Optimization of ingredients for Lactobacillus fermented beverages Impact of different fermentation parameters on the final product’s sensory and shelf life ___________________________________

Department of Food Technology Lund University Rebecka Olsson. Master Thesis 2017 Supervisors: Anna Andrys & Elisabeth Uhlig Examiner: Åsa Håkansson

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AbstractTheingredientsgenerallyusedinthefermentationofprobioticstendtogiveanoff-flavourtothefermentationproductwhichisnotidealwhenproducinge.g.fruitdrinks.Theaimofthismasterthesiswastoevaluateiftheconcentrationofsuchingredientscouldbeloweredandalsotoinvestigatewhetherthereareotheringredientsthatcouldactassubstitutestothoseingredientsgivingoff-flavours.Thegrowth,stabilityandtasteofthefermentationproductscontainingtheingredientsandprobiotics,aswellasthefermentationproductputinafruitmodelsystem,wasevaluated.Theexperimentalworkincludedchangingtheconcentrationoftheingredientgivingtheoff-flavourandexchangingitwithsubstitutingingredientsduringthefermentationofthreedifferentprobioticbacteria.Thebacterialcontentwasmeasuredusingaplatecountmethodandthestabilityofthefermentationproductwasevaluatedafteroneweekofstoragein4°C.Afterputtingthefermentationproductinafruitjuice,thestabilitywasmeasuredduringfourweeksofstorageandasimplifiedsensoryevaluationwasdone.Itwasfoundthattheingredientsthatgavethesignificantlyhighestgrowthwasgenerallytheingredientnormallyusedduringthefermentationandnoneofthesubstitutingingredientswereassuccessfulinsupportinggrowth.Itwasalsoconcludedthatthetypeofbacteriawillhaveaneffectonthefinalbacterialcontentinthefermentationproductandthesensorypropertieswheresomestrainshadabettertasteprofilethanothers.

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PrefaceandAcknowledgementTheideaforthismasterthesisoriginatedatProbiABandallexperimentalworkwasperformedthere.Theworkwasdoneduringspring2017andisthefinalsubmissionofmymaster’sdegreeinBiotechnologywithFoodTechnologyspecializationatLTH.Thisthesishadnotbeenpossiblewithoutthehelpofthefollowingpeople.

AnnaAndrys,mysupervisoratProbiAB,whograntedmetheopportunitytodomythesisatProbiAB.Iwouldalsoliketothankherforallhervaluableinputsandhelpwiththeexperimentalwork,Ihavelearnedsomuch.IwouldalsoliketothankJennyRudolfssonwhoactedasastandinforwhenAnnawasnotavailable,Iapologizetoyoubothformakingyoutastethesometimes-badtastingdrinks.DoingmythesisatProbiABhaveprovidedmewithmuchappreciatedexperienceofhowitworkswhendevelopingnewfoodproductsandIhaveenjoyedeveryminuteofit.

IwouldalsoliketothankmysupervisoratLTH,ElisabethUhlig,whoalwayshadgoodadviceinstore.Thankyouforyourhelpwiththestatistics,foryourdedicationandforallyourvaluablecomments.

Finally,Iwouldliketothankmyfriendsandfamilywhohavestoodbymeduringthisentireproject,Icouldnothavedonethiswithoutyou.

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Tableofcontent

Abstract 2

PrefaceandAcknowledgement 3

1 Introduction 6

2 TheLactobacillusgenus 72.1 Carbohydratemetabolism 7

2.1.1 Glucose 72.1.2 Maltose 82.1.3 Fructose 82.1.4 Pentose 82.1.5 Citrate 92.1.6 Pyruvateandendproducts 9

2.2 Malolacticfermentation(MLF) 92.3 Proteolysis 92.4 Lactobacillusrhamnosus 92.5 Lactobacillusparacasei 92.6 Lactobacillusplantarum 10

3 Substitutingingredientsandfermentationconditions 123.1 Plantseedpowder 123.2 PeaProtein 123.3 RiceProtein 123.4 IncreasingthetemperatureforL.rhamnosus 12

4 Materialandmethod 134.1 Organismsandinoculum 134.2 Cultivationconditionsandingredients 13

4.2.1 Varyingingredient2and3 144.2.2 Ingredient4 144.2.3 Ingredient5 144.2.4 Combiningingredient3and5 144.2.5 Ingredient6and7 144.2.6 Ingredient8 144.2.7 Ingredient9 144.2.8 IncreasingthefermentationtemperatureforL.rhamnosus 144.2.9 IncreasingthefermentationtimeforL.paracasei 14

4.3 Platecountanalysis 154.4 Storagestability 154.5 Sensoryevaluation 154.6 Statistics 15

5 Resultanddiscussion 165.1 pH 165.2 Growthandstability 195.3 Stabilityinjuice 24

5.3.1 Varyingtheconcentrationofingredient2and3 245.4 Sensoryevaluation 26

5.4.1 Varyingtheconcentrationofingredient2and3 265.4.2 Ingredient4 265.4.3 Ingredient5 265.4.4 Combiningingredient3withingredient5 27

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5.4.5 Ingredient6and7 275.4.6 Ingredient8 275.4.7 Ingredient9 275.4.8 IncreasingthefermentationtemperatureforL.rhamnosus 28

6 Conclusion 29

7 Futuresuggestions 30

8 References 31

9 Appendix 329.1 Platecountanalysis 329.2 Refrigeratortemperature 329.3 Varyingtheconcentrationofingredient2and3 339.4 Ingredient4 349.5 Ingredient5 359.6 Ingredient6and7 359.7 Ingredient8 36

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1 IntroductionThefieldofprobioticsisgrowingconstantlyandtheriseofconsumerawarenessforhealthyandtastyproductsareincreasingthedemandsonprobioticproducts.Fortheprobioticbacteriatogrowandsurviveintheharshenvironmentofe.g.afruitdrink,additionalingredientsmustbeaddedthatsupportsgrowth.Thoseingredientsarecomponentswhichmightprovideoff-flavours.Theaimofthisthesisistoinvestigatewhetheritispossibletolowertheconcentrationoftheingredientsthatarecreatingoff-flavoursorexchangethemforsomethingthathasabettertasteprofile.Thetaste,growthandstabilityofthefermentationproductcontainingdifferentLactobacillusspecieswillbeevaluatedaswellasputtingthefermentationproductinafruitmodelsystem.Lacticacidbacteria(LAB)isdefinedasafunctionalgroupoflacticacidproducingandharmlessbacteria,bothtohumanhealthandtothequalityoffood,thatarespontaneouslypresentinlacticacidfermentedfood.Theconsumptionoflacticacidbacteriamighthavebeeninvented1.5millionyearsago,assuggestedbyarchaeologists.(Molin,2013)ThepreservativefunctionofLABworkswhenthefoodproductcontainingcarbohydratesarestoredinanenvironmentwithoutoxygen.Thebacteriaexistingonthefoodproductwillstarttomultiplyresultinginanincreasedconcentrationofcarbondioxide,adecreaseinoxygentensionandadecreaseinpH.Attheendofthelacticacidfermentation,thefoodproductwillbedominatedbylacticacidbacteriaandhaveafinalpHofaround3.5-4.0.ThelowpHwillinhibitthegrowthofmostbacteria.Inaddition,LABcanproduceinhibitoryfactorssuchascarboxylicacids,nitrogenoxide,hydrogenperoxideandbacteriocins.(Molin,2013)ProbioticsaredefinedbyaworkinggroupfromFAOandWHOas“livemicroorganismswhichwhenadministratedinadequateamountsconferahealthbenefitonthehost”.(FAO/WHO,2002)Fortheprobioticstohaveaneffect,thedailydoseshouldbeatleast109CFUbut1010ispreferred.Noupperlimitoftheprobioticdoseexists.(Molin,2013)Probioticscanoftenbefoundindifferentkindsoffermentedmilk,suchasyoghurt.Thepossibilityofusingfruitjuicesarecurrentlybeingstudied.Pasteurizedfruitjuicesarebeneficialforthebacteriasinceitcontainsvariousnutrientsandnootherbacteriathatmaycompetewiththesupplementedprobiotics.Anotherbenefitisthatfruitjuicesoftencontainsoxygenscavengingingredients,e.g.ascorbicacid,thatwillpromoteananaerobicenvironment.Fruitjuicesarealsorichinsugar,whichpromotesthegrowthoftheprobiotics.(GarciaMaiaCosta,etal.,2013)Howwellbacteriaadapttofruitandvegetablesystemswillvaryaccordingtothestrainandspeciesoflactobacilli.Thereasonforthisisthatthenaturalenvironmentinwhichtheygrowdiffersgreatlyandwillthereforeaffecttheabilitytodistributetheenergyusedformetabolismbetweenmaintenanceandbiosynthesis,i.e.betweenforexampleresponsestostressandtheutilizationofalternativesubstrates.(Filannino,etal.,2014)Findingthebalancebetweengrowthandsurvivalisthereforeofgreatimportance.

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2 TheLactobacillusgenusThebacteriainthegenusLactobacillusarenon-motile,normallyrod-shapedanddonotformspores.Thegrowthtemperatureisbetween2and53°Cwheretheoptimaltemperatureisintherangeof30to40°CwhilethepHcanrangefrom3to8.Lactobacillusspeciestoleratesoxygenbutwillalsogrowanaerobicallyandthemainproductfromthefermentationofsugarislacticacid.(Holzapfel&Wood,2014)Lactobacillicanbedividedintothreedifferentfunctionalgroups;facultativelyheterofermentative,obligatelyhomofermentativeandobligatelyheterofermentative,whichwillgivedifferentendproductswhenfermentinghexoses:

• Obligatelyhomofermentative:glucoseàlacticacid• Obligatelyheterofermentative:glucoseàlacticacid,carbondioxide,ethanoland/oracetic

acid• Facultativeheterofermentative:

-Glucoseàlacticacid-Pentosesàlacticacidandaceticacid-Malicacidàlacticacidandcarbondioxide-Citrateàdiacetyl,acetoinandcarbondioxide

Lactobacillithataregrowingonglucoseandarefacultativeheterofermentativewill,inpresenceofoxygen,havelacticacid,diacetyl,aceticacidandacetoinasendproducts.(Molin,2013)2.1 Carbohydratemetabolism2.1.1 GlucoseThemetabolismofglucoseiseitherdonethroughtheEmbden-Meyerhof-Parnas(EMP)pathway,alsocalledglycolysis,orthepentosephosphatepathway,alsocalledthephosphogluconatepathway.ThehomofermentativelactobacilliusestheEMPpathwaywhenlacticacidisproducedfromhexoseswhiletheobligatelyheterofermentativebacteriausestheheterolacticphosphogluconatepathway.FacultativelyheterofermentativeLABusestheEMPpathway.(Holzapfel&Wood,2014)AsimplificationoftheglucosefermentationcanbeseeninFigure1wheretheEMPpathway/glycolysisislocatedtotheleftandthepentosephosphatepathwaytotheright.AllthebacteriausedintheexperimentareabletoutilizeGlucoseasacarbonsource.(ProbiAB,2017)

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Figure1.Glucosefermentationinlacticacidbacteria,theEMPpathway/glycolysistotheleftandthepentosephosphatepathwaytotheright.(Butler,etal.,2010)

2.1.2 MaltoseMaltoseiscleavedintoglucoseandb-glucose-1-phosphatewhichisconvertedtoglucose-6-phosphateandcanthenenterthepentosephosphatepathway.However,thehexokinaseresponsibleforthisconversionisnotpresentinexponentiallygrowingcellsinamediacontainingonlymaltose.Thismeansthattheglucosethatisun-phosphorylatedcannotbeutilizedandisexcretedintothemedium.Although,thehexokinaseactivityisbelievedtobeinducedwhenfructoseorglucoseisinthemedium.(Holzapfel&Wood,2014)ThethreebacteriausedintheexperimentsareallabletoutilizeMaltoseasacarbonsource.(ProbiAB,2017)2.1.3 FructoseL.sanfranciscensisandL.pontisareabletoutilizefructoseasacarbonsource.However,whenthereisanoxygendepletionandavailablemaltose,thefructoseismainlyusedasanelectronacceptorandmannitolwillbeproduced.Aceticacidisthemainmetabolicproductwhenthemolarratiobetweenfructoseandmaltoseis4:1.L.sanfranciscensisproducemannitolfromfructosewhileL.pontisproducesethanolandlacticacidinsmallamounts.(Holzapfel&Wood,2014)Thebacteriausedintheexperimentarealsoabletoutilizefructoseasacarbonsource.(ProbiAB,2017)2.1.4 PentoseThepentosesarephosphorylatedtoxylulose-5-phosphateorribulose-5-phosphatewhicharethenmetabolizedinthepentosephosphatepathway.(Holzapfel&Wood,2014)

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2.1.5 CitrateTheutilizationofcitratedependsonatransportergenepresentinanendogenousplasmid.Citratecanbeconvertedintooxaloacetateandacetatewhicharedecarboxylatedintopyruvateanditcanalsobetransformedintolactate.Carbondioxideisproducedduringthebreakdownofcitrate.(Holzapfel&Wood,2014)2.1.6 PyruvateandendproductsSinceLABcanaltertheirmetabolismdependingontheenvironmentinwhichtheylive,thiswillalsoresultindifferentendproducts.Pyruvateisgenerallyusedforthereductiontolacticacid,however,pyruvatecanbeconvertedintomanydifferentendproducts,e.g.diacetyl,ethanolandacetate.Dependingonthelactobacillistrainandtheconditionsinwhichitisunder,differentendproductswillbeproduced.(Axelsson,2004)2.2 Malolacticfermentation(MLF)ManyLABcanconvertmalatetolactateandcarbondioxidewhichisanimportantreactionwhenfermentingfruitsandvegetables,e.g.inwinemaking,sincetheycontainhighconcentrationsofmalate.LAButilizingtheMLFincombinationwiththefermentationofcarbohydratesaregenerallyshowinge.g.highergrowthrates.Thereasonforthisisnotentirelyknownbutithasbeensuggestedthatthedeacidificationoftheexternalenvironmentincombinationwithprovidingsmallamountsofelectronacceptorscreatestheenergybenefits.(Axelsson,2004)2.3 ProteolysisThecapabilityofLABtouseinorganicnitrogentosynthesizeaminoacidsislimitedanditisthereforenecessarytohavepre-formedaminoacidspresentasanitrogensourceinthemediumusedforgrowth.Whichaminoacidsthatneedstobesupplementeddiffersamongdifferentspeciesandstrains.(Axelsson,2004)2.4 LactobacillusrhamnosusLactobacillusrhamnosusbelongstothephylogeneticgroupcasei.Itisfacultativelyheterofermentativeandgrowsat15°Cand45°C.Theyarenon-motile,rodshapedwithasizeof0.8-1.0x2.0-4.0µmwithsquareendsandexistinchainsorsingly.Theydonothydrolysearginineandtheyareureasenegative.(Holzapfel&Wood,2014)L.rhamnosuscanbefoundinthedigestivetractofhumansandwill,aswellasL.paracasei,growspontaneouslyinmilkproductsduringlacticacidfermentation.(Molin,2013)AstudyonL.rhamnosusATCC10863concludedthattheaminoacids,outofthe20added,thathadthehighestutilizationduringgrowthwasCysteine,Serine,AsparagineandGlutamine.(Berry,etal.,1999)L.rhamnosusdoesnotpossesstheenzymetosynthesiseaminoacidsandvitaminBbyitselfwhichmeansthatthatmustbeaddedforsufficientgrowth.(Cui,etal.,2010)ForL.rhamnosusGG,growninawater-basedpuddingcontainingriceflour,maizeflour,fructose,waterandNaClandfermentedfor12hoursat37°C,theutilizationandproductionofcitricacid,acetionandethanolwasinvestigatedafter21daysofstorageat4-6°C.Thestudyshowedadecreaseincitricacidandanincreaseinacetoinandethanol.Theconcentrationofdiacetylduringthefermentationwasincreasedtoaround7mg/kgwhichisabovethethresholdoftastewhichis0.03mg/kg.Diacetylwillgiveaflavourofbutter.Lacticacidwasalsoproducedwhileoroticacidwasconsumed.(Helland,etal.,2004)2.5 LactobacillusparacaseiLactobacillusparacaseiisfacultativelyheterofermentativeandbelongstothephylogeneticgroupcasei.Itgrowsat10°Cand40°Cbutsomestrainsgrowat5°Cand45°C,isrodshaped(0.8-1.0x2.0-4.0µm)withsquaredendsandexistinchainsorsingly.(Holzapfel&Wood,2014)L.paracaseicanbe

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foundinthedigestivetractofhumansbutitalsooccursandgrowsspontaneouslyinthelacticacidfermentationofmilkproducts,e.g.incheese.(Molin,2013)L.paracaseistrain8700:2canutilizeandgrowrapidlyonlong-chaininulinandoligofructosewhichareconsideredprebiotics.Themetabolicendproductsduringgrowthontheseenergysourcesaremainlylacticacidbutalsoaceticacid,ethanolandformicacid.(Makras,etal.,2005)2.6 LactobacillusplantarumLactobacillusplantarumbelongstothephylogenicgroupplantarumwhichisdividedintosixspeciesandsubspecies;L.plantarumsubsp.argentoratensis,L.plantarumsubsp.plantarum,L.paraplantarum,L.xiangfangensis,L.fabifermentans,andL.pentosus.Theyarefacultativelyheterofermentative,growat15°Cbutnotat45°CandhasaG+Ccontentwithinthegroupof44and47mol%.Theyarestraightrodswithasizeof0.9-1.2x3-8µmwithroundedends,non-motileandexistsinglyorinpairsorchains.L.plantarumisrecognisedbyitspseudocatalaseactivity,thereductionofnitrateandalsoitsincapabilitytoutilisea-methyl-D-mannoside.(Holzapfel&Wood,2014)L.plantarumexistspontaneouslyinmostofthefoodsthatarelacticacidfermented,especiallyplantbasedfermentedfood,andwillthereforealsoexistinthedigestivetractofhumans.(Molin,2013)SinceL.plantarumcanpassthroughtheacidicstomachinhumansandoccurspontaneouslyinpHlessthan4.0,itishighlytoleranttolowpH.ThelargegenomeofL.plantarumanditsabilitytofermentalargenumberofcarbohydratesindicatethatitcanadapttodifferentenvironments.ManganeseisrequiredforgrowthofL.plantarumsinceitwillprovideprotectionagainstoxygenradicals.Theradicalswillbereducedtohydrogenperoxidewhichinturnwillbeconvertedtooxygenandwaterbymanganesecofactoredpseudocatalase.Themicroorganismcanbreakuptanninsintoflavonoidsandphenolicacidswhichhashealthbeneficialantioxidantproperties.(Molin,2013)ThecarbonmetabolismofL.plantarumisfairlysimple,generallyprovidinglacticacidasitsmainproduct.However,theyareveryflexibleregardingthesubstrates.AstudyinvestigatinggrowthinfruitandvegetablejuicesconcludedthatthegrowthofL.plantarumstrainsCIL6,C2,POM1,1MR20andCC3M8hadanegativecorrelationwiththeconcentrationsofcarbohydrates(fructoseandglucose)andmalicacid.Ahighconcentrationofcarbohydrates,ine.g.pineapplejuice,leadstoeitheraninefficientmetabolismorcataboliterepression(Filannino,etal.,2014),i.e.thepreventionofexpressionofcatabolicsystemsthatmakestheusageofsecondarysubstratespossiblewhenapreferredcarbonsourceispresent.(Görke&Stülke,2008)Thisisnotoptimalsincethebacterianeedstohaveanequilibriumbetweentheconcentrationsbothinsideandoutsideofthecell.Thestudyobservedthatthebacteriainvegetablejuiceshadsimilarconsumptionofcarbohydratestothebacteriainfavourableenvironments,suchasMRSbroth,comparedtojuiceswhichweremoreacidicandhadhigherconcentrationsoffructoseandglucose,e.g.pineapple.ThismeansthatthestrainsofL.plantarumwillchangeitsmetabolismfromonethatfavoursgrowth,e.g.carbohydratefermentation,toonethatfavoursthemaintenanceofthecells,e.g.malolacticfermentation.ThepresenceofmalicacidwillresultinadecreaseinpH,bothexternalandinternal,andachangeinthepermeabilityofthemembrane.However,theincreasedpHinthecellandthesynthesisofreducingpowerfromthedecarboxylationofmalicacidwillcreateenergyadvantages.(Filannino,etal.,2014) Thestudyalsoshowedthatduringthefermentation,theconcentrationofbranched-chainedaminoacids(Valine,LeucineandIsoleucine)decreasedandmighthavebeenconvertedtobranchedalcohols(2-methyl-1-butanol,2-methyl-1-propanoland3-methyl-1-butanol)sincetheconcentrationofthoseincreased.AlltheL.plantarumstrainshad,whenfermentedintomatojuice,anincreasedlevelofglutamicacidandGABA.ThestudydeterminedthatthefreeaminoacidcatabolismismorepronouncedinahigherpHenvironment,asinthevegetablejuices,whileinanenvironmentwith

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lowerpH,themalolacticfermentationismorepronounced,asinthecherryjuice.ItwasalsoshownthattheaminoacidTyrosineactasastimulatoryagentonL.plantarumgrowthandthatduringstress,thebacteriawillsynthesizealcohols,ketoacids,terpenesandketones.Thefermentedjuicesallcontainedaceticacidanddiacetyl.(Filannino,etal.,2014) GenomesequencingofL.plantarumWCFS1showedafermentationpatternalmostcompletelyhomolacticduringgrowthonglucoseasthecarbonsource.PyruvateisproducedviatheEMPpathwayfromglucoseandthepyruvateisthenconvertedintolactate.ItwasalsoshownthatthisstrainofL.plantarumhadgenesthatencodesenzymesrelatedtotheconversionofpyruvateintoe.g.acetoin,ethanol,formate,2,3-butanediolandacetate.SinceL.plantarumcanutilizeawidevarietyofcarbonsources,itcanbeconcludedthatthebacteriaareveryflexible,versatileandcanadapttomanydifferentenvironments.(Kleerebezem,etal.,2003)L.plantarumarevitaminandaminoacidauxotrophs,whichmeansthattheycannotsynthesizecertainvitaminsandaminoacidthatareessentialfortheirgrowth.AstudybyMaetal.(2016)showedthatforL.plantarumST-III,sixaminoacids(Leucine,Isoleucine,Valine,Methionine,TyrosineandPhenylalanine)andonepurine(guanineoradenine)areessentialforitsabilitytofermentmilk.Inaddition,mineralsaltshadastimulatingeffectongrowthbutwasnotessential.SinceL.plantarumisvitaminauxotrophs,theywillalsoneedvitaminsforgrowth.However,milkisveryrichinvitaminswhichmeansthatnovitaminsneededtobesupplemented.(Ma,etal.,2016)

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3 Substitutingingredientsandfermentationconditions3.1 PlantseedpowderTherehavebeenstudieswheretheuseofvegetalcarbonandnitrogensourceshavebeeninvestigatedtosubstituteconventionalMRSmedium.Plantseedpowderwasusedinsteadofpeptone,beefextractandyeast.ThestudywasdoneonL.lactisanditwasobservedthatthegrowthwasenhancedwhenusingthevegetalsourcescomparedtoMRS.Thesampleseedsusedweremungbeans,chickpea,lentil,Bengalgram,wheatandpeanutpowder.Therecipecontained10goftherespectiveseedpowder,20gGlucose,1gTween-80,2gK2HPO4,5gNa-acetate,2g(NH4)2citrate,0.2gMgSO4-7H2Oand0.05gMnSO4-H2O.Alltheingredientswerethendissolvedindistilledwater(850ml)andthepHwasthenadjustedto6.5afterwhichthemediumwasautoclaved.ThemediumcontaininglentilseedpowdershowedthebestresultforgrowingL.lactis,evenbetterthantheconventionalMRSmedium.(Pathak&Martirosyan,2012)3.2 PeaProteinTheuseofpeaseedasaproteinsourceisgettingmoreattention.Onereasonforthismightbethatithaslessanti-nutritivecomponents,e.g.phyticacid,andislessallergenicthane.g.soybean.Togetapeaproteinhydrolysate,thepeaproteinisolateishydrolyseduntilaspecificdegreeofhydrolysis(DH).TheDHisimportantsinceanexcessinhydrolysiswille.g.causeadeclineinsolubilitywhiletherightdegreeofhydrolysiswillimprovesolubility.Thepropertiesofthehydrolysatewilldependontheenzymedoingthehydrolysis.(Barać,etal.,2015) OnestudygrewL.acidophilus10inMRSbrothwithanadditionofpeaproteinhydrolysateinaconcentrationof1mg/ml.EventhoughthepeaproteinhydrolysatestimulatedthegrowthofL.acidophilusduringthefirstpartofthecultivation,itwasdirectlyfollowedbyadrasticdeclineintotalbacterialnumberattheendofthefermentation.(Świątecka,etal.,2010)3.3 RiceProteinRiceproteinhasahighnutritionalvalueandisahypoallergenicfoodcomponent.However,thereareonlyfewstudiesonthenutritionalvalueofricebranproteins.Onestudyconcludedthatthericebranproteinshowedanutritionalqualitythatwasconsideredsuperiortoothervegetableproteins,suchassoy.However,thebiologicalavailabilityislowerthanproteinsoriginatingfromanimals.(Han,etal.,2015)ThelacticacidproductionofL.rhamnosusNBRC3863wasinvestigatedwhilegrowingonricebran.Thericebranwashydrolysedwithacidandshowedaproductivitysimilarto8g/Lyeastextract.However,thelagphasewasprolongedasmuchasto40hoursforthericebranhydrolysedatpH0.5.AtpH1and2,thelagphasewasshorter.Analternative,suggestedbytheauthorsofthestudy,wouldalsobetocombinethericebranhydrolysatewithyeastextract,e.g.30g/Lricebranincombinationwith3g/Lyeastextract.(Gao,etal.,2008)3.4 IncreasingthetemperatureforL.rhamnosusThelacticacidproductionbyL.rhamnosusgrownonwheypermeatewasinvestigatedinonestudy.Thegrowthtemperaturethatobtainedthehighestcellcount(2.98*1010CFU/mL)were42°Ccomparedto~1.7*1010CFU/mLfor37°C.(Cui,etal.,2010)

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4 MaterialandmethodAlltrialswithdifferentcultivationcharacteristicsweredoneinduplicates,intwofermenters(ProbiAB,Lund,Sweden),withthesameingredients.Onesampleistakenfromeachfermenteronwhichonedilutionseriesisdone.Fromthedilutionseries,0.1mlofthedilutedsampleareputonplatesinduplicates,seeFigure2.

Figure2.Theexperimentalset-upofthefermentationdoneinduplicates,withthesameingredientsintwofermenters.

TheoriginalrecipeusedforfermentationatProbiABcanbeseeninTable1.Therecipeisyeastbased.Table1.TheingredientsusedintheoriginalrecipeatProbiABandthecorrespondingamounts.

Ingredients AmountIngredient1 39.5gIngredient2 1.5gIngredient3 1.5g

Waterwasthenaddedupto600ml.Thecultivationmediumwasthenheatedto90°Cfor15minutesafterwhichthetemperaturewasloweredtofermentationtemperature.ThejuicesusedinthetrialsareLOKAcrush,withstrawberryorraspberryflavour,andmangojuice(Rubicon).Thedoseofprobioticsinthejuicesare5*107/mland1.25*108/mlforLOKAcrushandmangojuicerespectively.ThereasonforthisisthattheestimateddailydoseoftheLOKAis200mlwhilethedoseofthemangojuiceis80ml,whichwillgiveacellcountof10billionprobioticbacteria.4.1 OrganismsandinoculumThebacteriausedinthefermentationwereL.plantarum299v(ProbiAB,Lund,Sweden),ProbiRhamnosus6594(ProbiAB,Lund,Sweden)andProbiParacasei13434(ProbiAB,Lund,Sweden).Thenumberofbacteriaaddeddependsonwhichstrainthatwasused.Avolumeof0.05mlofL.plantarumwasgoingtobeaddedtotheculturemediumcontaining600ml.However,sinceaddingsuchasmallvolumewillincreaseerrors,thebacteriaarediluted1:10whichmeansthat0.5mlshouldbeaddedinstead.ForL.rhamnosus,8.57µlshouldbeadded.Forthesamereasonaspreviouslymentioned,thesampleisdiluted1:100whichmeansthattheaddedvolumeis0.857ml.TheaddedamountofL.paracaseiwas1.333mlaftera1:10dilution.4.2 CultivationconditionsandingredientsThefermentationwasdonebatch-wiseandanaerobicallyinafermenterwithaworkingvolumeof600ml.ThepHandtemperatureweremeasuredwithapHmeter(Mettler-Toledo,Stockholm,Sweden)onlinewithLabVIEW8.6(NationalInstruments,Austin,USA)withtemperature,timeandstirringspeedaccordingtothestandardfermentationsettingsusedatProbiAB.Awaterbath(Julabo,Seelbach,Germany)wasusedtoadjustthetemperature.

Fermenter1

Sample1

Plate1bPlate1a

Fermenter2

Sample2

Plate2bPlate2a

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4.2.1 Varyingingredient2and3Ingredient2andIngredient3,whichareyeastbased,wereused.Therecipeforthecultivationmediumwasdoneaccordingtotheoriginalrecipe,seeTable1,exceptforthevaryingamountsofingredient2and3.ThetrialsweredoneinduplicatesasexplainedinTable2.Table2.Explanationofthetrialswiththeingredientsusedandtheamount.Thetrialsweredoneinduplicates.

Trial Ingredient2(g/600ml) Ingredient3(g/600ml)1 0.5 1.52 1.5 0.53 1.0 1.54 1.5 1.0

4.2.2 Ingredient4Ingredient4,ayeastbasedcomponent,hasbeenrecommendedasasubstitutetobothingredient2and3.Itwasthereforehypothesisedthatingredient4couldbeusedtostimulatesufficientgrowthonitsown.Thecompositionofingredient4canbeseeninTable11intheappendix,section9.4.Ingredient4wasusedinaconcentrationof3g/600mland1.5g/600mlforL.plantarumandtheconcentrationthatachievedthebestresultswasfurthertestedforL.rhamnosusandL.paracasei.Otherwise,theoriginalrecipeasdescribedinTable1wasused.4.2.3 Ingredient5Ingredient5,acomponentbasedonyeast,hasalsobeenrecommendedasasubstitutetobothingredient2and3.Thecompositionofingredient5canbeseeninTable14intheappendix,section9.5.Ingredient5wasusedinaconcentrationof3.0g/600mlforallthebacteriaincombinationwiththeingredients,exceptingredient2and3,presentedinTable1.4.2.4 Combiningingredient3and5Exchangingingredient2withingredient5incombinationwithingredient3hasalsobeenrecommended.1.5gingredient5incombinationwith1.5gingredient3wasusedduringtheexperiment.Duetolackoftime,thefermentationproductwasnotputinjuiceforstoragestabilitymeasurements. 4.2.5 Ingredient6and7Thestudydescribedinthebackground,section3.1,used10gofplantseedpowderinapproximately850ml,whichcorrespondsto7.0g/600mlofingredient6and7respectivelyandingredient1wasalsoaddedtothefermenter,seetheoriginalrecipeinTable1.ThecompositionoftheingredientscanbeseeninTable17intheappendix.4.2.6 Ingredient8Ingredient8,aplantbasedcomponent,wasusedinaconcentrationof3.0g/600ml.Ingredient8wasusedincombinationwithingredient1andwateraspresentedinTable1.4.2.7 Ingredient93.0g/600mlofingredient9,aplantbasedcomponent,wasaddedtothecultivationmedium.Ingredient2and3intheoriginalrecipeinTable1wassubstitutedwithingredient9.4.2.8 IncreasingthefermentationtemperatureforL.rhamnosusThetemperaturewasincreasedto42°C,whichisthetemperaturethatthestudyinthebackground,section3.4,suggested.Thefermentationwasdonewiththeoriginalrecipe,aspresentedinTable1.Duetolackoftime,thefermentationproductwasnotputinjuiceforstoragestabilitymeasurements.4.2.9 IncreasingthefermentationtimeforL.paracaseiDuringtheexperiments,itwasobservedthatthelag-phaseofL.paracaseiwasapproximately40%longerthanfortheothertwobacteria.Hence,anexperimentwithafermentationtimethatwas12.5%longerthanthestandardfermentationtimeusedatProbiABwasadded.Thefermentationwasdonewiththeoriginalrecipe,Table1.Duetolackoftime,nostabilitymeasurementsinjuicewasdoneforthisexperiment.

15

4.3 PlatecountanalysisWhenanalysingthebacterialcountinthefermentationproductandinthejuice,thebacteriaaredilutedusingDilucups(labrobot,Stenungsund,Sweden),containing9mlpeptonewater,where1mlofsampleistransferredbetweenthecupswith1mlpipettes(Mettler-Toledo,Stockholm,Sweden)and1mltips(Mettler-Toledo,Stockholm,Sweden)andmixedusingaDilushaker(labrobot,Stenungsund,Sweden)ataspeedof400rpmfor3seconds.Dilutions-5,-6and-7(105,106and107)forthefermentationproductand-4,-5and-6(104,105and106)forthejuiceisthenplatedwitha0.1mlpipette(Mettler-Toledo,Stockholm,Sweden)and0.1mltips(Mettler-Toledo,Stockholm,Sweden)onMRS(deMan,RogosaandSharpe)agarplates(Biomérieux,MarcyI’Etoile,France)andspreadwithaninoculationspreader(Sarstedt,Nümbrecht,Germany).TheplatesarethenputanaerobicallyusingananaerobicgeneratorcalledGENboxanaer(BioMérieux,MarcyI’Etoile,France)andanaerobicindicator(ThermoScientific,Basingstoke,UnitedKingdomorBioMérieux,MarcyI’Etoile,France)andincubatedat37°Cforapproximatelythreedays.Aftertheincubation,theplatesarecountedwheretheexactnumberofcoloniesarenotedif>400,andtheconcentrationofbacteriaiscalculatedontheplatesthathaveacolonycountbetween25and300.Iftwoplatesarewithin25-300colonies,thenumberofcoloniesisaddedanddividedby1.1,andtheresultcorrespondstothelowestdilution.Theresultisthendividedby0.1totakeintoaccounttheaddedamountontheplates.Anexampleofthecalculationcanbeseenintheappendix,section9.1.Theerrorofthemethodofanalysisis20%.4.4 StoragestabilityThestoragestabilityofthefermentationproductandthemixtureoffermentationproductandjuicewasinvestigated.ThestabilityinthefermentationproductisconsideredunacceptableifthelogCFU/mlhavedecreasedbelow7.0uponitsexpirationdatesincethatistheleastamountofbacteriasufficientforaprobioticdrinktobeeffective.Thehighnumberisbecauseamanyoftheprobioticbacteriadieduringthepassagethroughthebody.(Nualkaekul&Charalampopoulos,2011)ThecriteriaforthefermentationproducttobeputinjuiceisthattheCFU/mlis>5*108or8.7logCFU/ml.Thestabilityofthefermentationproductwasevaluatedatanaverageof3.7°C,seeTable7intheappendix,attime0and7dayswhilethejuicemixturewasevaluatedattime0,2and4weeks.Unfortunately,threeweeksintothelabwork,therefrigeratorinwhichthesampleswerestoredbrokeovertheweekend.Thismighthaveresultedinupto48hoursofstorageinroomtemperature(21.8°C).Toevaluatepossibleeffectsofthestorageinhighertemperature,allsampleswereevaluatedoncemoreupondiscoveryofthebrokenrefrigerator.Thismeansthatanotherdatapointotherthanthe0and7daysforthefermentationproductand0,2and4weeksforthejuicescanbeobservedforthesamplesaffectedbythebreakage.Allsampleswereafterthispointstoredatanaverageof4.3°C,seeTable8intheappendix,duringtheentireexperiment.4.5 SensoryevaluationAsimplesensoryevaluationwasdonewithtwoparticipantstoevaluateifthejuicescontainingthefermentationproductwasconsideredacceptabletoconsumers.Sincetheaimofthesensoryevaluationwastoevaluateconsumeracceptance,thetestfocusedonthreetastecategories,good,badandacceptable.4.6 StatisticsComparingthebacterialcontentinCFU/mlforthesamplescontainingthedifferentingredientswiththestandardrecipe,Table1,isdonebyusingstatistics.ThestatisticsisinturndonebyusingKruskal-WallisOneWayANOVAonRanks,whichisnon-parametricandcanthereforenotbeassumedtobenormallydistributed,wheninvestigatingifthereisasignificantdifferencebetweentheingredientsusedinthefermentationforallthreebacteriarespectively.Ifso,twoingredientsmustbecomparedwitheachothertoinvestigatewhichingredientwassignificantlydifferentfromtheoriginalrecipe.Thisisdonebyusingamultiplecomparisonprocedure,Student-Newman-Keuls.Whenp<0.05,thereisasignificantdifference.

16

5 ResultanddiscussionIngredient2and3inthefermentationofprobioticsmaygiveoff-flavourstothefruitdrink.Thefollowingtrialswillinvestigatewhetheritispossibletoeitherlowertheamountorexchangingthemforanotheringredientandstillgetanacceptablegrowthandstoragesurvival.5.1 pHThepHduringthefermentationofL.plantarumincultivationmediumcontainingthedifferentingredientscanbeseeninFigure3.Thevaluesareanaverageoffermentersdoneinduplicatesforeachoftheingredients.ThefermentationofL.plantarumwasdonewithdifferentratiosofingredient2and3asdescribedinTable2.

Figure3.ThepHduringthefermentationofL.plantarumincultivationmediumcontainingdifferentingredients.Thelinesareanaverageoftwofermentersdoneinduplicatesforeveryingredient.

Fromthefigure,itcanbeseenthattheingredient3andingredient4hasahigherpHthane.g.theingredient2,resultinginahigherstartingpHforthetrialsthatcontainhighercontentsofthoseingredients.DespitethehigherinitialpH,thefinalpHisapproximatelythesame(around3.4),indicatingthattheinitialpHdoesnotmatterthatmuch.Forthetrialscontainingingredient6or7,thepHdidnotdecreasetothesamelevelsasfortheothertrials.Thismaybeduetolesslacticacidbeingproducedwhichinturnindicatesthatthereisalowercontentofbacteriainthefermentationproduct.Thelag-phaseforthetrialcontainingingredient9islongerthanfortheothertrialsforL.plantarum,around58%longerthanthelag-phaseofingredient2,3,4,5,6and7and91%longerforthelag-phaseofingredient8.Thismaybeduetotheunavailabilityofnutrientsiningredient9orthattheutilizationofnutrientsismoredifficult. ThepHduringthefermentationofL.rhamnosusincultivationmediumcontainingdifferentingredientscanbeseeninFigure4.Thevaluesareaverageswhichoriginatesfromduplicatefermenters.SincethebacterialcontentofL.plantarumincultivationmediumcontainingacombinationofingredient2and3,wasthehighestforthetrialwith0.5gand1.0gingredient2incombinationwith1.5gingredient3(seesection5.2.1below)and3.0gingredient4(seesection5.2.2),thosearetheonesthatarefurthertestedonL.rhamnosus.

3

3,2

3,4

3,6

3,8

4

4,2

4,4

4,6

4,8

5

pH

Time(h)

pHduringthefermentationofL.plantarumwiththedifferentingredients

0.5gingredient2+1.5gingredient3

1.5gingredient2+0.5gingredient3

1.0gingredient2+1.5gingredient3

1.5gingredient2+1.0gingredient3

3.0gingredient4

1.5gingredient4

3.0gingredient5

7.0gingredient6

7.0gingredient7

3.0gingredient8

3.0gingredient9

1.5gingredient3+1.5gingredient5

17

Figure4.ThepHduringthefermentationofL.rhamnosusincultivationmediumcontainingdifferentingredients.Thelinesareanaverageoftwofermentersdoneinduplicatesforeveryingredient.

Forthetrialscontainingacombinationofingredient2and3,asmallincreasecanbeobservedattheendofthelag-phasebeforethemajordecreaseinpH.ThismightbeduetotheutilizationofnutrientsthathavealowpHortheproductionofalkalineproductsbythebacteria.Aspreviouslydiscussed,thepHforthetrialscontaininghighconcentrationsofingredient3ishigherduetothemorealkalinenatureofthesecomponentsbutthepHwilldespitethatbereducedtothesamelevelasfortheothertrialscontainingingredient2,3,4and5.Allthetrialscontainingingredients2,3,4and5reachesafinalpHofaround3.3-3.4whilethetrialscontainingingredients6,7,8and9areabithigherinfinalpH.Forthetrialscontainingingredient6and7,thefinalpHdidnotdecreasetothesamelevelasforL.plantarum,indicatingthegrowthandproductionofacidsbyL.rhamnosuswasnotasefficientintheseingredientsforL.plantarum.ThefermentationofL.rhamnosusiningredient7hadaslightlyhigherendpH(around3.85)thanthefermentationwithingredient6(around3.75).Forthetrialcontainingingredient9,therewasnodecreaseinpHduringthefermentationwhichmeansthatthebacteriadidnotproduceanyacid.Itwasthereforeassumedthattherewasnogrowthduringthefermentation.However,toverifytheseresultsandmakesurethattherehasnotbeenahumanerror,thetrialwasredone.ThesecondattemptonthefermentationofL.rhamnosusincultivationmediumcontainingingredient9showedthesamepatternforthepHasduringthefirstfermentationwhichverifiestheresultthattherewasnoproductionofacids. Thestudydescribedinthebackground,section3.4,suggestedthatanincreaseintemperatureto42°CwouldincreasethebacterialcontentforL.rhamnosus.However,lookingatthepH,thecurve

3

3,2

3,4

3,6

3,8

4

4,2

4,4

4,6

4,8

5

pH

Time(h)

pHduringthefermentationofL.rhamnosuswithdifferentingredients 1.5gingredient2+1.5gingredient3

0.5gingredient2+1.5gingredient3

1.0gingredient2+1.5gingredient3

3.0gingredient4

3.0gingredient5

7.0gingredient6

7.0gingredient7

3.0gingredient8

3.0gingredient9

1.5gingredient2+1.5gingredient3,increasedtemperature

1.5gingredient3+1.5gingredient5

18

looksalmostidenticaltothetrialdonewiththesameingredientsbutatthestandardtemperature,indicatingthattheproductionofacidshadnotincreasedduetotheincreaseintemperature. ThepHduringthefermentationofL.paracaseiincultivationmediumcontainingdifferentingredientscanbeseeninFigure5.Thevaluesoriginatefromtwofermentersandarethereforeaverages.AsforL.rhamnosus,theconcentrationsofingredient2and3thatareusedforL.paracaseiaretheonesthatgavethehighestbacterialcontentforL.plantarum,0.5gand1.0gingredient2incombinationwith1.5gingredient3and3.0gingredient4.

Figure5.ThepHduringthefermentationofL.paracaseiincultivationmediumcontainingdifferentingredients.Thelinesareanaverageoftwofermentersdoneinduplicatesforeveryingredient.

ThepHcurvesshowasimilarincreaseinpHattheendofthelag-phaseandbeforethemajordecreaseinpHasforL.rhamnosus.Asdiscussedpreviously,thismightbetheresultoftheutilizationofacidicnutrientsortheproductionofalkalineproductsbythebacteria.Inaddition,thelagphaseofL.paracaseiseemstobelongercomparedtotheotherbacteria,around40%longerthanthelag-phaseoftheotherbacteriaandthefinalpHseemstobeabithigher.ThefinalpHisaround3.6forthetrialscontainingingredient2,3,4and5andaround3.8foringredient6,7and8.Thelag-phaseinthetrialcontainingingredient9wassignificantlylongerthanfortheothertrials,resultinginthatthebacteriadidnothavetimetogrowforaslongtimeasfortheothertrials.Asdescribedpreviously,thelag-phaseduringthefermentationofL.paracaseiwasgenerally40%longerthanfortheotherbacteriaanditwasthereforeofinteresttotryatotalfermentationtimethatwas12.5%longerforL.paracaseithanfortheotherbacteria.Theprofileofthecurvelooksthesameasfortheotherfermentationswithingredient2and3andtheendpHwasslightlylower,indicatingthattheremighthavebeenanincreaseinbacterialnumbercomparedtothestandardfermentation.

3

3,2

3,4

3,6

3,8

4

4,2

4,4

4,6

4,8

5

pH

Time(h)

pHduringthefermentationofL.paracaseiwithdifferentingredients

1.5gingredient2+1.5gingredient3

0.5gingredient2+1.5gingredient3

1.0gingredient2+1.5gingredient3

3.0gingredient4

3.0gingredient5

7.0gingredient6

7.0gingredient7

3.0gingredient8

3.0gingredient9

1.5gingredient2+1.5gingredient3,12.5%longerfermentation1.5gingredient3+1.5gingredient5

19

5.2 GrowthandstabilityThebacterialcountforallthebacteriaandthestabilityafteroneweekofstoragecanbeseeninTable3.Alltrialsweredoneintwofermentersinwhichtheplatingwasdoneinduplicates,providinganaverageforeachingredient.Thetrialusing1.5gingredient2and1.5gingredient3,whichistheoriginalrecipe,Table1,forL.plantarumwaspreviouslydoneatProbiAB.Thesamplesthatareputinjuicearewritteninboldaswellasthesamplesthatshouldhavebeenputinjuicebutduetolackoftime,wasnot.SomeofthesamplescontainingL.plantarumandL.rhamnosuswereaffectedbythebrokenrefrigerator,resultinginanadditionalmeasurementatday4and3,respectively.SincethebacterialcontentofL.rhamnosusinthefermentationproductcontainingingredient9was0logCFU/ml,nostabilitymeasurementwillbedoneforthosesamples.ThestabilityofthesamplecontainingL.paracaseiand1.5gingredient3and1.5gingredient5after7dayswasmeasuredafter6daysinstead. Table3.TheaveragelogCFU/mlwiththestandarddeviationfromthefermentationofL.plantarum,L.rhamnosusandL.paracaseiforthedifferenttrialsandthestabilityafteroneweekofstorage.Theaverageisoftwofermentersonwhichduplicateplateswerecounted,i.e.4valuespercombination.Themeasurementsduringday3and4wasdoneduetothebreakageoftherefrigerator.Thesamplesthataresignificantlydifferentaremarkedwithastar,*=p£0.05,**=p£0.01and***=p£0.001comparedtotheoriginalrecipe.Thesamplesthatareputinjuiceaswellasthesamplesthatshouldhavebeenputinjuiceiftherewasmoretimeavailablearewritteninbold.

GrowthinlogCFU/ml±standarddeviation

L.plantarum L.rhamnosus L.paracaseiDay0 Day4 Day7 Day0 Day3 Day7 Day0 Day7

Originalrecipe:1.5gingredient2+1.5gingredient3

8.9 - - 8.8±0.11

8.7±0.10

8.8±0.09

8.4±0.03

8.4±0.05

0.5gingredient2+1.5gingredient3

8.8±0.07

- 8.5±0.13

8.7±0.05*

8.8±0.04

8.7±0.11

8.3±0.05***

8.3±0.04

1.5gingredient2+0.5gingredient3

8.5±0.05**

- 8.3±0.04

- - - - -

1.0gingredient2+1.5gingredient3

8.8±0.06

- 8.6±0.04

8.7±0.13*

- 8.7±0.08

8.3±0.02

8.3±0.10

1.5gingredient2+1.0gingredient3

8.7±0.08**

- 8.5±0.06

- - - -

1.5gingredient4 8.5±0.05**

- 8.7±0.01

- - - - -

3.0gingredient4 8.8±0.14

- 8.4±0.03

8.9±0.07

- 8.8±0.06

8.4±0.07

8.4±0.01

3.0gingredient5 8.9±0.06

- 8.7±0.09

8.6±0.13*

- 8.6±0.10

8.4±0.03

8.2±0.11

1.5gingredient3+1.5gingredient5

8.8±0.08

- 8.6±0.13

8.8±0.10

- 8.7±0.08

8.5±0.09

8.7±0.17

7.0gingredient6 8.4±0.07**

8.4±0.07

8.5±0.12

8.1±0.30*

- 8.2±0.15

8.5±0.07

8.6±0.10

7.0gingredient7 8.3±0.10**

8.2±0.53

8.5±0.06

8.6±0.13*

- 8.5±0.21

8.6±0.05

8.6±0.10

3.0gingredient8 8.7±0.15*

- 8.7±0.11

8.4±0.04*

- 8.3±0.02

8.2±0.01**

8.1±0.06

3.0gingredient9 8.6±0.07**

- 8.6±0.06

0±0**

- - 8.3±0.04

8.7±0.07

20

ThehighestcellcountforL.plantarum,otherthanthetrialwith1.5gingredient2and1.5gingredient3whichistheoriginalrecipe,wasobtainedwith1.0gingredient2and1.5gingredient3.Whendecidingwhichofthesamplestoputinjuice,thetwosampleswiththehighestcellcountwerechosen,i.e.thetrialwith0.5gingredient2and1.5gingredient3andtheonewith1.0gingredient2and1.5gingredient3,whicharealsotheonesthatareclearly>8.7logCFU/mlwhichisthecriteriaforputtinginjuice.Thetrialwith1.5gingredient2and1.0gingredient3seemstobeabove8.7logCFU/mlbutwasnot.Itappearsasthecontentofingredient3hasthehighesteffectonthegrowthofthebacteria,asseeninTable3forL.plantarum.I.e.theconcentrationofingredient2canbeloweredwhiletheconcentrationofingredient3shouldstaythesame.However,itcanalsobeseenthatahigherconcentrationofingredient2willhaveapositiveeffectonthegrowth.Thetrialsthatgavethehighestbacterialcountwasfurthertestedwiththeotherbacteria.ThestabilityofL.plantaruminallthefermentationproductscontainingingredient2,3,4and5isconsideredacceptablesincethebacterialcontentisabove7.0logCFU/ml,asdescribedsection4.4.Itseemslikethebacteriainthefirsttrial,i.e.theonewith0.5gingredient2and1.5gingredient3,hadthelowestsurvivalrate.However,oneoftheduplicateplatesfromthistriallostanunreasonablyhighamount,whichcanbeseenatthehighstandarddeviationandmightsuggestanerrorineithertheinitialorthefinalvalueforthatsample.Inaddition,thetwovaluesforday0andday7fromeachduplicatesampleoriginatingfromonefermentercamefromdifferenttesttubes,whichmayindicatethattherearesomedifferencesdependingonwhichtesttubethatisused.Asuggestionmightthereforebetohavealargersamplethatallsamplesformeasurementscanbeobtainedfrom. Thetrialwith1.5gingredient2and1.5gingredient3forL.rhamnosusishighenoughtobeputinjuiceaswellasoneoftheduplicatefermentersfortrial1.0gingredient2and1.5gingredient3.Thevaluesforday3havenotincreasedthatmuch,consideringthestandarddeviationandthelargeerrorofthemeasuringmethod,whichmeansthatfurthermeasurementscanbedoneonthesesamplesandcanthereforecontinueuntilday7.Thesampleshavestayedapproximatelythesameinbacterialcountduringthestorageweekandasdiscussedpreviously,thestoragestabilityisconsideredgood.Ascanbeseeninthetable,thetrialwith3.0gofingredient4hadhigherconcentrationofL.plantarumthanthetrialwith1.5gingredient4.Since3.0gingredient4gavethebestresults,thatwastheconcentrationthatwasfurtherusedfortheotherbacteria.Forthetrialwith3.0gingredient5,onlyoneduplicatefermentercontainingL.rhamnosusqualifiedtobeputinjuicewhiletheotherwasbelowthelimit,resultinginanaveragebelowthelimitaswell.Sincenoneofthefermentationproductshavedecreasedonelog,thesamplescanbeconsideredstableduringthestoragefor7days.L.paracaseididnotgrowaswellasexpected,seethetableabove.ItwashypothesizedthatthebacterialcontentwouldbeinapproximatelythesamerangeasforL.plantarumandL.rhamnosus.Thelongerlag-phaseofL.paracaseimightresultinalowerbacterialcontentwhenfermentedforaslongtimeasfortheotherbacterialspecies.Sincethelagphaseislonger,asuggestionmightbetoincreasethefermentationtimewith12.5%togetalog-phasethatisapproximatelythesameasfortheothertwobacteria.Thisistotrytogetthesamebacterialcountasfortheotherbacteria.AnotheralternativeisthattheseresultsindicatethattherearesomenutrientsessentialforgrowthforL.paracaseithatismissingintheculturemedium.ThecontentofL.paracaseiwasverylowforallingredientsandnoneofthesamplesqualifiedtobeputinjuice.Thestoragestabilityofthefermentationproductcanbeconsideredacceptable.Theincreaseofthebacterialcontentinsomeofthesamplescanbeconsiderednegligiblesincetheerrorofthemeasuringmethodissolarge.ThestudybyPathaketal.(2012)suggestthatacultivationmediumcontaininghighproteinvegetalsourcescanbeasubstituteforMRSmediumanditisthereforehypothesisedthatsuchsourcescan

21

beusedinsteadofingredient2and3inthemediumcurrentlyusedatProbiAB.However,ascanbeseeninthetable,noneofthesamplescontainingingredient6or7qualifiedtobeputinjuicesincethebacterialcontentwastoolow.Thereasonforthismightbethatthenutrientsintheseingredientsarenotasavailableforutilizationforthebacteriaasaretheothernitrogensourcesusedintheexperimentalwork,eventhoughtheproteincontentishigh.Inaddition,asmentionedabove,thelag-phasewaslongerwhichmeansthatahighercellcountmightbeobtainableifthefermentationcouldgoonforalongeramountoftime.However,ingredient6and7providedthehighestgrowthofL.paracasei,evenhigherthanthemediumcontainingingredients2,3,4and5.Generally,forL.plantarumandL.rhamnosus,theplantbasedingredients,i.e.ingredient6,7,8and9,seemstoresultinaslightlylowerbacterialcontent.Thiscanalsobeobservedsincetheseingredientsaresignificantlylowerthantheoriginalrecipe.ThereasonforthatmightbethatthereissomenutrientmissingthatisimportantforgrowthforL.plantarumandL.rhamnosusorthatthebiologicalavailabilityofthenutrientsislowerthanfortheotheringredients.InthestudybyPathaketal.(2012),theauthorsconcludedthatamediumcontainingvegetalseedpowderprovidedbettergrowththanconventionalMRSmedium.Inthisexperiment,nocomparisonbetweenMRSandtheusedingredientsweredone.Thestudyratherprovideevidencethatitwouldbepossibletogrowprobioticsinmediumcontainingingredient6and7,whichitevidentlyis.However,thegrowthwasnotasgoodashoped.Thismightbeduetothemanydifferencesbetweenthestudyandtheexperimentalwork.Forexample,theprobioticusedinthestudywasL.lactiswhichmostlikelybehavesdifferentlythanthebacteriausedinthisexperimentandtheingredientsinthemediumusedinthestudycontainedadditionalingredients,e.g.citrateandacetatewhichmightinfluencethegrowth.Inaddition,thefermentationwasdonefor72hoursandthegerminationoftheseedspriortopowderingwasmonitoredinthestudywhichwasnotthecasewiththeingredientsinthisexperiment,providingdifferentconditionsrightfromthestart.Allthesamplescontainingingredient6and7areconsideredstablesincethereisnosamplethatdecreasemorethanonelog-unit,andnonearebelow7.0logCFU/ml.ForL.plantaruminingredient7,thebacterialcontentduringday4decreased,whichwasnotexpected.However,thestandarddeviationisveryhigh,indicatingthattheremightnothavebeenadecreaseatall.Thereasonforthehighstandarddeviationcouldbethatoneoftheplatesforoneofthefermentershadasignificantlylowerbacterialcontent,i.e.7.3logCFU/mlcomparedto8.4,8.4and8.7fortheotherplates.Thetrialcontainingingredient6andL.rhamnosushasalsoahighstandarddeviation,higherthanisacceptable,i.e.>0.2.Ingeneral,thestandarddeviationofsomeofthevaluesinthetableareabithigh,whichisaresultofthatthefermentersdifferedbetweenoneanotherdespitecontainingthesameingredients.Thismightbeexplainedbytheadditiontheingredientswhich,duetotheaccuracyofthescale,willdifferslightlyamongthefermenters,providingdifferentamountsofnutrientsbeingadded. ThestudybyGaoetal.(2008)showedthattheproductionoflacticacidwith30g/Lacid-hydrolyzedricebrangaveasgoodresultsaswith8g/Lyeastextract.Inthisexperiment,3.0g/600mlingredient9wasusedtoinvestigatethepossibleimpactoftheingredientongrowth.Thegrowthwithingredient2,3,4and5wasslightlyhigherthaningredient9forL.plantarumwhenthesameconcentrationofeachingredientwasused.AssuggestedbyGaoetal.(2008)itmightbebeneficialtocombineingredient9withasmallamountofyeasttoincreasethegrowth,givinganevenhighergrowththanwhenonlyyeastisused.However,therearesomedifferencesbetweenthestudyandthisexperiment.Thericeusedinthestudywasacid-hydrolyzedricebranatpH0.5,1and2,whileingredient9usedinthisexperimentwasnotmadeofbran.Inaddition,thefermentationinthestudyweredoneat42°Cwithadditionofnitrogengasandthefocusofthestudywastheproductionoflacticacidandnotthegrowthofprobiotics.However,asthestudyconcluded,itispossibletogrowbacteriaonricebutahigherbacterialcountmightbeobtainedwhencombinedwithyeast.

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Thereasonforthelowernumberofbacteriainingredient9mightbethatnutrientsthatareessentialforgrowthwasnotpresentorthatthebiologicalavailabilityoftheproteinswasnotashighasinproteinsfromanimalsources,assuggestedbyHanetal.(2015).However,thecontentofL.rhamnosuswasunexpectedlylowandthefermentationwasthereforerepeatedtoverifytheresults.Thesecondfermentationdidnotshowanycolonies,asforthefirstfermentation,whendilutedaccordingtothedilutionseriesexplainedinmaterialandmethod.Theoretically,thereshouldbeasmanybacteriapresentinthefermentationproductasaddedbeforethefermentationifnogrowthoccurred.Sincetherewerelessbacteriapresent,thebacteriahaveprobablydiedoffduringthefermentation.Despitethelonglagphase,whichwasalsoobservedinthestudybyGaoetal.(2008),andtheshortperiodofactualgrowthduringthetrialwithL.paracaseiandingredient9,thebacterialcontentwasstillinthesamerangeasforingredient8whichdidnothaveaslonglagphase.ThismightsuggestthatthebacteriadidgrowbutdidnotproduceasmuchacidduringthattimetoreducethepHorthatingredient8wasnotaseffectiveasingredient9insupportinggrowth,resultinginashighgrowthforingredient9asfor8despitethelonglagphaseforingredient9.Thestabilityofthefermentationproductcontainingbothingredient8and9isconsideredgood.Thesamplecontainingingredient8andL.plantarumstaysapproximatelythesameduringthestorageweekwhilethesamplescontainingL.rhamnosusandL.paracaseidecreases.However,thedecreaseisverysmallconsideringthelargeerrorofthemeasuringmethod.ThecontentofL.paracaseiinthefermentationproductcontainingingredient9seemstoincrease,despiteconsideringthestandarddeviation,suggestingthatthebacteriamightcontinuetogrowduringthestorageorthatahumanerrorwasmade.ComparingalltheusedingredientswitheachotherwithANOVAforeachofthebacteriaseparately,therewasasignificantdifferencebetweenthesamplessincep<0.05.ForL.plantarum,thesamplesthathadasignificantdifferenceregardinggrowthcomparedtotheoriginalrecipewas1.5gingredient2+0.5gingredient3,1.5gingredient2+1.0gingredient3,1.5gingredient4,ingredient6,7,8and9.Whencomparingthesesamplesthatshowednosignificantdifferencefromtheoriginalrecipewitheachother,nosignificantdifferencecouldbefoundbetweenthem,indicatingthatthesesixsamplescanbeconsideredequallysuccessfulinsupportinggrowth.Aspreviouslydiscussed,itcanalsobeseenfromthestatisticsthatloweringtheconcentrationofingredient3and4willresultinasignificantlylowergrowthcomparedtoloweringtheconcentrationofingredient2.ForL.rhamnosus,3.0gingredient4and1.5gingredient3+1.5gingredient5gavenosignificantdifference,i.e.theseingredientsgaveagrowththatcanbeconsideredasgoodastheoriginalrecipewhiletherestofthesamplescanbeconsiderednotassuccessfulinsupportinggrowth.ForL.paracasei,itcanbeobservedthattheoriginalrecipewasonlysignificantlydifferentfrom0.5gingredient2+1.5gingredient3andingredient8.Thatmeansthatcomparedtotheoriginalrecipe,allotheringredientswereasgoodwhenconsideringgrowth.However,sincetheoriginalrecipeisnottheingredientthatgavethehighestbacterialcount,itisalsoofinteresttoalsoinvestigateifasignificantdifferenceisobtainedwhenthebestingredient,howevernotsignificantlythebest,iscomparedwiththeotheringredients.Theingredientthatgavethehighestbacterialcontent,i.e.ingredient7,hadasignificantdifferencewithingredient5,8,9,1.0gingredient2+1.5gingredient3and0.5gingredient2+1.5gingredient3.Thatmeansthatingredient7wassignificantlybetterthantheseingredients.Asfortheothersamples,nosignificantdifferencecouldbeobservedandtheingredientscanthereforebeassumedtobeasgoodatsupportinggrowthasingredient7.

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ThebacterialcontentandthestabilityafteroneweekofstorageinthefermentationproductcontainingL.rhamnosus,fermentedatthestandardtemperatureor42°CcanbeseeninTable4.Thecultivationmediumcontained1.5gingredient2and1.5gingredient3asaccordingtotheoriginalrecipeinTable1.Table4.TheaveragelogCFU/mlwiththestandarddeviationandthestoragestabilityforthefermentationproductcontainingL.rhamnosusfermentedatstandardtemperatureand42°Cincultivationmediumcontaining1.5gingredient2and1.5gingredient3.Thetrialwasdoneinduplicatesintwofermentersandinfromwhichtwoplateswerecounted.Thesamplesthataresignificantlydifferentaremarkedwithastar,*=p£0.05,**=p£0.01and***=p£0.001comparedtotheoriginalrecipeatstandardtemperature.

ConditionsduringthefermentationofL.rhamnosus

AveragelogCFU/ml±standarddeviation,day0

AveragelogCFU/ml±standarddeviation,day7

Standardtemperature 8.8±0.11 8.8±0.0942°C 8.3±0.10** 8.4±0.07

Thebacterialcontentofthefermentationproductwassignificantlylowerwhenfermentedat42°C.Itwasthereforenotadvantageoustoincreasethetemperatureduringthefermentation,despitetheresultsobtainedinthestudybyCuietal.(2010)Thatmightbeduetothedifferenceinbehaviourofthestrains,sincethestrainusedinthestudywasnotthesameasthestrainusedinthisexperiment.Inaddition,thebacteriainthestudygrewonwhey,whichhasacompletelydifferentnutritionalcontentthanthecultivationmediumusedinthisexperiment.Also,thedifferenceinbacterialcontentbetween37°Cand42°CinthestudybyCuietal.(2010)wasnotmorethanaround0.24logCFU/mlwhichisnotasignificantdifference.Inconclusion,thebacterialstrainusedinthisexperimentdidnotbenefitfromaraisedtemperaturebutgavealowerbacterialcontent.Thestabilityisacceptablesincethebacterialcontentdoesnotdecreasebelow7.0logCFU/ml.Theincrease,however,canbedisregardedsincethereisanoverlapbetweenday0and7whenthestandarddeviationofthevaluesisconsidered. ThecontentofL.paracaseiinmediumcontaining1.5gingredient2and1.5gingredient3accordingtotheoriginalrecipeinTable1thatwasfermentedfor12.5%longeraswellasforstandardtimecanbeseeninTable5.Thestabilityforthesamplefermentedforlongertimewasmeasuredafter6daysinsteadof7.Table5.TheaveragelogCFU/mlwiththestandarddeviationforthefermentationproductcontainingL.paracaseifermentedforstandardtimeand12.5%longerwith1.5gingredient2and1.5gingredient3.ThetrialwasdoneinduplicatesintwofermentersandtheaverageCFUoriginatedfromtwoplates.Thesamplesthataresignificantlydifferentaremarkedwithastar,*=p£0.05,**=p£0.01and***=p£0.001comparedtotheoriginalrecipe.

ConditionsduringthefermentationofL.paracasei

AveragelogCFU/ml±standarddeviation,day0

AveragelogCFU/ml±standarddeviation,day7/6

Standardtime 8.4±0.03 8.4±0.0512.5%longerfermentation 8.5±0.09 8.6±0.20

Asmallincreaseinbacterialcontentcanbeobservedwhenthetotalfermentationtimeisincreasedwith12.5%.Ifthestandarddeviationisconsidered,thereisnooverlapbetweenthesamples,suggestingthatthelongerfermentingwillincreasethebacterialcontent.However,thereisnosignificantdifferencebetweenthesamples,indicatingthatincreasingthefermentationwith12.5%willnotgiveasignificantlyhigherbacterialcontentbutacontentinthesamerangeasthestandardfermentation.Inaddition,theincreaseissosmallthatitwillnotbebeneficialtoincreasethefermentationtimeconsideringtheextratimeandcostthatwillbeaddedduringtheproduction.Theincreaseinbacterialcontentafter6daysofstoragecouldinfactbeadecreaseduetothelargestandarddeviation.

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5.3 Stabilityinjuice5.3.1 Varyingtheconcentrationofingredient2and3ThesurvivalofL.plantaruminthejuicecontainingvaryingconcentrationsofingredient2and3waseffectedbythebreakageoftherefrigerator,resultinginahighervalueforweek2comparedtoweek1,seeTable6.ThesurvivalofL.plantarumwithingredient4inthejuicewasalsoeffectedbythebreakageoftherefrigerator,resultinginanadditionalvalueatweek1.5.However,theincreaseisnotashigh,consideringthebigerrorinthemeasuringmethod,whichmeansthatthestorageanalysiscancontinuewiththelastmeasurementatweek4.ThefermentationproductcontainingL.rhamnosuscontainingvaryingconcentrationsofingredient2and3willnotbeaseffectedbythebrokenrefrigeratorsincetheinitialbacterialcountdoesnotmatterasmuchsincethestoragestabilitywillbeevaluatedrelativetotheinitialvalue.Sinceoneoftheduplicatefermentershadabacterialcontentbelowthelimittobeputinjuice,thevaluesfortrial1.0gingredient2and1.5gingredient3arebasedononefermenterwithduplicateplates.ThestabilityofthetrialscontainingL.plantarumorL.rhamnosusputinjuicecanbeseeninTable6.Table6.ThestabilityofthefermentationproductcontainingL.plantarumorL.rhamnosusandthedifferentingredientsinLOKAandMangojuiceduring4weeksofstorage,presentedinaveragelogCFU/mlwiththestandarddeviationforduplicatefermentersonwhichduplicateplateswerecounted,exceptforthetrialwith1.0gingredient2and1.5gingredient3andingredient5forL.rhamnosusonwhichonlyonefermenterwithduplicateplateswereputinjuice.Astandarddeviationof0meansthatthetwoduplicateplateshadthesamenumberofcolonies.Thebreakageoftherefrigeratorresultedinanadditionalmeasurementatweek1.5forL.plantarum.

Sampleputinjuice AveragelogCFU/ml±standarddeviation,week0

AveragelogCFU/ml±standarddeviation,week1.5

AveragelogCFU/ml±standarddeviation,week2

AveragelogCFU/ml±standarddeviation,week4

L.plantarum0.5gingredient2+1.5gingredient3inLOKA 7.5±0.06 - 8.0±0.04 7.7±0.411.0gingredient2+1.5gingredient3inLOKA 7.6±0.14 - 8.0±0.13 8.0±0.060.5gingredient2+1.5gingredient3inMango 7.9±0.12 - 7.9±0.24 8.1±0.051.0gingredient2+1.5gingredient3inMango 8.0±0.06 - 8.1±0.09 8.3±0.033.0gingredient4inLOKA 7.6±0.04 7.9±0.22 7.9±0.06 7.9±0.033.0gingredient4inMango 8.2±0.08 7.7±0.25 7.8±0.12 8.0±0.123.0gingredient5inLOKA 7.6±0.05 - 7.4±0.03 7.2±0.033.0gingredient5inMango 8.0±0.05 - 7.9±0.03 7.6±0.023.0gingredient8inLOKA 7.7±0.03 - 7.7±0.06 7.8±0.043.0gingredient8inMango 8.1±0.08 - 8.1±0.04 8.2±0.02

L.rhamnosus1.5gingredient2+1.5gingredient3inLOKA 7.7±0.13 - 8.0±0.36 7.5±0.071.0gingredient2+1.5gingredient3inLOKA 7.6±0.13 - 7.5±0.14 7.5±01.5gingredient2+1.5gingredient3inMango 8.1±0.13 - 8.2±0.13 7.9±0.071.0gingredient2+1.5gingredient3inMango 7.9±0.06 - 7.8±0.03 7.6±0.043.0gingredient4inLOKA 7.3±0.11 - 7.7±0.20 7.3±0.053.0gingredient4inMango 7.9±0.03 - 7.2±0.07 7.0±0.103.0gingredient5inLOKA 7.3±0.08 - 7.5±0.01 7.5±0.033.0gingredient5inMango 8.0±0.03 - 8.0±0.07 7.9±0

ThestabilityofthefermentationproductcontainingL.plantarumandvaryingconcentrationsofingredient2and3varies,butallareconsideredacceptablesincetheyareallabove7.0logCFU/ml.Sincethesamplesatweek2wasevaluatedafterthebreakageoftherefrigerator,anincreaseinthebacterialcontentwasexpected,whichwasthecaseforallsamplesexceptforthetrialcontaining0.5

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gingredient2and1.5gingredient3inMango.Consideringthestandarddeviationofthattrial,itappearstooverlap,suggestingthattheremighthavebeeneitheradecreaseoranincreaseduringweek2.Lookingatthesurvivalofthebacteriainthejuicesafterthebreakageoftherefrigerator,i.e.afterweek2,sometrialshaveincreased(bothtrialsinMango)andsomehavedecreasedorstayedthesame(bothtrialsinLOKA)inbacterialcontent.However,consideringthestandarddeviation,bothtrialsinLOKAandthetrialcontaining0.5gingredient2and1.5gingredient3inMangohaveanoverlapbetweenweek2and4,indicatingthattheincreasemightnotbeanincreaseatall.Thetrialwith1.0gingredient2and1.5gingredient3inMangoseemstohaveincreasedandthebacteriamighthavegrowninthejuice.Thehighstandarddeviationsthatcanbeseenine.g.thefourthweekoftrial0.5gingredient2and1.5gingredient3inLOKAcontainingL.plantarummightbeduetothedifferencebetweenthesamplesoriginatingfromdifferentfermenters.ThesurvivalofL.rhamnosusinjuicecontainingvaryingconcentrationsofingredient2and3isconsideredgoodsincenoneofthesamplesarebelow7.0logCFU/ml.Mostofthesamplesdecreasedorstayedthesameduringthefourweeksofstoragewhilethetrialswith1.5gingredient2and1.5gingredient3inbothLOKAandMangoseemstohaveincreased.However,whenconsideringthestandarddeviationofthevaluesfromthetrialsthathaveincreaseduntilweek2,boththeLOKAandMangohaveanoverlapbetweenthevaluesforweek0and2,whichindicatesthattheremightnotbeanincreaseatall.Inaddition,thestandarddeviationofthetrialcontaining1.5gingredient2and1.5gingredient3inLOKAduringweek2islarge,whichmightbeduetothelargedifferenceinbacterialcontentbetweentheduplicatefermenters.Thestabilityofthefermentationproductscontainingingredient4andL.plantarumputinjuiceareallconsideredacceptable.ForthetrialsputinLOKA,thebreakageoftherefrigeratorresultedinanincreaseinbacterialcontentwhileforthetrialswithMangojuice,thebreakageresultedinadecrease.Sincethestandarddeviationdoesnotoverlapbetweenweek0and1.5foranyofthetrials,itseemsliketheincreaseanddecreasemightbetrueunlessahumanerrorhasbeenmade.ThetrialwithL.plantarumputinMangohaveincreasedduringstorageafterweek1.5,suggestingthatthebacteriacangrowinthenutritiousmangojuice.ThetrialcontainingL.rhamnosusandingredient4putinLOKAseemstohaveincreasedduringthesecondweekofstoragewhilethetrialsputinMangoseemstohavedecreasedduringthefourweeks.Whenthestandarddeviationisconsidered,nooverlapbetweentheweekscouldbefound,suggestingthattheincreaseanddecreaseistrue.Allthesamplescontainingingredient5areconsideredstableconsideringthattheyareallabove7.0logCFU/ml.ThestabilityofL.plantarumdoesnotseemtobeasgoodasforL.rhamnosuswhenthevaluesandtheirstandarddeviationisstudied.However,thelargeerrorinthemeasuringmethodpreventsfromreachingthatconclusion.ThevalueofL.rhamnosusputinLOKAforweek0seemstobeabitlowwhichmightbetheresultofsomekindoferror.Thejuicescontainingingredient8canbeconsideredstable.ThesmallincreasethatcanbeobservedfortheMangojuiceafterfourweeksmightnotbeanincreaseduetoboththelargeerrorofthemeasuringmethodandtheroundingoffofthedecimals.

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5.4 Sensoryevaluation5.4.1 Varyingtheconcentrationofingredient2and3TheentireresultfromthesmallsensoryevaluationofthejuicescontainingL.plantarumcanbeseeninTable9intheappendix.BothtrialsinLOKA(0.5gingredient2+1.5gingredient3and1.0gingredient2+1.5gingredient3)tastedgoodwithnoumamioff-flavours,althoughthestrawberryflavourdisappearedabitwhenthefermentationproductwasadded,givingablandtaste.However,LOKAiscurrentlynotadaptedtohavingprobioticsaddedtoitandfurtherdevelopmentmightremovetheproblemwiththedisappearingfruitflavour.Thetrialcontaining0.5gingredient2and1.5gingredient3inMangodidalsotastegood,butaslightumamioff-flavourwasnoticedduringweek2and4.Forthetrialcontaining1.0gingredient2and1.5gingredient3inMango,theoff-flavourseemedtohavedisappearedinweek4. ThetablefortheentiresensoryevaluationforthefermentationproductcontainingL.rhamnosusputinjuicecanbeseeninTable10intheappendix.Forthejuicesinweek0,thetrialcontaining1.5gofbothingredient2and3wasconsideredacceptable/badsinceithadsomemeatandumamioff-flavourwhilethetrialcontaining1.0gingredient2and1.5gingredient3wasconsideredgoodinbothLOKAandMangojuice.Forweek2,bothtrialswereconsideredacceptable,wherethetrialwith1.5g+1.5ghadlostsomeofitsoff-flavourandthetrialwith1.0+1.5hadgainedsome.Afterfourweeksofstorage,trial1.5+1.5wasstillconsideredacceptableduetoitsoff-flavourwhiletrial1.0+1.5inbothLOKAandMangojuicewasconsideredgoodduetothattheoff-flavourhaddisappeared. SincethefermentationproductcontainingL.paracaseiwasnotputinjuice,thefermentationproductwastastedbyitself.Allfermentationproductshadasavouryandacidictasteandtheoff-flavourofingredient2and3wasclearlynoticed.5.4.2 Ingredient4TheresultsfromthesensoryevaluationofthejuicescontainingL.plantarumcanbeseeninitsentiretyinTable12intheappendix.ThefermentationproductthatwasputinLOKAwasduringtheentirestorageconsideredacceptable.Thereasonforthiswasthatthestrawberryflavourwaslostandasavouryandsaltyoff-flavourcouldbeidentifiedduringthefourthweekofstorage.Thefermentationproductthatwasputinmangojuicewaswelltasting.ThecompleteresultsfromthesensoryevaluationofL.rhamnosusputinjuicecanbeseeninTable13intheappendix.TheMangojuicewasconsideredwelltastingduringtheentirefourweeksofstorage,however,duringthetwofirstweeksanoff-flavourofumamiwasnoticedbutthejuicewasconsideredgooddespitethat.ThetrialputinLOKAwasconsideredacceptableduringweek0duetooff-flavoursandbadduringweek2duetothelackoffruitflavourandadistincttasteofmetal.Afterfourweeksofstorage,thetrialputinLOKAwasagainconsideredacceptableduetothatthemetaltastehaddisappeared.Sincethefermentationproductwasnotputinjuice,itwastastedbyitself.Ithadastrongsavouryandsaltyflavour.5.4.3 Ingredient5TheentireresultfromthesensoryevaluationofjuicecontainingL.plantarumcanbeseeninTable15intheappendix.Thesensoryevaluationofthesamplecontainingingredient5showedanacceptabletastewithanumamioff-flavourduringweek0forbothLOKAandMangojuice.After2weeksofstorage,theoff-flavourwasnotasapparentanymoreandbothsampleswereconsideredwelltasting.However,afterfourweeksofstoragethemangojuicewasstillwelltastingwhileanumamioff-flavourhadreappearedintheLOKA.

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ThesensoryevaluationforthejuicecontainingL.rhamnosusdoneononeoftheduplicatescanbeseeninitsentiretyinTable16intheappendix.Duringweek0,bothsampleshadaslightumamiaftertastebutwasstillconsideredwelltasting.After2weeksofstorage,thesampleinLOKAwasconsideredacceptableduetoastrongerumami/saltyaftertastewhichwasstillpresentafter4weeksofstorage.ThesamplecontainingMangohadaslightaftertasteofumamiduringtheentire4weeksofstoragebutwasstillconsideredgood.SincetheconcentrationofL.paracaseiinthefermentationproductwasbelowthecriteriatobeputinjuice,thefermentationproductalonewastasted.Ithadastrongtasteofumamianditwasveryacidic.Oneoftheparticipantsofthesensoryevaluationsaidthatthesesampleswerethemostdisgustingthingshehadevertasted.5.4.4 Combiningingredient3withingredient5Noneofthesampleswereputinjuiceandthesensoryevaluationwillbeonthefermentationproducts.Thesampleshadanacidictastewithaclearsensationofumamiandoneotherunidentifiedcomponent.Whencomparingwiththeoriginalrecipe,thiscombinationwasnotasgoodastheoriginal.5.4.5 Ingredient6and7SincenoneofthesamplescontainingL.plantarumqualifiedtobeputinjuice,therewillnotbeasensoryevaluation.However,thefermentationproductwastastedandwasquitenice.Thesourtastethatisusuallypresentinthefermentationproductswasnottherebutthesampleshadratheratasteofvegetables.However,thismightbemorepreferredwhendoingvegetabledrinkscontainingprobiotics.Inaddition,sincethesamplescontainedalargeamountoftheingredients,itsanktothebottomofthecontainersfast,resultinginanotsopleasantappearance.NoneofthesamplescontainedhighenoughamountofL.rhamnosustobeputinjuice.Asforthetasteofthefermentationproduct,ithadthesamecharacteristicsasthefermentationproductcontainingL.plantarum.ThetasteofthefermentationproductcontainingL.paracaseihadthesamesensorycharacteristicsasthefermentationproductcontainingtheotherbacteria.5.4.6 Ingredient8TheentiretablewiththeresultsfromthesensoryevaluationofjuicecontainingL.plantarumandingredient8canbeseeninTable18intheappendix.AllsampleswereconsideredgoodduringalltheweeksofstorageexceptforoneduplicateofLOKAduringthefourthstorageweekthathadaslightoff-flavourofmetal/mineralsandwasconsideredacceptable.SincethecontentofL.rhamnosusinthefermentationproductwasnothighenoughtobeputinjuice,thefermentationproductwastastedasitwas.Ithadasweettaste,suggestingthatthesugarsourceinthefermentationproductwasnotfullyutilized.Itwasnotasacidicandhadatasteofvegetables.AsforL.rhamnosus,thefermentationproductcontainingL.paracaseiwasnotacidic,asexpected,andhadatasteofcerealsandsoil.Sincetheacidictastewasavoided,thesefermentationproductsmightbeeasiertocoverinafruitdrink.5.4.7 Ingredient9SincethesamplescontainingL.plantarumdidnotqualifytobeputinjuice,thefermentationproductwastasted.Thetastewasmildandnotasacidicasfermentationproductsusuallyare.Ithadaslighttasteofvegetable.NosensoryevaluationwillbedoneonthesamplescontainingL.rhamnosussincethecontentofbacteriawasnon-existinginthefermentationproduct.ThefermentationproductscontainingL.

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paracaseididnotqualifytobeputinjuiceandtheywerethereforetastedastheywere.Thetasteprofilewasnotacidicwhichwasexpected.Ithadatasteofgrassandcerealswhichmightbeeasytocoverwhenputinafruitdrink. 5.4.8 IncreasingthefermentationtemperatureforL.rhamnosusDuetolackoftime,thefermentationproductwasnotputinjuicebutwastastedasitwas.ThetastewasacidicandremindedofSwedishsourmilk(filmjölk).Therewasnoclearoff-flavourofumami.

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6 ConclusionTheingredientsthatgavethesignificantlyhighestbacterialgrowthforL.plantarumis1.5gingredient2+1.5gingredient3,3.0gingredient5,1.5gingredient3+1.5gingredient5,1.0gingredient2+1.5gingredient3,3.0gingredient4and0.5gingredient2+1.5gingredient3.Alltheseingredientsgave,accordingtothestatistics,equallysuccessfulgrowthcomparedtotheoriginalrecipe.Inaddition,theywerealsoallgoodenoughtobeputinjuice.However,duetolackoftime,thetrialscontaining1.5gingredient2+1.5gingredient3and1.5gingredient3+1.5gingredient5wasnotputinjuiceandcannotbeevaluatedaccordingtotheirsensoryproperties.Theotheringredientsputinjuicewereallconsideredeitheracceptableorgood.ThetrialsthatgavethesignificantlyhighestgrowthofL.rhamnosusare1.5gingredient2+1.5gingredient3,1.5gingredient3+1.5gingredient5and3.0gingredient4.Thetrialcontaining1.5gingredient3+1.5gingredient5wasnotputinjuiceduetolackoftimeandwasnotsubjectedtosensoryevaluationandcouldthereforenotbeconsideredfurther.Theothertwotrialswereputinjuicebutwithlesssatisfactoryresults.Thetrialcontaining1.5gingredient2and1.5gingredient3inMangoandthetrialcontaining3.0gingredient5inLOKAwasconsideredbadduringoneofthetestweeks.Inaddition,forthetrialwith1.5gofbothingredient2and3,nosamplewaseverconsideredgood.NoneofthesamplescontainingL.rhamnosuscanthereforebeconsideredgoodenoughregardingthesensoryaspect.ForL.paracasei,thetrialsthatshowedsignificantlythehighestgrowthwere1.5gingredient2+1.5gingredient3,ingredient6,ingredient7,1.5gingredient3+1.5gingredient5,1.5gingredient2+1.5gingredient3for+2hoursand3.0gingredient4.However,comparingthebacterialcontentofL.paracaseiwiththeothertwobacteriacannotbeconsideredagoodideasincethegrowthisgenerallylowerforL.paracasei.ItwasthereforenotpossibletoputanysampleinjuiceduetothatthecriteriaforputtingfermentationproductinjuicewassetaccordingtoL.plantarumandshouldbereconsideredwhenfermentingL.paracasei.Inconclusion,theingredientsthatgavethehighestbacterialcontentandbestresultsfromthesensoryevaluationwouldbetheingredientsusedwithL.plantarum.DuetotheinsufficientgrowthofL.paracaseiandtheunsatisfactoryresultfromthesensoryevaluationofL.rhamnosus,neitherofthosebacteriaareconsideredasgoodasthesamplescontainingL.plantarum.

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7 FuturesuggestionsItwouldbeinterestingtoinvestigatewhatthedifferenceisbetweenL.paracaseiandtheotherbacteriaandwhyitdoesnotgrowaswellastheotherbacteria.Theremightbesomeessentialnutrientmissinginthecultivationmediumthatisimportantforgrowthanditwouldbebeneficialtoidentifythatcomponenttobeabletoincreasethegrowth.Anothersuggestionwouldbetotryotherconcentrationsofingredient4and5,e.g.2.0g/600ml,andinvestigatewhetherthatwillgiveabettergrowththanusing1.5g/600mlandabettertastethanusing3.0g/600ml.DuetotheexcellentsensoryevaluationofthefermentationproductcontainingL.plantarumandingredient8,itmightbeagoodideatodevelopthisfurther.However,thegrowthwasnotthathighwhenusingingredient8.Asuggestionmightthereforebetocombinethisingredientwithasmallamountofyeastextractoryeastpeptone.Inaddition,oneofthestudiespresentedinthebackgroundsuggestedthataddingasmallamountofayeastcomponenttoingredient9willdecreasethelag-phaseandgiveahigherbacterialcontentforL.plantarumandL.paracasei.

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8 ReferencesŚwiątecka,D.etal.,2010.Theimpactofpeaproteinhydrolysatesonbacterialphysiologicalactivity—Aninvitrostudy.InternationalJournalofFoodMicrobiology,1506,140(2-3),pp.263-270.Axelsson,L.,2004.LacticAcidBacteria:ClassificationandPhysiology.In:S.Salminen,A.vonWright&A.Ouwehand,eds.LacticAcidBacteriaMicrobiologicalandFunctionalAspects.Ås:CRCPress.Barać,M.B.etal.,2015.TECHNO-FUNCTIONALPROPERTIESOFPEA(Pisumsativum)PROTEINISOLATES-AREVIEW.Actaperiodicatechnologica,Issue46.Berry,A.R.,Franco,C.M.,Zhang,W.&Middelberg,A.P.,1999.GrowthandlacticacidproductioninbatchcultureofLactobacillusrhamnosusinadefinedmedium.Biotachnologyletters,Volume21,p.163–167.Butler,M.,Sparling,R.&Court,D.A.,2010.EnergyMetabolismofCellsUsedforIndustrialProduction.In:M.C.Flickinger,ed.Inbook:EncyclopediaofIndustrialBiotechnology:Bioprocess,Bioseparation,andCellTechnology.s.l.:Wiley.Cui,F.etal.,2010.Co-productionofLacticAcidandLactobacillusrhamnosusCellsfromWheyPermeatewithNutrientSupplements.FoodandBioprocessTechnology,05,5(4),pp.1278-1286.FAO/WHO,2002.GuidelinesfortheEvaluationofProbioticsinFood,LondonOntario:FAO/WHO.Filannino,P.etal.,2014.MetabolicResponsesofLactobacillusplantarumStrainsduringFermentationandStorageofVegetableandFruitJuices.AppliedandEnvironmentalMicrobiology,04,80(7),pp.2206-2215.Görke,B.&Stülke,J.,2008.Carboncataboliterepressioninbacteria:manywaystomakethemostoutofnutrients.NatureReviewsMicrobiology,08,Volume6,pp.613-624.Gao,M.-T.etal.,2008.Utilizationofricebranasnutrientsourceforfermentativelacticacidproduction.BioresourceTechnology,06,99(9),p.3659–3664.GarciaMaiaCosta,M.,VidalFonteles,T.,TibériodeJesus,A.L.&Rodrigues,S.,2013.SonicatedpineapplejuiceassubstrateforL.caseicultivationforprobioticbeveragedevelopment:Processoptimisationandproductstability.FoodChemistry,08,139(1-4),pp.261-266.Han,S.-W.,Chee,K.-M.&Cho,S.-J.,2015.Nutritionalqualityofricebranproteinincomparisontoanimalandvegetableprotein.FoodChemistry,0104,Volume172,p.766–769.Helland,M.H.,Wicklund,T.&Harvhus,J.A.,2004.Growthandmetabolismofselectedstrainsofprobioticbacteriainmilk-andwater-basedcerealpuddings.InternationalDairyJournal,11,14(11),p.957–965.Holzapfel,W.H.&Wood,B.J.,2014.LacticAcidBacteria:BiodiversityandTaxonomy.Chichester:JohnWiley&Sons.Kleerebezem,M.etal.,2003.CompletegenomesequenceofLactobacillusplantarumWCFS1.PNAS,1802,100(4),pp.1990-1995.Ma,C.etal.,2016.DeterminationoftheessentialnutrientsrequiredformilkfermentationbyLactobacillusplantarum.LWT-FoodScienceandTechnology,01,Volume65,pp.884-889.Makras,L.,VanAcker,G.&DeVuyst,L.,2005.Lactobacillusparacaseisubsp.paracasei8700:2DegradesInulin-TypeFructansExhibitingDifferentDegreesofPolymerization.AppliedandEnvironmentalMicrobiology,11,71(11),pp.6531-6537.Molin,G.,2013.LECTURESINPROBIOTICS.2nded.Lund:s.n.Nualkaekul,S.&Charalampopoulos,D.,2011.SurvivalofLactobacillusplantaruminmodelsolutionsandfruitjuices.InternationalJournalofFoodMicrobiology,3003,146(2),pp.111-117.Pathak,M.&Martirosyan,D.,2012.Optimizationofaneffectivegrowthmediumforculturingprobioticbacteriaforapplicationsinstrictvegetarianfoodproducts.FunctionalFoodsinHealthandDisease,2(10),pp.369-378.ProbiAB,2017.ResultsfromAPI.Lund:s.n.

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9 Appendix9.1 PlatecountanalysisBelowisanexampleofacalculationofthebacterialconcentrationfromthenumberofcolonies.Onedilutionwithintheinterval25-300:Dilution Numberofcolonies Sum Result-4 104 104 104*104/0.1=1.04*107-5 12Twodilutionswithintheinterval25-300:Dilution Numberofcolonies Sum Result-4 256 304 304*104/1.1=276*104à -5 48 276*104/0.1=2.76*1079.2 RefrigeratortemperatureThetemperatureoftherefrigeratorbeforethebreakagecanbeseeninTable7.Table7.Thetemperatureoftherefrigeratorbeforethebreakage.

Date Temperature(°C)2017-02-13 3.42017-02-15 4.12017-02-16 3.02017-02-17 3.82017-02-20 3.92017-02-22 3.42017-02-23 4.12017-02-24 3.5Average 3.7

The27thofFebruary,therefrigeratorbroke,resultinginameasurementof21.8°Cafterwhichanewrefrigeratorwasbeingusedandthetemperatureofthatrefrigeratorwasnotnoteduntilthe10thofMarch,ascanbeseeninTable8.Ascanbeseen,thethermometerusedfurtherondidnothaveanydecimals.Table8.Thetemperatureoftherefrigeratorafterthebreakage.

Date Temperature(°C)

Date Temperature(°C)

Date Temperature(°C)

2017-03-10 4 2017-03-29 4 2017-04-20 42017-03-13 4 2017-03-30 5 2017-04-21 42017-03-15 3 2017-04-03 5 2017-04-24 42017-03-16 4 2017-04-05 4 2017-04-26 52017-03-17 4 2017-04-06 4 2017-04-27 52017-03-20 5 2017-04-07 4 2017-05-02 42017-03-22 5 2017-04-10 4 2017-05-04 52017-03-23 4 2017-04-12 4 2017-05-08 42017-03-24 4 2017-04-18 5 Average 4.32017-03-27 4 2017-04-19 5

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9.3 Varyingtheconcentrationofingredient2and3ThesensoryevaluationofthejuicecontainingL.plantarumandvaryingconcentrationsofingredient2and3canbeseeninTable9where1representsthetrialcontaining0.5gingredient2and1.5gingredient3and3representsthetrialcontaining1.0gingredient2and1.5gingredient3.Table9.TheresultsfromthesensoryevaluationwherethefermentationproductcontainingL.plantarumandvaryingyeastconcentrations,where1representsthetrialcontaining0.5gingredient2and1.5gingredient3and3representsthetrialcontaining1.0gingredient2and1.5gingredient3,wereputineitherLOKAormangojuice.Thesensoryevaluationincludedtwoparticipants.

Sample Good Acceptable Bad CommentWeek01–LOKA X However,somestrawberryflavourwaslost1–Mango X 3–LOKA X However,somestrawberryflavourwaslost3–Mango X Anumamioff-flavourwasnoticedWeek21–LOKA X Didnothaveafruitflavouranymore1–Mango X X Oneoftheduplicateshadanumamioff-flavourwhile

theotherdidnot3–LOKA X 3–Mango X Anumamioff-flavourwasnoticedWeek41–LOKA X 1–Mango X X Oneoftheduplicateshadanumamioff-flavourwhile

theotherdidnot3–LOKA X 3–Mango X

TheentireresultfromthesensoryevaluationofjuicecontainingL.rhamnosuscanbeseeninTable10,where1representsthetrialcontaining1.5gingredient2and1.5gingredient3and3representsoneoftheduplicatesfromthetrialcontaining1.0gingredient2and1.5gingredient3.Table10.TheresultsfromthesensoryevaluationwherethefermentationproductcontainingL.rhamnosusandvaryingyeastconcentrations,where1representsthetrialcontaining1.5gingredient2and1.5gingredient3and3representsoneoftheduplicatesfromthetrialcontaining1.0gingredient2and1.5gingredient3,wereputineitherLOKAormangojuice.Thesensoryevaluationincludedtwoparticipants.

Sample Good Acceptable Bad CommentWeek01–LOKA X Someumamioff-flavour1–Mango X Hadalotofumamioff-flavour3–LOKA X Somestrawberryflavourwaslost3–Mango X Week21–LOKA X Loststrawberryflavourbuttheumamioff-flavourwas

notasapparentanymore1–Mango X Theumamioff-flavourwasnotasapparentanymore3–LOKA X Someoff-flavourwasidentified3–Mango X Someoff-flavourwasidentifiedWeek41–LOKA X Someoff-flavourwasidentified(umami)1–Mango X Someoff-flavourwasidentified(salty)

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3–LOKA X Verylittlestrawberryflavourbuttheumamioff-flavourwasnotpresentanymore

3–Mango X Theumamioff-flavourwasnotpresentanymore 9.4 Ingredient4Thecompositionofingredient4,canbeseeninTable11.Table11.Thecompositionofingredient4in%(g/g).

Component Contentin%(g/g)Drymatter 96.4Totalnitrogen 11.13Aminonitrogen 3.73Sodiumchloride 0.15

TheresultsfromthesensoryevaluationofthefermentationproductcontainingL.plantarumand3.0gingredient4putinLOKAandMangojuicecanbeseeninTable12.Table12.TheresultsfromthesensoryevaluationwherethefermentationproductcontainingL.plantarumandingredient4,wereputineitherLOKAormangojuice.Thesensoryevaluationincludedtwoparticipants.

Sample Good Acceptable Bad CommentWeek0LOKA X SomestrawberryflavourislostMango X AbitofasaltyaftertasteWeek2LOKA X Thestrawberryflavourwasabitstrongerwhenthe

carbondioxidehaddisappearedMango X Week4LOKA X Hadsomesavouryandsaltyoff-flavoursMango X

ThesensoryevaluationinitsentiretyforL.rhamnosusputinjuicecanbeseeninTable13.Table13.TheresultsfromthesensoryevaluationwherethefermentationproductcontainingL.rhamnosusandingredient4wereputineitherLOKAormangojuice.Thesensoryevaluationincludedtwoparticipants.

Sample Good Acceptable Bad CommentWeek0LOKA X Somestrawberryflavourwaslostandanaftertastecan

befeltMango X SomeaftertastecanbetastedWeek2LOKA X NofruitflavourpresentandatasteofmetalwasnoticedMango X Anoff-flavourwasbarelynoticedWeek4LOKA X NofruitflavourpresentandthemetaltastedisappearedMango X

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9.5 Ingredient5Thecompositionofingredient5canbeseeninTable14.Table14.Thecompositionofingredient5in%(g/g).

Component Contentin%(g/g)Drymatter 95.80Totalnitrogen 11.60Aminonitrogen 3.70Sodiumchloride 0.27

TheentiresensoryevaluationforL.plantarumcontaining3.0gingredient5inLOKAandMangojuicecanbeseeninTable15below.Table15.TheresultsfromthesensoryevaluationwherethefermentationproductcontainingL.plantarumandingredient5wereputineitherLOKAormangojuice.Thesensoryevaluationincludedtwoparticipants.

Sample Good Acceptable Bad CommentWeek0LOKA X Somestrawberryflavourwaslostandanaftertastecan

befeltMango X SomeaftertastecanbefeltWeek2LOKA X Mango X Week4LOKA X Someoff-flavourscanbefelt,umamiMango X

ThesensoryevaluationofL.rhamnosusputinLOKAorMangojuicecanbeseeninTable16below.Table16.TheresultsfromthesensoryevaluationwherethefermentationproductcontainingL.rhamnosusandingredient5wereputineitherLOKAormangojuice.Thesensoryevaluationincludedtwoparticipants.

Sample Good Acceptable Bad CommentWeek0LOKA X Aslightumamioff-flavourwasnoticedMango X Aslightumamioff-flavourwasnoticedWeek2LOKA X Aclearumami/saltyoff-flavourwasnoticedMango X Aslightumamioff-flavourwasnoticedWeek4LOKA X Aclearumami/saltyoff-flavourwasnoticedMango X Aslightumamioff-flavourwasnoticed

9.6 Ingredient6and7Thecompositionofingredient6and7usedinthefermentationcanbeseeninTable17.Table17.Theproximatecompositionin%ofingredient6and7.

Component(%) Ingredient6 Ingredient7Moisturecontent 8.0 12.0Protein 23.5 26.0Ash 2.8 2.6Fat 7.0 2.5Carbohydrates 66.0 68.0

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9.7 Ingredient8ThesensoryevaluationofL.plantarumand7.0gingredient8putineitherLOKAorMangojuicecanbeseeninTable18.Table18.TheresultsfromthesensoryevaluationwherethefermentationproductcontainingL.plantarumandingredient8wereputineitherLOKAormangojuice.Thesensoryevaluationincludedtwoparticipants.

Sample Good Acceptable Bad CommentWeek0LOKA X Mango X Week2LOKA X Mango X Week4LOKA X X Oneoftheduplicateshadaslightoff-tasteofminerals

andmetalMango X