Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

0.89 MB·English

by  Adewumi, Gbenga A.

ORIGINALRESEARCHARTICLE
published:07January2013
doi:10.3389/fmicb.2012.00436
Combination of culture-independent and
culture-dependent molecular methods for the
determination of bacterial community of iru,
a fermented Parkia biglobosa seeds
GbengaA.Adewumi1,2,3,FolarinA.Oguntoyinbo1*,SantoshKeisam2,WahengbamRomi2 and
KumaraswamyJeyaram2
1DepartmentofMicrobiology,FacultyofScience,UniversityofLagos,Akoka,Lagos,Nigeria
2MicrobialResourcesDivision,InstituteofBioresourcesandSustainableDevelopment,TakyelpatInstitutionalArea,Imphal,Manipur,India
3DepartmentofFoodScienceandTechnology,CollegeofFoodSciences,BellsUniversityofTechnology,Ota,Nigeria
Editedby: In this study, bacterial composition of iru produced by natural, uncontrolled fermentation
DaniloErcolini,UniversitàdegliStudi of Parkia biglobosa seeds was assessed using culture-independent method in
diNapoliFedericoII,Italy combination with culture-based genotypic typing techniques. PCR-denaturing gradient
Reviewedby: gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA
Jean-PierreGuyot,Institutde
extraction methods used and confirmed bacterial diversity in the 16 iru samples
RecherchePourleDévelopement,
France from different production regions. DNA sequencing of the highly variable V3 region
LucaCocolin,UniveristyofTurin, of the 16S rRNA genes obtained from PCR-DGGE identified species related to
Italy Bacillus subtilis as consistent bacterial species in the fermented samples, while other
*Correspondence: major bands were identified as close relatives of Staphylococcus vitulinus, Morganella
FolarinA.Oguntoyinbo,Department
morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus
ofMicrobiology,FacultyofScience,
UniversityofLagos,Akoka,Lagos, thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and
Nigeria. uncultured bacteria clones. Bacillus species were cultured as potential starter cultures
e-mail:[email protected] and clonal relationship of different isolates determined using amplified ribosomal DNA
restriction analysis (ARDRA) combined with 16S–23S rRNA gene internal transcribed
spacer(ITS)PCRamplification,restrictionanalysis(ITS-PCR-RFLP),andrandomlyamplified
polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants
from food-borne pathogens such as B. cereus and suggested the need for development
of controlled fermentation processes and good manufacturing practices (GMP) for iru
productiontoachieveproductconsistency,safetyquality,andimprovedshelflife.
Keywords:iru,fermentation,safety,Bacillus,DGGE
INTRODUCTION (Achi, 2005). Nonetheless, the production process is generally
Fermentedvegetableproteinseedsusedforcondimentsproduc- characterized by spontaneous solid substrate alkalinefermenta-
tioninWestAfricaincludeAfricanlocustbean[Parkiabiglobosa tion with increase in pH as a result of extensive hydrolysis of
(Jacq. Benth)], melon seeds [Citrullus vulgaris (Schrad)], castor the proteins into peptides, amino acids and ammonia, which
oilseeds(Ricinuscommunis),flutedpumpkinseeds(Telfariaocci- favorsthedominanceofBacillusspp.asthefermentingorganisms
dentale),Africanyambean(Stenophylisstenocarpa),cottonseeds (Kiers et al.,2000; Sarkar et al., 2002; Ouoba et al.,2003; Achi,
[Gossypiumhirsitium(L.)],roselleseeds(Hibiscussabdariffa),and 2005).Theartisalsocarriedoutinexclusiveuncontrollableenvi-
baobab seeds [Adansonia digitata (L.)] (Odunfa and Oyewole, ronmental conditions thereby yielding products with variation
1998;Ouobaetal.,2008;Parkoudaetal.,2010).Condimentscon- in qualityandorganoleptic properties (Sanni,1993; Steinkraus,
stituteasignificantproportionofAfricandietswheretheyserve 1997;Ouobaetal.,2007).Thisnecessitatestheneedforthedevel-
asflavorsomeandculinarycomponentsinvariousdishes(Achi, opmentofappropriatestarterculturestoinitiatefermentationfor
2005). They include iru or dawadawa in Nigeria and Ghana; theproductionofconsistentproductswithacceptablequalities.
soumbala, bikalga, and maari in Burkina Faso; afitin and sonru Theprocessing operations of Africanlocustbean to produce
in Benin Republic; nététou in Senegal; kinda in Sierra Leone; iruinvolvecleaningandsorting,boiling,dehulling,andfermen-
dawadawa botso in Niger Republic; datou in Mali; mbuja in tationatambienttemperature(Oyeyiola,1988).Investigationson
CameroonandfurunduinSudan(N’Diretal.,1994;Ouobaetal., the microbiology of iru using selective isolation andcultivation
2008,2010;Parkoudaetal.,2010). based on traditional culture-dependent methods including the
Though,thevegetableseedsusedfortheproductionofthese biochemical changes that occur during fermentation have been
condiments differ considerably from one region to another documented(Odunfa,1981,1985;Omafuvbeetal.,2004).These
www.frontiersin.org January2013|Volume3|Article436|1
Adewumietal. Moleculardeterminationofirubacterial
techniquesoftenfailtoaccountforminormicrobialpopulations; CULTURE-DEPENDENTANALYSIS
stressed andinjuredcells thatarepresentinlownumber (Fleet, A total of 280 bacterial isolates were isolated from 20 iru sam-
1999), and have been observed not to give a complete repre- ples and Reference bacterial strains obtained from Microbial
sentation of foodmicrobialcommunities (Kesmen et al.,2012). TypeCultureCollection(MTCC),Indiaweresubjectedtogeno-
Also,theyarebias,unreliableandlackaccuratedetectionofpop- typic characterization. Amplified ribosomal DNA restriction
ulation dynamics and microbial diversity (Amann et al., 1995; analysis (ARDRA) combined with PCR amplification of the
Hugenholtz et al., 1998; Giraffa and Neviani, 2001; Jany and 16S–23SrRNAgeneinternaltranscribedspacer(ITS),ITS-PCR-
Barbier,2008). restrictionfragmentlengthpolymorphism(ITS-PCR-RFLP),and
Culture-independent microbial techniques, such as PCR- randomly amplified polymorphic DNA (RAPD-PCR) as previ-
denaturing gradient gel electrophoresis (DGGE) has proven ouslydescribed (Oguntoyinboet al.,2010) were used for geno-
much more reliable, fast, and economical in profiling complex typic grouping, clonal relationship, and isolates diversity. The
bacterialcommunitystructure,dynamicsandevolutionofmicro- almostcomplete16SrRNAgenesofrepresentativeisolatesfrom
bialpopulationsoffermentedfoods (GiraffaandNeviani,2001; theclustersgeneratedfromdendrogramofgelfingerprintswere
Randazzo et al., 2002; Cocolin et al., 2007; Jany and Barbier, amplifiedandsequencedasdescribedpreviously(Jeyarametal.,
2008). PCR-DGGE has been successfully employed to study- 2010).Closestknownidentitiesofthe16SrRNAgenesequences
ing the microbial community structures and dynamics of fer- wereobtainedfromNCBIGenBankdatabase.
mented vegetal protein seeds such as doenjang and meju (Cho
and Seo, 2007; Kim et al., 2009; Lee et al., 2010) as found in CULTURE-INDEPENDENTANALYSIS
Asia. More recently, barcoded pyrosequencing of V1/V2 region DNAextractionandPCRamplification
of 16S rRNA gene was used in assessing the microbial com- Two methods of bacterial genomic DNA described previously
munity of doenjang (Nam et al., 2012). Meanwhile, there is by Ercolini et al. (2003) and Rantsiou et al. (2005) were used
dearth of informationon theapplicationof thesetechniques to to extract genomic DNA from 16 iru samples (no apprecia-
profile microbial community of fermented vegetable foods in ble DNA was obtained from remaining four samples). The V3
Africa. region of the 16S rRNA gene was amplified using the set of
(cid:3) (cid:3)
Studies involving the use of both culture-dependent and primers 338f (5-ACTCCTACGGGAGGCAGCAG-3) and 518r
culture-independentmolecularmethodsfortheidentificationof (5(cid:3)-ATTACCGCGGCTGCTGG-3(cid:3)) (Sigma-Aldrich). A GC-rich
dominantbacterialspecies; accuratedeterminationanddetailed clampwasattachedtotheforwardprimerasdescribedbyMuyzer
investigationofmicrobialcompositionoffermentedfoodprod- et al. (1993). Five-microliters PCR amplified product was anal-
ucts have been reported (Pulido et al., 2005; Vilela et al., ysed on 2% agarose (Promega, USA) containing ethidium bro-
2010; Alegría et al., 2012; Kesmen et al., 2012). In this study, mide(0.5µg/ml)(E1510,Sigma-Aldrich),using0.5×TBEbuffer
wecombinedbothculture-dependentand-independentmolec- (45mM Tris-borate, 1mM EDTA, pH 8.0) with running con-
ular techniques as a polyphasic strategy to first understand ditions of 80V for about 1h. DNA fragments of about 240bp
and characterize the microbial community profile of iru pro- obtained by comparing with PCR 100bp low ladder (P1473,
duced by natural, uncontrolled fermentation process; secondly, Sigma-Aldrich)wasusedforDGGEanalysis.
to have a deeper insight into the bacterial diversity, which
can be used to locate and identify the dominant bacteria with DGGEanalysisoffermentedirusamples
technological and functional potentials that can be further DGGE was performed using a DCODE™ Universal Mutation
developed as autochthonous starter cultures to improve indus- Detection System (BIO-RAD, USA) following manufacturer’s
trial production of condiments in W. Africa and guarantee instructions.PCRampliconsincludingaDGGEmarkerdesigned
productsafety. andusedasareferenceDNAladder,wereseparatedin8%poly-
acrylamidegelscontaining1×TAEwithdenaturinggradientsof
MATERIALSANDMETHODS 25–55% [100% denaturant corresponds to 7M urea and 40%
SAMPLECOLLECTION (v/v) formamide]. Electrophoresis was run initially at 20V for
◦
Fermenting P. biglobosa during iru production were obtained 10min and at a constant temperature of 60 C, and finally at
◦
fromlocalproducers aswell asretailmarkets samplesinsouth- 140Vfor5hat60 C.Thegelswerestainedwithethidiumbro-
west (Lagos, Ibadan, Abeokuta, Oyo, andAdo-Ekiti) and north mide, visualized under UV light and documented using Gene
central Nigeria (Kaduna and Ilorin). They were transported Snap(PerkinElmerGELIANCE200,USA).
immediatelyintothelaboratorywiththeaidoficepack,storedin
therefrigeratorat4◦Candanalyzedwithinfewdaysofcollection. PCR-DGGEgelimagesanalysis
TheDGGEgelimagesobtainedwereconvertedtodensitometric
ISOLATIONOFMICROORGANISMS profilesusingthesoftwareTL120v2006(Phoretix1DAdvanced
Samples of fermented iru were homogenized in 90ml Ringer’s Software, NonLinear Dynamics, Newcastle, UK). The similarity
solution. Serial dilutions were made and 0.1ml of appropriate between theDGGElaneswas investigated byconstructing non-
dilutionswasspreadplatedonnutrientagar(Oxoid,Hampshire, metricmultidimensionalscaling(non-metricMDS)scatterplots
◦
UK). Plates were subsequently incubated at 37 C for 18–24h. as described elsewhere (Boon et al., 2002; McOrist et al., 2008)
Bacterialcoloniesobtainedwerepurifiedbyrepeatedstreakingto using the software package P A S T (PAleontological STatistics,
obtainpurecultures. http://folk.uio.no/ohammer/past/). The Shannon–Weaver index
FrontiersinMicrobiology|FoodMicrobiology January2013|Volume3|Article436|2
Adewumietal. Moleculardeterminationofirubacterial
of H diversity and R richness index were determined based on Similarity and variation among DGGE gel patterns of the vari-
thenumberpresence/absenceinthefragments.Theimportance ous fermentedcondimentswereestablishedbasedoncombined
probabilityPiandH werecalculatedusingPAST(Shannonand analysis of Dice similarity coefficient and nMDS. The results
Weaver,1963). obtainedshowedthatthe16irusamplesanalysedclusteredinto
five groups—a, b, c, d, and e (Figure1). Noticeable similarity
ExcisionofDGGEbandsandsequencingofamplicons can be saidto exist in thebacterial community structure of the
DGGE major bands of interest were excised from the poly- different samples of iru obtained in the two geographical zones
acrylamide gels, eluted in 50µl sterile deionized water (Milli under study (southwest and north central Nigeria). Apart from
◦
Q, Millipore, Bangalore) and incubated overnight at 4 C as twoirusamplesfromLagossouthwesternNigeria,thatclustered
describedbyCocolinetal.(2001).Onemicroliterofeacheluate into the same group “a” variation was observed among sam-
wasused asDNAtemplate forPCRreamplification.PCRprod- ples within the same geographical location especially southwest
uctsoriginatingfromtheexcisedbandswereprofiledandchecked Nigeriansamples.
forqualityinagarose;presenceandpositionofthebandsofinter-
estinDGGEandcomparisonwithparentDGGEprofiles.Elution IDENTIFICATIONOFMAJORBACTERIALPCR-DGGEBANDS
andreamplificationwerecarriedoutatleasttwiceoruntilasingle DNA sequencing was carried out on the major bacterial bands.
bandthatco-migrateatthesamepositionwiththeparentDGGE Theclosestknownidentities,percentagesimilarityandfrequency
band is obtained. Sequences of major bands obtained from the of occurrence of each bandin thefermented condiments areas
DGGE gel fragments were compared with the NCBI GenBank showninTable3.Amongthemall,B.subtilisoccurredmostfre-
databasetodetermineclosestrelatives. quently(band10),Figure2).Itwasfoundtobepresentin94%
of the iru samples, which confirms its consistency, dominance,
RESULTS and viability in the fermented condiments. B. licheniformis and
CULTURE-BASEDMOLECULARAPPROACH Brevibacillusspeciespreviouslyisolatedintheculture-dependent
Intheculturedanalysis,similarcolonialmorphologyandpopu- study were also found present in at least a sample of the fer-
lationprofilerangingfrom106–107CFU/mlwereobtainedfrom mented condiment (Table3). Other bands identified include
fermented samples as well as final product. A total of 280 iso- closerelativesofS.vitulinus,Morganellamorganii,B.thuringien-
lates selected for analysis by ARDRA restriction endonucleases sis, S. saprophyticus,T. halophilus, Ureibacillus thermosphaericus,
especiallyCfoIshoweddifferentiationoftheisolatesintovarying Salinicoccus jeotgali,Brevibacterium sp.,andunculturedbacteria
species,inparticular,B.subtilisandB.cereusphylotypes.Further clones.
characterizationwithITS-PCR-RFLPrevealedintraspeciesdiver-
genceamongtheBacillusisolates.ThegeldataofARDRA,ITS- DISCUSSION
PCR,ITS-PCR-RFLP,andRAPD-PCRwerecombinedtogenerate In W. Africa traditional fermented condiments form an impor-
polyphasic gel fingerprint. Representative of the major clusters tantcomponentofthedietaryproteinrequirementofthepeople,
obtainedwereidentifiedbysequencedataof16SrRNAgenethat where they are added generously to various dishes to supple-
identified two major bacilli groups; B. subtilis and B. amyloliq- mentproteinintake,improvenutritivevalue,andimpartsensory
uefaciensasthedominant,aswellasB.cereus groupasmember attributes. This study combined molecular techniques to reveal
ofthecultivatablebacteriaassociatedwithfermentedP.biglobosa microbiotaoftraditionalfermentedcondimentssampletestedat
cotyledon(Table1). differentfermentationtimeregimes withconsiderationforgeo-
graphical spread in Nigeria. This is aimed at future objectives
ANALYSISOFBACTERIALCOMMUNITIESANDDIVERSITYOFiru of developing predictable process through the use of identified
USINGPCR-DGGE bacteria that can be applied as starter cultures to dominate the
The two genomic DNA extraction protocols- enzymatic and fermentation,impactdesirablebiochemicalchangesandimprove
chemicalprovedtobeefficientinterms ofquantity,purity,and safetyquality.
amplifiabiltiy,andthusproducedDNAsuitableforPCRamplifi- Theresultsofculture-dependentanalysisand16SrRNAgene
cation.Theresultsof thePCR-DGGEgelfingerprintsoftheV3 sequencing showed that iru is composed of clonally related
regionofamplified16SrRNAgeneshowedidenticalDGGEpat- Bacillus species identified as B. subtilis, B. amyloliquefaciens, B.
ternsandhighdegreeofsimilarityinDNAfragmentswiththetwo cereus,B.licheniformis,andBrevibacillusformosusinorderoffre-
DNAextractionmethods.ThePCR-DGGEgelprofileswerealso quencyofincidence.Also,thecombinationofARDRA,ITS-PCR,
usedtoassessthebacterialcommunityprofile;richness,biodiver- ITS-PCR-RFLP,andRAPD-PCRrevealedhighdegreeofdiversity
sity, anddominanceindexes of naturallyfermented iru samples amongtheBacillusisolatesandidentifiedthedominantspeciesto
obtained from different geographical locations in Nigeria. The beB.subtilisandB.amyloliquefaciens.Theresultsclearlyshowed
species richness index (R) determined was highest for DGGE sub-typingofautochthonousclonallyrelatedspeciesofbacilliand
profiles of iru samples from Ilorin (R 19), thus exhibiting the geneticallyidentifieddominantgroups,thisisinagreementwith
highest number of bands/bacterial species than those of Lagos previous reports in W. Africa and India duringfermentation of
(16<R<18); Ado-Ekiti (R♦16); Ibadan (11<R<14); Oyo similarvegetableproteinsstudies(Sarkaretal.,2002;Ouobaetal.,
(R(cid:4)10) and Abeokuta (9<R<17) (Table2). Bacterial diver- 2004; Jeyarametal.,2008; Kimet al.,2010; Oguntoyinboetal.,
sity index (H) calculated on thebasis of number of bands on a 2010;Parkoudaetal.,2010).
gel trackwasalsohighestforirusamples fromIlorin(H 2.92) Culture-independenttechniqueshavebeenpreviouslyusedto
with the lowest being Abeokuta (2.19<H <2.80) (Table2). describemicrobiotaoffermentedfoodsinAfrica(Humblotand
www.frontiersin.org January2013|Volume3|Article436|3
Adewumietal. Moleculardeterminationofirubacterial
o. d)
n 1 e
ncing NCBIacc. JN255703 JN165753. JN255715 JN255725 JN255727 JN255710 JN255726 JN255717 JN255722 JN255707 (Continu
e
u
q y
genese Similarit 99% 98% 100% 97% 100% 99% 99% 100% 99% 98%
A
N
SrR own e- e-
16 Closestknrelative B.subtilis B.subtilis B.subtilis B.subtilis B.subtilis B.subtilis B.pumilus B.pumilus B.amyloliqufaciens B.amyloliqufaciens
e
v
ati
nt
preseains EBA 04 54B 91A 33 22A 13A 58 84B 9BA
Restr U4 U1 U1 U1 U2 U1 U2 U1 U1 U7
s
ofate
No.isol 93 26 1 3 1 1 2 62 37
a.
s
o
mentedParkiabiglob R-RFLPsub-groupsnbp) 7,339,278,260,214, 6 9,280,141 1,322,263,247,218,9 9,271,231,137 6,330,269,255,7,120 9,268,223,134 9259,149 0,343,274,5,137 4,384,369,343,0,147,138
onalfer ITS-PC(sizesi IA1. 502,41 143,13 IA2. 506,33 IIA1. 485,40138,12 IIA2. 489,32 IIA3.494,40225,12 IIA4.494,32 IIA5. 490,33 IIIAl.504,42259,14 IIIA2.506,42274,26
diti b-es 63
a uz 8
fromiru,tr ITS-PCRsgroups(si inbp) IA. 1065,945, 869, IIA. 1036,845 IIIA.1057,939,
d
e
isolat RsaI 525 477 429 149 515 470417 146 515424147
es p)
speci sinb TaqI 573 458 349 214 590 412361 221 722375232
s e
u z
Bacill file(si HinfI 581 355 317 177 592 362324 183 609374335 190
aracterizationof SrRNA-RFLPpro CfoIDdeI 908508 451465 248237 150 122 904504 445,381243280 147 117 510912392451286247 236122
h 6
Genomicc 1 HaeIII a627,473 318 156 622,467 314,151 627,472,318,156
1| As
Table ARDRgroup I II III
FrontiersinMicrobiology|FoodMicrobiology January2013|Volume3|Article436|4
Adewumietal. Moleculardeterminationofirubacterial
o.
n
ncing NCBIacc. JN255724 JN255711 JN255721 JN255728 JN255723
e
u
q y
genese Similarit 98% 100% 96% 92% 99%
A
N
rR wn mis mis
16S Closestknorelative B.lichenifor B.lichenifor B.cereus B.cereus Brevibacillusformosus
e
v
ati
nt
e
presains 86 26 75 43 85B
Restr U1 U1 U1 U2 U1
s
ofate
No.isol 3 1 49 1
9 5,
s 2 03
p 1 23
grou 142 271, 225,42,1
RFLPsub-bp) 272,229, 414,335, 168,136 294,262,167,157,1
R-n 6, 5, 1, 5,3,
ITS-PC(sizesi IVAl. 496,33 IVB1. 489,43 VA1. 506,31 VIA1.461,35198,19
b-es
ITS-PCRsugroups(sizinbp) IVA. 1024,845 IVB. 1044,926, 842,339 VA. 1078,827 VIA.1359,1161,1103,1020,910
RsaI 498 459 409 142 494 411 355 142 452411353131107
p)
inb TaqI 570 395 345 211 600 481 134 575389365196
s
e
z
(si nfI 1 4 7 6 3 2 0 3447
file Hi 58 34 30 16 94 31 17 97302316
o
FLPpr DdeI 518 288 277 237 132 115 516 287 275 172 150 115 546441238167120
R
A-
N oI 4 6 8 2 7 7 2 5 2482
rR Cf 41 32 22 17 56 41 33 17 41322117
S
6
Continued 1 HaeIII 622,475 324,156 595,477 322,161 627,477,228,157 mentsizes.
g
Table1| ARDRAgroups IV V VI aDNAfra
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Adewumietal. Moleculardeterminationofirubacterial
Table2|Speciesrichnessestimates(R)andShannon’sindexof Guyot, 2009; Oguntoyinbo et al., 2011). Although none of this
diversity(H)ofDGGEprofilesoffermentedirusamplesobtained studiesreportedmicrobiotaoffermentedvegetableproteinused
fromvariousgeographicallocationsinNigeria. ascondiments.Inthisstudy,duringprofilingofbacterialcommu-
nityoffermentediru,twomethods ofgenomicDNAextraction
Samples R H
were employed. No significant differences in the DNA yields,
fragmentsandDGGEbandpatternswereobserved.Mafraetal.
iruAbeokuta1 12 2.44
(2008)reportedsimilarfindingsinastudyinvolvingcomparison
iruAbeokuta2 13 2.57
of four DNA extraction methods for downstream processing,
iruAbeokuta3 17 2.80
comparison such as this guarantees better microbiome profil-
iruAbeokuta4 12 2.49
ing. Results of PCR-DGGE gel analysis of iru sampled from
iruAbeokuta5 11 2.34
Ilorin, north central Nigeria had the highest number of bacte-
iruAbeokuta6 11 2.35
rialspecies thanothers,includingthehighestbacterial diversity
iruAbeokuta7 15 2.70
index;thevariableproductionprocesscouldpossiblyberespon-
iruAbeokuta8 9 2.19 sibleforthisobservation.Theintraspecificvariationobservedin
iruLagos1 18 2.88 thefermentedirusamplesaspredicatedbyDicesimilaritycoef-
iruLagos2 16 2.74 ficientandnMDSisanindicationthatthebacterialcommunity
iruIlorin1 19 2.92 profileofthiscondimentisdiverse;havinggroupedintodistinct
iruIlorin2 19 2.92 clusters with varying diversity indexes. Iru from different geo-
iruIbadan1 11 2.40 graphicalzoneswereproducedfromthesamesubstrateP.biglo-
iruIbadan2 14 2.60 bosaseedsbutvariablefermentationperiods.Therefore,diversity
iruOyo 10 2.25 observed in the microbial communities could be due to differ-
iruAdo-Ekiti 16 2.77 enceinfermentationconditions,whichisalsospontaneous and
MeaniruAbeokuta(±STDEV) 12.50±2.35 2.49±0.19 uncontrolled, without the use of any known specific isolate as
MeaniruLagos(±STDEV) 17.00±1.00 2.81±0.05 starterculture.Otherworkershavealsoreportedsimilarvariation
inthemicrobialcompositionofnaturallyfermentedfoodmate-
MeaniruIlorin(±STDEV) 19.00±0.00 2.92±0.00
rials produced using same substrate (Ampe and Miambi, 2000;
MeaniruIbadan(±STDEV) 12.50±1.50 2.50±0.10
Oguntoyinboetal.,2011).
FIGURE1|Non-metricMultidimensionalScalinganalysisofDGGEdata (b):iruAbeokuta4southwestNigeria((cid:3)).Group(c):iruAbeokuta1southwest
Group(a):iruAbeokuta2southwestNigeria(+);iruAbeokuta3 Nigeria((cid:4));iruAbeokuta5southwestNigeria( );iruAbeokuta8
southwestNigeria((cid:2));iruLagos1southwestNigeria((cid:2));iruLagos2 southwestNigeria( );iruOyosouthwestNigeria((cid:4));iruAdo-Ekitisouthwest
southwestNigeria(•);iru Ilorin1northcentralNigeria((cid:3));iruIlorin2 Nigeria(♦).Group(d):iruAbeokuta6southwestNigeria((cid:6));irulbadan2
northcentralNigeria( );iru Ibadan1southwestNigeria( ).Group southwestNigeria((cid:7)).Group(e):iruAbeokuta7southwestNigeria( ).
FrontiersinMicrobiology|FoodMicrobiology January2013|Volume3|Article436|6
Adewumietal. Moleculardeterminationofirubacterial
Table3|IdentitiesofmajorbacterialbandsexcisedfromPCR-DGGEgelsoffermentedParkiabiglobosaseeds.
Bands Source/locationsa 16SrRNAgeneclosestknownrelative %Similarity
1 Oyo(1),Abeokuta(4),Ado-Ekiti(1),Lagos(1),Ibadan(1),Ilorin(2) Staphylococcusvitulinus 98
2 Ado-Ekiti(1),Ibadan(2),Abeokuta(4),Lagos(2),Ilorin(2) Morganellamorganii 98
3 Abeokuta(2),Ibadan(1),Lagos(1) Bacillusthuringiensis 99
4 Abeokuta(2),Ibadan(1),Ilorin(1) B.licheniformis 98
5 Oyo(1),Abeokuta(1),Lagos(1),Ilorin(1) S.saprophyticus 99
6 Oyo(1),Abeokuta(7),Ado-Ekiti(1),Lagos(2),Ilorin(2) Unculturedbacteriumclone 97
7 Abeokuta(3),Ibadan(1),Lagos(1) Tetragenococcushalophilus 95
8 Oyo(2),Ibadan(2),Abeokuta(1),Lagos(1) Ureibacillusthermosphaericus 100
9 Ado-Ekiti(1),Ibadan(1),Abeokuta(1),Lagos(1) Bacillussp. 98
10 Oyo(1),Abeokuta(7),Ado-Ekiti(1),Ibadan(2),Lagos(2),Ilorin(2) B.subtilis 99
11 Abeokuta(1) Brevibacillusparabrevis 97
12 Ibadan(1),Abeokuta(1) Salinicoccusjeotgali 99
13 Ado-Ekiti(1),Lagos(1) Brevibacteriumsp. 97
BandsareasshowninFigure2.
aValuesinparenthesisrepresentnumberoffermentedirusamples,withtotalnumberofirusampledbeing16.
S.saprophyticus,B.thuringiensis,Tetragenococcus halophilus,etc.
Of interest is T. halophilus, a halophilic lactic acid bacterium,
predominantlyfoundinfermentedfoodswithhighsaltconcen-
trationsuchassoysauce,misoanddoenjang (Tanasupawatetal.,
2002; Onda et al., 2003; Kim et al., 2009). The source of this
bacterium in iru can somewhat be attributed to the salt added
as a preservative at the end of the fermentation process. The
detectionofB.cereus,B.thuringiensis,andStaphylococcusspecies
clearly raises doubt regarding the microbiological safety of this
fermentedcondiment.Informationonthetoxigenicpotentialof
food-bornepathogenintraditionalAfricanfermentedfoodsexist
(OguntoyinboandOni,2004;Abrioueletal.,2007;Oguntoyinbo
etal.,2010).
In conclusion, the results indicated that combination of
culture-dependent and culture-independent techniques can be
usedtoprofilebacterialmicrobiota,examinedynamicsanddiver-
sityofnaturallyfermentedfood.Ontheoverall,thisstudyshowed
that bacterial composition of iru is significantly more diverse
than earlier reported and justified the need to develop process
across production sites to facilitate safety quality and consis-
FIGURE2|DGGEprofilesofPCR-amplified16SrRNAgenefragments
ofsixteen(16)fermentedirusamplesshowingmajorbacterial tency. This study confirmed dominance of Bacillus species and
amplicons.SamplesA:iruOyo;B:iruLagos1;C:iru Ilorin2;D:iru Ilorin occurrenceofotherbacteriawithpathogenictraitandunknown
1;E:iru Ibadan1;F:iruAdo-Ekiti;G:iruAbeokuta2;H:iruAbeokuta1;I: functions. Condiments can support dietary need of the people
iru Ibadan2;J:iruAbeokuta3;K:iruAbeokuta4;L:iruAbeokuta5;M:iru
ifbetter understandingofthefunctionalrolesofallmembersof
Abeokuta6;N:iruLagos2;Î:iruAbeokuta7;P:iruAbeokuta8;“m”is
thecommunityduringfermentationcanbedetermined,thiswill
DGGEreferenceDNAladder.Bands1:Staphylococcusvitulinus;2:
Morganellamorganii;3:Bacillusthuringiensis;4:B.licheniformis;5:S. supportdevelopmentofpredictableprocess, eliminatevariation
saprophyticus;6:Unculturedbacteriumclone;7:Tetragenococcus andinconsistency.
halophilus;8:Ureibacillusthermosphaericus;9:Bacillussp.;10:B.subtilis;
11:Brevibacillusparabrevis;12:Salinicoccusjeotgali;13:Brevibacteriumsp.
ACKNOWLEDGMENTS
This project was funded by the Department of Biotechnology
PCR-DGGE bands sequencing identified B. subtilis as the (DBT), Government of India and the academy of Sciences for
major bacterial species associated with iru, which is in agree- the Developing World (TWAS), Italy through the award of a
mentwiththeresultofculture-basedPCRanalysis.Earlierstudies DBT-TWAS Postgraduate Sandwich fellowship to Gbenga A.
using traditional culture-dependent methods and biochemical Adewumi and SfAM New Lecturer Grant, UK to Folarin A.
characterization havealsoreported thedominanceof B. subtilis Oguntoyinbo. The leave of absence also granted to Gbenga A.
duringirufermentation(AntaiandIbrahim,1986; Odunfaand Adewumi by Bells University of Technology, Ota, Nigeria that
Oyewole, 1986). Other DGGE bands corresponded to potential facilitatedhiscollaborationwithIBSD,Imphal,Indiaisgratefully
food-borne pathogens and contaminants such as S. vitulinus, acknowledged.
www.frontiersin.org January2013|Volume3|Article436|7
Adewumietal. Moleculardeterminationofirubacterial
REFERENCES to monitor dynamic changes in by whole-cell proteinpatterns and inMicrobiologyofFermentedFoods,
Abriouel,H.,BenOmar,N.,López,R. thebacterialpopulationduringfer- 16S rRNA gene sequence anal- 2nd Edn, Vol. 2, ed B. J. B. Wood
L., Cañamero, M. M., Ortega, mentationofItaliansausages.Appl. ysis. J. Microbiol. Biotechnol. 20, (London, UK: Blackie Academic
E., and Gálvez, A. (2007). Environ.Microbiol.67,5113–5121. 1210–1214. andProfessional),712–752.
Differentiation and characteri- Ercolini, D., Hill, P. J., and Dodd, Kim,T.-W.,Lee,J.-H.,Kim,S.-E.,Park, Oguntoyinbo, F. A., Huch, M., Cho,
zation by molecular techniques of C.E.R.(2003).Bacterialcommu- M.-H., Chang, H. C., and Kim, G.-S.,Schillinger,U.,Holzapfel,W.,
Bacillus cereus group isolates from nitystructureandlocationinstilton H.-Y. (2009). Analysis of micro- Sanni,A.I.,etal.(2010).Diversity
potopotoanddegue,twotraditional cheese.Appl.Environ.Microbiol.69, bial communities in doenjang, a of Bacillus species isolated from
cereal-based fermented foods of 3540–3548. Korean fermented soybean paste, okpehe,atraditionalfermentedsoup
Burkina Faso and Republic of Fleet, G. H. (1999). Microorganisms usingnestedPCR-denaturinggradi- condimentofNigeria.J.FoodProt.
Congo.J.FoodProt.70,1165–1173. in food ecosystems. Int. J. Food entgelelectrophoresis. Int.J.Food 73,870–878.
Achi, O. K. (2005). Traditional fer- Microbiol.50,101–117. Microbiol.131,265–271. Oguntoyinbo, F. A., and Oni, O.
mented protein condiments in Giraffa, G., and Neviani, E. (2001). Lee, J.-H., Kim, T.-W., Lee, H., M. (2004). Incidence and charac-
Nigeria. Afr. J. Biotechnol. 4, DNA-based, culture-independent Chang, H. C., and Kim, H.-Y. terization of Bacillus cereus iso-
1612–1621. strategies for evaluating microbial (2010).Determinationofmicrobial lated from traditional fermented
Alegría, A., Szczesny, P., Mayo, B., communities in food-associated diversity in meju, a fermented meals in Nigeria. J. Food Prot. 67,
Bardowski, J., and Kowalczyk, M. ecosystems. Int. J. Food Microbiol. cooked soya beans, using nested 2805–2808.
(2012). Biodiversity in oscypek, 67,19–34. PCR-denaturing gradient gel elec- Oguntoyinbo, F. A., Tourlomousis,
a traditional Polish cheese deter- Hugenholtz, P., Goebbel, B. M., and trophoresis. Lett. Appl. Microbiol. P., Gasson, M., and Narbad,
mined by culture-dependent and Pace, N. R. (1998). Impact of 51,388–394. A. (2011). Analysis of bacterial
in-dependent approaches. Appl. culture-independentstudiesonthe Mafra, I., Silva, S. A., Moreira, E. J. communities of traditional fer-
Environ.Microbiol.78,1890–1898. emergingphylogeneticviewofbac- M. O., da Silva, C. S. F., Beatriz, mented West African cereal foods
Amann,R.I.,Ludwig,W.,andSchleifer, terial diversity. J. Bacteriol. 180, M., and Oliveira, P. P. (2008). using culture-independent meth-
K.-H. (1995). Phylogenetic iden- 4765–4774. ComparativestudyofDNAextrac- ods. Int. J. Food Microbiol. 145,
tification and in situ detection Humblot,C.,andGuyot,J.P.(2009). tion methods for soybean derived 205–210.
of individual microbial cells with- Pyrosequencingoftagged16SrRNA food products. Food Control 19, Omafuvbe, B. O., Falade, O. S.,
out cultivation. Microbiol. Rev. 59, geneampliconsforrapiddecipher- 1183–1190. Osuntogun,B.A.,andAdewusi,S.
143–169. ing of the microbiomes of fer- McOrist,A.L.,Abell,G.C.J.,Cooke, R. A. (2004). Chemical and bio-
Ampe, F., and Miambi, E. (2000). mented foods such as pearly mil- C.,andNyland,K.(2008).Bacterial chemicalchangesinAfricanlocust
Clusteranalysis,richnessandbiodi- letslurries.Appl.Environ.Microbiol. population dynamics and faecal bean(Parkiabiglobosa)andmelon
versityindexesderivedfromdena- 75,4354–4361. short-chainfattyacid(SCFA)con- (Citrullus vulgaris) seeds during
turing gradient gel electrophoresis Jany, J. L., and Barbier, G. (2008). centrationsinhealthyhumans.Br.J. fermentationtocondiments.Pak.J.
fingerprints of bacterial commu- Culture-independent methods for Nutr.100,138–146. Nutr.3,140–145.
nities demonstrate that traditional identifying microbial communi- Muyzer, G., De Waal, E. D., and Onda, T., Yanagida, F., Uchimura, T.,
maize fermentations are driven by ties in cheese. Food Microbiol. 25, Uitterlinden,A.G.(1993).Profiling Tsuji,M.,Ogino,S.,Shinohara,T.,
transformationprocess.Int.J.Food 839–848. of complex microbial populations etal.(2003).Analysisoflacticacid
Microbiol.60,91–97. Jeyaram, K., Romi, W., Singh, T. A., by denaturing gradient gel elec- bacterialfloraduringmisofermen-
Antai, S. P., and Ibrahim, M. H. Devi, A. R., and Devi, S. (2010). trophoresis analysis of polymerase tation. Food Sci. Technol. Res. 9,
(1986). Microorganisms associated Bacterialspeciesassociatedwithtra- chainreaction-amplifiedgenescod- 17–24.
with African locust bean (Parkia ditionalstarterculturesusedforfer- ing for 16S rRNA. Appl. Environ. Ouoba, L. I. I., Diawara, B., Amoa-
biglobosa Welw) fermentation for mented bamboo shoot production Microbiol.59,695–700. Awua, W. K., Traoré, A. S., and
dawadawa production. J. Appl. in Manipur state of India. Int. J. Nam, Y.-D., Lee, S.-Y., and Lim, S.-I. Møller, P. L. (2004). Genotyping
Bacteriol.61,145–148. FoodMicrobiol.143,1–8. (2012).Microbialcommunityanal- of starter cultures of Bacillus sub-
Boon,N.,DeWindt,W.,Verstraete,W., Jeyaram,K.,Singh,W.M.,Premarani, ysis of Korean soybean pastes by tilis and Bacillus pumilus for fer-
and Top, E.M.(2002).Evaluation T., Devi, A. R., Chanu, K. S., next-generation sequencing. Int. J. mentation of African locust bean
of nested PCR-DGGE (denaturing Talukdar, N. C., et al. (2008). FoodMicrobiol.155,36–42. (Parkiabiglobosa)toproducesoum-
gradient gel electrophoresis) with Molecular identification of dom- N’Dir, B., Hbid, C. L., Cornelius, bala. Int. J. Food Microbiol. 90,
group-specific 16S rRNA primers inant microflora associated with C., Roblain, D., Jacques, P., 197–205.
for the analysis of bacterial com- hawaijar–atraditional fermented Vanhentenryck, F., et al. (1994). Ouoba,L.I.I.,Diawara,B.,Jespersen,
munitiesfromdifferentwastewater soybean [Glycine max (L.)] food Proptiétés antifongiques de la L., and Jakobsen, M. (2007).
treatment plants. FEMS Microbiol. of Manipur of India. Int. J. Food microflora sporulée du nététu. Antimicrobial activity of Bacillus
Ecol.39,101–112. Microbiol.122,259–268. CahiersAgric.3,23–30. subtilisandBacilluspumilusduring
Cho, K. M., and Seo, W. T. (2007). Kesmen, Z., Yetiman, A. E., Gulluce, Odunfa,S.A.(1981).Microorganisms the fermentation of Africanlocust
BacterialdiversityinaKoreantra- A., Kacmaz, N., Sagdic, O., Cetin, associated with fermentation of bean (Parkia biglobosa) for soum-
ditional soybean fermented foods B., et al. (2012). Combination of African locust bean (Parkia fil- balaproduction.J.Appl.Microbiol.
(doenjang and ganjang) by 16S culture-dependent and culture- icoidea) during iru preparation. 102,963–970.
rRNAgenesequenceanalysis.Food independent molecular methods J.PlantFoods3,245–250. Ouoba, L. I. I., Nyanga-Koumou, C.
Sci.Biotechnol.16,320–324. for the determination of lactic Odunfa, S. A. (1985). Biochemical A. G., Parkouda, C., Sawadogo,
Cocolin, L., Diez, A., Urso, R., microbiota in sucuk. Int. J. Food changes in fermenting African H., Kobawila, S. C., Keleke, S.,
Rantsiou,K.,Comi,G.,Bergmaier, Microbiol.153,428–435. locust bean (Parkia biglobosa) et al. (2010). Genotypic diver-
I., et al. (2007). Optimization of Kiers, J. L., Van Laeken, A. E. A., during iru fermentation. J. Food sity of lactic acid bacteria isolated
conditions for profiling bacterial Rombouts, F.M., and Nout,M.J. Technol.20,295–303. from African traditional alkaline-
populations of food by culture- R. (2000). In vitro digestibility of Odunfa, S. A., and Oyewole, O. B. fermentedfoods.J.Appl.Microbiol.
independent methods. Int. J. Food Bacillusfermentedsoyabean.Int.J. (1986). Identification of Bacillus 108,2019–2029.
Microbiol.120,100–109. FoodMicrobiol.60,163–169. species from iru, a fermented Ouoba,L.I.I.,Parkouda,C.,Diawara,
Cocolin,L.,Manzano,M.,Cantoni,C., Kim,T.-W.,Kim,Y.-H.,Kim,S.-E.,Lee, Africanlocustbeanproduct.J.Basic B., Scotti, C., and Varnam, A. H.
and Comi, G. (2001). Denaturing J.-H., Park, C.-S., and Kim, H.-Y. Microbiol.2,101–108. (2008). Identification of Bacillus
gradient gel electrophoresis analy- (2010).Identificationanddistribu- Odunfa, S. A., and Oyewole, O. B. spp. from bikalga, fermented
sisofthe16SrRNAgeneV1region tionofBacillusspeciesindoenjang (1998). “Africanfermented foods,” seeds of Hibiscus sabdariffa:
FrontiersinMicrobiology|FoodMicrobiology January2013|Volume3|Article436|8
Adewumietal. Moleculardeterminationofirubacterial
phenotypic and genotypic charac- molecular and culture-dependent kinema) and locust beans (soum- commercial or financial relationships
terization. J. Appl. Microbiol. 104, methods. Appl. Environ. Microbiol. bala). Int. J. Food Microbiol. 77, thatcouldbeconstruedasapotential
122–131. 71,7872–7879. 175–186. conflictofinterest.
Ouoba, L. I. I., Rechinger, K. B., Randazzo, C. L., Torriani, S., Shannon, C. E., and Weaver, W.
Barkholt, V., Diawara, B., Traore, Akkermans, A. L. D., de Vos, (1963). The Mathematical Theory Received:28September2012; accepted:
A. S., and Jakobsen, M. (2003). W.M.,andVaughan,E.E.(2002). ofCommunication.Urbana,IL:The 17December2012;publishedonline:07
Degradation of proteins dur- Diversity, dynamics and activity UniversityofIllinoisPress. January2013.
ing the fermentation of African of bacterial communities during Steinkraus,K.H.(1997).Classification Citation: Adewumi GA, Oguntoyinbo
locust bean (Parkia biglobosa) production of an artisanal Sicilian of fermented foods: worldwide FA, Keisam S, Romi W and Jeyaram
by strains of Bacillus subtilis and cheese as evaluated by 16S rRNA review of household fermenta- K (2013) Combination of culture-
Bacillus pumilus for production of analysis. Appl. Environ. Microbiol. tion techniques. Food Control 8, independent and culture-dependent
soumbala. J. Appl. Microbiol. 94, 68,1882–1892. 311–317. molecularmethodsforthedetermination
396–402. Rantsiou, K., Urso, R., Iacumin, L., Tanasupawat,S.,Thongsanit,J.,Okada, of bacterial community of iru, a fer-
Oyeyiola, G. P. (1988). Microbiology Cantoni,C.,Cattaneo,P.,Comi,G., S.,andKomagata,K.(2002).Lactic mented Parkia biglobosa seeds. Front.
of iru pete and iru woro obtained et al. (2005). Culture-dependent acidbacteriaisolatedfromsoysauce Microbio. 3:436. doi: 10.3389/fmicb.
fromlocalproducers.MIRCENJ.4, and -independent methods to mash in Thailand. J. Gen. Appl. 2012.00436
439–445. investigate the microbial ecology Microbiol.48,201–209. This article wassubmitted to Frontiers
Parkouda,C.,Thorsen,L.,Compaore, of Italian fermented sausages. Vilela,D.M.,Pereira,G.B.M.,Silva, in Food Microbiology, a specialty of
C.S.,Nielsen,D.S.,Tano-Debrah, Appl. Environ. Microbiol. 71, C. F., Batista, L. B., and Schwan, FrontiersinMicrobiology.
K., Jensen, J. S., et al. (2010). 1977–1986. R. F. (2010). Molecular ecology Copyright © 2013 Adewumi,
Microorganisms associated with Sanni,A.I.(1993).Theneedforprocess and polyphasic characterization Oguntoyinbo, Keisam, Romi and
maari, a baobab seed fermented optimization of Africanfermented of the microbiota associated with Jeyaram. This is an open-access article
product.Int.J.FoodMicrobiol.142, foods and beverages. Int. J. Food semi-dry processed coffee (Coffee distributed under the terms of the
292–301. Microbiol.18,85–95. arabica L.). Food Microbiol. 27, CreativeCommonsAttributionLicense,
Pulido,R.P.,BenOmar,N.,Abriouel, Sarkar,P.K.,Hasenack,B.,andNout, 1128–1135. which permits use, distribution and
H., López, R. L., Cañamero, M. J. R. (2002). Diversity and reproduction in other forums,provided
M. M., and Gálvez, A. (2005). functionalityofBacillusandrelated Conflict of Interest Statement: The theoriginalauthorsandsourcearecred-
Microbiological study of lactic generaisolatedfromspontaneously authors declare that the research itedandsubjecttoanycopyrightnotices
fermentation of caper berries by fermented soybeans (Indian was conducted in the absence of any concerninganythird-partygraphicsetc.
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