional analysis has shown ha , in e ms o
adequacy, he indi iduals included in he NW g oup accomplish in highe p opo ion he
nu i ional objec i es and he DRI o he Spanish popula ion han he OW and OB g oups
( o example, he % o adequacy o he g oup o OW and OB is 0, whe eas 15% o he
indi iduals in he NW g oup mee he RDI)
To in es iga e alpha-di e si y, he Chao1 ichness and Shannon di e si y ( ichness
and abundance) indices we e de e mined. No s a is ical di e ences we e ound be ween
he NW and OW/OB g oups o Chao1 (ANOVA, p= 0.1953) no o Shannon (p= 0.711).
Fo be a-di e si y, calcula ed by B ay-Cu is, no s a is ical di e ences we e ound be ween
g oups (F- alue: 0.7111; R-squa ed: 0.0079266; p- alue: 0.768) ei he . See Figu e 1.
3.2.2. Rela i e Abundance o Bac e ia
The ela i e equency (Figu e 2) a he phylum le el (a) showed an inc ease in he
Bac e oide es phlylum in he OW/OB (35.5%) g oup in compa ison wi h he NW g oup
(33.2%), whe eas he Fi micu es phylum dec eased in he OW/OB g oup (53.3% s. 56.3%)
as well as Ac inobac e ia (3.54 s. 3.90%). A he genus le el (Figu e 1b), he main iden i ied
bac e ia we e Bac e oides and P e o ella, con ibu ing o he Bac e oide es phylum, while
Blau ia and Ruminoccocus o med he Fi micu es phylum, and Bi idobac e ium con ibu es
o Ac inobac e ia phylum; in he P o eobac e ia phylum, he main genus was Su e ella
and Succini ib io. Finally, a he species le el, 50 species we e iden i ied, wi h he main
iden i ied ones being P e o ella cop i,Bac e oides uni o mis,Bi idobac e ium adolescen is, and
Bi idobac e ium longum, among o he s.
Nu ien s 2023,15, 3418 8 o 19
Nu ien s 2023, 15, x FOR PEER REVIEW 8 o 21
( o example, he % o adequacy o he g oup o OW and OB is 0, whe eas 15% o he
indi iduals in he NW g oup mee he RDI)
To in es iga e alpha-di e si y, he Chao1 ichness and Shannon di e si y ( ichness
and abundance) indices we e de e mined. No s a is ical diffe ences we e ound be ween
he NW and OW/OB g oups o Chao1 (ANOVA, p = 0.1953) no o Shannon (p = 0.711).
Fo be a-di e si y, calcula ed by B ay-Cu is, no s a is ical diffe ences we e ound be-
ween g oups (F- alue: 0.7111; R-squa ed: 0.0079266; p- alue: 0.768) ei he . See Figu e 1.
Figu e 1. (a) Shannon index and (b) Chao1 index (alpha-di e si y), in ed o no mal-weigh indi-
iduals (NW) and, in blue o o e weigh /obese (OW/OB); (c) be a-di e si y de e mined by p inci-
pal coo dina es analysis (PCoA) based on he B ay-Cu is dissimila i y index; ed NW g oup and
blue OW/OB g oup.
3.2.2. Rela i e Abundance o Bac e ia
The ela i e equency (Figu e 2) a he phylum le el (a) showed an inc ease in he
Bac e oide es phlylum in he OW/OB (35.5%) g oup in compa ison wi h he NW g oup
(33.2%), whe eas he Fi micu es phylum dec eased in he OW/OB g oup (53.3% s. 56.3%)
as well as Ac inobac e ia (3.54 s. 3.90%). A he genus le el (Figu e 1b), he main iden i-
ied bac e ia we e Bac e oides and P e o ella, con ibu ing o he Bac e oide es phylum,
while Blau ia and Ruminoccocus o med he Fi micu es phylum, and Bi idobac e ium con-
ibu es o Ac inobac e ia phylum; in he P o eobac e ia phylum, he main genus was Su -
e ella and Succini ib io. Finally, a he species le el, 50 species we e iden i ied, wi h he
main iden i ied ones being P e o ella cop i, Bac e oides uni o mis, Bi idobac e ium adolescen is,
and Bi idobac e ium longum, among o he s.
Rega ding he s a is ical diffe ences, he analysis using he so wa e STAMP showed
s a is ical diffe ences o 18 ASVs (see able in Supplemen a y Ma e ial). In Figu e 3, i is
possible o see he box plo s o h ee iden i ied bac e ial gene a and 1 o he species. In he
g aphics, i can be obse ed as he gene a Anae os ipes and Faecalibac e ium dec eased in
Figu e 1.
(
a
) Shannon index and (
b
) Chao1 index (alpha-di e si y), in ed o no mal-weigh indi iduals
(NW) and, in blue o o e weigh /obese (OW/OB); (
c
) be a-di e si y de e mined by p incipal coo dina es
analysis (PCoA) based on he B ay-Cu is dissimila i y index; ed NW g oup and blue OW/OB g oup.
Nu ien s 2023, 15, x FOR PEER REVIEW 9 o 21
he OW/OB g oup whe eas he genus Oscillospi a inc eased. Fo he species, R. aecis, he
p opo ion o sequence ob ained was s a is ically highe in NW indi iduals han in
OW/OB.
(a) (b)
Figu e 2. Rela i e abundance o diffe en bac e ial phyla and gene a. Bac e ial composi ion ( ela-
i e abundance, %) was de e mined using 16S RNA amplicon sequencing a he phylum (a) and
genus (b) le els. The x-axis shows he diffe en g oups e alua ed (NW s. OW/OB). Due o he la ge
numbe o epo ed bac e ia, only he op 19 mos abundan gene a we e included in he legend.
NW: no mal weigh ; OW/OB, o e weigh /obese indi iduals.
Figu e 2. Rela i e abundance o di e en bac e ial phyla and gene a
. Bac e ial composi ion ( ela i e
abundance, %) was de e mined using 16S RNA amplicon sequencing a he phylum (
a
) and genus
(
b
) le els. The x-axis shows he di e en g oups e alua ed (NW s. OW/OB). Due o he la ge numbe
o epo ed bac e ia, only he op 19 mos abundan gene a we e included in he legend. NW: no mal
weigh ; OW/OB, o e weigh /obese indi iduals.
Nu ien s 2023,15, 3418 9 o 19
Rega ding he s a is ical di e ences, he analysis using he so wa e STAMP showed
s a is ical di e ences o 18 ASVs (see able in Supplemen a y Ma e ial). In Figu e 3, i is
possible o see he box plo s o h ee iden i ied bac e ial gene a and 1 o he species. In
he g aphics, i can be obse ed as he gene a Anae os ipes and Faecalibac e ium dec eased in
he OW/OB g oup whe eas he genus Oscillospi a inc eased. Fo he species, R. aecis, he
p opo ion o sequence ob ained was s a is ically highe in NW indi iduals han in OW/OB.
Nu ien s 2023, 15, x FOR PEER REVIEW 10 o 21
Figu e 3. Box plo s ob ained in he s a is ical analysis wi h STAMP o he gene a Anae os ipes,
Faecalibac e ium, Oscillospi a, and he species R. aecis. Blue ep esen s he NW g oup, and o ange is
he OW/OB g oup.
3.2.3. Co ela ion Analysis: Mic obio a-Fibe , Mic obio a-MUFAs
Since s a is ical diffe ences we e ound o ibe and MUFAs in ake in he nu i ional
analysis, a co ela ion analysis was ca ied ou wi h hese wo ac o s o es ablish a linea
ela ionship among he iden i ied species. Indi iduals we e, in his case, classi ied de-
pending on hei adequa ion o he ecommended in ake, eaching o no eaching he
ecommenda ion s anda ds o an adequa e, lowe , o high consump ion in he case o
MUFAs. Figu e 4a,b show he ob ained esul s. As can be seen in Figu e 4a, P. cop i was
mo e abundan in indi iduals achie ing ecommenda ions o ibe , and his species was
posi i ely co ela ed wi h F. p ausnizii, B. o a us, B. caccae, B. uni o mis, and R. aecis. A
nega i e co ela ion was ound o B. adolescen is, Collinsella ae o aciens, Do ea o micigen-
e ans, Pa abac e oides dis asonis, Ruminococcus b omii, and B. longum. The bac e ia posi i ely
co ela ed wi h ibe we e hose mo e abundan in NW indi iduals (see Supplemen a y
Ma e ial Figu e S1).
Rega ding MUFAs (Figu e 4b), P. cop i was mo e abundan in hose indi iduals wi h
adequa e consump ion o ecommenda ions, ollowed by indi iduals wi h low consump-
ion, and he lowes abundance was obse ed o hose indi iduals wi h high consump-
ion. Fo his species, a posi i e co ela ion was obse ed wi h B. caccae, F. p ausni zii, R.
aecis, and B. o a us.
Figu e 3.
Box plo s ob ained in he s a is ical analysis wi h STAMP o he gene a Anae os ipes,
Faecalibac e ium,Oscillospi a, and he species R. aecis. Blue ep esen s he NW g oup, and o ange is
he OW/OB g oup.
3.2.3. Co ela ion Analysis: Mic obio a-Fibe , Mic obio a-MUFAs
Since s a is ical di e ences we e ound o ibe and MUFAs in ake in he nu i ional
analysis, a co ela ion analysis was ca ied ou wi h hese wo ac o s o es ablish a linea ela-
ionship among he iden i ied species. Indi iduals we e, in his case, classi ied depending on
hei adequa ion o he ecommended in ake, eaching o no eaching he ecommenda ion
s anda ds o an adequa e, lowe , o high consump ion in he case o MUFAs. Figu e 4a,b
show he ob ained esul s. As can be seen in Figu e 4a, P. cop i was mo e abundan in
indi iduals achie ing ecommenda ions o ibe , and his species was posi i ely co ela ed
wi h F. p ausnizii,B. o a us,B. caccae,B. uni o mis, and R. aecis. A nega i e co ela ion was
ound o B. adolescen is,Collinsella ae o aciens,Do ea o micigene ans,Pa abac e oides dis asonis,
Ruminococcus b omii, and B. longum. The bac e ia posi i ely co ela ed wi h ibe we e hose
mo e abundan in NW indi iduals (see Supplemen a y Ma e ial Figu e S1).
Rega ding MUFAs (Figu e 4b), P. cop i was mo e abundan in hose indi iduals wi h
adequa e consump ion o ecommenda ions, ollowed by indi iduals wi h low consump-
ion, and he lowes abundance was obse ed o hose indi iduals wi h high consump ion.
Fo his species, a posi i e co ela ion was obse ed wi h B. caccae,F. p ausni zii,R. aecis,
and B. o a us.
Nu ien s 2023,15, 3418 16 o 19
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/nu15153418/s1, Figu e S1: Co ela ion analysis. Figu es show he
ob ained esul s o he co ela ion analysis be ween iden i ied bac e ial species and BMI o indi idual
(NW in ed and OW/OB in g een, g oups) Red lines showed a posi i e co ela ion among bac e ial
species whe eas blue lines showed a nega i e co ela ion; Table S1: Pa icipan s’ die quali y by sex, in
e ms o calo ic p o ile and lipid quali y: adequacy o nu i ional objec i es o he Spanish popula ion.;
Table S2: Mic onu ien in ake and adequa ion o he Die a y Re e ence In akes (DRI) o he Spanish
popula ion in women and men.
Au ho Con ibu ions:
Concep ualiza ion, A.C. and A.C.-C.; me hodology, L.S.-L. and P.A.-L.; so -
wa e, A.C.-C.; alida ion, A.C.-C.; o mal analysis, J.M.M.; w i ing—o iginal d a p epa a ion,
L.S.-L. and B.I.V.; w i ing— e iew and edi ing, A.C.-C., J.M.M. and A.C.; supe ision, A.C.; unding
acquisi ion, A.C. 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 Minis y o Economy and Compe i i eness h ough
he S a e P og am o I+D+I O ien ed o he Challenges o Socie y 2017–2020 (In e na ional Join
P o-g amming 2018). P ojec PCI2018-093245.
Ins i u ional Re iew Boa d S a emen :
E hical app o al o his s udy was ob ained om The
Regional E hics Commi ee o Clinical Resea ch (Galician Heal h Se ice, SERGAS, n
◦
2018/270) in
compliance wi h he Decla a ion o Helsinki o 1964 ega ding p i acy, con iden iali y, and in o med
consen . All expe imen s we e ca ied ou in acco dance wi h app o ed guidelines and egula ions.
In o med Consen S a emen :
In o med consen was ob ained om all subjec s in ol ed in he s udy.
Da a A ailabili y S a emen :
The da a p esen ed in his s udy a e a ailable on eques om he
co esponding au ho .
Acknowledgmen s:
The au ho s hank he CyTED and each Na ional O ganism o Science and
Technology o unding he IBEROBDIA p ojec (918PTE0540). In his ega d, Spain speci ically
hanks he Minis y o Economy and Compe i i eness o he inancial suppo o his p ojec
h ough he S a e P og am o I+D+I O ien ed o he Challenges o Socie y 2017–2020 (In e na ional
Join P og amming 2018). P ojec PCI2018-093245.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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