Screening and characterization of lactic acid bacterial strains that produce fermented milk and reduce cholesterol levels

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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 /

Food

Microbiology

Screening

and

characterization

of

lactic

acid

bacterial

strains

that

produce

fermented

milk

and

reduce

cholesterol

levels

Xuefang

Guan,

Qingxian

Xu,

Yi

Zheng,

Lei

Qian,

Bin

Lin

∗

InstituteofAgriculturalEngineeringTechnology,FujianAcademyofAgriculturalSciences,Fuzhou,Fujian,People’sRepublicofChina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11July2016

Accepted16February2017

Availableonline2June2017

AssociateEditor:SusanaSaad

Keywords: Fermentedmilk Lactobacillusplantarum Cholesterol Rat

a

b

s

t

r

a

c

t

Objective:Toscreenforandcharacterizelacticacidbacteriastrainswiththeabilityto

pro-ducefermentedmilkandreducecholesterollevels.

Methods:ThestrainswereisolatedfromtraditionalfermentedmilkinChina.Invitroand

invivoevaluationofcholesterol-reductionwereusedtoidentifyandverifystrainsofinterest.

Characteristicswereanalyzedusingspectrophotometryandplatecountingassays.

Results:TheisolateHLX37consistentlyproducedfermentedmilkwithstrong

cholesterol-reducingpropertieswasidentiﬁedasLactobacillusplantarum(accessionnumber:KR105940)

andwasthusselectedforfurtherstudy.ThecholesterolreductionbystrainHLX37was

45.84%.Theisolateswereacid-tolerantatpH2.5andbile-tolerantat0.5%(w/v)insimulated

gastricjuice(pH2.5)for2handinsimulatedintestinalﬂuid(pH8.0)for3h.The

auto-aggregationrateincreasedto87.74%after24h,whiletheco-aggregationwithEscherichiacoli

DH5was27.76%.StrainHLX37wasintrinsicallyresistanttoantibioticssuchaspenicillin,

tobramycin,kanamycin,streptomycin,vancomycinandamikacin.Comparedwithratsin

themodelhyperlipidemiagroup,thetotalcholesterolcontentintheserumandtheliver

aswellastheatherogenicindexofratsintheviablefermentedmilkgroupsigniﬁcantly

decreasedby23.33%,32.37%and40.23%,respectively.Fewerfatvacuolesandotherlesionsin

livertissuewerepresentinboththeinactivatedandviablefermentedmilkgroupscompared

tothemodelgroup.

Conclusion:ThesestudiesindicatethatstrainHLX37ofL.plantarumdemonstratesprobiotic

potential,potentialforuseasacandidateforcommercialuseforpromotinghealth.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Corresponding}_author_at:_Institute_of_Agricultural_Engineering_Technology,_Fujian_Academy_of_Agricultural_Sciences,_Fuzhou,_Fujian

350003,People’sRepublicofChina.

E-mail:binlin591@163.com(B.Lin).

http://dx.doi.org/10.1016/j.bjm.2017.02.011

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Introduction

Cholesterolisanindispensablenutrientinthehumanbody,1

asit serves as acomponent of the cell membraneand as

aprecursor forcholicacid and steroidhormonesynthesis.

However,excesscholesterolisamajorcontributorto

hyper-lipidemiaandcardiovascularandcerebrovasculardiseases.2,3

AccordingtothestatisticsfromtheNationalHealthand

Fam-ilyPlanningCommissionofChina(2015),cardiovascularand

cerebrovascular diseases have become a leading cause of

deathworldwide,withasmanyas 15millionpeople dying

fromthesediseasesannually.Researchshowsthatthe

prob-abilityofsufferingfromcardiovasculardiseasedecreasesby

2–3%4_with_every_1%_decrease_in_the_serum_cholesterol_level.

Therefore,aneffectivemethodforreducinghighcholesterol

toahealthylevelcouldprotectagainstthesediseases.

Studies haveshown that sometherapeutics, suchas

3-hydroxy-3-methylglutarylcoenzymeAreductase inhibitors,

effectivelyreduceserumcholesterol.5_Along_with_these

ther-apeutics,aperson’sdietshouldalsoberegulatedtoinclude

foodseffectiveatpreventingandreducingcholesterol.

Numer-ousexperimentshavedemonstratedthatprobioticlacticacid

bacteriacanalsoreducecholesterolviaadsorption,

conver-sion,co-precipitationandbilesalthydrolase(BSH)enzymatic

degradation.5,6 _However,_the _oral_intake_of_probiotics_is_not

palliativeformostpeople.

Fermented milk obtained through the lactobacillus

fer-mentationoffreshmilkisadairyproductwithanumberof

nutritionalandhealthadvantages.Thefermentationprocess

also involves the growth of lactic acid bacteria that

con-vertthelactoseandmilkproteinintolacticacid,galactose,

aminoacids and small peptides, whichare easilydigested

andabsorbedbythehumanbody.Moreover,thenutritional

valueoffermentedmilkproductsgeneratedbyvariouslactic

acidbacteriacanalsobedifferent. Inadditiontosatisfying

nutritionalrequirements,somefermentedmilkproductscan

improvetheabsorptionanduptakeofnutritivesubstances,

suchascalcium-containingsubstances.Therearenumerous

lacticacidbacteriathatcaninhabittheinternalwallofthe

intestinaltractandformmicrobialcommunitiesand a

bio-logicalbarrier.Inhibitionofthegrowthofpathogenicbacteria

intheintestinaltract7,8_and_the_regulation_of_the_balance_of

intestinalmicroﬂorainsuchawaymayhaveeffectssimilarto

thoseofprobioticsandmetabolites.9,10

Forthesereasons,wesetouttoscreenforlacticacid

bac-terial(LAB)strainscapableofproducingfermentedmilkand

reducing cholesterollevels foruse asstarter cultures.

Fur-thermore,thisstudyinvestigatedtheabilityofthesestrains

infermentedmilktoreducecholesterolinhigh-fatrats,and

evaluatedthesestrainsascandidatecommercialprobioticsto

promotehealth.

Materials

and

methods

Samplesandmedia

Traditionalfermentedmilksampleswerescreenedforlactic

acidbacteriastrainsabletoreducecholesterollevels.Selected

strainswerecultivatedinfreshmilkcollectedfromafarmerin

MangdangTown,YanpingDistrict,NanpingCity,intheFujian

ProvinceofChina(26.510◦N,118.090◦E).Traditionalfermented

milkwasmadeasfollows:thefreshmilkfromChinese

Hol-steincowswasincubatedat15◦Cfor35daystoferment;when

thepHvaluereachedapproximately4.4,fermentedmilkhad

beenproduced.Man,RogosaandSharpe(MRS)mediumwas

usedforculturingthe LAB.Abasaldietwasobtainedfrom

theCenterforDiseaseControlandPrevention(Fujian,China)

andcontained10%moisture,22%crudeprotein,4%crudefat,

5%crudeﬁber,and8%crudeash.Ahypercholesterolforage

consistedof57%basaldiet,13%lard,5%peanuts,2%sesame

oil, 20% sucrose,3% cholesterol (Zhengzhou Tianjian Food

TechnologyCo.,Ltd,HunanProvince,China),and0.1%sodium

cholate(ZhengzhouTianjianFoodTechnologyCo.,Ltd.,Hunan

Province,China).

Animals

Sprague-Dawley(SD)malerats(weighing150–170g)were

pur-chased from the Hayes Lake Laboratory Animal Co., Ltd.

(Shanghai, China) [license number: SCXK (Shanghai

2007-0005)]. The rats were housed under standard controlled

laboratoryconditions(temperatureof24±3◦C,humidityof

50±10% and 12h light/12h dark cycle), with unrestricted

accesstofoodandwater.Allanimalswerehandledin

accor-dancewiththeanimalcareanduseguidelinesestablishedby

theFujianUniversityofTraditionalChineseMedicine(Fujian,

China), which entailed minimizing both the number and

sufferingoftheanimalsusedfortheexperiments.All

experi-mentalproceduresinvolvingratswereapprovedbytheFujian

AssociationforLaboratoryAnimalScience(FALAS).

Screeningforstrainscapableoffermentingmilkand

reducingcholesterollevels

Naturalfermentedmilksamples(1mL)wereseriallydiluted,

and 10−5, 10−6 and 10−7 dilutions insalinewere platedon

solidMRSagarplates.Theplateswereincubatedat42◦Cfor

2days.Colonieswithphenotypesandcharacteristicssimilar

tothoseofLactobacilluswereselectedandsub-inoculatedinto

MRSmediumthreetimes.Theisolated strainswere

inocu-latedinto10mLofMRSmediumandincubatedanaerobically

at42◦Cfor1day.A1.0-mLaliquotoftheculturebrothwas

sub-inoculated into a fermenter containing 100mLof

pas-teurizedmilk(ChangfuDairyGroupCo.,Ltd.FujianProvince,

China), which had been sterilizedat 115◦C for20min and

incubatedat42◦Cfor12h.Followingfermentation,anystrain

thatconsistently resultedinsolidiﬁedfermentedmilkwith

apleasingsmell andapHvalue≤4.4wasselected, labeled

and stored in 20% glycerol at −20◦_C _until _further

analy-sis.Toscreenforstrainsthatcouldreducemilkcholesterol

levels, a 1-mLaliquot of theculture brothfrom the tested

fermentationstrains wastransferredintoafermenter

con-taining100mLofcholesterol(SinopharmChemicalReagent

Co.,Ltd.,Shanghai,China)screeningmediumandincubated

at 42◦C for 2 days. Any strain that produced turbidity in

the culturebrothwas testedforcholesterolreduction

abil-ity.LABculturesweregrowninMRSmediumsupplemented

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24 to 72h and then centrifuged at 9000×g for 15min at

4◦C.Thecholesterolinthesupernatantwasquantiﬁedusing

theO-phthaldehydemethoddescribedbyRudeletal.,11_and

cholesterol was used for the standard curve. The control

brothwasuninoculatedMRSbrothcontainingapproximately

200mg/L of water-soluble cholesterol. The ability of each

straintoreducetheamountofcholesterolwascalculatedwith

thefollowingequation:

%ofcholesterolreducingrate=

C0−C

C0

×100,

whereC0isthecontrolbrothandCisfermentedbroth.The

strainthatresultedinthehighestyogurtfermentationand

cholesterolreductionwasselectedforfurtherstudy.

Identiﬁcationofstrainsabletobothfermentmilkand

reducecholesterol

The colony morphology of the isolates incubated on MRS

mediumplatesat42◦Cfor2dayswasevaluated.Gram

stain-ingwasperformed,andthebacteriawereexaminedusinga

YS100typemicroscope(Nikon,Japan).Cellmorphologywas

determined using a JSM-6380/LV scanning electron

micro-scope(ElectronOpticsLaboratoryCo.,Ltd.,Japan).Inaddition,

the16SrRNAandRNApolymerasealphasubunit(rpoA)genes

of the strains were partially sequenced using the primers

F9–27/R1525–1542 (5-GAGTTT GAT CCT GGC TCAG-3/5-AGA

AAGGAGGTGATCCAGCC-3)andrpoA-21-F/rpoA-22-R(5

-ATGATYGARTTTGAAAAACC-3/5-ACYTTVATCATNT

CWGVY-TC-3),as described byNaser etal.12 _{respectively.}_The _PCR

reactionmixturecomprised2.5␮Lof10×PCRbuffer(Tiangen

BiotechCo.,Ltd.Beijing,China),2.5␮Leach of2mMdNTPs

(dATP,dCTP,dGTP,anddTTP)(TiangenBiotechCo.,Ltd.Beijing,

China),1␮Lof50mMMgCl2(TiangenBiotechCo.,Ltd.Beijing,

China),2␮Lofeachprimer(5pmol/L),0.5␮LofBIOTaqDNA

polymerase(2.5U/L),1␮Loftemplate,anddistilledddH2Oto

aﬁnalvolumeof25␮L.ThePCRprogramonanEppendorfAG

included35ampliﬁcationcyclesof1minat94◦Cfor

denatur-ation,1minat55◦C(16SrRNA)or50◦C(rpoA)forannealing,

and1minat72◦Cforextension,followed byaﬁnal

exten-sioncycleat72◦Cfor10min.Inaddition,the16SrDNAand

rpoApartialsequencesobtainedwereanalyzedusingBLAST

ontheNCBIserver(http://www.ncbi.nlm.nih.gov/).

Phyloge-neticanalysesofthe16SrRNAandrpoAgenesequenceswere

performedusingtheClustalWprogramwiththeMEGA4

soft-wareversion14.0.0.162.13

Acidtoleranceandbiletolerancetest

Theacidtoleranceofthestrainswasevaluatedasdescribed

byArcheretal.14_with_slight_{modiﬁcations.}_Brieﬂy,_MRS

pre-paredatpHvaluesof1.5,2.0,2.5,3.0,3.5and4.0,inoculated

witha5%overnightbacterialsuspension,andgrown

anaero-bicallyat37◦Cfor24h.Aliquotsweretakenatintervalsof0,2

and4h,seriallydiluted,andplatedontoMRSagarplates.After

incubatinganaerobicallyat37◦Cfor48h,thenumberofviable

colonies was counted. Similarly, MRS broth supplemented

with0.1%,0.2%,0.3%,0.4%and0.5%bilesalts(w/v)(Huankai

MicrobialSciCo.,Ltd.China)wasfreshlypreparedand

inocu-latedwitha5%overnightbacterialsuspension.Aliquotswere

takenatintervalsof0,2and4h, andthenumberofviable

coloniesonMRSagarplateswascounted.Samplestakenat

0hwereusedasacontrol.

Simulatedgastricjuiceandintestinalﬂuidtolerancetests

Gastricjuicewassimulatedusingasolutionof125mMNaCl,

7mMKCl, 45mMNaHCO3 and 3g/Lpepsin adjustedtopH

valuesof2.5and3.0andpreparedaccordingtothemethod

describedbyArcheretal.14_Bacterial_cultures_were_grown_for

24h,centrifugedfor5000×gfor15minat4◦Candaddedto

thesamevolumeofnormalsaline(0.9%)toreplacethebroth,

while10%wasinoculatedintothegastricﬂuidandincubated

at37◦Cinanincubator(MS-2102,LNB,Shanghai,China)with

shakingat180rpm.Samplesweretakenat0,1,2,3and4hof

incubation,andthenumberofbacterialcoloniesonMRSagar

plates wascounted.Intestinalﬂuidconsistingof0.1%(w/v)

pancreatin and0.15%(w/v)ox-bilewasadjustedtopH8or

7.5.Inamannersimilartothegastricjuiceassay, the

bac-terialculturewasusedtoinoculateintestinalﬂuidandthen

incubated.Viablecellcountswereobtainedbyplatingsamples

takenat0,3and6hofincubation.Samplestakenat0hwere

usedascontrols.Thepercentsurvivalwascalculatedusing

thefollowingformula:

Bacterialsurvival (%)=_CFUcontrolCFUassay ×100,

whereCFUassayisthecellcountafterincubationinsimulated

gastricjuiceorintestinalﬂuid,andCFUcontrolisthecellcount

afterincubationinnormalMRSat0h(control).

Aggregationactivity

Cultures ofHLX37and Escherichia coli DH5␣ were grownin

MRS and Luria-Bertani medium (LB, Canspec, Inc., China)

overnight,respectively,and thencentrifugedat3000×gfor

10minat4◦C.Thecellswerewashedtwice,resuspendedin

phosphate-bufferedsolution(PBS),andadjustedtoaninitial

OD600of0.4±0.1(A0).Thecellsuspensionswerethen

incu-batedatroomtemperature at25◦Cfor24htoallowphase

separation, and the ﬁnal OD600 value(A24) ofthe aqueous

phasewasmeasured.Thepercentageofauto-aggregationwas

expressedasfollows:Aauto%=(A0−A24)/A0×100.15Similarly,

equalvolumesofLABandE.coliDH5␣weremixed,adjustedto

aninitialOD600of0.6±0.1(A0)inPBSandincubatedatroom

temperaturefor24hwithoutagitation.TheOD600value(A24)

oftheaqueousphasewasmeasured.Theco-aggregation

per-centagewasexpressedasfollows:Aco%=(A0−A24)/A0×100.

Antibioticresistance

TheantibioticresistancepatternsofstrainHLX37were

deter-mined by a disk diffusion method using the Kirby-Bauer

technique.16 _MRS_agar _plates _with_a _thickness_of₄_±₁_mm

wereevenlyspreadwith100␮LofstrainHLX37(8.0lgcfu/mL)

using a sterile swab. Antibiotic discs (Hangzhou Microbial

ReagentCo.,Ltd.,GuangdongProvince,China)wereplacedon

theplates.Theplateswerethenincubatedfor36hat37◦C.

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Verniercaliper,andtheantibioticresistancewasdetermined

accordingtoCLSI.17

Cholesterolreductionabilityinarathyperlipidemiamodel

AfermentedmilksamplewithHLX37waspreparedby

fer-mentingfreshmilk(wholemilk,ChangfuDairyGroupCo.,Ltd.,

FujianProvince,China)withLactobacillusplantarumHLX37at

42◦C for12huntilaviablecount(8.30±0.01lgcfu/mL)was

achievedandtheaciditywas82.32±0.99◦T(asmeasuredby

titrationwithaGB5409-85,China).Fermentedmilkwas

pro-ducedevery7daysandstoredat4◦C,sothestorageperiodof

thefermentedmilkwas7days.Onthe7thday,theviablecount

was8.39±0.04lgcfu/mL(F=13.73,p=0.021>0.05),theviable

countdidnotchangesigniﬁcantlyduringstorage.Atotalof

40ratswererandomlyassignedtothefollowingfourgroups

(n=10):(1)aninactivatedfermentedmilkgroup(Inactivated)

inwhichtheratswerefedafermentedmilksamplethatwas

inactivatedina100◦Cwaterbathfor30minanda

hyperc-holesterolchow,(2)aviablefermentedmilkgroup(Viable)in

whichtheratswerefedafermentedmilksampleand

hyper-cholesterolchow,(3)ahyperlipidemiamodelgroup(Model)in

whichtheratswerefedasalinesolutionanda

hypercholes-terolchow,and(4)anormalcontrolgroup(Normal)inwhich

theratswerefedsalineandonlyabasaldietprovidedbythe

CenterforDiseaseControlandPrevention,Fujian,China.The

fermentedmilk(1mL/100(dg)ofratbodyweight)orsalinewas

fedtotheratsfor62dbygavage.Theratswereallowedfree

accesstotheexperimentaldietandwater;foodconsumption

andbodyweightweremonitored.Thebloodandliverwere

collected after12h offasting underanesthesia byinjected

sodiumpentobarbital(40mg/kgbodyweightofrat,Shanghai

ChemicalReagentsProcurementSupplyStation,China).Blood

collectedfromthepostcavaserumwasisolatedby

centrifug-ingthebloodsamplesat3000×gfor10min.Aportionofthe

liverwasimmediatelyﬁxedin10%formalinbufferforfreezing

andliquidhistologicalsectioning,andtheremainingtissues

wereﬂashfrozeninliquidnitrogenandstoredat−80◦_C_until

use. Thetotalcholesterol(TC)levelinthe serum and liver

weredeterminedusingatotalcholesterolassaykit(Jiancheng,

Nanjing,China).High-densitylipoproteincholesterol(HDL-C)

and low-densitylipoproteincholesterol(LDL-C) inthe liver

weremeasuredusinghigh-densityandlow-density

lipopro-tein cholesterolassay kits,respectively (Nanjing Jiancheng

Bioengineering Institute,China).Theatherogenic index(AI)

wasexpressedasfollows:AI=(TC-HDL-C)/HDL-C.18

Statisticalanalysis

All experimentalsampleswere testedintriplicate,and the

resultingvaluesareexpressedasthemean±standard

devi-ation(sd).ThestatisticalanalysiswasperformedusingSPSS

17.0software.Aone-wayANOVAwasperformedacross

mul-tiplegroupstotestsigniﬁcance,andp<0.05wasconsidered

signiﬁcant.

Results

Screeningandisolationofbacterialstrainsabletoferment

milkandreducecholesterol

Strainscapableoffermentingmilkandreducingcholesterol

werescreenedandisolatedfromtraditionalfermentedmilk

samples collected from a farmer. A preliminary screening

yielded22isolatesthathadaphenotypeandcharacteristics

similartoLactobacillusspp.onsolidMRSplates.Theseisolates

weretransferredtopasteurizedmilk.Ofthese22isolates,8

isolatesfermentedmilkandwereselectedanddesignatedas

HLX31-HLX38.Ofthese8isolates,2strains(HLX33andHLX37)

producedturbidityinacholesterolscreeningmedium,sothey

werefurthertestedfortheirabilitytoreducecholesterol.The

cholesterolcontentandtheresidualcholesterolintheculture

brothduringgrowthareshowninTable1.StrainsHLX33and

HLX37Aconstantlyreducedcholesterolinthebroth

through-outthefermentationprocess.After72hofincubation,strain

HLX37showedthehighestcholesterolreduction,45.84%,in

aculturebrothcontaining123.1␮g/mgofcholesterol;the F

valuebetweenstrains=21.016,andp=0.000<0.01,which

indi-cates that the ability ofstrain HLX37 to lower cholesterol

was signiﬁcantlybetterthan strainHLX33.thus,HLX37was

selectedforfurtherstudy.

CharacterizationandidentiﬁcationofstrainHLX37

ThemorphologyofstrainHLX37isshowninFig.1.Colonies

on MRS plates were white, round, opaque, Gram-positive,

androd-shapedwithacellsizeof(1.8–3.5)×(0.7–0.9)␮m.LAB

Table1–Abilityoflacticacidbacterialstrainstolowercholesterol.Thedataareexpressedasx±sd(n=3foreach sample).Note:TheLABstrainsweregrowninacholesterolmedium(MRSmedium+approximately200␮g/mL

cholesterol).TheFvaluebetweenstrains=21.016,andp=0.000<0.01,whichindicatesthattheabilityofstrainHLX37to lowercholesterolwassigniﬁcantlybetterthanstrainHLX33.

Incubationtime(h) Incubationtemperature(◦C) HLX33 HLX37

Cholesterol content(␮g/mL) Cholesterol reduction(%) Cholesterol content(␮g/mL) Cholesterol reduction(%) 0 42 227.30±5.80 227.30±5.80 24 42 172.80±9.84 23.88±4.33 143.69±1.80 36.78±0.79 48 42 168.59±6.74 25.83±2.97 143.12±9.39 37.04±4.13 72 42 158.88±0.77 30.10±0.34 123.10±23.05 45.84±10.14

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A

B

C

D

Fig.1–ColonycharacteristicsandmorphologyofL.plantarumstrainHLX37.Note:(A)Colonycharacteristicsinoculatedin MRSmedium;(B)Gramstaining;(C)ScanningElectronMicroscopy(SEM);(D)TransmissionElectronMicroscope(TEM).

A

8.4

V

iable count (lg cfu/mL)

V

Survival percentage (%) Survival percentage (%) 8.2 8 7.8 7.6 7.4 1.5 2 2.5 3 3.5 (pH) 0 8 7 6 0.1 0.2 0.3 0.4 0.5 30 0 60 90 120 150 (%) 7.5 6.5 30 60 90 120 150 4

B

Fig.2–Theviablecount(lgcfu/mL)andsurvival(%)ofLactobacillusplantarumHLX37incubatedin(A)acidicand(B) bile-containingmediumwithtime.Theviablecountsweremeasuredafter,0h;,2h;and䊉,4h.Thesurvivalwas measuredafter,2hand,4h.

strainswereidentiﬁedbyBLASTsequencesofthe16SrRNA

geneandwerefoundtohave>99%homologywithLactobacillus

plantarumandLactobacilluspentosus.L.plantarumandL. pen-tosusweredifferentiatedbyarpoApartialsequence(861bp)

thatwasalsoanalyzedbyBLASTandwerefoundtohave99%

homologywithL.plantarumbutonly97%homologywithL.

pentosus.Thisresultwasfurtherconﬁrmedbymappingthe

strainphylogeny;thus,strainHLX37wasidentiﬁedasL.

plan-tarum.The16SrDNAsequence (1425bp)forthisstrainwas

depositedintheGenBankdatabaseundertheaccession

num-berKR105940. Additionally, HLX37wasadded tothe China

General Microbiological Culture Collection Center (CGMCC)

collectionunderthenumber10632.

Acidandbiletolerance

TheeffectofacidicconditionsontheviabilityofL.plantarum

HLX37isshowninFig.2.StrainHLX37survivedwellunder

acidicconditions(pH≥2.5),andtheviablecellcountreached

7.43±0.05lgcfu/mL. Thesurvival rate was 56.84±0.57% at

pH=2.5 after 4h of incubation at 37◦C. Survival reached

122.86% and 143.81%after2 and 4hincubation periodsat

a pH of 4.0. The bile tolerance of the strain was

consid-eredanimportantcharacteristicbecauseitenablesthestrain

to surviveandremain functional followinggastrointestinal

transit.15 _Strain _HLX37_was_grown _in_the_presence_of_0.1%,

0.2%,0.3%,0.4%and0.5%bilesalts andwasdeterminedto

haveahighbiletolerance.Therateofsurvivalin0.3%bilesalts

after2hwas53.24±4.09%andthatafter4hwas54.68±2.03%,

withaviablecountof7.57±0.03lgcfu/mL.Thisstraincould

alsotolerateupto0.5%bilesalts,andtheviablecountrange

was6.40±1.10to6.5±0.16lgcfu/mLafter2–4hofincubation.

ThisresultsuggeststhatHLX37cantoleratealowpHanda

highconcentrationofbilesalts.

Simulatedgastricandintestinalﬂuidtolerancetests

The effect of simulated gastric ﬂuidsand intestinal ﬂuids

on the viability of strain HLX37 is shown in Fig. 3. Strain

HLX37couldsurviveatpH3.0for2hwithasurvivalrateof

33.41%. Strain HLX37also had asurvival rategreater than

13.66%atapH2.5after2hingastricﬂuidwithaviablecount

8.40 8.50 8.00 7.50 7.00 6.50 0 3 6 (h) 150 100 50 0 1 0 2 3 4 0 50 100 150 (h) 7.90 6.90 V

V

Survival percentage (%) Survival percentage (%) 7.40

A

B

Fig.3–Viablecount(lgcfu/mL)andsurvival(%)ofLactobacillusplantarumHLX37afterexposuretosimulatedgastricjuice(A) andintestinalﬂuid(B).Theviablecountwasmeasuredinthepresenceof䊉,pH2.5inAorpH8.0inB;and,pH3.0inAor pH7.5inB.Thesurvivalwasmeasuredinthepresenceof,pH2.5inAorpH8.0inB;and,pH3.0inAorpH7.5inB.

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Table2–AdhesiveabilityofL.plantarumHLX37(n=3,x±sd).

Tests A0 A24 Aggregationpercentage(%)

Auto-aggregation 0.5131±0.0045 0.0629±0.0369 87.74

Co-aggregationwithEscherichiaDH5␣ 0.5133±0.0082 0.3708±0.0355 27.76

greater than 7.47±0.03lgcfu/mL (Fig. 3A) and greater than

40.05±7.09%atpH8.0after3hwithaviablecountgreater

than7.90±0.07lgcfu/mLinintestinalﬂuid(Fig.3B).Thisresult

suggestsstrainHLX37couldtoleratesimulateddigestivejuice.

Aggregationactivity

Theresultsofassaystomeasureaggregation areshown in

Table2. Auto-aggregation increasedto 87.74%after 24h at

roomtemperature(25◦C),whiletheco-aggregation

percent-age was 27.76% for Escherichia DH5␣ after24h ina mixed

suspension.

Antibioticresistance

ThesusceptibilitypatternsofL.plantarumHLX37areshown

inTable3.Noinhibitionzonewasobservedinthepresence

of tobramycin. Furthermore strain HLX37was resistant to

kanamycin,streptomycinand vancomycinaccordingtothe

Table3–AntibioticsusceptibilityofL.plantarumHLX37 usingtheKirby-Bauermethod(n=3,x±sd.)Note:(S),(I) and(R)denotesensitive,intermediaryandresistant strainsaccordingtothebreakpointsestablishedbyCLSI (2007).

Antibioticdisks Paper content ␮g/piece Diameterof zoneof inhibition (cm) Antimicrobial susceptibility type Beta-amide/penicillin Penicillin(P) 10 23.00±2.83 R Cephalosporins Cephalexin(CA) 30 23.17±5.103 S Ceftriaxone(CTR) 30 44.67±0.473 S Aminoglycosides Amikacin(AK) 30 13.33±0.24 R Gentamicin(GM) 10 12.33±2.17 I Tobramycin 10 – R Kanamycin(K) 30 – R Streptomycin(S) 10 – R Tetracyclines Tetracycline(TE) 30 19.43±0.50 S Beta-lactam/Beta-inhibitor Amoxicillin(AMX) 20 33.17±1.18 S Phenicols Chloroamphenicol(C) 30 21.33±4.11 S Glycopeptides Vancomycin(VA) 30 – R Macrolides Erythromycin(E) 15 32.50±0.41 S

CLSI withStaphylococcus aureusATCC25923 asthe indicator bacterium.Thediametersoftheinhibitionzoneofpenicillin andamikacinwerelessthan2.8cmand1.4cm,respectively, indicating resistance. The antimicrobial susceptibility type was Intermediary(I)withgentamicin.Noresistance tothe other antibiotics tested was evident, and the clear zones rangedfrom19.43to44.67cm.

Cholesterolreductionabilityofthebacterialstrainsinrats

EffectsofHLX37fermentedmilkinrats

Food consumption was significantlygreater in the normal group than in the other groups, but no significant differ-enceswerenotedfortheinactivated,viableandmodelgroups. Meanwhile,thevalueofbody finalweightwassignificantly greaterinthenormalgroupthanintheinactivatedandmodel groups. No significant differenceswere foundbetween the viablegroupandtheothergroups(Table4).TheTClevelsin

therat’sserumandliverareshowninFig.4A.Comparedtothe

hyperlipidemiamodelgroup,theTClevelsintheserumand

liveroftheratsintheviablegroupweresigniﬁcantlylessby

23.33%and32.37%,respectively,andasigniﬁcantdecreaseof

27.46%intheserumwasobservedfortheinactivatedgroup.

TheorderoftheTCcontentvaluesintheratsforeach

treat-mentwereModel>Inactivated>Viable>Normal.Ratsinthe

inactivatedgroupshowedasigniﬁcantdecreaseinserum

LDL-Cof15.19%despitetheirhigh-fatdiet(Fig.4B),butthiswasnot

observedinviablegroup.Nosigniﬁcantdifferenceswerefound

in the HDL-C inthe inactivated,viable and model groups.

Additionally,theviablegroupshowedasigniﬁcantdecrease

inAI(40.23%;Fig.4C).

Microscopemorphologicalanalysis

Morphological differences were noticed in the treated rat

liver tissue biopsiescompared to the healthy tissuesfrom

normalrats(Fig.5).Inthemodelgroup,numerousfat

vac-uoles,hepaticlobulescontainingdebris,loosecytoplasmand

afoldedmembranethatwaspushedtoonesidewerefound

inthe liverbiopsies, conﬁrming thatthe modelgroup was

unhealthycomparedtothenormalgroup.Onlyafewfat

vac-uoles were notedin the rats in the inactivated and viable

bacterial fermented milk groups, which were notably less

thaninthehyperlipidemiamodelgroup.Furthermore,inthe

inactivated andviablebacterialfermentedmilkgroups, the

hepaticlobuleswereclear,thecellswereneatlyarranged,and

theliversinusoidwasnormalwithnosigniﬁcantlesions.The

occurrenceoffattyliverlesionswassigniﬁcantlyreducedin

the high-fatrats,and liveradipose degenerationwas

ame-lioratedtosomedegreeintheinactivatedandviablegroups,

(7)

Table4–Foodconsumptionandfinalbodyweightofratsfor62d(x±sd,n=10).Note:Valueswiththesameletterarenot significantlydifferent(p>0.05),whiledifferentsuperscriptlettersindicatesignificanceatp<0.05.

Diettreatment Inactivatedgroup Viablegroup Modelgroup Normalgroup

Consumptionofdiet[g/(onerat·d)] 24.94±2.26b _25.34_±_1.91b _25.64_±_2.02b _36.74_±_1.36a Bodyﬁnalweight(g/onerat) 345.61±54.03b _376.41_±_38.51ab _361.27_±_41.25b _431.73_±_42.83a

C

B

A

5 6 3 2 1 0 Inactivated

Viable Model _Normal

Inactivated

Viable Model _Normal

Inactivated

Viable _{Model Normal} 4 5 6 2 1.20 0.90 0.60 0.30 0.00 1.6 1.2 0.8 0 3 2 1 0 4 b bc d a Serum Liver LDL-C HDL-C b ab ab ab a a c c c Al Content (mmol/L) TC (mmol/g protein) TC (mmol/L) cd a a a cd ab d

Fig.4–EffectsofL.plantarumHLX37fermentedmilkinrat.(A)Totalcholesterol(TC)levelsinserum(mmol/L)andliver (mmol/gprotein),(B)LDL-CandHDL-Clevelsinserumfollowingtreatment,(C)theatherogenicindex

(AI)=(TC-HDL-C)/HDL-C.Note:(1)Inactivated:thefermentedmilksamplewasinactivatedina100◦Cwaterbathfor30min andtheratswerefedthehypercholesterolchow;(2)Viable:thefermentedmilksampleandtheratswerefedthe

hypercholesterolchow;(3)Model:salineandtheratswerefedthehypercholesterolchow;(4)Normal:salineandtherats werefedabasaldiet.Valueswiththesameletterswerenotsigniﬁcantlydifferentp>0.05,whiledifferentsuperscriptletters indicatesigniﬁcanceatp<0.05.

Discussion

Traditional fermented foods such as cabbage, cucumber andmilk,whichprovideproductswithdeﬁnedconsistency andhaveadditionalhealth-promoting(functional)properties, have usually been used as a source for screening various LAB.19,20 _Screening _for _strains _with _the _ability _to _ferment

milkandreducecholesterollevelsidentiﬁedaLABstrain

des-ignated as HLX37, which was subsequently cultured. This

strain could ferment milkand reducethe cholesterollevel

inMRSmediumby45.84%;consequently,itbecamethe

sub-jectforfurtherstudy.Amorphologicalanalysisand16SrDNA

sequencingidentiﬁedtheisolateHLX37asLactobacillusspp.To

furtheridentifyHLX37tothespecieslevel,weusedrpoAgene

sequencesthatofferareliableidentiﬁcationsystemfornearly

all species of the genus Lactobacillus,12 _and _strain _HLX37

wasidentiﬁedasL.plantarum(Accession no.KR105940).We

foundthatL.plantarumHLX37couldlowercholesterolandwas

suitableforthermophilicfermentationtoproducefermented

milk.ThisstrainwaslistedintheStrainListApplicabletoFood

speciﬁedinFileNo.[2010]65bythehealthofﬁceoftheChina

MinistryofHealth,whichsupervisesediblemicroorganism.

AnFAO/WHOworkinggroupconﬁrmedthatsomebacterial

speciessuchasL.fermentumcanbedesignatedasprobiotics

whendeliveredinfoodandfoodsupplementsataminimum

levelof9lgcfuviablecellsperserving.21_This_indication_means

thatconsumingenoughfermentedmilk(i.e.,morethan10mL

fermentedmilkthatcontains8lgcfu/mL)perservingnotonly

tastesexcellentandprovidesahighnutritionalvaluebutalso

is agood deliverysystem forprobiotics. Humanscan

con-sumesufﬁcientfermentedmilktoexertabeneﬁcialprobiotic

effecttoregulatetheintestinalmicroecology.However,before

theseprobioticscanreachthedistalpartoftheintestinaltract

Inactivated Viable Model Normal

Fig.5–Ratlivermorphologyasindicatedbystainingwithhematoxylinandeosin(20×10magniﬁcation).Note:(1) Inactivated:thefermentedmilksamplewasinactivatedina100◦Cwaterbathfor30minandtheratswerefedthe hypercholesterolchow;(2)Viable:thefermentedmilksampleandtheratswerefedthehypercholesterolchow;(3)Model: salineandtheratswerefedthehypercholesterolchow;(4)Normal:salineandtheratswerefedabasaldiet.

(8)

andexertaprobioticeffect,theLABmustpassthroughthe

highlyacidic(approximatelypH3)gastricﬂuidinthe

stom-achandtheweaklybasic(pHof7.8–8.4)intestinaljuicethat

contains0.3–2%(w/v)bile-saltsintheupperpartofthe

intesti-naltract,includingaresidencetimeof1–2h.22–24 _Therefore,

itwasnecessarytodetermineifthestraincouldfunctionas

desiredafterovercomingthestressfromacidicgastricjuicein

additiontostressfromweaklybasicandbile-salt-containing

intestinaljuice.StrainHLX37wasacidtolerant,asindicated

bytestinginsimulatedgastricjuice(pH2.5)for2h.

Speciﬁ-cally,insimulatedgastricjuice(pH=3for2h)orinacid(pH

3.0,for2h),thesurvivalrateswere33.41%or85.45%,

respec-tively,whilehumangastricjuiceshowedsurvivalratesof15

and45%.20_Similar_results_were_obtained_by_other_researchers

suchasZieli ´nskaetal.,20_who_noted_that_the_survival_rate_of

bacteriaofthegenusLactobacillusinalowpHrangedfrom30

to100%.Meanwhile,HLX37wasalsoabletotoleratea

simu-latedintestinalﬂuid(pH8.0)for3handbile-salts0.3%(w/v)

for2hwithasurvivalraterangingfrom53.27to95.83%.This

resultwasalsoconﬁrmedbyMatharaetal.,19_who_found_that_L.

plantarumstrainsisolatedfromfermentedmilkproductswere

resistantto0.1–0.5%bilesalts.Thisresultsuggeststhatour

isolatescantoleratelowpHaswellasasuitablebile

concen-tration.

Somelacticacidbacteriacanproducecellsurfaceproteins,

whichoftennotonlydegradesubstratesandingestproducts24

butalsoaidinbindingthebacteriatotheepitheliumofthe

humangastrointestinaltract.Thisbindingphenomenon

pro-vides the required conditions for the immunoregulation25

necessary to remove pathogenic bacteria26 _from _the _host.

Therefore,theadhesivecapacityofabacterialstraininthe

intestinalmucosaisanimportantindex forevaluating the

invivonichesofaprobiotic.Inthisstudy,theauto-aggregation

rateofstrainHLX37and the coagulationrateofEscherichia

DH5␣ were 87.74% and 27.76%, respectively. These results

demonstrated thatthe HLX37strain hasa strongadhesive

capacityconducivetostrongadherenceintheintestinaltract.

Researchhasshownthatanumberofproteinsoccuronthe

surfaceofL.plantarum. Severalsingleproteinscould

medi-atethe in vitroepithelialadhesive capacityofthe bacteria.

Importantly,amannose-speciﬁcadhesivefactorparticipates

inthebindingofstrainstotheintestinaltract.27_Further

stud-iesarerequiredtodeterminewhetherspeciﬁcproteinsand/or

amannose-speciﬁcadhesivefactorispresentonthesurface

ofstrainHLX37.

Antibioticresistancestudiesindicatedthatlactobacilliare

usually resistant to classes of antibiotics such as

amino-glycosides, beta-lactams, cephalosporins, quinolones, and

glycopeptides.28–30_Kattla_et_al.31_conﬁrmed_that_{Lactobacillus}

issensitivetotetracyclineandchloramphenicol.L.salivarius

strainMTC1026wasfoundtobesensitivetopenicillin,

strep-tomycinandvancomycin.32_In_this_study,_strain_HLX37_was

alsoresistantto antibiotics,including almostall

aminogly-cosidesaswell aspenicillinandvancomycin testedinthis

study,whichwasalsoreportedinpreviousstudies.Previous

studiesalsoindicatedthatantibioticresistance,suchasthat

observedforL.plantarumHLX37,wasconsideredtobeintrinsic

ornaturalnon-transmissibleresistance.20,33–35

Thesigniﬁcantlylower foodconsumptionandﬁnalbody

weightshowedthatratsfedwithhypercholesterolchowthat

contains higherenergyandismoredifﬁculttodigest could

causetheratstoreducetheirintakesigniﬁcantly.Thisresult

wasaccompaniedbyaslowerweightgain,exceptintheviable

group, which showed thatfeeding withstrain L.plantarum

HLX37couldincreasetheabsorptionandutilizationoffood

inrats.InadditiontoTC,theLDL-CandHDL-Ccontentand

theAIsareimportantindicatorsthatmeasurethe

predispo-sitionforcardiovascularandcerebrovasculardiseases.HDL-C

ismainlysynthesizedintheliver.Itmovessystemic

choles-teroltotheliverforprocessing,whichreducestheprobability

ofcardiovasculardisease,soitisknownasgoodcholesterol.

However,thefunctionofLDL-CopposesthatofHDL-C,and

LDL-Clevelsareindirectlycorrelatedwithgoodhealth.36_The

conceptofAIwasproposedbyBhanetal.29 _in_a_study_that

had a large sample, and it was positively correlated with

atherosclerosis,whichsuggestedthatatherosclerosisoccurs

in individuals, and increases the risk ofindividual

cardio-vascular and cerebrovascular disease. Theeffect ofHLX37

fermentedmilkoncholesterolinratswasinvestigatedinthis

study.Wefoundthatviablefermentedmilkratgroupsshowed

alowercholesterollevelintheserumandliverfollowingthe

intakeofahigh-fatdietaswellasalowerAIcomparedwiththe

modelgroup,whichindicatedthatL.plantarumstrainHLX37

couldlowercholesterollevelinvivo.Thisresulthasalsobeen

conﬁrmedbyEl-Gawadetal.37 _and_Fazeli_et_al.38_No

signiﬁ-cantdifferenceintheHDL-Clevelbetweentheviablegroup

andmodelgroupwasobservedattheendofthe62d

exper-imentalperiod.Endo etal.39andEl-Gawadetal.37 reported

thattheadditionofaprobiotictothediethadnoeffectonthe

HDL-Clevel.Ourdataagreedwiththisﬁnding.Inaddition,we

alsofoundthatdeadbacteriahadnoeffectontheHDL-Clevel.

HDL-Cconsistsofcholesterol,lipoprotein,phospholipidand

asmallamountoffatty acid.29 _The_HDL-C_level_in_the

nor-malgroupwaslowerthanintheotherexperimentalgroup,

possiblybecausetheexperimentalgroupsreceiveda

hyperc-holesteroldietthatcontainedlard,peanut,sesameoil,sucrose

andcholesterol,whichcouldleadtoanincreaseintheHDL-C

levelcomparedwiththebasaldiet.StudiesbyLiongetal.,40

Kimotoetal.41_and_Jiao_et_al.2_noted_that_dead_bacteria_may_be

abletolowerthecholesterollevel.Inthisstudy,wealsofound

thatratsfedinactivatedfermentedmilkshowedanobviously

lowercholesterollevelintheliverandthepresenceofLDL-C

intheserum.Thisstrainmayreducethecholesterolinthe

mediathroughvariousmeans,suchasco-precipitation.

Meanwhile,amicroscopicpathomorphismanalysisnoted

fewerfatvacuolesandlesionsintheliverininactivatedand

viablegroupscomparedtotheuntreatedmodelcontrolgroup.

Both inactivated and viablefermentedmilkimproved lipid

metabolismwithsigniﬁcantdecreasesinbloodfatand

inhi-bitionofhighcholesterolinratsandincreasedresistanceto

arteriosclerosis and repair of hepatic steatosis in the rats.

Thus,it ispossiblethatthis straincanbeusedtocreatea

fermentedmilkproductthatefﬁcientlyreducescholesterol.

Nguyenet al.42_found_that_L._plantarum_PH04_had _a_high

bilesalthydrolaseenzymaticactivity,whichreducesthetotal

cholesterol ina hyperlipidemia rat model, but Tok et al.43

foundthatL.delbrueckiisp.hadnoBSHactivity.The

capac-itytoreducecholesterolintheculturemediumwasdirectly

correlatedwiththebacterialexopolysaccharide.Inthisstudy,

(9)

reducethe TCintheliverandthe LDL-Clevel.These

char-acteristics ofstrain HLX37 are combined with a very high

coagulationrate.Furtherstudiestoinvestigatewhetherthe

metabolicsubstancesinfreshmilkfermentedbythisstrain

can reducecholesteroland to elucidatethe mechanism of

actionunderlyingthisreductionarenecessary.

In conclusion, inthis study,we isolated the LAB strain

L.plantarum HLX37, which could ferment milkand reduce

cholesterol,fromtraditionalfermentedmilk.Aninvitrostudy

conﬁrmed that strain HLX37 could tolerate acid and bile

saltsandsurviveundersimulatedgastrointestinalconditions.

StrainHLX37wasintrinsicallyresistant tothe usual

antibi-oticstestedcomparedtootherL.plantarumforwhichprevious

reports exist, but more information is required to

estab-lishwhetherstrainHLX37containanytransferableantibiotic

resistancegenes.StrainHLX37wasalsofoundtohavestrong

adhesiveproperties,butmorestudiesabouttheadhesive

fac-torare required.Speciﬁcally, strainL.plantarumHLX37can

lowercholesterol,asconﬁrmedbyaninvivostudythatshowed

asigniﬁcantreductionoftheTClevelandAIinrats.

Addition-ally,fewerfatvacuolesandliverlesionswerefoundinratsfed

viablefermentedmilkcontainingstrainHLX37.L.plantarum

strainHLX37showsacommercialprobioticpotentialforuse

inpromotinghealth.

Compliance

with

ethics

requirements

Allofanimalsinthisstudywerehandledinaccordancewith

theanimalcareanduseguidelinesestablishedbytheFujian

University of Traditional Chinese Medicine (Fujian, China),

whichentailsminimizingboththenumberandsufferingof

theanimalsusedfortheexperiments.Allexperimental

proce-duresinvolvingratswereapprovedbytheFujianAssociation

forLaboratoryAnimalScience(FALAS).

Conﬂicts

of

interest

Nonedeclared.

Acknowledgments

Thisworkwassupportedbygrantsfromthewelfareprojects

oftheScienceandTechnologyDepartmentofFujianProvince,

China[2015R1015-8]; the YouthInnovation Fund Project of

AcademyofAgriculturalSciences ofFujianProvince, China

(2014CX-21); the SpecialScienceand Technology Project of

Provincial Department of Finance Fujian Province, China

[2011]211and[2012]1025;andthesurplusfundsofinstituteof

AgriculturalEngineeringTechnology,FujianAcademyof

Agri-culturalSciences[2016-1].

Appendix

A. Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound,

intheonlineversion,atdoi:10.1016/j.bjm.2017.02.011.

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c

e

s

1.WangWM,XuL.Lacticacidbacteria:mechanismsfor cholesteroldegradationin-vitroandin-vivoandits application.JAnimNutr.2014;26(2):295–303(inChinese).

2.JiaoYH,ZhangLW,YiHX,ZhangLL,MaW.Cholesterol removingabilityandbiletoleranceoflacticacidbacteria isolatedfromfermentedyakmilk.JNortheastAgricUniv.

2012;43(2):6–12(inChinese).

3.LiuX,FangYL,DuZL,DaiMX.Screeningofanacinetobacter strainproducingcholesteroloxidaseandstudyonits characteristicandfermentationconditions.Biotechnology.

2010;20(3):80–82(inChinese).

4.LiN,SuGC,ZhouCY,SuWJ.Theprogressinresearchonthe applicationandmechanismofLacticacidbacteriain reducingcholesterol.JMicroecol.2012;24(5):460–464(in Chinese).

5.ShobharaniP,HalamiPM.Invitroevaluationofthe

cholesterol-reducingabilityofapotentialprobioticBacillus

spp.AnnMicrobiol.2015,

http://dx.doi.org/10.1007/s13213-015-1146-6.

6.JeunJ,KimS,ChoSY,etal.Hypocholesterolemiceffectsof

LactobacillusplantarumKCTC3928byincreasedbileacid excretioninC57BL/6mice.Nutrition.2010;26(3):321–330.

7.TomasMSJ,BruE,Nader-maciasME.Differentcombinations ofsaltsaffectthegrowthandbacteriocinproductionby

LactobacillussalivariusCRL1328.JChemTechnolBiotechnol.

2010;85(1):91–99.

8.ArakawaK,KawaiY,FujitaniK,etal.Bacteriocinproduction ofprobioticLactobacillusgasseriLA39isolatedfromhuman fecesinmilk-basedmedia.AnimSciJ.2008;79(5):634–640.

9.WangCY,WuSC,NgCC,ShyuYT.Effectof Lactobacillus-fermentedadldy-basedmilkonlipid metabolismofhamstersfedcholesterol-enricheddiet.Food ResInt.2010;43(3):819–824.

10.LiuYY,YuCQ,WangRH.EffectofLactobacillusplantarumon

fermentationoncholesterolcontentinyogurt.JHeilongjiang BayiAgricUniv.2011;23(6):56–59(inChinese).

11.RudelLL,MorrisMD.Determinationofcholesterolusing O-phtaldealdehyde.JLipidRes.1973;14:364–366.

12.NaserSM,DawyndtP,HosteB,etal.Identiﬁcationof lactobacillibypheSandrpoAgenesequenceanalyses.IntJ SystEvolMicr.2007;57:2777–2789.

13.TamuraK,DudleyJ,NeiM,KumarS.MEGA4:molecular evolutionarygeneticanalysis(MEGA)softwareversion4.0.

MolBiolEvol.2007;24:1596–1599.

14.ArcherAC,HalamiPM.ProbioticattributesofLactobacillus fermentumisolatedfromhumanfecesanddairyproducts.

ApplMicrobiolBiotechnol.2015;99:8113–8123.

15.BaoY,ZhangYC,ZhangY,etal.Screeningofpotential probioticpropertiesofLactobacillusfermentumisolatedfrom traditionaldairyproducts.FoodControl.2010;21:

695–701.

16.BauerRW,KirbyMDK,SherrisJC,TurckM.Antibiotic susceptibilitytestingbystandardsinglediscdiffusion method.AmJClinPathol.1966;45:493–496.

17.CLSI(2007)PerformanceStandardsforAntimicrobial

SusceptibilityTesting;SeventeenthInformational

Supplement.ClinicalandLaboratoryStandardsInstitute:

M100-S17(ISBN1-56238-652-5):27(1)(inUSA).

18.HaCH,SwearinginB,JeonYK,LeeM.Effectsofcombined exerciseonHOMA-IR,HOMAb-cellandatherogenicindexin Koreanobesefemale.SportSciHealth.2015;11:49–55.

19.MatharaJM,SchillingerU,KutimaPM,etal.Functional propertiesofLactobacillusplantarumstrainsisolatedfrom MaasaitraditionalfermentedmilkproductsinKenya.Curr Microbiol.2008;56:315–321.

(10)

20.Zieli ´nskaD,RzepkowskaA,RadawskaA,Zieli ´nskiK.Invitro screeningofselectedprobioticpropertiesofLactobacillus strainsisolatedfromtraditionalfermentedcabbageand cucumber.CurrMicrobiol.2015;70:183–194.

21.HillC,GuarnerF,ReidG,etal.TheInternationalScientiﬁc AssociationforProbioticsandPrebioticsconsensus statementonthescopeandappropriateuseoftheterm probiotic.NatRevGastroenterolHepatol.2014;11(8): 506–514.

22.ChenW,TanHZ,HuB,LiuXM.Themechanismofacidand biletoleranceofprobioticsinGI-trac.JChinInstFoodSci Technol.2010;12(6):1–6(inChinese).

23.JiangXJ.Effectofintestinalﬂoraandmicroecologicalin paediatrics.JPhysician.2005;35(3):79–82(inChinese).

24.NavarreWW,SchneewindO.Surfaceproteinsof

gram-positivebacteriaandmechanismsoftheirtargetingto thecellwallenvelope.MicrobiolMolBiolRev.

1999;63(1):174–229.

25.Cunningham-RundlesS,AhrnéS,BengmarkS,Johann-liang R,MarshallF.Probioticsandimmuneresponse.AmJ Gastroenterol.2000;95(1):S22–S25.

26.MackDR,MichailS,WeiS,McdougallL,HollingsworthMA. ProbioticsinhibitenteropathogenicE.coliadherenceinvitro byinducingintestinalmucingeneexpression.AmJPhysiol.

1999;276(4Pt1):G941–G950.

27.AdlerberthI,AhrneS,JohanssonML,MolinG,HansonLA, WorldAE.Amannose-speciﬁcadherencemechanismin

Lactobacillusplantarumconferringbindingtothehuman coloniccelllineHT-29.ApplEnvironMicrobiol.

1996;62(7):2244–2251.

28.KaurIP,ChopraK,SainiA.Probiotics:potential

pharmaceuticalapplications.EurJPharmSci.2002;15:1–9.

29.BhanV,YanRT,LeiterLA,etal.Relationbetweenobesityand theattainmentofoptimalbloodpressureandlipidtargetsin highvascularriskoutpatients.AmJCardiol.2010;9:

1270–1276.

30.SinghTP,KaurG,MalikRK,SchillingerU,GuigasC,KapilaS. CharacterizationofintestinalLactobacillusreuteristrainsas potentialprobiotics.ProbioticsAntimicrobProteins.

2012;4:47–58.

31.KatlaAK,KruseH,JohnsenG,HerikstadH.Antimicrobial susceptibilityofstarterculturebacteriausedinNorwegian dairyproducts.IntJFoodMicrobiol.2001;67:147–152.

32.TinratS,SarayaS,ChomnawangMT.Isolationand characterizationofLactobacillussalivariusMTC1026asa potentialprobiotic.JGenApplMicrobiol.2011;57:365–378.

33.KumarM,GhoshM,GanguliA.Mitogenicresponseand probioticcharacteristicsoflacticacidbacteriaisolatedfrom indigenouslypickledvegetablesandfermentedbeverages.

WorldJMicrobiolBiotechnol.2012;28:703–711.

34.MatharaJM,SchillingerU,GuigasC,etal.Functional characteristicsofLactobacillusspp.fromtraditionalMaasai fermentedmilkproductsinKenya.IntJFoodMicrobiol.

2008;126:57–64.

35.TamangJP,TamangB,SchillingerU,FranzC,GoresM, HolzapfelWH.Identiﬁcationofpredominantlacticacid bacteriaisolatedfromtraditionallyfermentedvegetable productsoftheeasternHimalayas.IntJFoodMicrobiol.

2005;105:347–356.

36.YuP,WangXH.Cholesterol-degradinginmicebyLactobacillus plantarumLpT1andLpT2invivo.ActaMicrobiolSin.

2012;52(1):124–129.

37.EI-GawadIAA,EI-SayedEM,HafezSA,El-ZeiniHM,SalehFA. Thehypocholesterolaemiceffectofmilkyoghurtand soy-yoghurtcontainingbiﬁdobacteriainratsfedona cholesterol-enricheddiet.IntDairyJ.2005;15(1):37–44.

38.FazeliH,MoshtaghianJ,MirlohiiM,ShirzadiM.Reductionin serumlipidparametersbyincorporationofanativestrainof

LactobacillusplantarumA7inmice.IranianJDiabetesLipid Disord.2010;9:17.

39.EndoT,NakanoM,ShimizuS,FukushimaM,MiyoshiS. Effectsofaprobioticonthelipidmetabolismofcocksfedon acholesterol-enricheddiet.BiosciBiotechnolBiochem.

1999;63:1569–1575.

40.LiongMT,ShahNP.Acidandbiletoleranceandcholesterol removalabilityoflactobacillistrains.JDairySci.

2005;88:55–66.

41.KimotoH,OhmomoS,OkamotoT.Cholesterolremovalfrom mediabyLactococci.JDairySci.2002;85:3182–3188.

42.NguyenTDT,KangJH,LeeMS.Characterizationof

LactobacillusplantarumPH04,apotentialprobioticbacterium withcholesterol-loweringeffects.IntJFoodMicrobiol.

2007;113:358–361.

43.TokE,AslimB.Cholesterolremovalbysomelacticacid bacteriathatcanbeusedasprobiotic.MicrobiolImmunol.