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Food
Microbiology
Fermentation
process
for
production
of
apple-based
kefir
vinegar:
microbiological,
chemical
and
sensory
analysis
Roberta
Oliveira
Viana
a,1,
Karina
Teixeira
Magalhães-Guedes
a,1,
Roberto
Alves
Braga
Jr.
b,
Disney
Ribeiro
Dias
c,
Rosane
Freitas
Schwan
a,∗ aFederalUniversityofLavras(UFLA),BiologyDepartment,Lavras,MG,BrazilbFederalUniversityofLavras(UFLA),DepartmentofEngineering,Lavras,MG,Brazil cFederalUniversityofLavras(UFLA),DepartmentofFoodScience,Lavras,MG,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received28June2016 Accepted13November2016
AssociateEditor:AdalbertoPessoa
Keywords: Yeast Saccharomyces Lactobacillus Apple MALDI-TOF BiospeckleLaser Vinegar
a
b
s
t
r
a
c
t
Theaimofthisstudywastodevelopakefirapple-basedvinegarandevaluatethis fermen-tationprocessusingnewmethodologywithBiospeckleLaser.Braziliankefirgrainswere inoculatedinapplemustforvinegarproduction.Inthisstudy,themicrobialcommunity presentinkefir,andcorrespondentvinegar,wasinvestigatedusingMatrixAssistedLaser Desorption/Ionization–TimeofFlightMassSpectrometry(MALDI-TOFMS)technique. Sac-charomycescerevisiae,Lactobacillusparacasei,Lactobacillusplantarum,Acetobacterpasteurianus andAcetobactersyzygiiwerethemicrobialspeciesidentified.S.cerevisiae,L.plantarum,A. pasteurianusandA.syzygiiwerefoundinsmallerquantitiesatthebeginningofthealcoholic fermentation,butwerefoundthroughoutthealcoholicandaceticfermentation.Kefirgrains wereabletoutilizeapplemustassubstratetoproduceethanol,andaceticacid.Acetate, volatilealcoholsandaldehydesinthevinegar-basedkefirwerealsoproduced.Theyield ofaceticacidinthekefirvinegarswas∼79%.Theaceticacidconcentrationwas∼41gL−1, reachingtherequiredstandardfortheBrazilianlegislationacceptsitasvinegar(4.0%acetic acid).Kefirvinegarshowedgoodacceptanceinthesensoryanalysis.Thetechnology pro-posedhereisnovelbytheapplicationofimmobilized-cellbiomass(kefirgrains)providinga mixedinoculaandeliminatingtheuseofcentrifugeattheendofthefermentativeprocess. Thisstepwillsaveenergydemandandinvestment.Thisisthefirststudytoproduceapple vinegarusingkefirgrains.
©2017PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
∗ Correspondingauthorat:BiologyDepartment,FederalUniversityofLavras(UFLA),37.200-000Lavras,MG,Brazil.
E-mail:rschwan@dbi.ufla.br(R.F.Schwan).
1 Theseauthorshavecontributedequally.
http://dx.doi.org/10.1016/j.bjm.2016.11.006
1517-8382/©2017PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Apple(Malusspp.)isprobablytheoldestfruitknowntoman andisfavoredbymillionsofpeoplearound theglobe.10 In
Brazil,someoftheseapplefruitsareconsumedbylocal popu-lations, but the majorityis wastedduring harvestbecause ofthe high productionper tree,the short shelflife ofthe freshfruit,andthe lackofuse ofthesefruitsasprocessed products.1,22Fruitswineand/orvinegarproduction,couldbe
agoodsolutionbecausetheyallowproductstobemaintained inalcohol or acetic acid.1,9 Althoughthe number of
stud-iesoffruitwines1,5,7,21 and fruitvinegars9,10,12 hasrecently
increased,nostudyhasfocusedontheproductionofapple kefirvinegar.
Kefirisacultureemployedtoproducebeverages,suchas the traditional Russian beverage alsonamed “kefir” which isfrom milk,and haslowalcoholcontent.23,13–17 Thekefir
is a mixed culture of various yeast species of the genus Kluyveromyces, Candida, Saccharomyces and lactic acid from bacteriaofthegenusLactobacilluscombinedinamatrixof pro-teinsandpolysaccharide‘kefiran’,whichareformedduring cellgrowthunderaerobicconditions.23Thegrainsofkefirare
irregularlyshaped,withyellowish-whitecolor,andhard gran-ules, which resemble miniature cauliflower blossoms.15 In Brazil,thegrainsofkefirareusedinprivatehousehold,15and
theyareaddedtodifferenttypesofmilk,suchascow,goator sheep,coconut,riceandsoymilk.21,23Thegrainsare
respon-sible for the fermentation that produce lactic acid, acetic acid,CO2, alcohol(ethyl alcohol)and aromaticcompounds. Thesecompoundsprovidekefir’suniquesensory characteris-tics:fizzy,acidtaste,tartandrefreshingflavor.15Thebeverage
containsvitamins,mineralsandessentialaminoacidsthat helpthebodywithhealingandmaintenancefunctionsand containseasilydigestiblecompleteproteins.15
Anopticaltechniquetoevaluatethebiologicalactivityof kefir grainswas used, the Laser Biospeckle.Thetechnique defineswhenalaserbeamisscatteredbyabiologicalsample; thescatteredwavesgeneratedintheilluminatedsample cre-atethespecklepatternthatchangesitsimageinaccordance withthechangesinthemonitoredmaterial.Thus,thesurface appearstobecoveredwithtinybrightdotsthatfluctuateina seeminglyrandomwayasforaboilingliquid.3,8
Theaimofthis study was todevelopanapple vinegar-basedkefir andstudy this fermentationprocessusing new methodBiospeckleLasertoevaluatethebiologicalactivityof kefirgrains.
Materials
and
methods
Brazilianmilkkefirgrains(Stock-cultureoftheMicrobial Fer-mentation Laboratory of the Federal University of Lavras, Brazil)wereusedintheexperiments.Theapplefruitswere obtainedfromLavrascitymarket,MinasGeraisStateinBrazil. Theseapplefruits,whichfailtomeetthequalitystandards requiredformarketing,werewashedincleanwatertoremove residues.Thepulpwasextractedusinganautomatic depulp-ingmachine(ITAMETAL0.5DS,Itabuna,BA,Brazil).The◦Brix
was analyzed,and the juicewasdivided into three500mL Erlenmeyerflaskstostartbatchfermentation.
Theprocessofvinegarproduction
Alcoholicfermentation
Themust was inoculated withkefir grainsin aproportion of10%(w/v).Allfermentationswereperformedintriplicate. Theflaskswereincubatedstaticallyatatemperatureof28◦C and fermentation was monitoreddaily to observe the end ofthefermentation(stabilityoftheconsumptionofsugars –Brix).Thefermentationmustwasfilteredbykitassato fil-ters(0.5m).Thekefirgrainswererecovered.Thealcoholic must wasusedforapple kefirvinegarproduction. Microbi-ological analysis (grains and must), measurement of Brix, reducing sugar analysisand pH 11 were carriedout during
fermentation.
Todeterminefermentationperformancewere calculated the substrateconversion factorsinethanol(Yp/s),substrate conversion in glycerol(Yg/s), substrate conversion in acetic acid (Yac/s),ethanolvolumetricproductivity ofethanol(Qp), biomassproductivity(Px)andconversionefficiency(Ef).The totalconcentrationofsugarswascalculatedconsideringthe conversionforeachmoleofsucrose(342g)in1molofglucose (180g)and1moloffructose(180g).
Aceticfermentation
Theapplefermentedmustobtainedintheprevioussection wereacetifiedin500mLErlenmeyerflasks,whichwere con-trolled temperature of28◦C and agitation of150rpm. The aceticfermentationwasconductedusingthefollowing treat-ments:(1) kefirgrains 10%(w/v);(2) kefirgrains 20%(w/v). Duringaceticfermentation,dailysamplesweretaken(6days) intriplicateforacidityanalysis(pHmeter)andalcohol (alco-holometer).Theaceticfermentationwasfinishonthesixth daywhenthevinegarpresentedalcoholcontentbelow1.0% (v/v).
Theyieldwas calculatedasthe acetic acid producedin relationtothetheoreticalyield.Thetheoreticalyieldwas cal-culated asthe amount ofethanol convertedto aceticacid, inwhich 1.0gofethanolyields1.304gofaceticacid.5 The
ethanol,methanol,higheralcoholswereanalyzedaccording toMagalhãesetal.15usingaJascochromatographequipped
with a refractive index (RI) detector (Jasco 830-RI, Madrid, Spain).AChrompackcolumn(30cm×6.5mm)at60◦C,using 5mMsulphuricacidastheeluent,ataflowrateof0.5mL/min. Glycerol, organic acids (lactic, acetic, tartaric, malic, cit-ric and succinic acid) and carbohydrates (glucose, sucrose and fructose) contents were quantified according to Puer-ari et al.21 using a Jasco chromatograph equipped with a
refractiveindex(RI)detector(Jasco830-RI,Madrid,Spain)and UV–visibledetector(Jasco870-UV-visible).AChrompack col-umn(300×6.5mm)at60◦C,using5mMsulphuricacidasthe eluent,ataflowrateof0.5mL/minandasamplevolumeof 20Lwasused.Allthesampleswereexaminedintriplicate.
Microbiologicalanalysis
Yeast,lacticacidbacteria(LAB),andaceticacidbacteria(AAB) counts were performed on Yeast Extract Peptone Glucose [YEPG:1%yeastextract,2%peptone(Himedia,Mumbai,India),
2%glucoseatpH3.5,containing100mgL−1chloramphenicol (Sigma,St.Louis,Missouri,USA),and2%agar(Merck, Darm-stadt,Germany)]foryeast,DeManRogosaSharpeagar[MRS (Merck),containing0.4%(v/v)nystatin(Merck)]forLAB count-ing,GlucoseYeastExtract[GYC:5%glucose(Merck),1gyeast extract(Merck),3gcalciumcarbonate(Merck)forAAB,and2g agar(Merck)perliter,pH5.6].
Morphologicalandbiochemicalcharacterization
A total of 358 isolates were subjected to macroscopic and micro-morphologicalanalysesand biochemicalassays. Yeast strains were characterized by determining spore formation and by testing fermentation on different car-bon sources (glucose, fructose, and sucrose), as described elsewhere.15,17
All bacterial strains were characterizedmorphologically andbiochemicallyviaGramstaining,catalasereaction, motil-ity test, sporulation, and oxidase activity. Bacterial cells were grownon MRSmediumand underwent fermentation testsusingdifferentcarbonsources:glucose,maltose, man-nitol, and sorbitol. Furthermore, the bacterial cells were grownonGYCmedium.Strainsthatweregram-negativeand oxidase-negativewereassessedusingtheBactrayIandIIkits forclusteranalysis. For gram-negativebut oxidase-positive strains,theBactrayIIIkitwasused.
MALDI-TOFsamplepreparation,measurement,anddata
analysis
Basedonthe resultsfrom themorphologicaland biochem-ical characterization, strains were selected for MALDI-TOF MSanalysis.11Cellsweregrownonplatesusingspecific
cul-turemediumforeachtaxonomicgroup,asdescribedabove. Cellswereincubatedat28◦Cfor18handthenapproximately 7.11logCFU/mL (107 cells) of each strain were aseptically transferredtomicrotubes.Subsequently,3Loforganic solu-tion(water/acetonitrile/trifluoro-aceticacid,50:47.5:2.5)was addedtoeachmicrotubecontainingthebacterialisolates,and 3Lofformicacid/acetonitrile(25:75)wasaddedtoyeast iso-lates.
The microtubes were immediately and vigorously vor-texed for 1min and then 1L ofthe resulting suspension wastransferredtoa96-wellMALDIflextargetplate(Bruker Daltonics, Bremen, Germany). When the liquid phase was almostevaporated,1Lofmatrixsolution[saturatedsolution of␣-cyano-4-hydroxy-cinnamicacid(CHCA)in50% acetoni-trile/2.5%trifluoro-aceticacid]wasaddedandthesolutionwas gentlymixed.11
AnEscherichiacoliK12colonywasobtainedfromthe Pub-licPortugueseCultureCollection Micoteca da Universidade doMinho(MUM,www.micoteca.deb.uminho.pt)andusedfor insituextractionofproteins,whichwereusedasstandardfor theMALDI-TOFMSexternalcalibration.CellsofE.coliK12were grownonLuria-Bertanimediumagar(LB;1%Bacto-tryptone, 0.5%Bacto-yeastextract,and1%NaClperliter)at37◦Cfor 18h,aspreviouslydescribedbyLimaetal.11Briefly,
approxi-mately1gofcellularmaterialfromasingleE.coliK12colony wastransferredfromtheplatetotheMALDIflexplate,and
CHCAmatrixsolutionwasaddedfollowedbygentlemixing. EachMALDI-TOFsamplewasspottedintriplicatetoevaluate reproducibility. Samples were then analyzed in a MALDI-TOF microflex LTspectrometer (Bruker Daltonics, Bremen, Germany),usingthe MALDIBiotyper3.0automaticsystem. For MALDI-TOF microbial identification, reference strains werefromtheCultureCollectionofAgriculturalMicrobiology (CCMA-UFLA,https://sites.google.com/site/ccmaufla/).
AssessmentofmetabolicactivitybyBiospeckleLaser
DailyanalysesofkefirgrainsweredonebyBiospeckleLaser toobservethebiologicalactivity.Thegrainsofkefirwere illu-minatedbyaHeNelaser,wavelengthof632nm,and10mW power,enlargedbyaplaneconcavelensinordertocoverthe entiresample.Theinterferencepatternsformedonthemwere captured byaCCD camera 640×486pixels, withashutter speedof1/60sandinarateof0.08screatingacollectionof 128images.Theanalysisoftheimages,fromthelaser illumi-nation,wasperformedaccordingtoGuedesetal.8 Thekefir
grainswereilluminatedbyalaserHeNe17mW632nmand analyzedbythenumericalSpeckleLasermethod. Organolep-tic properties were evaluated individually via the senses, including taste, sight, smell, and touch. Alcoholmeter was usedforalcohol,thetitrationmethodforacidityandsugars byFehling’ssolution
Vinegartreatment
Thekefir applevinegarwasthenfilteredthrough diatoma-ceous earth, bottled and pasteurized at 65◦C for 20min. Samples were analyzed by optical microscopy (National Optical 131Microscope)toevaluatethe clarityofthe vine-gar.Analyses(organolepticevaluation,real-alcoholicdegree, volatileacidityandtotalsugar)wereperformedfollowingthe “operatingmanualofbeveragesandvinegars”asinstruction No.24.
Sensoryevaluation
Thefinalvinegarwasevaluatedinasensorytest.Thetasters were asked to indicate how much they liked or disliked each productona9-pointhedonicscale(9=likeextremely; 1=dislikeextremely)according tooverall acceptability18 by
twenty-five trained tasters, bothmales and females,25–35 years ofage(studentsandstaffoftheBiologyDepartment, Federal University of Lavras, Brazil). The evaluationof the appearance,color andodorattributeswerealsoconducted. Randomized,refrigerated(10◦C)10mLsampleswereserved inclear,tulip-shapedglasseswithavolumeof50mL.These samplesweremarkedwithathree-digitrandomnumberand coveredwithPetridishes.
Statisticalanalyses
Each fermentation process (alcoholic and acetic) was con-ducted in duplicate and the mean values±standard devi-ations are reported. TheTukey’s testwas performed using StatgraphicsPlusforWindows4.1software(Statistical Graph-icsCorp.Software–(Freedownload))toevaluatethestatistical significance(levelofp<0.05).
Results
Microbiologicalanalysis
MicrobiologicalanalysisisinFig.1.LABcountsstartedwith apopulationof8.10logCFU/mL(approximately108cells)on theseconddayoffermentation,therewasaslightdecrease, where the population was 7.55logCFU/mL (107 cells). The largest LAB population was observed on the fourth day, where8.45logCFU/mL was reached, comingto the end of the fifthdaywitha populationof8.22logCFU/mL. Accord-ingtothedatapresented,thepopulationofLABperformed between7.73logCFU/mL107 and 8.522logCFU/mL 108 cells throughoutthe fermentation. Theacetic fermentationwas dividedintotwotreatments,oneconductedwith10% inocu-lum (treatment 1) and another 20% (treatment 2). At the beginningofaceticfermentation,thepopulationofLABwas thesameforbothtreatments,8.22logCFU/mL(108cells),the second to the fifth day, the population has remained sta-ble,with108cells.Thefinalfermentationshowspopulation of9.75logCFU/mL-(treatment1)and10.73logCFU/mL (treat-ment2).Theinitialyeastpopulationwasof4.56logCFU/mL, increasing in third day to 7.97logCFU/mL (107 cells). The populationdeclinedinthefourthdayto7.89logCFU/mL.In thetreatment1ofaceticfermentationtheyeastpopulation increasedto8.81logCFU/mL.Inthesixthdaythepopulation decreaseto3.98logCFU/mL.Thetreatment2wassimilarto treatment1.AABwerefoundinbothfermentations.Atthe beginningofthealcoholicfermentationthepopulationwas
12 10 8 6 4 2 0 12 10 8 6 4 2 0 1 2 3 4 5 1 2 3 4 5 6
Fermentation time in days
Number of cells log CFU mL
–1
Number of cells log CFU mL
–1
Fermentation time in days
a
b
Fig.1–Numberofcells(LogCFU/mL)duringthe fermentationprocess.(A)Alcoholicfermentation,
Lactobacillus( ),Acetobacter( ),Yeasts( ).(B)
Aceticfermentation,10%inoculum(Lactobacillus( ),
Acetobacter( ),Yeasts( )),20%inoculum
(Lactobacillus( ),Acetobacter( ),Yeasts( )).
low(4.01logCFU/mL)increasingto6logCFU/mL(treatment1) and9logCFU/mL(treatment2)infinalfermentation.Inboth aceticfermentationtreatments(1and2)thepopulationofAAB increasedto109and108cells,respectively.
Atotalof358organismswereisolated,and31(9%)were yeastsand 327 (91%)were bacteria(divided into lactic and aceticbacteria’s).TheisolateswereidentifiedbyMALDI-TOF MStechnique(Fig.2).AccordingtoFig.2a,thebeginningof fermentation(0h)showedhigherpopulationofLactobacillus paracasei (106CFU/mL). Saccharomyces cerevisiae, Lactobacillus plantarum,AcetobacterpasteurianusandAcetobactersyzygiiwere foundinsmallerquantities(105CFU/mL)atthebeginningof thealcoholicfermentation.Thesemicroorganismswerefound throughoutthealcoholicand aceticfermentation(10% and 20%kefir).AccordingtoFig.2b,attheendof264h(11days) offermentationthepopulationofS.cerevisiae,remainedthe same initialpopulationof105CFU/mL.ThepopulationofL. paracaseiandL.plantarumincreased,comparedtotheinitial timereaching108CFU/mLand107CFU/mL,respectively.The populationofA.pasteurianusand A.syzygiihadthehighest increase109CFU/mLattheendofaceticfermentation.
KefirgrainswereanalyzeddailyusingBiospeckleLaserand thebiologicalactivityvalueobtainedwas14.21.Thisvaluewas consideredtheinitialfermentationtime(0h).Thetestof sen-sorialanalysesofthegrainsofkefircanbeseeninFig.3,where inFig.3atheimageofthekefirgrainispresentedwithan illus-trationofawindowwherethelaserwasilluminatedandthe imagesassembled.
The resultofthe biological activity testis presentedin
Fig. 3(b andc). Inthe alcoholicfermentationthe biological activityofthekefirgrainsdecreasedintheearlydaysofthe fer-mentationprocess(Fig.3b).Thedecreaseinbiologicalactivity persisteduntilthethirddayoffermentation.Onthefourthday thebiologicalactivityofkefirgrainsinapplemustincreased to11.51.Onthelastdayoffermentationthebiologicalactivity ofkefirgrainsdecreasedtovalue7.12.
Theacetic fermentationoccurred simultaneouslyas the alcoholicfermentation.Intheaceticfermentationthe biolog-icalactivityofthekefirgrainsdecreasedintheearlydaysof thefermentationprocessfortreatment1and2(Fig.3c).The decreaseinbiologicalactivitypersisteduntilthethirddayof fermentation.Onthefifthday,thebiologicalactivityofkefir grainsincreasedto8.01(treatment1)and8.79(treatment2). Onthelastdayoffermentation,thebiologicalactivityofkefir grainsdecreasedto6.36(treatment1)and6.77(treatment2).
Chemicalanalysis
Applealcoholicmustproduction
Theapplealcoholicmustwasproducedandwhereaslactic, acetic,citric,succinicandmalicacidswerefoundintheapple alcoholic must (1.37, 0.92, 1.37,1.71 and 1.00gL−1, respec-tively)(Table1).Tartaric,butyricandpropionicacidswerenot detected.
Thefermentationtemperatureselectedwas28◦Candthe kefirgrainswerechosenastheinoculumforthefermentation process.Aninoculum of10%kefir inoculum wassufficient becausethe ethanolconcentrationat120hoffermentation wasapproximately97.0g/L(∼12.3GL(GayLussac))(Table1). Small amounts of sucrose(0.09g/L), fructose (0.06g/L) and
Saccharomyces cerevisiae
a
b
Acetobacter pasteurianus and acetobacter syzygii
Lactobacillus paracasei
Lactobacillus plantarum
Saccharomyces cerevisiae
Acetobacter pasteurianus and acetobacter syzygii Lactobacillus paracasei Lactobacillus plantarum 0 101 102 103 104 105 CFU mL–1 106 107 108 108 110 0 101 102 103 104 105 CFU mL–1 106 107 108 108 110
Fig.2–MicrobialIdentificationbyMALDI-TOFMStechnique.(A)inoculumkefirgrainsand(B)kefirvinegar.
reducingsugars(0.04g/L)weredetectedintheapplealcoholic must.Methanol was notdetected inthe samples ofapple alcoholicmust.Thedecreaseinthetotalsolublesolidsand the increase inthe content of ethanolduring the fermen-tativeprocessareshowninTable1. ThepHvaluewas3.82 and ◦Brix was 4.37 inthe final alcoholicmust. Oxalicacid wasdetectedintheapplealcoholicmustbeforetheaerobic phaseofacetic fermentation.Theconcentrationofglycerol waslow(5.10g/L)(Table1).Thevaluesofthekinetic parame-tersobtainedfromtheapplealcoholicmustaredescribedas ethanolproductivity(Yp/s)/g0.47g,yieldglycerol(Yg/s)0.05g/g, ethanol yield (qp) 0.37g/L/h and fermentation efficiency (Ef)93.12%.
After the alcoholic fermentative process, the alcoholic mustwasprocessedasdescribedinthemethodologyforthe subsequentproductionofapplevinegar.
Kefirvinegar
The kefir vinegar was produced and lactic, citric, succinic andmalicacids weresimilar inthetreatment1and treat-ment2(Tables2and3).Theyieldofaceticacidinthekefir vinegarswas∼79%.Thetotalaceticacidconcentrationwas
∼41.00gL−1, reachingthe required standard forthe Brazil-ianlegislationacceptsitasvinegar(4.0%aceticacid)(Brasil, 20094).Inourstudy,fiveflavor-activecompounds,including
threealcohols,oneacetateandonealdehydewereidentified (Table4).Thevaluesofthesecompoundsweresimilarforthe kefirvinegarsintreatment1andtreatment2.Thekefir vine-garsshowedtheclearappearanceandhadagoodcolor(pale yellow).
Sensoryanalysis
Sensoryanalyseswereconductedtokefirvinegar(10%and 20% of kefir grain inoculum) and the commercial apple vinegar. Using the check-all-that-apply (CATA) testing, the vinegarsweresubjectedtosensoryevaluationtoassesstheir acceptanceandpreference.Vinegarsat10%,20%and commer-cialhadgoodacceptance(scores7.1,7.4and7.2,respectively). Consumers classified vinegars in “liked slightly”. Vinegars were evaluated as “sour, acetic acid, limpid and translu-centappearance”(Fig.4).Nosignificantdifferencesobserved betweenallevaluatedvinegars.Theapplekefirvinegars pro-ducedwerewellacceptedbytheevaluators(95%ofconsumers wouldbuykefirvinegar).
10mm 12 10 8 6 4 2 0 10 9 8 7 6 5 4 3 2 1 0 0 1 2 3
Fermentation time in days
Biological activity
Biological activity
4 5
0 1 2 3
Fermentation time in days
4 5 6
a
b
c
Fig.3–BiologicalactivityofkefirgrainsbyBiospeckleLasertechnique.(A)KefirgrainsandimageformedbyBiospeckle Laser.(B)Alcoholicfermentation().(C)Aceticfermentation,( )10%kefirinoculum,()20%kefirinoculum.
Discussion
Theapplepulpwasprocessedtoobtainafermentablemust from which the alcoholic must was produced. The inocu-lumforthefermentationprocess(kefirgrains)wassufficient becausetheethanolconcentrationat120hoffermentation wasapproximately 97.0gL−1 (∼12.3GL(Gay Lussac)).These resultsarecomparabletothatobtainedbyDiasetal.,5who
foundavalueof89.5gL−1whenfermentingcocoapulpwith aninitial22◦Brix.
Smallamountsofsucrose,glucose,fructoseandreducing sugars were detectedinthefinal alcoholicmustofapples. The rapid decrease in the sugar content and increase in theconcentrationofethanolduringaninoculated fermenta-tion(74.78gL−1)wasalsoobservedbyDomizioetal.6during
fermentation of grape must under controlled temperature conditions. Nurgelet al.19 foundthatfermentationof
non-pasteurized grapeusing the selected yeast(6.7logCFU/mL) was completed in 6 days, while indigenous fermentation lasted10days.TheseauthorsalsofoundthatthepHvaluesof thefermentersweresimilar(approximately3.08).
Table1–Chemicalandphysicalcharacteristicsoftheapplealcoholicmust.
Periodof
fermentation
Acids(gL−1)
Aceticacid Lacticacid Citricacid Tartaric
acid
Butyric acid
Succinic acid
Malicacid Oxalicacid Propionic
acid
0h 0.11±0.01a 0.10±0.01a 1.99±0.01a n.d. n.d. 0.25±0.01a 0.11±0.01a 0.09±0.01a n.d.
120h 0.92±0.01b 1.37±0.01b 1.37±0.01b n.d. n.d. 1.71±0.01b 1.00±0.02b 0.29±0.01b n.d.
Periodof
fermentation
Sugars(gL−1)
Sucrose Glucose Fructose Reducing
sugars
0h 92.60±0.11a 12.60±0.08a 23.47±0.21a 36.46±0.31a
120h 0.09±0.01b n.d.b 0.06±0.01b 0.04±0.01b
Periodoffermentation Alcohols(gL−1)
Ethanol Glycerol Methanol
0h n.d.a n.d.a n.d.
120h 96.78±0.41b 5.10±0.01b n.d.
Periodoffermentation pH Brix
0h 4.05±0.02a 13.5±0.02a
120h 3.82±0.02a 4.37±0.02b
Thedataarethemeanvaluesoftriplicatemeasurements±standarddeviation.Differentlettersintherowandcolumnindicatesignificant
differences(p<0.05).n.d.,notdetected.
Aceticacidwasformedduringthefermentationofapple alcoholic must. Acetic acid provides a pleasant taste and inhibits the development of undesirable or pathogenic microorganisms,duetothehighacidity.Duringthe fermen-tationprocess,thecitricacid concentrationwasalmostthe same as at 0h; the citric acid concentration decreased of 1.99–1.37g/L.Itispossiblethatcitricacidwasmetabolizedby S.cerevisiae(presentinkefirgrains)asacarbon andenergy source; which have the ability to ferment/assimilate this organic acid,causingthe increase inthe pHvalue.15 Citric
and malic acids are commonly found in fermented fruit beverages,wheretheyactaspreservativeswithantimicrobial properties.20Theorganicacidsproducedbyyeastand
bacte-rialspeciescontributetotherefreshingflavor,uniquearoma and the texture, in addition to controlling the growth of food-spoilagemicroorganisms.2Theconcentrationofglycerol
intheapplealcoholicmustwaslow(5.10g/L).Thisvaluewas consistent with the value ofless than ∼10.0g/Lsuggested byDiasetal.5 toconferthecharacteristicbodyandtexture
ofthe beverage.Glycerolisthe mainsecondaryproductof
Table2–Chemicalandphysicalcharacteristicsoftheapplekefirvinegarintreatment1(10%kefirinoculum).
Periodof
fermentation
Acids(gL−1)
Aceticacid Lacticacid Citricacid Tartaric
acid
Butyric acid
Succinicacid Malicacid Oxalicacid Propionic
acid
0h 0.96±0.01a 1.12±0.01a 1.39±0.01a n.d. n.d. 5.52±0.01a 1.07±0.01a 0.22±0.01a n.d.
144h 41.66±0.31b 1.11±0.01a 6.67±0.01b n.d. n.d. 5.60±0.01a 7.02±0.01b 0.18±0.01a n.d.
Periodoffermentation Alcohols(gL−1)
Ethanol Glycerol Methanol
0h 94.02±0.01a 5.32±0.01a n.d.
144h 1.76±0.01b 5.36±0.01a n.d.
Periodoffermentation PH
0h 3.08±0.01a
144h 2.74±0.11b
Thedataarethemeanvaluesoftriplicatemeasurements±standarddeviation.Differentlettersinthesamecolumnindicatesignificant
Table3–Chemicalandphysicalcharacteristicsoftheapplekefirvinegarintreatment2(20%kefirinoculum).
Periodof
fermentation
Acids(gL−1)
Aceticacid Lacticacid Citricacid Tartaric
acid
Butyric acid
Succinicacid Malicacid Oxalicacid Propionic
acid
0h 0.96±0.01a 1.11±0.01a 1.42±0.01a n.d. n.d. 4.59±0.01a 1.01±0.01a 0.16±0.01a n.d.
144h 40.06±0.31b 1.09±0.01a 5.27±0.01b n.d. n.d. 4.90±0.01a 6.04±0.01b 0.11±0.01a n.d.
Periodoffermentation Alcohols(gL−1)
Ethanol Glycerol Methanol
0h 94.02±0.01a 4.82±0.01a n.d.
144h 2.16±0.01b 4.76±0.01a n.d.
Periodoffermentation PH
0h 3.09±0.01a
144h 2.42±0.11b
Thedataarethemeanvaluesoftriplicatemeasurements±standarddeviation.Differentlettersinthesamecolumnindicatesignificant
differences(p<0.05).n.d.,notdetected.
Viscous 100 90 80 70 60 50 40 30 20 10 0 Limpid Translucent Sour Astringent Bitter Acetic acid Wine Fruity
10% Kefir inoculum 20% Kefir inoculum Commercial vinegar
Fig.4–Kefirvinegarsensoryanalysis.
alcoholicfermentationsbyS.cerevisiae,which ispresentin thekefirinoculuminthisstudy.Theglycerolconcentrationof approximately5.1g/Lwasclosetothevaluesof6and10g/L suggestedbyVogt etal.24 toconfer the characteristicbody
andtextureofthebeverage.
Atotalof358organismswereisolated,and31wereyeasts and 327werebacteria(divided intolactic andacetic bacte-ria).TheisolateswereidentifiedbyMALDI-TOFMStechnique (Fig.2).ThepopulationwereL.paracasei,S.cerevisiae,L. plan-tarum,A.pasteurianusandA.syzygii.Thesemicroorganisms arecommonlyfoundinkefirgrains.23,13–17Rapiddiagnosisof
microorganismsisdecisivetoguaranteeadequate identifica-tion insamples.Culturemethodsarepreciseandsensitive, butratherslow.Newresourcesareavailabletoenablefaster diagnosis,and the mostpromisingisMALDI-TOFMS tech-nologyappliedtomicrobiologicaldiagnosis.Timesasfastas 10–15mintoetiologicaldiagnosisarepossibleafterapositive bloodcultureresult.20
Kefir grainswere alsoanalyzed bythe Biospeckle Laser technique. The results presented the activity of the kefir
Table4–VolatilecompoundsinthekefirvinegaridentifiedbyGC-FID.
Compounds Finalconcentration(mgL−1)
Treatment1a Treatment2b
Aldehyde
Acetaldehyde 21.33±0.24a 20.33±0.54a
Alcohols
1-Hexanol 9.01±0.06a 9.38±0.56a
2-Methyl-1butanol 7.01±0.06a 7.38±0.56a
3-Methyl-1-butanol 12.76±10.08a 12.23±12.68a
Acetate
Ethylacetate 9.24±0.32a 9.94±0.45a
Thedataarethemeanvaluesoftriplicatemeasurements±standarddeviation.Differentlettersinthesamelineindicatesignificantdifferences (p<0.05).
a 10%kefirinoculum. b 20%kefirinoculum.
grainsduringfermentation(Fig.3),whereitwaspossibleto observethesensitivityofthetechniquetofollowtheexpected activity.Theproposed techniqueissimple,relativelycheap andfast, easytoimplement,and requiresonlyalaserand standarddigitalimagingprocessinghardwarecomponents.8
TheapplicationofBiospeckleLasermethodtothekefir via-bilitydetection wasoneofobjectives ofthiswork,but the opticaltechniquecould alsobe appliedtocharacterizethe kefirgrainsinothertypesofprocesses.BiospeckleLaser tech-niqueshowedanefficienttoolformonitoringandquantifying biologicalactivityofkefir grains,showingtheviabilitytime of these grains in a fermentation process, which demon-stratesthetechniquepotentialforevaluatingandmonitoring ofkefir grains in productionofkefir vinegars inindustrial scale.
Volatile compounds are important contributors to the flavorsofbeverages,andareresponsiblefordifferent desir-ablesensorycharacteristics.14–16Previousstudieshaveshown
that the formation of volatile higher alcohols and esters duringkefir fermentationisinfluenced bythecomposition of the medium.15 Five flavor-active compounds, including
threealcohols, oneacetateandonealdehyde,were identi-fiedby gaschromatography coupledwith flame ionization detection(GC-FID)inkefirvinegars.Theconcentrationthese compounds were particularly high (Table 4) in compara-tivewithstudy of15 showing the biochemicalchanges and
volatilecompoundformationsduringtheproductionofnovel whey-based kefir beveragesand traditionalmilk kefir. The valuesofthesecompounds weresimilar forthe kefir vine-garsintreatment1andtreatment2;i.e.,fermentationwith 10%and20% inoculumkefirproducedsimilarquantitiesof volatiles.Thehigheralcoholsidentifiedduringkefirvinegar fermentations were 2-methyl-1-butanol (active amyl alco-hol)and 3-methyl-1-butanol(isoamyl alcohol).Thevolatile higheralcoholidentified,2-methyl-1-butanolisproduced dur-ingthecatabolismofthebranchedchainaminoacid(BCAA) isoleucine,or issynthesized denovoduringthe biosynthe-sisoftheAAB.13-methyl-1-butanolhasapositiveinfluence
on the aromaofthe fermentedbeverage. On the contrary, increasedconcentrationofthesealcohols,havingavolatile descriptionof“coconut-like”,“harsh”and“pungent”,can con-tribute negatively to the product aroma.1 Only one ester,
characterizedbyfruityattributes,namelyethylacetatewas detectedin kefir vinegar. Kourkoutas et al.,10 showed that
yeastswerecapableofproducingethylacetatefrom fermen-tationprocessinawiderangeofconcentrations(fromtraces to 95mgL−1). According to these authors,such concentra-tionsaretypicaloffermentedbeverages.Acetaldehyde,which impartsnuttyandpungentaromas,wasalsofoundinkefir vinegar.
Thetasteofthefilteredvinegarwasfruityandanicesmell wasnoted.Variousorganicacidsinvinegarareimportantfor impartingasuitabletasteandflavor.Thetotalaceticacid con-centrationintheapplevinegarproduced usingkefirgrains inoculumwas ∼41.0g/L, whichisapproximately twotimes theconcentrationinonionvinegar(∼25.0g/L)producedusing cell-freeofAcetobacteraceti.9Thekefirgrainswerehighly
sat-isfactoryforaceticfermentationusingapplealcoholicmust asthemedium.Thissystemachievedhigheraceticacid pro-ductivitythanthatoffermentationsbyfreecells.Ourresults
indicatedthattheuseofkefirgrainsmightbeoneofthebest fermentingstrategiesemployedtoovercomesubstrate limita-tionandachievehighproductyield.
Theperiodfortheoxidationofethanoltoyieldaceticacid was 140h. According to the stoichiometry of reaction that convertsethanoltoacetic acid,1.0gofethanolcanprovide 1.304gofaceticacid.12Inindustries,theconversionof1.0g
ethanolto1.0gofaceticacid (yieldof76.6%)isconsidered economic.12Theproductionwasfavorable,reachingvaluesof
∼79.0%yield.Therefore,wecanconcludethattheevaporation ofvolatilecompoundswaslow,whichmightbeattributedto usingappropriateaerationandthermalconditions.Methanol wasnotdetectedintheapplekefirvinegarandtheglycerol concentrationremainedlowinbothtreatments.
Basedontheanalysesofthevinegarandthelimitsrequired bylaw,9 thefinalproductofkefirvinegarhasanacceptable
aceticacidlevel,ofapproximately5.0%(w/v),andanethanol concentrationoflessthan1.0%(v/v).Thekefirvinegarproved tobeaverypromisingproduct.
Conclusions
Vinegarcouldbesuccessfullyproducedfromapplealcoholic must using kefir grains. Thisis the first study to produce kefirvinegar.Thechemicalanalysesrevealedthatkefir vine-garhashighcontentsoforganicacids,whichaddfunctional valuetothevinegar. Kefirvinegarhadagoodperformance duringtheacetylation(∼79%),reachingtherequiredstandard fortheBrazilianlegislationacceptsitasvinegar(4.0%acetic acid).4 Microorganisms were foundpreviously described in
kefirgrainsandthemicrobiotaremainedstandardizedduring thefermentationprocess.Therewasnomicrobial contami-nationduringthefermentationprocess.Anewmethodology tomeasurethemetabolicactivityofkefirgrainsbyBiospeckle Laserwaspresented.Thisfactfacilitatesthemicrobiological controloverafermentationprocess.Kefirgrainsshowed effi-ciencyintheproductionofapplevinegar.Kefirvinegarwas wellacceptedbysensoryanalysis.
Thenoveltechnologyproposeduseofkefirgrainsforthe productionofvinegarwassuccessfullydone. Thekeypoint forindustrialapplicationoftheproposedtechnologyisthe promotion offermentationbyanimmobilized-cellbiomass (kefirgrains)thateliminatetheuseofcentrifugalseparators, whichhaveahighenergydemandandrequirehighindustrial investment.
Conflicts
of
interest
Noconflictofinteresttodeclare.
Acknowledgements
The authors thank the Conselho Nacional de Desenvolvi-mento CientíficoeTecnológico(CNPq),theCoordenac¸ão de Aperfeic¸oamentodePessoaldeNívelSuperior(CAPES)andthe Fundac¸ãodeAmparoàPesquisadoEstadodeMinasGerais (FAPEMIG)forfinancialsupportandscholarships.
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