Megazyme/DLactic Acid (DLactate) (Rapid) Assay Kit/KDATE/50 assays (manual) / 500 assays (micropl

Itiswithoutdoubtthattestingplaysapivotalrolethroughoutthewholeofthevinificationprocess.ToproducethebestpossIBLequalitywineandtominimiseprocessproblemssuchas“stuck”fermentationortroublesomeinfections,itisnowrecognisedthatifpossibletestingshouldbeginpriortoharvestingofthegrapesandcontinuethroughtobottling.TrADItionalmethodsofwineanalysisareoftenexpensive,timeconsuming,requireeitherelaborateequipmentorspecialistexpertiseandfrequentlylackaccuracy.However,enzymaticbio-analysisenablestheaccuratemeasurementofthevastmajorityofanalytesofinteresttothewinemaker,usingjustonepieceofapparatus,thespectrophotometer(seepreviousissueNo.116foradetailedtechnicalreview).Grapejuiceandwineareamenabletoenzymatictestingasbeingliquidstheyarehomogenous,easytomanipulate,andcangenerallybeanalysedwithoutanysamplepreparation.

ManyoftheenzymatictestkitsareofficialmethodsofprestigiousorganisationssuchastheAssociationofOfficialAnalyticalChemicals(AOAC)andtheAmericanAssociationofCerealChemists(AACC)inresponsetotheinterestfromoenologists.Megazymedecidedtouseitslonghistoryofenzymaticbio-analysistomakeasignificantcontributiontothewineindustry,bythedevelopmentofarangeofadvancedenzymatictestkits.Thistaskhasnowbeensuccessfullycompletedthroughthestrategicandcomprehensiveprocessofidentifyinglimitationsofexistingenzymaticbio-analysistestkitswheretheyoccurred,andthenusingadvancedtechniques,suchasmolecularBIOLOGy(photo1),torapidlyovercomethem.Noveltestkitshavealsobeendevelopedforanalytesofemerginginteresttotheoenologist,suchasyeastavailablenitrogen(YAN;seepages2-3ofissue117article),orwherepreviouslyenzymesweresimplyeithernotavailable,orweretooexpensivetoemploy,suchasforD-mannitolanalysis.

Thefreshwatermicroalga,Hydrodictyonreticulum,thatcontained47.5%reducingsugarsincluding35%glucosewasusedassubstratefortheproductionofL-lacticacid(LA)byLA-producingbacteria.LactobacillusparacaseiLA104wasselectedforfermentationina5-lfermentorsinceitwasabletogrowatpH3,60gLA/l,200gglucose/l,125gNaCl/l,and45°Candproducedover97.3%opticallypureL-lacticacidwithglucoseasasubstrate.SimultaneoussaccharificationandcofermentationfromH.reticulumtoL-LAusingLA104wasinvestigatedinajarfermentor.Theyieldreached46g/100gH.reticulumdrymaterial,withafinalconcentrationof37.11g/landaproductivityof1.03g/l/h.ThisisthefirstreportoftheproductionofL-LAfromamicroalga,andH.reticulumcouldbeapotentialfeedstockforlarge-scaleproductionofL-LAbyLA104.

D-Lacticacidproductionfromdrybiomassofthemicroalga,Hydrodictyonreticulatum,wascarriedoutina5-ljarfermentor(initialpH6,34°CusingCaCO₃asaneutralizingagent)throughsimultaneoussaccharificationandco-fermentationusingtheLactobacilluscoryniformissubsp.torquens.After36h,36.6glacticacid/lwasproducedfrom80gH.reticulatum/linthemediumcontaining3gyeastextract/land3gpeptone/lintheabsenceofmineralsalts.Themaximumproductivity,averageproductivityandyieldwere2.38g/lh,1.02g/lhand45.8%,respectively.TheopticalpurityofD-Lacticacidrangedfrom95.8–99.6%.H.reticulatumisthusapromisingbiomassmaterialfortheproductionofD-Lacticacid.

AmylolyticlacticacidbacteriumLactobacillusamylovorusDSM20531^Tutilisedglucose,sucroseandstarchasasolecarbonandenergysource.ThethreesubstrateswerecompletelydepletedfromMRSmediumduringbatchcultivationscarriedoutinalaboratoryscalestirredtankbioreactoratconstanttemperature(40°C)andpHvalue(5.5).Underthetestedconditions,thebacteriumwascapableofconductingsimultaneouslystarchhydrolysisandfermentation.Amixtureoftwostereoisomers,D-(-)-andL-(+)-lacticacid,wasproducedinallcasesbyhighlyefficienthomofermentativebioprocesswith0.93to1goflactateproducedpergoftotal(consumed)substrate.Theeffectoftemperatureonthekineticsofcellgrowthandlacticacidproductionbytheamylolyticstraininthestarch-containingmediumwasalsoinvestigated.Efficientsimultaneoussaccharificationandfermentation(SSF)wasobtainedat35,40and45°Cwithcompletelydegradedcomplexcarbohydratein8to12handtheproductyieldcoefficientintherangefrom0.91to0.93g/g.Maximumvaluesforsubstrateconsumptionrate(0.89h^-1),maximumspecificgrowthrate(0.87h^-1),productformationrate(2.01h^-1),andproductivityoflacticacid(1.45g/(Uh))wereobtainedat45°C,whilemaximumbiomassconcentration(4.38g/L)wasattainedat40°C.Theratioofthetwostereoisomericformsofproducedlacticacidwasstronglyaffectedbythetemperature.Unstructuredkineticmodelwasusedtodescribetheconsumptionofthethreesubstrates,bacterialbiomassformationandlacticacidproductionbyL.amylovorusDSM20531^T.Thedependenceofbiokineticparametersontemperaturewasdescribedbycardinaltemperaturemodel.Theappliedmodelssuccessfullypredictedallexperimentaldata.

Metabolicengineeringofmicroorganismshasbecomeaversatiletooltofacilitateproductionofbulkchemicals,fuels,etc.Accordingly,CO₂hasbeenexploitedviacyanobacterialmetabolismasasustainablecarbonsourceofbiofuelandbioplasticprecursors.HereweextendedtheseobservationsbyshowingthatintegrationofanldhgenefromBacillussubtilis(encodinganL-lactatedehydrogenase)intothegenomeofSynechocystissp.strainPCC6803leadstoL-lacticacidproduction,aphenotypewhichisshowntobestableforprolongedbatchculturing.Coexpressionofaheterologoussolubletranshydrogenaseleadstoanevenhigherlactateproductionrateandyield(lacticacidaccumulatinguptoaseveral-millimolarconcentrationintheextracellularmedium)thanthoseforthesingleldhmutant.Theexpressionofatranshydrogenasealone,however,appearstobeharmfultothecells,andamutantcarryingsuchageneisrapidlyoutcompetedbyarevertant(s)withawild-typegrowthphenotype.FurThermore,ourresultsindicatethattheintroductionofalactatedehydrogenaserescuesthisphenotypebypreventingthereversion.

Sourdoughbreadhasbeenreportedtoimproveglucosemetabolisminhealthysubjects.Inthisstudypostprandialglycaemicandinsulinaemicresponseswereevaluatedinsubjectswithimpairedglucosetolerance(IGT)whohadamealcontainingsourdoughbreadleavenedwithlactobacilli,incomparisontoareferencemealcontainingbreadleavenedwithbakeryeast.SixteenIGTsubjects(agerange52–75,averageBMI29.9±4.2kg/m²)wererandomlygivenamealcontainingsourdoughbread(A)andamealcontainingthereferencebread(B)intwoseparateoccasionsatthebeginningofthestudyandafter7days.Sourdoughbreadwasleavenedfor8husingastartercontainingautochthonousSaccharomycescerevisiaeandseveralbacilliabletoproduceasignificantamountofD-andL-lacticacid,whereasthereferencebreadwasleavenedfor2hwithcommercialbakeryeastcontainingSaccharomycescerevisiae.Plasmaglucoseandinsulinlevelsweremeasuredattime0,30,60,120,and180min.InIGTsubjectssourdoughbreadinducedasignificantlylowerplasmaglucoseresponseat30minutes(p=0.048)andasmallerincrementalareaundercurve(AUC)Δ0–30andΔ0–60min(p=0.020and0.018respectively)incomparisontothebreadleavenedwithbakeryeast.Plasmainsulinresponsetothistypeofbreadshowedlowervaluesat30min(p=0.045)andasmallerAUCΔ0–30min(p=0.018).ThisstudyshowsthatinsubjectswithIGTglycaemicandinsulinaemicresponsesaftertheconsumptionofsourdoughbreadarelowerthanafterthebreadleavenedwithbakeryeast.Thiseffectislikelyduetothelacticacidproducedduringdoughleaveningaswellasthereducedavailabilityofsimplecarbohydrates.Thus,sourdoughbreadmaypotentiallybeofbenefitinsubjectswithimpairedglucosemetabolism.

Caseinwheypermeate(CWP),alactose-enricheddairywasteeffluent,isaviablefeedstockfortheproductionofvalue-addedproducts.TwolacticacidbacteriawerecultivatedinasyntheticcaseinwheypermeatemediumwithorwithoutpHcontrol.LactobacilluslactisATCC4797producedD-lacticacid(DLA)at12.5gl^-1inabioreactor.ThevaluesofLeudking–Piretmodelparameterssuggestedthatlactatewasagrowth-associatedproduct.BatchfermentationwasalsoperformedemployingCWP(35glactosel^-1)withcaseinhydrolysateasanitrogensupplementinabioreactor.After40h,L.lactisproduced24.3glacticacidl^-1withanopticalpurity>98%.ThusCWPmayberegardedasapotentialfeed-stockforDLAproduction.

ThisresearchwasconductedtoexaminetheinherentpropertiesofyogurtcontaminatedwithstaphylococcalenterotoxinB(SEB).Twotypesofyogurtswereproducedforthisstudy.TypeIyogurtswereproducedbyaddingSEBatthestartofyogurtproduction,andtypeIIyogurtswereproducedbyaddingSEBafterthemilkbasehadbeenboiled.Biochemicalcharacteristicsinherenttoyogurt,includingpH,lacticacidandacetaldehydeconcentrations,wereanalyzedweeklyforeachbatchbeginningatatimejustafterproductionandthroughoutastorageperiodofatleast4weeks.Thepresenceoftoxinduringyogurtproductiondidnotresultinanysignificantbiochemicalorphysicalchangesinyogurt.However,wewereunabletodetectSEBtoxinintypeIyogurtusingacommerciallyavailableenzyme-linkedimmunosorbentassay(ELISA).Incontrast,SEBwaseasilydetectablebyourELISAintypeIIyogurtsamples.HigherlevelsofSEBwererecoveredfromtypeIIyogurtthathadbeenstoredfor1weekthanfromtypeIIyogurtthathadbeenstoredforanyotherlengthoftime.Theseresultsindicatethatthebiochemicalcharacteristicsofyogurtdidnotchangesignificantly(relativetocontrolyogurt)inthepresenceofeitherthermallyprocessedSEBornativeSEB.However,theabilitytodetectSEBbyELISAwasdependentonwhetherthetoxinhadbeenprocessed.

Theeffectoftheciliates,Eudiplodiniummaggii,DiploplastronaffineandEntodiniumcaudatum,andnaturalprotozoalfaunaontheruminalmureinconcentrationandmureinolyticactivitywasexaminedonthreerams,repeatedlydefaunatedandrefaunatedwithEudiplodiniummaggii,Diploplastronaffine,Entodiniumcaudatumandnaturalprotozoalfauna.Thenumberofciliatesvariedfrom18(E.maggii)to334x10³/grumencontent(naturalfauna).Themureinconcentrationfluctuatedbetween180and277mg/gdrymatter(DM).Theestablishmentofciliatesintherumenofdefaunatedsheepdecreasedthemureincontentby28-35%(P<0.05). mureinolytic="" activity="" varied="" from="" 2.2="" and="" 5.7="" μg/g="" dm="" of="" rumen="" fluid/min="" and="" was="" the="" lowest="" in="" defaunated="" sheep="" and="" the="" highest="" in="" animals="" faunated="" with="">E.caudatum.Theprotozoaincreasedthisactivityfrom32(E.maggii)to159%(E.caudatum).Allexaminedparametersshoweddiurnalvariations.Theciliatenumberwasthegreatestjustbeforefeedingandthesmallest4hthereafter.Thefluctuationpatterninmureincontentwasinversetothatofprotozoaconcentrationandmureinolyticactivity.

BACKGROUND:Theabilityofbacteriocin-likeinhibitorysubstance(BLIS)-producinglacticacidbacteria(LAB)todegradebiogenicaminesaswellastoproduceL(+)andD(−)-lacticacidduringsolidstatefermentation(SSF)oflupinandsoyabeanwasinvestigated.Inaddition,theproteindigestibilityandformationoforganicacidsduringSSFoflegumewereinvestigated.RESULTS:Proteindigestibilityoffermentedlupinandsoyabeanwasfoundhigheronaverageby18.3%and15.9%,respectively,comparedtountreatedsamples.TestedLABproducedmainlyL-lacticacidinsoyabeanandlupin(D/Lratio0.38–0.42and0.35–0.54,respectively),whilespontaneousfermentationgavealmostequalamountsofbothlacticacidisomers(D/Lratio0.82–0.98and0.92,respectively).TestedLABstrainswereabletodegradephenylethylamine,spermineandspermidine,whereastheywereabletoproduceputrescine,histamineandtyramine.CONCLUSIONS:SSFimprovedlupinandsoyabeanproteindigestibility.BLIS-producingLABinlupinandsoyabeanmediumproducedamixtureofD-andL-lacticacidwithamajorexcessofthelatterisomer.Mosttoxichistamineandtyramineinfermentedlupinandsoyabeanwerefoundatlevelslowerthosecausingadversehealtheffects.Selectionofbiogenicaminesnon-producingbacteriaisessentialinthefoodindustrytoavoidtheriskofamineformation.

Colorectalcancerisaclinicalconditionwhosetreatmentofteninvolvesintestinalresection.Suchtreatmentfrequentlyresultsintwomajorgastrointestinalcomplicationsaftersurgery:anastomoticleakageandprolongedileus.Anastomoticleakageisaseriouscomplicationwhich,moreoftenthannot,isdiagnosedlate;todate,C-reactiveproteinistheonlyavailablediagnosticmarker.Amonocentric,Prospective,opencase-controlstudywasperformedinpatients undergoingcolorectalsurgery.Intestinalfattyacidbindingprotein(i-FABP),citrulline,D-lactate,exhaledhydrogen,EscherichiacoligenomicDNA,andischemiamodifiedalbumin(IMA)weredeterminedpreoperatively,postoperatively,andonthefollowingfourconsecutivedays.BacterialDNAwasnotdetectedinanysample,andi-FABPandD-lactatelackedanydistinctpotentialtodetectpostoperativebowelcomplications.Exhaledbreathhydrogencontentshowedunacceptablylowsensitivity.However,citrullineturnedouttobeaspecificmarkerforprolongedileusonpostoperativedays3-4.Usingacut-offvalueof20 µmol/L,asensitivityandspecificityof~75%wasachievedonpostoperativeday4.IMAwasfoundtobeanefficientpredictorofanastomosisleakbycalculatingthedifferencebetweenpreoperativeandpostoperativevalues.Thistesthad100%sensitivityand80%specificityand100%negativeand20%positivepredictivevalue.

Background:LacticacidhasbeenapprovedbytheUnitedStatesFoodandDrugAdmiNISTrationasGenerallyRegardedAsSafe(GRAS)andiscommonlyusedinthecosmetics,pharmaceutical,andfoodindustries.Applicationsoflacticacidhavealsoemergedintheplasticsindustry.Lacticacidbacteria(LAB),suchasLeuconostocandLactobacillus,arewidelyusedaslacticacidproducersforfood-relatedandbiotechnologicalapplications.Nonetheless,industrialmassproductionoflacticacidinLABisachallengemainlybecauseofgrowthinhibitioncausedbytheendproduct,lacticacid.Thus,itisimportanttoimproveacidtoleranceofLABtoachievebalancedcellgrowthandahightiteroflacticacid.Recently,adaptiveevolutionhasbeenemployedasoneofthestrategiestoimprovethefitnessandtoinduceadaptivechangesinbacteriaunderspecificgrowthconditions,suchasacidstress.Results:Wild-typeLeuconostocmesenteroideswaschallengedlongtermwithexogenouslysuppliedlacticacid,whoseconcentrationwasincreasedstepwise(forenhancementoflacticacidtolerance)during1 year.Inthecourseoftheadaptiveevolutionat70 g/Llacticacid,threemutants(LMS50,LMS60,andLMS70)showinghighspecificgrowthratesandlacticacidproductionwereisolatedandcharacterized.MutantLMS70,isolatedat70 g/Llacticacid,increasedD-lacticacidproductionupto76.8 g/L,whichwastwicethatinthewildtype(37.8 g/L).Proteomic,genomic,andphysiologicalanalysesrevealedthatseveralpossiblefactorsaffectedacidtolerance,amongwhichamutationofATPaseεsubunit(involvedintheregulationofintracellularpH)andupregulationofintracellularammonia,asabufferingsystem,wereconfirmedtocontributetotheobservedenhancementoftoleranceandproductionofD-lacticacid.Conclusions:Duringadaptiveevolutionunderlethalstressconditions,thefitnessofL.mesenteroidesgraduallyincreasedtoaccumulatebeneficialmutationsaccordingtothestresslevel.TheenhancementofacidtoleranceinthemutantscontributedtoincreasedproductionofD-lacticacid.Theobservedgeneticandphysiologicalchangesmaysystemicallyhelpremoveprotonsandretainviabilityathighlacticacidconcentrations.

Therelationshipbetweenfoodandhealthhasbeeninvestigatedformanyyears,andtherefore,thedevelopmentoffoodsthatpromotehealthandwell-beingisakeyresearchpriorityofthefoodindustry.Fruitsandvegetablesareanessentialpartofhumannutrition.Unfortunately,thedailyintakeoffruitsandvegetablesisestimatedtobelowerthantherecommendationoftheWorldHealthOrganization(WHO),whosuggestadietaryintakeof450and500goffruitsandvegetables,respectively.Vegetablesarestronglyrecommendedinthehumandietbecausetheyarerichinantioxidants,vitamins,dietaryfibresandminerals.Themajorityofvegetablesconsumedinthehumandietarefresh,minimallyprocessed,pasteurisedorcookedbyboilinginwaterormicrowaving,andvegetablescanbecanned,dried,orjuicedormadeintopastes,salads,sauces,orsoups.Freshvegetablesorthosethathavebeenminimallyprocessedhaveaparticularlyshortshelf-lifebecausetheyaresubjectedtorapidmicrobialspoilage.Inaddition,theabovecookingprocessescancauseanumberofpotentiallyundesirablechangesinphysicalcharacteristicsandchemicalcomposition.

Synovialsepsisinhorsesislifethreateningandaccuratediagnosisallowingprompttreatmentiswarranted.ThisstudyassessedthediagnosticvalueofserumamyloidA(SAA)andD-lactateinbloodandsynovialfluid(SF)asdiagnosticmarkersofsynovialsepsisinhorsesandcorrelatedthemwithtotalnucleatedcellcount(TNCC),percentageofneutrophils(%N)andtotalprotein(TP)inSF.BloodandSFSAAandD-lactateconcentrationsweredeterminedinacase–controlobservationalstudyincluding112horses(38withsynovialcontaminationorsepsis(SCS),66withnon-septicintra-synovialpathology(NSISP)and8controls).BloodandSFSAAweresignificantlyhigherinSCSthaninNSISPandcontrolhorses.SAAvaluesweresimilarinNSISPandcontrolhorses.SFSAAwasmoderatelycorrelatedwithsynovialTNCC,TPandbloodSAA.BloodandSFSAAwere82.4percentand80percent sensitiveand88.9percentand73percent specificfordiagnosisofSCS,withcut-offvaluesof60.7and1.14 µg/ml,respectively.BloodandSFD-lactateconcentrationswerenotsignificantlydifferentbetweengroups.ThisstudyshowsthatbloodandSFSAAconcentrationscanaidtodistinguishSCSfromnon-septicsynovialpathology;however,D-lactatewasnotuseful.