Megazyme/D-Mannitol/L-Arabitol Assay Kit/K-MANOL/60 assays (manual) / 600 assays (microplate) / 580 assays (auto-analyser)

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.

Lipopolysaccharide(LPS)isknowntoproduceendotoxicshockbytriggeringsystemicinflammatoryresponses.Here,weevaluatedtheprotectiveeffectsofthreefermented/re-fermentedherbs,RhizomaAtractylodisMacrocephalae,MassaMedicataFermentata,andDolichorisSemen,inanLPS-mediatedinflammatoryinsult,eitherindividuallyinvitrousingRAW264.7cellsorincombinationininvivousingrats.Ingeneral,eachofthefermentedherbsshowedappreciableinvitroanti-inflammatoryactivity,althoughthedegreeofthisactivityvariedwiththeherbused.Moreover,amixtureoffermentedherbalextractsincombinationwithprobioticssignificantlyattenuatedthebloodendotoxinandCRPlevels,aswellasthegutpermeability,andsignificantlyaugmentedtheintestinalLactobacillusspp.colonisationinLPS-treatedrats.However,theseeffectswerenotobservedfollowingtheadmiNISTrationofthecorrespondingmixtureofunfermentedherbalextracts.Thus,ourresultshighlightthebeneficialimpactsoftheuseoffermentedherbproductswithprobioticstocombatLPS-mediatedinflammatoryinsults.

Botrytiscinereapresentshighvariabilityinseveralbiologicaltraits,whichcanbeexplainedbythehighdegreeofgenotypicdiversityamongisolates.Becausethisgeneticvariabilitymightberelatedtophenotypicdifferencestherequirementsforconidiagerminationofthreenaturalisolates(G1,G5andG11)obtainedfromgrapesandbelongingtothesamegeneticgroupwereanalyzed.Theresultsshowedthatcontactwithasolidsurfacewasacommonrequisiteforconidiagerminationoftheisolatesbuttheydifferedintheirnutritionalrequirementstogerminate.IsolateG11wasabletogerminateintheabsenceofacarbonornitrogensource.G1andG5requiredthepresenceofacarbonsourcesuchasglucose,fructoseorsucrose.InG11andG5isolatesamuchhigherrateofgerminationwasobtainedinthepresenceofsucrose.ItwasshownwithapharmacologicalapproachthatthecAMPstimulatedthegerminationonlyinthoseisolatesrequiringacarbonsource.ConidiagerminationofG1andG5wasinhibitedbyEGTA,acalciumchelator.IsolateG11germinatedinthepresenceofthiscompound.OntheotherhandthegerminationofthreeB.cinereaisolatesrequiredproteinsynthesisanddidnotrequireRNAsynthesis.ToexplaintheabilityofisolateG11togerminateinwaterthecontentoftotalandreducingsugars,mannitol/L-arabitol,trehalose,andproteinsinthenongerminatedconidiaofthethreeisolateswascompared.Theisolatespresentedsimilaramountsoftotalandreducingsugars.Inthethreeisolatestheamountofmannitol/L-arabitolwashigherthanthatoftrehalose.InisolateG11totalproteincontentwastwicehigherthanintheotherisolates.

Backgrounds&aims:Probioticshelpmaintainbalanceincompositionofthegutmicrobiota,andhavebeenconsideredasapotentialtreatmentforobesity.Thisstudywasconductedinordertoassesstheeffectsofprobioticswhencombinedwithherbalmedicineintreatmentofobesity.Probioticsweretestedfortheabilitytomodulategutmicrobiota,gutpermeability,andendotoxinlevel,whichmayhavecorrelationwithfactorsinvolvedinobesity.Methods:Arandomized,double-blind,placebocontrolledstudywasconducted,inwhichpatientswithhigherBMI(>25kg/m²)andwaistcircumference(>85cm)wereenrolledandrandomlyassignedtoreceiveBofutsushosanwitheitherprobioticsorplacebocapsulesforaperiodofeightweeks.Assessmentofbodycompositionparameters,metabolicbioMarkers,endotoxinlevel,gutpermeability,andfecalbacteriainstoolwasperformedatbaselineandatweek8.ThestudywasregisteredattheClinicalResearchInformationService,approvedbytheKoreaNationalInstituteofHealth(KCT0000386).Results:Althoughbothgroupsshowedasignificantreductioninweightandwaistcircumference(p=0.000),nosignificantdifferencesinbodycompositionandmetabolicmarkerswereobserved.Incorrelationanalysis,changeinbodycompositionshowedpositivecorrelationwithendotoxinlevel(r=0.441,p<0.05=""for=""bw;=""and="">r=0.350,p<0.05=""for=""fat=""mass)=""and=""the=""population=""of=""gut="">Lactobacillusplantarum(r=0.425,p<0.05=""for=""bw;=""and="">r=0.407,p<0.05=""for=""bmi).=""the=""gram=""negative=""bacterial=""population=""in=""gut=""also=""exhibited=""positive=""correlation=""with=""changes=""in=""body=""composition=""(wc)=""and=""total=""cholesterol=""level="">r=0.359,and0.393,fortheformerandlaterparameters,respectively,p<0.05=""for=""both).=""while,=""the=""profile=""of=""gut="">Bifidobacteriumbrevepopulationshowednegativecorrelationwithendotoxinlevel(r=−0.350,p<0.05).="">Conclusions:Correlationsbetweengutmicrobiotaandchangeinbodycompositionindicatethatprobioticsmayinfluenceenergymetabolisminobesity.Correlationbetweenendotoxinlevelandweightreductionindicatesthatprobioticsmayplayanimportantroleinpreventionofendotoxinproduction,whichcanleadtogutmicrobiotadysbiosisassociatedwithobesity.

D-Mannitol(hereafterdenotedmannitol)isusedinthemedicalandfoodindustryandiscurrentlyproducedcommerciallybychemicalhydrogenationoffructoseorbyextractionfromseaweed.Here,themarinecyanobacteriumSynechococcussp.PCC7002wasgeneticallymodifiedtophotosyntheticallyproducemannitolfromCO₂asthesolecarbonsource.Twocodon-optimizedgenes,mannitol-1-phosphatedehydrogenase(mtlD)fromEscherichiacoliandmannitol-1-phosphatase(mlp)fromtheprotozoanchickenparasiteEimeriatenella,incombinationencodingabiosyntheticpathwayfromfructose-6-phosphatetomannitol,wereexpressedinthecyanobacteriumresultinginaccumulationofmannitolinthecellsandintheculturemedium.Themannitolbiosyntheticgeneswereexpressedfromasinglesyntheticoperoninsertedintothecyanobacterialchromosomebyhomologousrecombination.Themannitolbiosynthesisoperonwasconstructedusinganoveluracil-specificexcisionreagent(USER)-basedpolycistronicexpressionsystemcharacterizedbyligase-independent,directionalcloningoftheprotein-encodinggenessuchthattheinsertionsitewasregeneratedaftereachcloningstep.Geneticinactivationofglycogenbiosynthesisincreasedtheyieldofmannitolpresumablybyredirectingthemetabolicfluxtomannitolunderconditionswhereglycogennormallyaccumulates.Atotalmannitolyieldequivalentto10%ofcelldryweightwasobtainedincellculturessynthesizingglycogenwhiletheyieldincreasedto32%ofcelldryweightincellculturesdeficientinglycogensynthesis;inbothcasesabout75%ofthemannitolwasreleasedfromthecellsintotheculturemediumbyanunknownmechanism.Thehighestproductivitywasobtainedinaglycogensynthasedeficientculturethatafter12daysshowedamannitolconcentrationof1.1gmannitolL^-1andaproductionrateof0.15gmannitolL^-1day^-1.ThissystemmaybeusefulforbiosynthesisofvaluablesugarsandsugarderivativesfromCO₂incyanobacteria.

Thehepatoprotectiveeffectsoffermentedblackricebranextracts(FF1andFF2:blackricebranfermentedbyLentinusedodesderivedfrommyceliumsupplementedwithsoybeanorHoveniadulcis)andtheirassociatedmechanismswereevaluated.Inaninvitroexperiment,FFscausedsignificantameliorationofthemetabolicfunctionofrathepatocytestreatedwithNH₄Cl.Inaddition,administrationofFFstoratswithchronicliverinjuryinducedby12-weekcontinualalcoholconsumptionresultedinsignificantrestorationofbodyweightshrinkage,notableattenuationofexcessiveaspartateaminotransferase,alkalinephosphataseandendotoxininserum,malondialdehydeinliverandthelactulose/mannitolratioinurine.FurThermore,FF1orFF2alsocausedsignificantdownregulationofgeneexpressionofseveralcriticalinflammatorymediators(interleukin-6,tumornecrosisfactor-alpha,cyclooxygenase-2andinduciblenitricoxidesynthase).HistopathologicalfindingsalsoindicatedthatFFsreducedinflammation,necrosisandfattyinfiltrationinlivertissue.Takentogether,FFsexerthepatoprotectiveeffectsthroughanti-inflammatoryandanti-lipidperoxidativepropertiesandregulationofintestinalpermeability.

Cost-effectiveapplicationoflignocellulolyticenzymesholdsthekeytowardscommercializationofenzymatichydrolysisoflignocellulosicbiomass.Carrierfreeimmobilizationofenzyme(s)offersalucrativeProspect.Combined-crosslinkedenzymeaggregates(combi-CLEAs)areanovelprospectiveandthispresentstudyaddressesthepreparation,characterizationandapplicationofxylanase–mannanasecombi-CLEASonlime-preteatedsugarcanebagasseandmilledcornstover.X6-CLEAs,X7-CLEAs,L1-CLEAsandL7-CLEAswerepreparedafterelaborativeoptimizationoftheprecipitatingagentandglutaraldehydeconcentration.Thehighestactivityafterprecipitationwasobservedwithacetonebutfollowingcross-linkingwithglutaraldehydelessthan60%activitywasretained,whilemorethan60%activitywasretainedafterprecipitationwithammoniumsulphateandcross-linkingwithglutaraldehyde.Accessoryenzymeactivitiesincludingα-arabinofuranosidase,β-xylosidase,esterases,β-mannosidase,α-galactosidaseandβ-glucosidasewerealsodetermined.Morethanan1.5foldincreaseinthermostabilitycomparedtothefreeenzymewasobservedoverabroadtemperaturerange(50–70°C).Tri-synergystudiesandquadsynergystudieswereusedtogeneratecombi-CLEAswithdifferentproteinratios.Hydrolysisoflimepre-treatedbagassewithcombi-CLEAsatproteinratioscorrespondingtoX6(33.0%):X7(17.0%):L1(17.0%):L7(33.0%)resultedina1.68foldhighersugarreleasecomparedtothequadsynergymodelusingfreeenzymes.Similarly,hydrolysisofcornstoverwithcombi-CLEAsatproteinratioscorrespondingtoX6(40.0%):X7(10.0%):L1(10.0%):L7(40.0%)resultedinan1.58foldhighersugarreleasecomparedtothesugarreleaseobservedwiththequadsynergymodelusingfreeenzymes.Monomericsugarsconstituted70–75%ofreducingsugarsreleasedduringhydrolysis.Theroleofaccessoryenzymesinimprovingenzymesynergywasclearlyshown.Theefficiencyofcombi-CLEAscomparedtofreeenzymesmakesthemidealcandidatesfortheprudentandcost-effectivecommercializationoflignocellulolyticenzymes.