Thispaperfocusesonfermentationoflactosefromamodelsystem(blacktea)andfromtwotypesofmilk(0.9%w/wand2.2%w/woffat)byapplicationofKombucha.QuantitiesoftheappliedKombuchastarterwere10%v/vand15%v/v.Allfermentationswereperformedat42°C.TheprocesstoachieveadesirablepH=4.5wasslowerinthemodelsystem(16h)thaninmilks(9-10h).Regardingstarterquantity,10%v/vprovedtheoptimal.Regardingtypesofmilk,higherfatcontentguaranteesshorterfermentationandhigheryieldofmetabolites.Utilizationoflactosewasfoundatalevelof≈20%and≈30%inmilkswith0.9%w/wand2.2%w/woffat,respectively.Thiswascorrelatedwithanappearanceofintermediatesand/orproducts.Glucoseunderwentfurthertransformationsalmostentirely,whilegalactoseshowedmuchlowerreactivity.Seventotwelvetimeshighercontentsoflacticacidwerefoundcomparedtoaceticacid.Milk-basedbeveragefromthereducedfatsample,inoculatedwith10%v/vofKombuchastarter,hasthebestphysicalcharacteristics(syneresisandwaterholdingcapacity).Italsodevelopedagoodtexture(especiallycohesivenessandindexofviscosity).Milklactosefermentationwasaprocessthatcouldhavebeenusedforobtainingnewmilk-basedproducts.

Theaimofthisstudywastoinvestigatetheeffectoftransglutaminase(TG)additiononrheologicalproperties,texturalcharacteristicsandmicrostructureoffermentedmilkproductsmanufacturedbydifferentstarters(probioticsandkombuchainoculum).Rheologicalanalysisrevealedthatallmanufacturedfermentedmilkproductshadhigherstoragemodulusthanlossmodulusandexhibitedthixotropicandatypicalshearthinningbehavior.TheadditionofTGinmilkincreasedapproximately10.5%hysteresislooparea,39%firmnessand48%consistencyinsampleproducedwithprobioticstarterandhadmorefirmandstablegelstructurethankombuchafermentedmilkproducts.ThescanningelectronmicroscopymicrographsshowedthatcaseinmatrixoffermentedmilkproductscontainingTGiscontinuousanduninterruptedexceptforvoidspacesoccupiedbymilkserumandstarterculturecell.

Twoα-L-arabinofuranosidaseswithdifferentsubstratespecificitieswereusedtomodifythearabinose-to-xyloseratioofcerealarabinoxylans:oneenzyme(AXH-m)removedtheL-arabinofuranosylsubstituentsfromthemonosubstitutedxylopyranosylresiduesandtheother(AXH-d3)the(1→3)-linkedL-arabinofuranosylunitsfromthedisubstitutedxylopyranosylresidue.Inthisstudy,wenoticedthatnotonlythearabinose-to-xyloseratiobutalsothepositionofthearabinofuranosylsubstituentsaffectsthewater-solubilityofarabinoxylans.TheAXH-d3treatmenthadnosignificanteffectonthesolutionconformationofarabinoxylans,butthedensityofthearabinoxylanmoleculesdecreasedinDMSOsolutionafterAXH-mmodification.ThepossIBLeheterogeneityofarabinoxylanscomplicatedtheinterpretationofdatadescribingthemacromolecularpropertiesoftheenzymaticallymodifiedsamples.

Arabinoxylanarabinofuranohydrolases(AXAHs)arefamilyGH51enzymesthathavebeenimplicatedintheremovalofarabinofuranosylresiduesfromthe(1,4)-β-xylanbackboneofheteroxylans.FivegenesencodingbarleyAXAHsrangeinsizefrom4.6kbto7.1kbandeachcontains16introns.ThebarleyHvAXAHgenesmaptochromosomes2H,4H,and5H.AsmallclusterofthreeHvAXAHgenesislocatedonchromosome4Handthereisevidenceforgeneduplicationandthepresenceofpseudogenesinbarley.TheCDNAscorrespondingtobarleyandwheatAXAHgeneswerecloned,andtranscriptlevelsofthegeneswereprofiledacrossarangeoftissuesatdifferentdevelopmentalstages.TwoHvAXAHcDNAsthatweresuccessfullyexpressedinNicotianabenthamianaleavesexhibitedsimilaractivitiesagainst4-nitrophenylα-L-arabinofuranoside,butHvAXAH2activitywassignificantlyhigheragainstwheatflourarabinoxylan,comparedwithHvAXAH1.HvAXAH2alsodisplayedactivityagainst(1,5)-α-L-arabinopentaoseanddebranchedarabinan.Westernblottingwithananti-HvAXAHantibodywasusedtodefinefurtherthelocationsoftheAXAHenzymesindevelopingbarleygrain,wherehighlevelsweredetectedintheouterlayersofthegrainbutlittleornoproteinwasdetectedintheendosperm.Thechromosomallocationsofthegenesdonotcorrespondtoanypreviouslyidentifiedgenomicregionsshowntoinfluenceheteroxylanstructure.ThedataarethereforeconsistentwitharoleforAXAHindepolymerizingarabinoxylansinmaternaltissuesduringgraindevelopment,butdonotprovidecompellingevidenceforaroleinremodellingarabinoxylansduringendospermorcoleoptiledevelopmentinbarleyaspreviouslyproposed.

Thereisanincreasinginterestinsubstitutingtoday"sfilmsforfoodpackagingapplicationswithfilmsbasedonrenewableresources.Forthispurpose,ryearabinoxylans,unmodifiedandenzymaticallydebranched,werestudiedforthepreparationofneatfilmsandcompositefilmsreinforcedwithbacterialcellulose(BC).Mixinginahomogenizerproducedopticallytransparent,uniformfilms.Physicalandmechanicalcharacteristicsofsuchfilmsareherereported.Debranchingofthearabinoxylancausedanincreaseinitscrystallinityof20%.DebranchingaswellasreinforcementwithBCresultedinadecreaseofthemoisturesorptionofthefilms.ThedebranchingalsoresultedinareducedbreakingstrainwhilethereinforcementwithBCincreasedstiffnessandstrengthofthefilms.

Thisstudyassessedandcomparedthephysicochemical,microbiological,andsensorialcharacteristicsofCaciocavallocheeses,madefromcowmilkandamixtureofcowwitheweorgoatmilk,duringripening.Differentcheese-makingtrialswerecarriedoutonanindustrialscalefollowingthestandardprocedureofpastafilatacheeses,withsomemodifications.Thepercentageofthedifferentaddedmilktocowmilkinfluencedcompositionalandnutritionalcharacteristicsoftheinnovativeproducts,leADIngtoasatisfactorymicrobiologicalandsensorialquality.

Lignocellulosicwasteisanaturallyabundantbiomassandisthereforeanattractivematerialtouseinsecondgenerationbiorefineries.Microbialgrowthonthemonosaccharidespresentinhydrolyzedlignocelluloseishoweverassociatedwithseveralobstacleswhereofoneisthelackofsimultaneousuptakeofthesugars.WehavestudiedtheaerobicgrowthofEscherichiacolionD-glucose,D-xylose,andL-arabinoseandforsimultaneousuptaketooccur,boththecarboncataboliterepressionmechanism(CCR)andtheAraCrepressionofxyloseuptakeandmetabolismhadtoberemoved.ThestrainAF1000isaMC4100derivativethatisonlyabletoassimilatearabinoseafteraconsiderablelagphase,whichisunsuitableforcommercialproduction.ThisstrainwassuccessfullyadaptedtogrowthonL-arabinoseandthisledtosimultaneousuptakeofarabinoseandxyloseinadiauxicgrowthmodefollowingglucoseconsumption.Inthisstrain,adeletioninthephosphoenolpyruvate:phosphotransferasesystem(PTS)forglucoseuptake,theptsGmutation,wasintroduced.Theresultingstrain,PPA652arasimultaneouslyconsumedallthreemonosaccharidesatamaximumspecificgrowthrateof0.59 h^-1,55%higherthanfortheptsGmutantalone.Also,noresidualsugarwaspresentinthecultivationmedium.ThepotentialofPPA652araisfurtheracknowledgedbytheperformanceofAF1000duringfed-batchprocessingonamixtureofD-glucose,D-xylose,andL-arabinose.Theconclusionisthatwithouttheremovalofbothlayersofcarbonuptakecontrol,thisprocessresultsinaccumulationofpentosesandleadstoareductionofthespecificgrowthrateby30%.

Recentlylactosemediatedauto-inductioninEscherichiacolihasgainedalotofinterestbecausehigherproteintitercouldbeachievedwithouttheneedtomonitorgrowthandaddinduceratthepropertime.Inthisstudyahighleveltherapeuticproteinproductionbyauto-inductionwasobservedinE.coliBL21usingeitherT7ortacpromotersinthemodifiedLuriaBertani(mLB)mediumcontainingsoypeptoneinsteadoftryptoneinLuriaBertani(LB)medium.Basedonmediumanalysisandspikingexperimentsitwasfoundthat0.4mMgalactosefromthesoypeptonecausedtheauto-induction.E.coliculturesinducedbygalactosecansaturateatconsiderablyhigherdensitythanculturesinducedbyIPTG.GalactoseisnotconsumedbyE.coliBL21.Finallyithasbeendemonstratedthatauto-inductioncanbeeffectivelyusedinfed-batchfermentationfortheindustrialproductionofatherapeuticprotein.Theprincipleofgalactosemediatedauto-inductionshouldbeabletoapplytohighthroughputmicroplates,shakeflasksandfed-batchfermentorsforclonescreeningandtherapeuticproteinexpressioninE.coligal^-strainssuchasmostcommonlyusedBL21.

Ourobjectivewastodevelopaprocesstoproduceahigh-concentrationliquidmicellarcaseinconcentrate(18%protein,MCC18)withalongrefrigeratedshelflife.TheMCC18isanovelmilkproteiningredientproducedbyfractionatingskimmilkusingMICROFILtration(MF).Toachievealongrefrigeratedshelflife,theprocessingofMCC18wasdesignedtomaximizetheremovaloflow-molecularweightcompounds[e.g.,lactoseandnonproteinnitrogen(NPN)]thatcanbeeasilymetabolizedbymicrobes,whileminimizingthemicrobialcountinthefinalproduct.TheproductionofMCC18wasdoneoveraperiodof5d.Theexperimentwasreplicated3timesindifferentweekswithadifferentbatchofrawmilk.Rawwholemilkwaspasteurizedandseparatedtoproduceskimmilk.Skimmilkwasultrafiltered(UF)toremovemorethanhalfofthelactoseandNPN.TheUFmilkretentatewasdilutedwithwaterandthenMFin3stagestoremoveapproximately95%oftheserumproteinandfurtherremovelactoseandNPN.TheretentatefromthelaststageofMFwasUFtoconcentratetheproteinto18%andbatchpasteurized.TheMCC18wascollectedimmediatelyafterprocessinginsterileplasticvialsandstoredat4°C.TheaverageMCC18contained21.78%totalsolids,18.27%trueprotein,0.31%NPN,and0.13%lactose.TheMCC18atthedayofprocessingcontainedameanaerobicbacterialcountof2.1logcfu/mLandmeanaerobicsporecountof2.3logcfu/mL.TheMCC18formedasolidgelattemperatures<22°c, but="" the="" mcc18="" reverted="" back="" to="" a="" liquid="" when="" warmed="" from="" 4°c="" temperature="" to="">22°C.Thisprovidesauniqueopportunityiningredienthandlingandpackagingandeliminatesthechallengesencounteredinreconstitutionofdriedmilkproteiningredients.TheMCC18producedinthisstudymaintainedabacteriacount<20,000 cfu/ml="" for="" 16="" wk="" when="" stored="" at="" a="" refrigeration="" temperature="" of="" 4°c.="" further="" study="" is="" needed="" to="" determine="" if="" changes="" occur="" in="" the="" organoleptic="" and="" functional="" properties="" of="" mcc18.="" we="" envision="" that="" the="" conversion="" of="" skim="" milk="" to="" mcc="" and="" its="" coproducts="" (serum="" protein="" concentrate="" and="" lactose="" concentrate)="" could="" be="" used="" as="" an="" alternative="" to="" the="" production="" of="" nonfat="" dry="" milk="" to="" balance="" milk="" production="" seasonality,="" specifically="" the="" components="" of="" skim="" milk="" portion.="">

Wheyandscottaareeffluentscomingfromcheeseandricottaprocessingrespectively.Wheycontainsminerals,lipids,lactoseandproteins;scottacontainsmainlylactose.Wheycanbereusedinseveralways,suchasproteinextractionoranimalfeeding,whilenowadaysscottaisjustconsideredasawaste;moreover,duetoveryhighvolumesofwheyproducedintheworld,itposesseriousenvironmentalanddisposalproblems.Alternativedestinationsoftheseeffluents,suchasbiotechnologicaltransformations,canbeawaytoreachbothgoalsofimprovingtheaddedvalueoftheagroindustrialprocessesandreducingtheirenvironmentalimpact.Inthisworkweinvestigatedthewaytoproducebioethanolfromlactoseofwheyandscottaandtooptimizethefermentationyields.Kluyveromycesmarxianusvar.marxianuswaschosenaslactose-fermentingyeast.Batch,aerobicandanaerobic,fermentationsandsemicontinuousfermentationsindispersedphaseandinpackedbedreactorwerecarriedoutofrowwhey,scottaandmix1:1whey:scottaatalaboratoryscale.Differenttemperatures(28–40°C)werealsotestedtocheckwhethertheThermotoleranceofthechosenyeastcouldbeusefultoimprovetheethanolyield.Thebestperformanceswerereachedatlowtemperatures(28°C);hightemperaturesarealsocompatiblewithgoodethanolyieldsinwheyfermentations,butnotinscottafermentations.Semicontinuousfermentationsindispersedphasegavethebestfermentationperformances,particularlywithscotta.Thenbotheffluentscanbeconsideredsuitableforethanolproduction.Thegoodyieldsobtainedfromscottaallowustotransformthiswasteinasource.

Theselectivityandbeneficialeffectsofprebioticsaremainlydependentoncompositionandglycosidiclinkageamongmonosaccharideunits.Thisisthefirststudytouseprebioticgalacto-oligosaccharides(GOS)thatcontainsβ-1,6andβ-1,3glycosidiclinkagesandthenovelcombinationofGOSandinulinincancerprevention.TheobjectiveofthepresentstudyistoexploretheroleofnovelGOSandinulinagainstvariousbiomarkersofcolorectalcancer(CRC)andtheincidenceofaberrantcryptfoci(ACF)ina1,2-dimethylhydrazinedihydrochloride(DMH)-inducedrodentmodel.PrebiotictreatmentsofcombinedGOSandinulin(57mgeach),aswellasindividualdoses(GOS:76–151mg;inulin114mg),weregiventoDMH-treatedanimalsfor16weeks.OurdatarevealthesignificantpreventiveeffectoftheGOSandinulincombinationagainstthedevelopmentofCRC.ItwasobservedthatinhibitionofACFformation(55.8%)wassignificantly(p ≤0.05)higherusingtheGOSandinulincombinationthanGOS(41.4%)andinulin(51.2%)treatmentsalone.Thiscombinationalsorenderedbetterresultsonshort-chainfattyacids(SCFA)andbacterialenzymaticactivities.Dose-dependenteffectsofprebiotictreatmentswerealsoobservedoncecumandfecalbacterialenzymesandonSCFA.Thus,thisstudydemonstratedthatnovelcombinationofGOSandinulinexhibitedstrongerpreventiveactivitythantheirindividualtreatmentsalone,andcanbeapromisingstrategyforCRCchemoprevention.

Asaprotectivefactorforlipopolysaccharide(LPS)-inducedinjury,14-3-3γhasbeenthesubjectofrecentresearch.Nevertheless,whether14-3-3γcanregulatelactationindairycowmammaryepithelialcells(DCMECs)inducedbyLPSremainsunknown.Here,theanti-inflammatoryeffectandlactationregulatingabilityof14-3-3γinLPS-inducedDCMECsareinvestigatedforthefirsttime,andthemolecularmechanismsresponsiblefortheireffectsareexplored.TheresultsofqRT-PCRshowedthat14-3-3γoverexpressionsignificantlyinhibitedthemRNAexpressionoftumornecrosisfactor-α(TNF-α),interleukin-6(IL-6),interleukin-1Β(IL-1β)andinduciblenitricoxidesynthase(iNOS).Enzyme-linkedimmunosorbentassay(ELISA)analysisrevealedthat14-3-3γoverexpressionalsosuppressedtheproductionofTNF-αandIL-6incellculturesupernatants.Meanwhile,CASY-TTAnalyserSystemshowedthat14-3-3γoverexpressionclearlyincreasedtheviabilityandproliferationofcells.Theresultsofkitmethodsandwesternblotanalysisshowedthat14-3-3γoverexpressionpromotedthesecretionoftriglyceridesandlactoseandthesynthesisofβ-casein.Furthermore,theexpressionofgenesrelevanttonuclearfactor-κB(NF-κB)andmitogen-activatedproteinkinase(MAPKs)andlactation-associatedproteinswereassessedbywesternblot,andtheresultssuggestedthat14-3-3γoverexpressioninactivatedtheNF-κBandMAPKsignalingpathwaysbydown-regulatingextracellularsignalregulatedproteinkinase(ERK),p38mitogen-activatedproteinkinase(p38MAPK)andinhibitorofNF-κB(IκB)phosphorylationlevels,aswellasbyinhibitingNF-κBtranslocation.Meanwhile,14-3-3γoverexpressionenhancedtheexpressionlevelsofΒ-casein,mammaliantargetofrapamycin(mTOR),ribosomalproteinS6kinase1(S6K1),serine/threonineproteinkinaseAkt1(AKT1),sterolregulatoryelementbindingprotein1(SREBP1)andperoxisomeproliferator-activatedreceptorgamma(PPARγ).Theseresultssuggestthat14-3-3γwasabletoattenuatetheLPS-inducedinflammatoryresponsesandpromoteproliferationandlactationinLPS-inducedDCMECsbyinhibitingtheactivationoftheNF-κBandMAPKsignalingpathwaysandup-regulatingmTORsignalingpathwaystoprotectagainstLPS-inducedinjury.

Prebioticgalacto-oligosaccharides(GOS)areimportant“functionalfoods”ofcurrentscenarioandusedforvarioushealthbenefitsincludingimprovedmineralabsorption.Inthepresentstudy,itwashypothesizedthatnovelGOSmixture,producedthroughtransgalactosylation,withsignificantamountofmonoanddisaccharidesmayenhancemineralabsorptioninSprague-Dawleyrats.Thenon-purifiedGOShavingβ-(1 → 6)andβ-(1 → 3)glycosidiclinkage,wereevaluatedforapparentabsorptionofcalcium,magnesium,ironandzinc.Theratsweredividedintotwomaingroups(n = 12pergroup,6male/6female)fedoncontrolandGOS(5 g/100 g)diet.Thefeceswerecollectedafter7 daysintervalfor28 days.Theweightgain,feedandwaterintakewerestatisticallysimilar(p < 0.05)in=""both=""groups=""irrespective=""of=""gender.=""similarly,=""the=""absorption=""of=""minerals=""was=""statistically=""not=""different=""in=""both=""genders=""during=""whole=""study.=""the=""gos=""diet=""significantly="">< 0.05)improved=""absorption=""of=""ca=""(34.55–39.93%),=""mg=""(51.22–58.05%)=""and=""fe=""(31.58–39.21%)=""as=""compared=""to=""control=""diet=""at=""the=""end=""of=""study.=""however,=""no=""impact=""on=""zn=""absorption=""was=""observed=""during=""the=""whole=""study.=""it=""can=""be=""inferred=""that=""the=""use=""of=""non-purified=""gos=""for=""3–4=""weeks=""may=""enhance=""ca,=""mg=""and=""fe=""absorption.="">

Milkisimportantforhumannutrition,andenhancedmilkqualityhasbecomeamajorselectioncriterionforthegeneticimprovementoflivestock.Epigeneticmodificationshavebeenshowntobeinvolvedinmammaryglanddevelopment;butthemechanismsunderlyingtheireffectsremainunknown.MicroRNAsareinvolvedintheregulationofmilksynthesisandinmammaryglanddevelopment.OurstudyisthefirsttoinvestigatetherolesofmiR-29sandepigeneticregulationindairycowmammaryepithelialcells(DCMECs).OurresultsshowthatmiR-29sregulatetheDNAmethylationlevelbyinverselytargetingbothDNMT3AandDNMT3BinDCMECs.TheinhibitionofmiR-29scausedglobalDNAhypermethylationandincreasedthemethylationlevelsofthepromotersofimportantlactation-relatedgenes,includingcaseinalphas1(CSN1S1),E74-likefactor5(ElF5),peroxisomeproliferator-activatedreceptorgamma(PPARγ),sterolregulatoryelementbindingprotein-1(SREBP1),andglucosetransporter1(GLUT1).TheinhibitionofmiR-29sreducedthesecretionoflactoprotein,triglycerides(TG)andlactosebyDCMECs.Moreover,thetreatmentofDCMECswith5-aza-2′-deoxycytidine(5-Aza-dC)decreasedthemethylationlevelsofthemiR-29bpromoterandincreasedtheexpressionofmiR-29b.ThelinkbetweenmiR-29sandDNMT3A/3BenhancesourunderstandingoftherolesofmiRNAsinmammaryglandfunction,andourdatawillinformmoreexperimentallyorientedstudiestoidentifynewmechanismsofregulatinglactation.Wepresentnewinsightsregardingtheepigeneticregulationoflactationperformance.Improvedunderstandingofthemolecularbasisoflactationwillaidinthedevelopmentofstrategiesforoptimizingmilkqualityindairycowsandmodifyingthelactationperformanceofoffspring.

TheEscherichiacolilacoperonisregulatedbyapositivefeedbackloopwhosepotentialtogenerateanall-or-noneresponseinsinglecellshasbeenaparadigmforbistablegeneexpression.However,sofarbistablelacinductionhasonlybeenobservedusinggratuitousinducers,raisingthequestionaboutthebiologicalrelevanceofbistablelacinductioninthenaturalsettingwithlactoseastheinducer.Infact,theexistingexperimentalevidencepointstoagradedratherthananall-or-noneresponseinthenaturallactoseuptakesystem.Incontrast,predictionsbasedoncomputationalmodelsofthelactoseuptakepathwayremaincontroversial.Althoughsomeargueinfavorofbistability,othersargueagainstit.Here,wereinvestigate lac operonexpressioninsinglecellsusingacombinedexperimental/modelingapproach.Tothisend,weparameterizeawell-supportedmathematicalmodelusingtransientmeasurementsofLacZactivityuponinductionwithdifferentamountsoflactose.Theresultingmodelpredictsamonostableinductioncurveforthewild-typesystem,butindicatesthatoverexpressionoftheLacIrepressorwoulddrivethesystemintothebistableregime.Bothpredictionswereconfirmedexperimentallysupportingtheviewthatthewild-typelacinductioncircuitgeneratesagradedresponseratherthanbistability.Moreinterestingly,wefindthatthelacinductioncurveexhibitsapronouncedmaximumatintermediatelactoseconcentrations.Supportedbyourdata,amodel-basedanalysissuggeststhatthenonmonotonicresponseresultsfromsaturationoftheLacIrepressoratlowinducerconcentrationsanddilutionofLacenzymesduetoanincreasedgrowthratebeyondthesaturationpoint.WespeculatethattheobservedmaximuminthelacexpressionlevelhelpstosavecellularresourcesbylimitingLacenzymeexpressionathighinducerconcentrations.

Standard-calcium(SCa)andreduced-calcium(RCa)half-fat(16%)Cheddar-stylecheeseswithfull-salt(1.9%)orhalf-salt(0.9%)weremadeintriplicate,ripenedfor270d,andanalysedforcompositionandchangesinlactosemetabolism,pH,proteolysis,water-sorption,fracturepropertiesandheat-inducedflowabilityduringmaturation.Thepressingloadappliedtothemouldedcheesewasmodifiedtoensureequalmoistureinallcheesesdespitethedifferencesinsaltandcalciumlevels.TheRCacheeseswerecharacterizedbyhigherprimaryproteolysis(α_s1-caseindegradation,pH4.6-solubleNdevelopment),lowersecondaryproteolysis(concentrationoffreeaminoacids),higherwater-holdingcapacityonreducingrelativehumidityfrom85to5%,lowerfracturestressandstrain,andmoreextensiveflowonheating.Overall,theusecalciumreduction,whenusedinconjunctionwithmoisturenormalization,provedaneffectivemeansofcounteractingtheadverseeffectsoffatreductionontextureandcookingpropertiesinhalf-fat,half-saltcheese.

AnnexinA2(AnxA2)hasbeenshowntoplaymultiplerolesingrowth,developmentandmetabolism,butthefunctionsofAnxA2andthesignalingpathwaysassociatedwithAnxA2arestillnotfullyunderstood.Inthisstudy,weaimtorevealwhetherandhowAnxA2couldbeinvolvedinmilksynthesisandproliferationofbovinemammaryepithelialcells(BMECs).Usinggenefunctionstudyapproaches,wefoundthatAnxA2positivelyregulatesPIP3level,phosphorylationofmTORandproteinlevelsofSREBP-1candCyclinD1leadingtomilksynthesisandcellproliferation.WefurtherobservedthatbothAnxA2-36kDphosphorylatedformandAnxA2-33kDproteincouldbeinducedfromAnxA2-36kDproteininBMECsundermethionine,leucine,estrogenorprolactinstimulation.TheseaboveresultsstronglydemonstratethatAnxA2functionsasacriticalregulatorforaminoacidorhormone-inducedmilksynthesisandcellproliferationviathePI3K-mTOR-SREBP-1c/CyclinD1signalingpathway.

Theevolutionoffreeaminoacids(FAA)inCaciocavallocheeses,madewithcowmilk(CC)andcowmilkmixedwithewe(CE)andgoat(CG)milk,wasstudiedthroughoutripening.InallCaciocavallocheesesproduced,thetotalfreeaminoacid(TFAA)contentincreasedduringripening.Ingeneral,thehighestTFAAcontentwasfoundincowcheeses,andthelowestinCGcheeses,whereasCEcheesesrangedoveranintermediatelevel.Inalltheanalyzedsamples,duringripening,thecontentoftheindividualFAAincreasedwiththeexceptionofarginine.TyrosineandhistidinewerefoundonlyinCEsamplesfromthemiddletotheendofripening.ThemajorFAAfoundthroughoutthewholeripeningperiod,inalltypesofcheese,wereleucine,phenylalanine,lysine,valine,asparagine,γ-aminobutyricacid,andornithine.TheTFAAandseveralAAshowedsignificantdifferencesinripeningtime,whereastyrosineandhistidineshowedsignificantdifferencesinkindsofmilk.