2.Evaluation of the treatment of Helicobacter pylori infection with herb residue fermentation supernatant by urease activity test 1, positive control; 2, negative control; 3–8, samples a
Trang 1Contents lists available atScienceDirect
Journal of Infection and Public Health
j o u r n a l h o m e p a g e :h t t p : / / w w w e l s e v i e r c o m / l o c a t e / j i p h
Reclamation of Chinese herb residues using probiotics and evaluation
of their beneficial effect on pathogen infection
Fanjing Menga, Shaoguo Yanga, Xin Wanga, Tingtao Chena,∗, Xiaolei Wanga,
a Institute of Translational Medicine, Nanchang University, Nanchang, Jiangxi 330031, PR China
b Department of Obstetrics and Gynecology, Shandong Provincial Hospital Affiliated with Shandong University, PR China
a r t i c l e i n f o
Article history:
Received 21 July 2016
Received in revised form 6 October 2016
Accepted 18 November 2016
Keywords:
Herb residue
Probiotics
DGGE
Lactobacilli
ELISA
a b s t r a c t
Environmentalpollutioncausedbyherbresiduesandthehugewasteofmedicinalingredientscontained
inherbresidueshinderthedevelopmentoftraditionalChinesemedicineenterprises.Tosolvethis prob-lem,severalprobioticsweretested,andLactobacillusplantarum(HM218749)wasfinallyselectedfor thereuseofherbresiduesofJianweixiaoshitablets.AmousemodelofHelicobacterpyloriinfectionwas developedtoevaluatetheanti-H.pyloriinfectionactivityoftheherbresiduefermentationsupernatant usingaureaseactivitytest,histologicalimaging,anenzyme-linkedimmunosorbentassay(ELISA)and denaturinggelgradientelectrophoresis(DGGE).Theresultsdemonstratedthattheherbresidue fer-mentationsupernatantsuccessfullyinhibitedureaseactivity,slowedcellinfiltrationinthegastricarea andsignificantlyreducedtheproductionofinterleukin-6(IL-6),IL-8andTNF-␣inthetreatmentgroup (p<0.01).Inaddition,theDGGEresultsindicatedthattheherbresiduefermentationsupernatantwas beneficialfortherecoveryofthedisturbedmicrobiotaintheinfectedmodeltothenormalcondition,in whichL.gasseri(GU417842.1)andL.johnsonii(HQ828141.1)weredominantinallgroups.Therefore,the probioticsexhibitedstrongpotentialforthedevelopmentofherbresiduesinthisstudy,andtheproducts showedstrongpotentialincuringH.pyloriinfections
©2017TheAuthors.PublishedbyElsevierLimited.ThisisanopenaccessarticleundertheCC
BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Introduction
Chineseherbalmedicine(TCHM)isamongtheimportant
tradi-tionalindustriesinChinaandisatreasureoftheChinesenation
Inrecentyears,thepoorresultsofsingle-targetdrugsinthe
treat-mentofchronicdiseases(e.g.,diabetesandcardiovasculardisease)
andtheurgentneedforgreenandsafeformulashavemadeTCHM
ahottopicworldwide[1–3].However,thehugeamountsofherb
residuesproducedbythecontinuousdevelopmentoftheChinese
herbalmedicineindustryhavebecomeaseriousproblemforlarge
pharmaceuticalcompanies
InChina,theannualyieldofherbresiduesisapproximately30
milliontons,mostlydisposedofthroughstackingintheopen,
san-itaryburialorburning,causingseriousenvironmentalpollution,
especiallyinwaterquality.Althoughsomestudieshavereusedherb
residuesforfeedadditives,thepreparationofactivatedcarbon,raw
materialforpapermaking,cultivationofediblefungiorpreparation
∗ Corresponding author.
E-mail address: chentingtao1984@163.com (T Chen).
ofethanol,thesemethodsconsideronlythenutritionalbenefitsand textureofmedicalplants,ignoringtheirpreciousmedicinal ingre-dients[4,5].Asiswellknown,herbresiduesaretheby-products
oftraditionalChinesemedicinalmaterialsextractedbywateror ethanol,andapproximately30%–50%ofthemedicinallyactive sub-stancesarestillcontainedinthem
ThemicroorganismfermentationtheoryinTCHMsuggeststhat thedigestiveenzymes(e.g.,cellulase,protease,pectinaseandlignin enzymes and lipase) produced by microorganismscould effec-tivelydegradeplantcellwalls,expandtheintercellularregionand improvetheextractionyieldofactiveingredients[6].Inaddition, microorganismscoulddegrademacromolecularmaterialtosmall moleculesfordirectabsorptionbythehumanbody,reducingthe sideeffectsofdrugsbydegradingtoxicsubstancesand introduc-ingnewmedicinaleffectsbybiologicalmodification[7].Probiotics arenowacceptedasusefulinthepreventionand/ortreatmentof certainpathologicalconditions[8].Atpresent,themoststudied probiotics are lactic acid-producing bacteria, particularly Lacto-bacillusspecies[9],whichhaveproventobeusefulinthetreatment
ofseveralgastrointestinaldiseasessuchasacuteinfectious
diar-http://dx.doi.org/10.1016/j.jiph.2016.11.013
1876-0341/© 2017 The Authors Published by Elsevier Limited This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
Trang 2Treatment group 6 85.87 ± 16.64 b 76.34 ± 16.31 b 97.45 ± 16.89 a
a p < 0.05.
b p < 0.01 (Compared with the Model group).
Table 2
Sequencing results of typical bands of DGGE patterns from Fig 4
Strain No Closest relatives Similarity (%) GenBank No.
Bacterial DGGE
a L gasseri 99 GU417842.1
b L johnsonii 100 HQ828141.1
c L plantarum 100 EF439684.1
Lactobacilli DGGE
a L gasseri 99 GU417842.1
b L johnsonii 100 HQ828141.1
c L lactis 100 FJ171327.1
rheaorpouchitis.Theintakeofprobioticscanalsobebeneficialin
Helicobacterpylori-infectedsubjects[8]
H.pylori,firstrecognizedin1982,isnowregardedasamajor
causeofgastritisandpepticulcersandisariskfactorforpeptic
ulcers,chronicgastritis,andgastricmalignancy.Thisorganismcan
befoundin70%–90%ofthepopulationindevelopingcountriesand
in25%–50%of thepopulationindevelopedcountries[8,10–12]
Atpresent,combinationtherapyconsistingof2antibioticsanda
protonpumpinhibitor(PPI)6isregardedasthetreatmentofchoice
toeradicateH.pyloriinfection,andthisregimenis90%effective
However,itisexpensiveandsubjecttosideeffectsandantibiotic
resistance[13]
Inthisstudy,theherbresiduesofJianweixiaoshitablets,which
consistofhawthorn,malt,pseudostellariaroot,Chineseyam,and
orange peeland have beenapproved by theMinistryof Public
Healthasbothamedicineandafood,werechosenforreuseby
pro-biotics,andtheanti-H.pyloriinfectioneffectwasevaluatedbased
ontheinvigorationofthestomachandpromotionofdigestionby
theJianweixiaoshitabletsinadditiontotheanti-H.pylorieffectsof
theprobiotics(Tables1and2)
Materials and methods
Acid,saltandantibacterialtestingoftheprobiotics
TheLactobacillusplantarum(HM218749),L.reuteri(EU547310),
L.rhamnosus (RS05630), L johnsoni (RS03965) and L paracasei
(ATCC334)weregrownin deManRogosaSharpe(MRS)media
at37◦C overnightand weresub-cultured3 times.The cultures
werethencentrifugedat4500×gfor10mintoobtainapure
cul-ture.Totestacidtolerance,eachisolatewasdiluted1:100(v/v)in
phosphate-bufferedsaline(PBS)atpH1.5,3.5,4.5,5.5and7.0for
4h.Totestsalttolerance,freshlypreparedcultureswereinoculated
intocorrespondingmediacontaining0.1–0.5%(w/w)bilesaltsand
wereincubatedat37◦Cforanother4h.Allbacterialcolonieswere
enumeratedusingtheplatecountmethod
For antimicrobial activity, overnight (12–16h) cultures of
pathogenicmicro-organisms,includingShigelladysenteriae301,Sh
dysenteriae2457,StaphylococcusaureusCOWAN1,S.aureusCMC,
Enterobactersakazakii45402,CandidaalbicansSC5314andH.pylori
SS1,werespread onthesurfacesof theircorrespondingplates,
andtheculturesupernatant(200L)oftheprobioticswasloaded
intoanOxfordcup(outerdiameter:7.8±0.1mm; inner
diame-Preparationofherbresidueextractandfermentationsupernatant TheherbresidueofJianweixiaoshitabletswasobtainedfrom River PharmaceuticalCo., Ltd and wasmashed using a pulper within 2h The probiotics L plantarum (HM218749), L reuteri (EU547310), L.rhamnosus (RS05630),L Johnsoni(RS03965) and
L.paracasei(ATCC334)(108cfu/mL)wereusedasaninoculumto preparetheherb residuefermentation supernatant,which was incubatedfor24–36hat37◦C,andthebacterialnumberwas eval-uatedusingtheplatecountmethod
H.pyloriinfectionmodelandtreatment ThestudywasapprovedbytheEthicalCommitteeoftheSecond AffiliatedHospitalofNanchangUniversity,andallmethodswere conductedinaccordancewiththeapprovedguidelines
The H pylori SS1 strain was routinely cultured under microaerophilicconditions(85%N2,5%H2,10%CO2)at37◦C on Wilkins-Chalgrenagarenrichedwith7%(vol/vol)horsebloodand 1%(vol/vol)VITOX(Oxoid,Basingstoke,UnitedKingdom) Specific-pathogen-free6-to8-week-oldmaleC57BL/6micewerehoused andfedacommercialdiet,withwateradlibitum.H.pylori infec-tionsbytheSS1strainwereconductedaspreviouslydescribed[15] Briefly,freshlypreparedaliquots(500l,109CFU)oftheH.pylori SS1straininbrainheartinfusionbroth(Oxoid)wereadministered
tomiceviaorogastricinoculationfivetimes(days1,3,5,7and9) Allnoninfectedcontrolanimalswereinoculatedwiththesame vol-umeofplainbrainheartinfusionbroth.Eightweeksafterthelast gavageofH.pylori,ureaseactivityandhistologicalimagingwere examinedtoconfirmsuccessfulmodeling
Next, the mice were divided into 3 groups: control group (N=12):normalmiceonlygivenPBS;modelgroup(N=12):the infectiousmodel, onlygiven PBS;treatmentgroup(N=12):the infectiousmodel,givenherbresiduefermentationsupernatant.Ten weeksafterthelastgavageofH.pyloriSS1,0.5mlofPBS(control groupandmodelgroup)orherbresiduefermentationsupernatant (treatmentgroup)wasadministeredtomicefor3weeks,andthen thestomachsof6miceineachgroupweresterilelyobtainedand dividedinto4partsfortheureaseactivitytest,histological imag-ing,ELISAtesting(IL-6,IL-8andTNF-␣),andDGGEanalysis.Two weekslater,thestomachsof5miceineachgroupwereobtained forDGGEanalysisintherecoverystage
DeterminationofureaseactivityandELISA Urease activity was determined by a method based onthe commercialrapidureasetest(SanqiangBiochemicalIndustry Cor-porationinFujian,China)withasensitivityof102bacteria[16] Followingthemanufacturer’sinstructions,eachstripofthe stom-achantrum and body washomogenized andplaced in1mLof reactionsolution(1gofurea/mL(wt/vol)containing850gof phe-nolred/mL(wt/vol)asapHindicator).Thesolutionbecamepinkred
ordarkredwithin5minasapositiveresultandremainedyellow
asanegativeresults
TheproductsofIL-1,TNF-␣andIL-6incellsupernatantswere determinedusingtheELISAkitforIL-1(eBioscience),TNF-␣ (eBio-science)andIL-6(eBioscience)
Trang 3Fig 1. Evaluation of the probiotic characteristics of selected Lactobacillus plantarum (HM218749), L reuteri (EU547310), L rhamnosus (RS05630), L Johnsoni (RS03965) and
L paracasei (ATCC 334) A, pH tolerance; B, bile salt tolerance; C, pathogen inhibition capability; D, growth in herb residues.
Histopathologicalanalysisofgastrictissuesamples
Excisedstomachswereopenedalongthelessercurvature,and
thelongitudinalhalfwasfixedin10%neutral bufferedformalin
solution,embeddedinparaffin,andprocessedfor
histopathologi-calanalysis[17].Allsampleswerestainedwithhematoxylin-eosin
(H&E)fortheevaluationofgastricinflammationandbythe
May-Grünwald Giemsastainmethodfor theassessment of H.pylori
SS1colonization.Histopathologicalevaluationwasperformedby
ahistopathologistwithnopriorknowledgeoftheidentityofthe
samples
DGGEanalysis
DNAwasisolatedbyabead-beatingmethod,andthebacterial
primersandLactobacillusprimerswereusedfortheDGGE
anal-ysis[18].ThebandsofinterestinDGGEgelswereexcisedusing
asterilebladeandwereincubatedovernightat4◦CinTEbuffer
(pH8.0)toallowDNAdiffusionforfurtheramplifications.ThePCR
productsforsequencingwerepurified usingtheQIAquick PCR
purificationkitandweresub-clonedusingthepMD18-Tvector
sys-temI(Takara)accordingtothemanufacturer’sinstructions,andthe
transformantswererandomlypickedandsequencedinInvitrogen
(Shanghai,China)
Results
Acid,saltandantibacterialtestoftheprobiotics
The populations of L plantarum (HM218749), L reuteri
(EU547310),L rhamnosus (RS05630),L Johnsoni(RS03965) and
L.paracasei (ATCC 334) afterexposureto acidand bile salt are
showninFig.1AandB.Asshowninthefigures,themostsuitable
conditionforallstrainswaspH7.0withoutbilesalt supplementa-tioninthemedium.WhenthepHwasloweredandthebilesalt concentrationwasincreased,anobvious reductionin microbial biomasswasobservedforallstrains.Inaddition,thehigh popu-lationof6.0logCFU/mLatpH1.5and8.5logCFU/mLatpH3.5(the actualpHinthestomachis2.0–4.0)forL.plantarum(HM218749) andthatof7.5logCFU/mLatabilesaltconcentrationof0.3% sug-gestedstrongsurvivalduringpassagethroughthegastrointestinal tract(GI).Moreover,thestronginhibitionoffood-bornepathogens (especiallyH.pyloriSS1,24mm)andhighgrowthnumberinherb residueswithoutanyotheradditives(8.8logCFU/g)suggestedthat
L.plantarum(HM218749)couldbeusedasapotentialstrainforthe developmentofherbresidues(Fig.1 andD)
EvaluationoftheH.pyloriSS1infectionmodel TheureaseexperimentsshowedthatthemiceadministeredH pyloriSS1 wereureasepositive (S-1), andthehistopathological evaluationrevealed asignificantincreasein mucosal inflamma-tioninthegastricbody(H&E).TheGiemsastainalsoindicateda highcolonizationnumberofH.pyloriSS1(S-1BandS-1C).Allthe datashowedthatwehadsuccessfullyestablishedtheH.pyloriSS1 infectionmodel
TreatmentofH.pyloriSS1-infectedmicewithherbresidue fermentationsupernatant
Beforetheformalexperiment,0.1ml,0.3ml,0.5mland0.7ml
ofherbresiduefermentationsupernatantswereusedtoevaluate thesuitabledoseforSS1-infectedmice,andthedoseof0.5mlwas chosen forits soundtreating effect.Fig 3shows that theherb residuefermentationsupernatantpossessedhighureaseinhibitory activity,andalltheH.pyloriSS1-infectedmiceinthetreatment
Trang 4Fig 2.Evaluation of the treatment of Helicobacter pylori infection with herb residue fermentation supernatant by urease activity test (1, positive control; 2, negative control; 3–8, samples) and histological image of gastric area in mice (B, HE staining, original magnification ×100; C, Giemsa stain, original magnification ×100).
groupshowednegativeresults(Fig.2A).Inaddition,the
histologi-calimageofthegastricareaindicatedthatthegavageoftheherb
residuefermentationsupernatanthadclearlyloweredthechronic
inflammationofthegastricmucosaandreducedthecolonization
ofH.pyloriSS1(Fig.2BandC),and theELISAresultsconfirmed
thattheherbresiduefermentationsupernatanthadsignificantly
reduced the production of IL-6 (123.43±20.52–85.87±16.64),
IL-8 (110.83±18.23–76.34±16.31) and TNF-␣
(132.44±25.65–97.45±16.89)inthegastricmucosa
Effectsoftheherbresiduefermentationsupernatantonthe
microbialdiversityinthestomach
AsshowninFig.4,thedominantbacteriainthestomachwere
relatively low in number, and only severallactobacilli species,
mostly L gasseri (GU417842.1) and L johnsonii (HQ828141.1),
maintaineddominanceregardlessoftheadministrationofH.pylori
SS1orherbresiduefermentationsupernatant(Fig.4).The
lacto-bacilliDGGEresultsalsoconfirmedthesametrends(Fig.4)
Discussion
In China, Jianweixiaoshi tablets bring more than 1.2 billion
RMB income for the enterprise each year, but they also
pro-duceapproximately100000tofherbresidue.Thehawthorn,malt tangerinepeel,radixpseudostellariaeandyamcontainedinthe Jianweixiaoshitabletsareusefulfordigestion,anorexia, abdomi-naldistension,invigoratingthestomachandtonifyingthespleen, andtheJianweixiaoshitabletsareclaimed topromote gastroin-testinalperistalsis,promotethegastricsecretionofdigestivejuices, enhance theactivityof pepsin, enhance physique and enhance immunefunction.Therefore,themedicinalcomponentsremaining
intheherbresidueshouldbeusefulforstomachdiseases
Inourpreviousstudies,L.plantarum(HM218749)wasisolated fromthehumanintestineandwasshowntotoleratelowpHand highconcentrationsofsaltbile,aswellastopossesshigh adher-encecapabilityandanti-adherencepropertiesagainstfood-borne pathogensbothinvivoandinvitro[19].Inthisstudy,itshigh sur-vivalrateinextremeacid(pH=1.5–3.5)andbilesaltconditions (0.3%)ensureitssurvivalinthestomachtoexertitsprobioticeffect andstronganti-H.pyloriSS1effect(Fig.1).Moreover,thehigh num-berofL.plantarum(HM218749) inherb residue(8.5 LGCFU/g) withoutanyadditivesoffersusmoreoptionsforthedevelopment
ofdrugsorhealthcareproducts
Toverifytheanti-H.pylorieffectoftheherbresidue fermenta-tionsupernatant,weemployedtheSS1H.pylorimousemodeland verifieditsreliabilityusingureaseactivitytestingandhistological imaging(S-1).H.pyloriisauniquetypeofbacteriathatcan
Trang 5sur-Fig 3.DGGE profiles of bacterial V3 16S rRNA gene fragments amplified from the
mouse stomach A, negative group; B, positive group; C, treatment group 1–5,
treat-ment period; 7–10, recovery period.
Fig 4.DGGE profiles of LAB species-specific fingerprints amplified from the mouse
stomach A, negative group; B, positive group; C, treatment group 1–5, treatment
period; 7–10, recovery period.
viveintheacidicenvironmentoftheanimalstomachbecauseof
itsabilitytoneutralizegastricacidswiththeammoniaproduced
byurease Theureaseactivity test is animportant indicatorof
H.pyloriinfection,andourresultsshowedthattheherbresidue
fermentationsupernatantsuccessfullyinhibitedureaseactivityin
thetreatmentgroup(Fig.2).Moreover,H.pyloriachievedthe
sub-stantialinfiltrationoflymphocytesandneutrophilsintheinfected
group,while theherb residuefermentation supernatant clearly
slowed the inflammatory response of the gastric mucosa and
reducedthecolonizationofH.pyloriSS1(Fig.2 whichwas
con-firmed bythe IL-6, IL-8 and TNF-␣ proteinlevels evaluatedby
ELISA.ExcesslevelsofIL-6,IL-8andTNF-␣inducedbyactivated
inflammatorycells(e.g.,eosinophils,macrophages,mononuclear
phagocytes, neutrophils) will lead to the damage of cells and
tissues,eventuallycausingtheinflammation-associateddiseases
Therefore,theoverexpressionofIL-6,IL-8andTNF-␣isstrongly
connectedtothegastricmucosalinflammationcausedbyH.pylori
SS1[20]
Asthemicrobialdiversityinthestomachiscrucialfor
stom-achdisease,thepolymerase chainreaction denaturinggradient
gelelectrophoresis(PCR-DGGE)methodwasusedtoinvestigate theeffectofherbresiduefermentationsupernatantonmicrobial diversity.DGGEisapowerfultoolbasedonthedirectanalysisof theextractofDNAfromthemicrobialenvironmentanddoesnot requirecellcultivation[14].Thismethodhasbeenused success-fullytoevaluatethebacterialcompositionofprobioticproducts andtoidentifyorprofilebacteriaindairyproducts[19,21–23].As DGGEcouldonlyidentifybacteriarepresentingmorethan1–3%of totalbacteria,thismethodonlyidentifiedthreedominantbacteria, namelyL.gasseri,L.johnsoniiandL.plantarum,ofwhichL.gasseri andL.johnsoniicouldtoleratetheextremelylowpHinthestomach andweredominantinallgroupswithorwithoutthe administra-tionofH.pyloriSS1orherbresiduefermentationsupernatant,both
inbacterialDGGEandlactobacilliDGGE(Figs.3and4).L.gasserihas longbeenusedasaprobioticandinthetreatmentofH.pyloriSS1 infection[9].Moreover,althoughoutsideinterferenceexhibited lit-tleinfluenceonthedominantlactobacillispecies,itseemedthatthe additionofH.pyloriSS1haddisturbedthemicrobialdiversityinH pyloriSS1infectionmodel,andtheadministrationofherbresidue fermentationsupernatanthelpedtorestoretheformermicrobial balanceinthemousestomachs(Fig.3)
Inconclusion,wehaveshownintheH.pyloriSS1mousemodel that herb residue fermentation supernatant can inhibit urease activity,reducethelevelofthegastricinflammatorycytokinesIL-6, IL-8andTNF-␣,alleviatehistologicallesionsandhelptorecoverthe disturbedmicrobiotatoanormallevel.Therefore,thecombination
ofJianweixiaoshiherbresidueandL.plantarum(HM218749) pro-videsanovelmethodfortheuseofherbresiduetobetteraddress theenvironmentalpollutionproblemandwasteofmedicinal ingre-dientscontainedinherbresidues
Funding
Nofundingsources
Competing interests
Nonedeclared
Ethical approval
Notrequired
Acknowledgments
ThisworkwassupportedbygrantsfromtheNationalNatural ScienceFoundationofChina(No.81503364,31560264)andJiangxi Province(20151BAB205001)
Appendix A Supplementary data
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.jiph.2016.11.013
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