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

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h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

Analysis

of

microbial

diversity

in

Shenqu

with

different

fermentation

times

by

PCR-DGGE

Tengfei

Liu,

Tianzhu

Jia

∗

,

Jiangning

Chen,

Xiaoyu

Liu,

Minjie

Zhao,

Pengpeng

Liu

LiaoningUniversityofTraditionalChineseMedicine,CollegeofPharmacy,KeyLaboratoryofProcessingTheoryAnalysisofState AdministrationofTraditionalChineseMedicine,Dalian,China

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Articlehistory:

Received16September2014 Accepted8December2015 Availableonline22January2017 AssociateEditor:WelingtonLuizde Araújo Keywords: Microbialdiversity PCR-DGGE Shenqu Molecularcloning

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

Rhizopus,Aspergillusoryzae,andRhizopusoryzae.Inaddition,pathogenicbacteria,suchas

Enterobactercloacae,Klebsiellaoxytoca,Erwiniabillingiae,andPantoeavaganweredetectedin Shenqu.DGGEanalysisshowedthatbacterialandfungaldiversitydeclinedoverthecourse offermentation.Thisdeterminationofthepredominantbacterialandfungalstrains respon-sibleforfermentationmaycontributetofurtherShenquresearch,suchasoptimizationof thefermentationprocess.

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

Introduction

Shenqu, also known as Liushenqu, is commonly used in Chinesemedicineclinicstoprotectthestomachandspleen and stimulates appetite and digestion. Current research effortshaverevealedthatsomedigestiveenzymes(amylase enzymes, protease enzymes, glucoamylase), vitamins and other substances play a main role in stimulating appetite anddigestion.1_Resistance_theory_is_the_earliest_work_to

men-tion Shenqu. Shenqu is traditionally processed as follows: wheatbran,ﬂour,ricebeanpowder(Vignaumbellata[Thunb.] Ohwi and Ohashi), and bitter apricot seed powder (Prunus

∗ _{Corresponding}_author.

mandshurica [Maxim.] Koehne) are blended in a particular ratio. VariousChinesemedicinedecoctionsarethen added, including Polygonum pubescens (Blume), Xanthium sibiricum

(Patr.),andArtemisiaannua(L.).Themixtureisthenkneaded anddividedintobricks,whichareputintoamold.Finally,the bricksarecoveredwithadhesive-bondedclothandplacedin aboxatconstanttemperatureandhumidity.Afterafewdays offermentation,theproductiscutintosmalllumpsanddried atalowtemperature.ThequalityoftheresultingShenqucan varyduetodifferencesintheamountofthemixedbacteria and fungithatarepresent duringfermentation. Itisworth notingthat thefungusAspergillusﬂavusproducesaﬂatoxin,

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

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acarcinogen,duringfermentation.Thisisoneofthereasons whyShenquisnot includedinthe ChinesePharmacopeia. However,the current theoretical support endorsesShenqu forstimulatingappetiteanddigestion.abetterunderstanding ofthemicrobesinvolvedinShenqufermentationmayleadto 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,2–4_such_as_PCR-SSCP

(singlestrandconformationpolymorphism)5andDGGE.6 PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis)is aculture-independent method designed toanalyze the geneticdiversity ina sample. Itovercomes thedisadvantagesofculture-dependentmethods,7_making_it

acommon toolfor molecularbiological investigations into microbialcommunities.PCR-DGGEhasbeenusedwidelyto analyzemicrobialcommunitystructureacrossdifferentﬁelds, suchasfoodmicrobiology,oralmicrobiology,soil microorgan-isms,environmentalmicrobiology,andotherareas.8–11_In_this

study,weusedculture-independentPCR-DGGEandTAcloning todeterminethemicrobialdiversityofShenquacross differ-entfermentationperiods.Theaimofthisstudywasto inves-tigateeubacteriamicrobialdiversityduringfermentationand identifyseveraldominantfermentationbacteriaandfungus.

Materials

and

methods

Shenqusamplecollection

Shenqufermentationparameterswere based onour previ-ousstudyandresponsesurfacemethodology.12_Raw_materials

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◦_C_until_they_were_analyzed.

DNAextraction

FivegramsofeachShenqusampleweresuspendedin50mL of phosphate buffered saline (PBS, 0.1mol/L, pH 8.0) and shakenfor10min.Themixedsuspensionwascentrifugedat 10,000×gfor10minandwashedthreetimesusingthesame PBS buffer.Totalgenomic DNAwasextractedfromthe 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.

PCRampliﬁcation

AllprimersusedinthisstudyarelistedinTable1. 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.

DGGEanalysis

ThePCRproductsofbacteriaandfungiwereanalyzedusing DGGE and the D-code Universal Mutation Detection Sys-tem (Bio-rad, USA). For assessing bacterial diversity, 10%

Table1–Primersusedinthisstudy.

Targets Primers Sequence(5–3) References

Bacteria 338Fa _{CCTACGGGAGGCAGCAG} _Muyzer_G13

518R ATTACCGCGGCTGCTGG MuyzerG13

Fungi

FirstPCRround NS1 GTAGTCATATGCTTGTCTC VainioEJ14

FR1 AICCATCAATCGGTAIT VainioEJ14

SecondPCRround NS1 GTAGTCATATGCTTGTCTC MayLA15

Fungb _{ATTCCCCGTTACCCGTTG} _May_LA15

F,forwardprimer;R,reverseprimer.

a _Primer_with_a_41-bp_GC_clamp_{(CGCCCGGGGCGCGCCCCGGGGCGGGGCGGGGGCGCGGGGGG).} b _Primer_with_a_40-bp_GC_clamp_{(CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC).}

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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).

SequencingofDGGEbands

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-fiedwithPCRusingtheprimersM13-4716_and_M13-48_(Sangon,

Shanghai, China). Samples were then sent to a sequenc-ingcompanyforsequencing(Sangon,Shanghai,China).The resultinggenesequenceswere alignedwiththose inaGen Bank withtheBlastprogramtoidentifythe closestknown relatives.

Statisticalanalyses

QuantityOnesoftware(Bio-rad,USA)wasusedtoanalyzethe DGGEproﬁlesandperformclusteranalysis.Statistical anal-ysis ofthe data sets was performed using MATLAB 2013a software(Mathworks,USA).TheShannon–Wienerindexwas determinedbytherelativeintensityofbands.

Results

Bacterialandfungalcommunitydiversity

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.

Bacterialandfungaldiversityaftervaryingdurationsof fermentation

ThesequencingofbacterialDGGEbandshighlightedthe pres-enceofvariousbacterialstrains,includingEnterobactercloacae

(band 1, 100%identity to NCBI accession KM408606), Kleb-siellaoxytoca(bands2and10,100%identitytoKM408607and KM408615), Erwiniabillingiae(bands3and11,100%identity

1d 2d 3d 4d 5d 6d 7d 8d 1d 2d 3d 4d 5d 6d 7d 8d 8 7 1 10 9 2 3 4 5 6 1 2 3 12 11

A

B

Fig.1–TouchdownPCR-DGGEandnestedPCR-DGGEproﬁleofbacterialcommunitydiversityofShenqufromthe16srDNA

and18srDNAobtainedfromShenquaftervaryingdurationsoffermentation.Lanes1–8drefertosamplesderivedfromthe

1sttothe8thdayoffermentation,respectively.(A)A40–70%denaturinggradientwasused.(B)A25–40%denaturing

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toKM408608 and KM408615), Escherichia hermannii(band 4, 99%identitytoKM408609),Paenibacilluspolymyxa(band5,99% identitytoKM408610),Pantoeavagans(band6,100%identity to KM408611), Acinetobacter baumannii (band 7, 100% iden-titytoKM408612),Desulfotomaculumthermocisternum(band8, 100%identitytoKM408613),P.acidilactici(band9,99%identity toKM408614),and Citrobacterkoseri(band 12,100% identity toKM408617) (Fig. 1A).Notably, P. acidilactici (band 9,100% identityto KM408614))was detectedthroughout the entire fermentationprocess.

The sequencing of fungal DGGE bands highlighted the presenceofthreestrains:unculturedRhizopus(band1,100% identitytoNCBIaccessionKM408618),Aspergillusoryzae(band 2,100%identitytoKM408619), andRhizopusoryzae(band3, 100%identitytoKM408620)(Fig.1B).Again,onespecies,the unculturedRhizopus(band 1),was detectedthroughoutthe entirefermentationprocess,followedbyband2,3(A.oryzae, R.oryzae).

Discussion

Inthisstudy,PCR-DGGEwas appliedtoanalyzethe micro-bial communitystructure oftheTCM supplement Shenqu. Shenquisanaturalculturemediumcontainingvarious nutri-ents.Conventionalculturemethodsareunabletoreﬂectits fullnutritional contents. Therefore, our study adopted the culture-independentmethodofPCR-DGGEtoinvestigatethe bacterialandfungalcommunitystructureofShenqu.The bac-terialDGGEﬁngerprintsshowedthatthePediococcusacidilactici

strain(band9,Fig.1A)wasthepredominantbacterialspecies presentduringfermentation.Likewise,thepredominant fun-gusduringfermentationwas unculturedRhizopus,followed byA.oryzae,andR.oryzae.FromBerger’sbacterial identiﬁca-tionmanualandrelatedliterature,17–19 _we_know_that_these

bacteriacanproduceamylase,proteaseenzymessuchas glu-coamylase,anddigestiveenzymes.Theseproductsarelikely tobeassociatedwiththeappetitestimulatinganddigestive functionsofShenqu.

Thesequencingresultsshowedthatthebacterial commu-nityincluded10typesofpathogenicbacteria,includingseven

E.cloacaestrains,K.oxytoca,20_E._billingiae,_and_P._vagan.21_This

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 _also_investigated_the

microbialcommunityofShenqubyPCR-DGGEandfoundthat thedominantmicrobesbelongedtothegeneraEnterobacter, Pediococcus,Pseudomonas,Mucor,andSaccharomyces,whichare resultsthataresomewhatdifferentfromours.Thisoutcome isprobablyduetothedifferentproportionsofingredientsand fermentationparametersusedinthetwostudies.

In conclusion, the aim ofthis study was toinvestigate themicrobesofShenquovervaryingdurationsof fermenta-tionbyPCR-DGGE.TheresultsrevealedthatP.acidilactici,A. oryzae,andR.oryzaewerethepredominantmicrobespresent. TheseresultsmaycontributetofurtherstudyofShenqu,such asstudiesfocusingonoptimizingthefermentationprocess orpurebredfermentationofShenqu.Onlybypurifyingthe

predominantmicrobesofShenquwillwebeabletoexamine themicrobialbiologicaltransformationsthatoccurinShenqu. Thus,inthisstudy,wesuggestthatPCR-DGGEshouldbe con-sideredasapreliminarytoolforinvestigatingthemicrobial community structureofShenqu.Becauseoftechnical deﬁ-cienciesofthePCR-DGGEmethod,however,someelementsof themicrobialcommunitymayinevitablygoundetected.Other newtechnologies,suchasT-RLFP,MLSTandhigh-throughput sequencing,couldthereforebeadoptedforfurtherstudies.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

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

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