Analysis of microbial diversity in Shenqu with different fermentation times by PCR-DGGE

Analysis of microbial diversity in Shenqu with different fermentation times by PCR DGGE B B A d TQ1 L A a A R A A A A K M P S M I S n a e e o d S b h 1 u 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1[.]

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Pleasecitethisarticleinpressas:LiuT,etal.AnalysisofmicrobialdiversityinShenquwithdifferentfermentationtimesbyPCR-DGGE.Braz J

h tt p : / / w w w b j m i c r o b i o l c o m b r /

Tengfei Liu, Tianzhu Jia∗, Jiangning Chen, Xiaoyu Liu, Minjie Zhao, Pengpeng Liu

Q1

Liaoning University of Traditional Chinese Medicine, College of Pharmacy, Key Laboratory of Processing Theory Analysis of State

Administration of Traditional Chinese Medicine, Dalian, China

a r t i c l e i n f o

Article history:

Received16September2014

Accepted8December2015

Availableonlinexxx

AssociateEditor:WelingtonLuizde

Araújo

Keywords:

Microbialdiversity

PCR-DGGE

Shenqu

Molecularcloning

a b s t r a c t

ShenquisafermentedproductthatiswidelyusedintraditionalChinesemedicine(TCM)

totreatindigestion;however,themicrobialstrainsinthefermentationprocessarestill unknown.TheaimofthisstudywastoinvestigatemicrobialdiversityinShenquusing dif-ferentfermentationtimeperiods.DGGE(polymerasechainreaction-denaturinggradientgel electrophoresis)proﬁlesindicatedthatastrainofPediococcus acidilactici(band9)isthe pre-dominantbacteriaduringfermentationandthatthepredominantfungiwereuncultured

Rhizopus, Aspergillus oryzae,andRhizopus oryzae.Inaddition,pathogenicbacteria,suchas

Enterobacter cloacae, Klebsiella oxytoca, Erwinia billingiae,andPantoea vaganweredetectedin Shenqu.DGGEanalysisshowedthatbacterialandfungaldiversitydeclinedoverthecourse

offermentation.Thisdeterminationofthepredominantbacterialandfungalstrains respon-sibleforfermentationmaycontributetofurtherShenquresearch,suchasoptimizationof thefermentationprocess

creativecommons.org/licenses/by-nc-nd/4.0/)

Introduction

Shenqu,alsoknownasLiushenqu,iscommonlyusedin

Chi-nese medicine clinics to protect the stomach and spleen

and stimulates appetite and digestion Current research

effortshaverevealedthatsomedigestiveenzymes(amylase

enzymes, protease enzymes, glucoamylase), vitamins and

othersubstancesplayamainroleinstimulatingappetiteand

digestion.1Resistancetheoryistheearliestworktomention

Shenqu.Shenquistraditionallyprocessedasfollows:wheat

bran,ﬂour,ricebeanpowder(Vigna umbellata[Thunb.]Ohwi

∗ Corresponding author.

and Ohashi),and bitter apricot seedpowder (Prunus mand-shurica [Maxim.]Koehne) are blended ina particular ratio VariousChinesemedicinedecoctionsarethenadded, includ-ingPolygonum pubescens(Blume),Xanthium sibiricum(Patr.),and

Artemisia annua(L.).Themixtureisthenkneadedanddivided intobricks,whichareputintoamold.Finally,thebricksare coveredwithadhesive-bondedclothand placedinaboxat constanttemperatureandhumidity.Afterafewdaysof fer-mentation,theproductiscutintosmalllumpsanddriedat

alowtemperature.ThequalityoftheresultingShenqucan varyduetodifferencesintheamountofthemixedbacteria and fungithat arepresent duringfermentation.Itisworth

http://dx.doi.org/10.1016/j.bjm.2017.01.002

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notingthatthefungusAspergillus ﬂavusproducesaﬂatoxin,a

carcinogen,duringfermentation.Thisisoneofthereasons

whyShenquis notincludedinthe ChinesePharmacopeia

However,thecurrenttheoreticalsupportendorsesShenqufor

stimulatingappetiteanddigestion.abetterunderstandingof

themicrobesinvolvedinShenqufermentationmayleadto

improvedmethodsoffermentation

There are two main types of methods for assessing

bacterial diversity, traditional culture-dependent methods

andculture-independentmethods.Thusfar,studiesonthe

microbial diversity of Shenqu have been mainly based on

traditionalculture-dependentmethods,2–4suchasPCR-SSCP

(singlestrandconformationpolymorphism)5andDGGE.6

PCR-DGGE (polymerase chain reaction-denaturing gradient gel

electrophoresis)is aculture-independent method designed

toanalyze the geneticdiversity in asample It overcomes

thedisadvantagesofculture-dependentmethods,7makingit

acommon toolfor molecularbiological investigations into

microbialcommunities.PCR-DGGEhasbeenusedwidelyto

analyzemicrobialcommunitystructureacrossdifferentﬁelds,

suchas food microbiology, oralmicrobiology, soil

microor-ganisms,environmentalmicrobiology,andotherareas.8–11In

thisstudy,weused culture-independentPCR-DGGEand TA

cloningtodeterminethemicrobialdiversityofShenquacross

differentfermentationperiods.Theaimofthisstudywasto

investigateeubacteriamicrobialdiversityduringfermentation

andidentifyseveraldominantfermentationbacteriaand

fun-gus

Shenqu sample collection

Shenqufermentationparameterswere based onour

previ-ousstudyandresponsesurfacemethodology.12Rawmaterials

were crushed in a grinder Fourteen grams of Polygonum

pubescens (Blume), Xanthium sibiricum (Patr.) and Artemisia

annua(L.)weremixedwithwateranddecoctedfor1hat32◦C

and75%relativehumidityandthenmixedwith60gofﬂour,

140gofwheatbran,8gofbitterapricot,and5.2gofricebean

Eightsampleswereprocessedanddesignatedas1–8for

fer-mentationforvaryinglengthsoftime,representingdays1–8,

respectively.EachShenqusample,ofapproximately100g,was

collectedduringdays1–8.Allsampleswerecollectedina ster-ileenvironment,transferredtosterilepolyethylenebagsand storedat−70◦Cuntiltheywereanalyzed.

DNA extraction

FivegramsofeachShenqusampleweresuspendedin50mL

of phosphate buffered saline (PBS, 0.1mol/L, pH 8.0) and shakenfor10min.Themixedsuspensionwascentrifugedat 10,000×gfor10minandwashedthreetimesusingthesame PBS buffer.TotalgenomicDNAwasextractedfromthe pel-letsusingaONE-4-ALLGenomicDNAMini-PrepsKit(Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions.Thesamplesweregroundusingliquidnitrogen andlysisbuffer,thenrapidlythawedinawater-bathat65◦C foranhour.Thesampleswereshakenevery10minduring lysis.ThecrudeDNAwaselectrophoreticallyanalyzedon1.2% (w/v)agarosegels;sampleswerethenkeptinaclean0.5-mL microcentrifugetubeandstoredat−20◦C.

PCR ampliﬁcation

All primersusedinthisstudyarelisted inTable1 General bacterial16SrRNAgeneprimers338Fand518Rwereusedto assessbacterialdiversity.Atouch-downPCRtechniquewas employedinordertoincreasesensitivity.Thethermalcycling conditions were as follows: 5min denaturation at 95◦C;5 cyclesof30sat94◦C,30sat62◦C(witheachcyclereduced

by2◦C),and90sat72◦C;25cyclesof30sat94◦C,30sat50◦C, and90sat72◦C;andﬁnalextensionfor10minat72◦C.AGC clamp(5-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCA CGGGGGG-3)wasattachedtothe5endofprimer338Ffor theDGGEanalysis

AnestedPCRtechniquewasemployedinordertoincrease sensitivity PCRampliﬁcationofgeneralbacterial18S rRNA wasperformedusinguniversalgeneprimersNS1andFR1in theﬁrststep,followedbynestedPCRusingNS1andGC-Fung Thethermalcyclingconditionswereasfollows:5min dena-turationat95◦C;30cyclesof30sat94◦C,30sat50◦C,and

90sat72◦C;andﬁnalextensionfor10minat72◦C.PCR prod-uctsfromtheﬁrststepweredilutedwith10timestheamount

ofddH2Oandservedasthetemplateforthesecondroundof nestedPCR

Table 1 – Primers used in this study.

Fungi

FirstPCR

round

SecondPCR

round

F,forwardprimer;R,reverseprimer

a Primerwitha41-bpGCclamp(CGCCCGGGGCGCGCCCCGGGGCGGGGCGGGGGCGCGGGGGG)

b Primerwitha40-bpGCclamp(CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC)

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DGGE analysis

ThePCRproductsofbacteriaandfungiwereanalyzedusing

DGGE and the D-code Universal Mutation Detection

Sys-tem (Bio-rad, USA) For assessing bacterial diversity, 10%

of the polyacrylamide gradient (acrylamide:bisacrylamide,

37.5:1)wasused.Theoptimalseparationwasachievedbya

40–70%denaturantgradient.Forassessingfungaldiversity,8%

polyacrylamideand25–40%denaturantgradientwereused

Electrophoresiswasthenperformedfor1hat60Vand15h

at100V(60◦C).Afterelectrophoresis,gelswerestainedwith

SYBRGreenI(MolecularProbes,BBI,Candia)for30min.The

gelswereobserved,andphotographsweretakenusingaKETA

GseriesImageSystem(Wletch,USA)

Sequencing of DGGE bands

Representativebands were excisedfromgels withasterile

blade.Thegelpiecesweregroundusingtissue-grinding

pes-tles(Sangon,Shanghai,China)andthenincubatedovernight

at4◦CinTEbuffer(pH8.0).TheDNAsolutionwithTEwas

thenampliﬁedwithprimerswithnoGCclamp.PuriﬁedPCR

productswereligatedintoapUCm-Tvectorandthen

trans-formedinto Trans5␣ChemicallyCompetent Cells(Transgen

Biotech,Beijing,China).Individualwhitecolonieswere

ampli-ﬁedwithPCRusingtheprimersM13-4716andM13-48(Sangon,

Shanghai, China) Samples were then sent to a

sequenc-ingcompanyforsequencing(Sangon,Shanghai,China).The

resultinggenesequenceswerealigned withthose inaGen

Bankwiththe Blastprogramtoidentifythe closestknown

relatives

Statistical analyses

Quantity Onesoftware (Bio-rad,USA) was usedto analyze the DGGE proﬁles and perform cluster analysis Statistical analysisofthedatasetswasperformedusingMATLAB2013a software(Mathworks,USA).TheShannon–Wienerindexwas determinedbytherelativeintensityofbands

Results

Bacterial and fungal community diversity

TheDGGEproﬁleforthebacterialcommunityoffermenting ShenquisshowninFig.1.Notably,thebacterialcommunity differed over the courseof fermentation, whilethe fungal community did notdiffer Diversity indicesof microbesin ShenquwerecalculatedbasedontheDGGEproﬁle.The bac-terialdiversityindicesover8daysoffermentationwereas follows:day1,21bands,Shannon–Wienerindex2.38;day2,23 bands,index2.56;day3,13bands,index2.07;day4,13bands, index2.05;day5,18bands,index2.19;day6,18bands,index 2.15;day7,19bands,index2.35;andday8,7bands,index1.52 Thefungaldiversityindicesoverthe8dayswereasfollows: day1,8bands,Shannon–Wienerindex1.69;day2,10bands, Shannon–Wienerindex1.92;day3,4bands,Shannon–Wiener index1.36;day4,8bands,Shannon–Wienerindex1.77;day

5, 7 bands, Shannon–Wiener index 1.77; day 6, 7 bands, Shannon–Wienerindex1.35;day7,7bands,Shannon–Wiener index1.59;andday8,7bands,Shannon–Wienerindex1.71 Thespeciesrichnessvariedovertheeightsamples,andmost bandswereobservedinthesamplefromday2(Fig.1AandB) Thesamplefromday2alsohadthehighestShannon–Wiener indices(2.56and1.92)ofthePCR-DGGEproﬁles

1d 2d 3d 4d 5d 6d 7d 8d 1d 2d 3d 4d 5d 6d 7d 8d

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Fig 1 – Touchdown PCR-DGGE and nested PCR-DGGE proﬁle of bacterial community diversity of Shenqu from the 16s rDNA

Q5

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Bacterial and fungal diversity after varying durations of

fermentation

ThesequencingofbacterialDGGEbandshighlightedthe

pres-enceofvariousbacterialstrains,includingEnterobacter cloacae

(band 1, 100% identity to NCBI accessionKM408606),

Kleb-siella oxytoca(bands2and10,100%identitytoKM408607and

KM408615),Erwinia billingiae(bands 3and 11,100%identity

toKM408608 and KM408615), Escherichia hermannii(band 4,

99%identitytoKM408609),Paenibacillus polymyxa(band5,99%

identitytoKM408610),Pantoea vagans(band6,100%identity

to KM408611), Acinetobacter baumannii (band 7, 100%

iden-titytoKM408612),Desulfotomaculum thermocisternum(band8,

100%identitytoKM408613),P acidilactici(band9,99%identity

toKM408614),and Citrobacter koseri(band 12,100% identity

toKM408617) (Fig 1A).Notably, P acidilactici (band 9, 100%

identityto KM408614))was detected throughoutthe entire

fermentationprocess

The sequencing of fungal DGGE bands highlighted the

presenceofthreestrains:unculturedRhizopus(band1,100%

identitytoNCBIaccessionKM408618),Aspergillus oryzae(band

2,100%identitytoKM408619), andRhizopus oryzae(band3,

100%identitytoKM408620)(Fig.1B).Again,onespecies,the

unculturedRhizopus (band1),was detectedthroughoutthe

entirefermentationprocess,followedbyband2,3(A oryzae,

R oryzae).

Discussion

Inthis study,PCR-DGGE wasappliedtoanalyzethe

micro-bial communitystructure oftheTCM supplement Shenqu

Shenquisanaturalculturemediumcontainingvarious

nutri-ents.Conventionalculturemethodsareunabletoreﬂectits

fullnutritional contents Therefore, our study adopted the

culture-independentmethodofPCR-DGGEtoinvestigatethe

bacterialandfungalcommunitystructureofShenqu.The

bac-terialDGGEﬁngerprintsshowedthatthePediococcus acidilactici

strain(band9,Fig.1A)wasthepredominantbacterialspecies

presentduringfermentation.Likewise,thepredominant

fun-gusduringfermentationwas unculturedRhizopus,followed

byA oryzae,andR oryzae.FromBerger’sbacterial

identiﬁca-tionmanualandrelatedliterature,17–19 weknow thatthese

bacteriacanproduceamylase,proteaseenzymessuchas

glu-coamylase,anddigestiveenzymes.Theseproductsarelikely

tobeassociatedwiththeappetitestimulatinganddigestive

functionsofShenqu

Thesequencingresultsshowedthatthebacterial

commu-nityincluded10typesofpathogenicbacteria,includingseven

E cloacaestrains,K oxytoca,20E billingiae,andP vagan.21This

studyconﬁrmedthatpathogenicbacteriaexistinthe

tradi-tionalChinesemedicineShenqu.Theexistenceofpathogenic

bacteria is likely to affect the quality of various batches

of Shenqu compared with batches of Shenqu that have

undergonepurebred fermentation22,6 alsoinvestigatedthe

microbialcommunityofShenqubyPCR-DGGEandfoundthat

thedominantmicrobesbelongedtothegeneraEnterobacter,

Pediococcus, Pseudomonas, Mucor,andSaccharomyces,whichare

resultsthataresomewhatdifferentfromours.Thisoutcome

isprobablyduetothedifferentproportionsofingredientsand fermentationparametersusedinthetwostudies

Inconclusion,theaimofthisstudywastoinvestigatethe microbesofShenquovervaryingdurationsoffermentation

byPCR-DGGE.TheresultsrevealedthatP acidilactici, A oryzae,

andR oryzaewerethepredominantmicrobespresent.These resultsmay contributetofurtherstudyofShenqu,suchas studies focusingonoptimizingthefermentationprocessor purebredfermentationofShenqu.Onlybypurifyingthe pre-dominant microbesof Shenquwill webe able toexamine themicrobialbiologicaltransformationsthatoccurinShenqu Thus,inthisstudy,wesuggestthatPCR-DGGEshouldbe con-sideredasapreliminarytoolforinvestigatingthemicrobial community structureofShenqu.Becauseoftechnical deﬁ-cienciesofthePCR-DGGEmethod,however,someelementsof themicrobialcommunitymayinevitablygoundetected.Other newtechnologies,suchasT-RLFP,MLSTandhigh-throughput sequencing,couldthereforebeadoptedforfurtherstudies

Theauthorsdeclarenoconﬂictsofinterest

Acknowledgments

ThisworkwasﬁnanciallysupportedbytheNationalScience Q3

andTechnologyMajorProjectsConstructionoftheIncubator (Benxi)BaseofNationalInnovationDrugsinLiaoningProvince (20102X09401-304-105A)

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