牛奶和其他 食品的技术应用Word格式.docx

牛奶和其他 食品的技术应用Word格式.docx

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Availableonline3April2010.

Abstract

Severalpreviousreviewshavedescribeddifferentwaystoenhancetheflavorandtextureofcheese,includinguseoflivecellsandnonviableattenuatedcellsasadjunct

cultures.

However,comparisonsbetweenviableandnonviable

cultures

wereneverdiscussedinthesereviews.Inaddition,recentpublicationsonadjunct

havenotbeencoveredinpreviousreviews.Thisarticlewillsurveythemorerecentworkonadjunct

—withparticularattentiontowhethertheadjunctscontainedviableornonviablecells—andproposeareaswhereadditionalresearchisneeded.

Keywords:

adjunctlactobacilli;

attenuation;

autolysis;

cheeseripening

Abbreviations:

LAB,lacticacidbacteria;

NSLAB,nonstarterlacticacidbacteria

Correspondingauthor.

1 

PartofthisreviewwaspresentedbyMorsiElSoda,recipientofthe1998MarschallRhodiaInternationalDairyScienceAwardatthe93rdADSAmeeting,Denver,Colorado.

JournalofDairyScience

Volume83,Issue4,April2000,Pages609-619

Anovelreal-timepolymerasechainreaction-basedmethodforthedetectionandquantificationoflactose-fermentingEnterobacteriaceaeinthedairyandotherfoodindustries

M.C.Martí

n1,a,N.Martí

nez1,a,B.delRioa,V.Laderoa,M.Ferná

ndezaandM.A.Alvarez

a,

aInstitutodeProductosLá

cteosdeAsturias,CSIC,33300Villaviciosa,Asturias,Spain

Received12June2009;

accepted25November2009. 

Availableonline19February2010.

Thepresenceoflactose-fermentingEnterobacteriaceaeandcoliformsisroutinelyassessedtodeterminethehygienicqualityofwaterandfoods,particularlydairyproducts.ThispaperreportstheuseoflacZ-specificprimersinanSYBRgreenI-basedreal-timePCRmethodfortheeasyandrapiddetectionofcoliformsindairyproducts.Alargenumberofbacterialspecieswereassayedtoestablishthespecificityofthemethod.Thesensitivityofthemethodwasassessedusingartificiallycontaminatedcheeses.Thelimitofdetectionwas1coliformcellincheesesamplesenrichedfor8 

hina

culture

medium.Theentireprocedure,includingsampleprocessing,enrichment,DNAextraction,andreal-timePCRamplification,canbecompletedwithin10to12 

h,makingitasingle-dayassay.

Enterobacteriaceae;

coliform;

real-timepolymerasechainreactiondetection;

cheese

ArticleOutline

Introduction

MaterialsandMethods

BacterialStrains

ExtractionofDNAfortheRT-qPCRAssay

lacZSequencingandNucleotideSequenceAnalysis

RT-qPCRConditions

DataAnalysis

ArtificialContaminationandEnrichmentofCheese

NucleotideSequenceAccessionNumbers

Results

SequencingofthelacZGenesandPrimerDesign

OptimizationofRT-qPCR

SpecificityandSensitivity

MeltingCurveAnalysisoftheAmplifiedDNA

RT-qPCRDetectionLimits

Discussion

Acknowledgements

References

Figure1. 

Alignmentofa63-bpregionoftheβ-galactosidase（lacZ）genefrom22representativecoliformstrains（Citrobacterfreundii,Escherichiacoli,Enterobactercloacae,Klebsiellapneumoniae,Enterobactersakazakii,Citrobacterkoseri,Citrobacteramalonaticus,andRaoultellaplanticola）usingClustalWsoftware（Altschuletal.,1997）.Thelocationsoftheprimersareshownbyarrows,anasteriskdenotesidenticalsequences,andadashindicatesamismatchwiththeconsensussequence.

ViewWithinArticle

Figure2. 

Standardcurvesforthelognumberofcoliformcellsperreactionversusthecyclethreshold（Ct）valueforthefluorescentsignalforA）EscherichiacoliCECT515,B）EnterobactercloacaeCECT194,C）CitrobacterfreundiiCECT401,andD）Klebsiellapneumoniaessp.pneumoniaeCECT143.Theerrorbarsindicatestandarddeviationsfor3independentexperiments.CECT 

= 

Colecció

nEspañ

oladeCultivosTipo,Burjasot,Valencia,Spain.

Table1.

Strainsusedinthiswork

CECT 

oladeCultivosTipo,Burjasot,Valencia,Spain;

LSP 

LaboratoriodeSaludPú

blica,PrincipadodeAsturias,Spain;

CNRZ 

CentreNationaldeRecherché

Zootechniques,Jouyen-Josas,France;

LMD 

LaboratoryofMicrobiology,TechnicalUniversity,Delft,theNetherlands;

ATCC 

AmericanTypeCultureCollection,Rockville,Maryland.

2 

RT-qPCR 

real-timequantitativePCR;

+ 

indicatesapositiveRT-PCRresult,–indicatesanegativeresult.

Table2.

Detectionofcoliformsincheesebycultureenrichmentandreal-timequantitativePCR1

1,10,and100indicatethecontaminationlevelofcoliforms（cfu/mL）beforeenrichment;

Theseauthorscontributedequallytothiswork.

Volume93,Issue3,March2010,Pages860-867

Biochemistry,Genetics,andApplicationsofExopolysaccharideProductioninStreptococcusthermophilus:

AReview1

J.R.Broadbent*,†,

D.J.McMahon*,†,D.L.Welker*,‡,C.J.Oberg*,§

andS.Moineau||

*WesternDairyCenter,DepartmentofUtahStateUniversity,Logan84322-8700

†NutritionandFoodSciencesandDepartmentofUtahStateUniversity,Logan84322-8700

‡BiologyUtahStateUniversity,Logan84322-8700

§

DepartmentofMicrobiology,WeberStateUniversity,OgdenUT84408-2506

||Dé

partementdeBiochimieetdeMicrobiologieUniversité

Laval,Qué

bec,Canada,G1K7P4

Received10May2002;

accepted25June2002. 

Availableonline27March2010.

ManystrainsofStreptococcusthermophilussynthesizeextracellularpolysaccharides.Thesemoleculesmaybeproducedascapsulesthataretightlyassociatedwiththecell,ortheymaybeliberatedintothemediumasalooseslime（i.e.,“ropy”polysaccharide）.AlthoughthepresenceofexopolysaccharidedoesnotconferanyobviousadvantagetogrowthorsurvivalofS.thermophilusinmilk,insituproductionbythisspeciesorotherdairylacticacidbacteriatypicallyimpartsadesirable“ropy”orviscoustexturetofermentedmilkproducts.Recentworkhasalsoshownthatexopolysaccharide-producingS.thermophiluscanenhancethefunctionalpropertiesofMozzarellacheese,buttheyarenotphage-proof.Asourunderstandingofthegenetics,physiology,andfunctionalityofbacterialexopolysaccharidescontinuestoimprove,novelapplicationsforpolysaccharidesandpolysaccharide-producingcultures

arelikelytoemergeinsideandoutsidethedairy

industry.

Thisarticleprovidesanoverviewofbiochemistry,genetics,andapplicationsofexopolysaccharideproductioninS.thermophilus.

Streptococcusthermophilus;

exopolysaccharide;

lacticacidbacteria

CPS,capsularexopolysaccharide;

CPS+/−,ability（+）orinability（−）toproducecapsularexopolysaccharide;

EPS,exopolysaccharide;

EPS+/−,ability（+）orinability（−）toproducecapsularorsecretedexopolysaccharide;

LAB,lacticacidbacteria

HeteropolysaccharideStructureandBiosynthesis

GeneticsofEPSProductioninS.thermophilus

PhysiologicalRoleofEPS

BacteriophageResistance

ApplicationsinYogurtandFermentedMilks

ApplicationinMozzarellaCheese

Conclusions

BasicrepeatingstructuresofStreptococcusthermophilusextracellularheteropolysaccharides.Fuc,fucose;

Gal,galactose;

GalNAc,N-acetyl-D-galactosamine;

Glc,glucose;

Rha,rhamnose;

Ac,O-acetylgroup;

p,pyranoseconfiguration;

andf,furanoseconfiguration.

ModelforassemblyoftheStreptococcusthermophilusSfi6exopolysaccharidebasicrepeatingunit.Gal,galactose;

UDP,uridine-diphosphate.AdaptedfromStingeleetal.（1996,1999）.

Figure3. 

PhysicalmapsofthegeneticregionsassociatedwithexopolysaccharidebiosynthesisinStreptococcusthermophilus.Boldlettersidentifystrainnames,andfillpatternsidentifygenesthatareatleast90%identicalamongstrains.Putativeorestablishedfunctionsforindividualgeneproductsarelistedundereachcluster.GTF,glycosyltransferase;

MTr,membranetranslocation;

Pol,polymerization;

Reg,regulation;

SB,sugarbiosynthesis;

Unk,unknown.Mapsarenottoscale.1StrainCNRZ368doesnotproduceexopolysaccharidedue,presumably,tomutationsinseveralcpsgenes（Bourgoinetal.,1999;

Broadbentetal.,2001）.2Sequencedataforthe3′regionoftheSfi6epsclusterdoesnotextendbeyondorf14.9（Stingeleetal.,1996）.3Sequencedataforthe3′regionoftheSfi39epsclusterdoesnotextendbeyondorf14.9（Germondetal.,2001）.4Sequencedataforthe3′regionoftheFI9186epsclusterdoesnotextendbeyondepsG（I’Anson,2002）.

F