Saccharomyces cerevisiae populations and other yeasts associated with indigenous beers (chicha) of Ecuador

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

Saccharomyces

cerevisiae

populations

and

other

yeasts

associated

with

indigenous

beers

(chicha)

of

Ecuador

Fernanda

Barbosa

Piló

_,

_Enrique

_Javier

_{Carvajal-Barriga}

_,

Maria

Cristina

Guamán-Burneo

_,

_Patricia

_{Portero-Barahona}

_,

Arthur

Matoso

Morato

Dias

_,

_Larissa

_Falabella

_Daher

_de

_Freitas

_,

Fátima

de

Cássia

Oliveira

Gomes

_,

_Carlos

_Augusto

_Rosa

a_{UniversidadeFederaldeMinasGerais,InstitutodeCiênciasBiológicas,DepartamentodeMicrobiologia,BeloHorizonte,MG,Brazil} b_{PontificiaUniversidadCatólicadelEcuador,EscueladeCienciasBiológicas,CentroNeotropicalparaInvestigacióndelaBiomasa,}

ColeccióndeLevadurasQuitoCatólica(CLQCA),Quito,Ecuador

c_{CentroFederaldeEducac¸ãoTecnológicadeMinasGerais,DepartamentodeQuímica,BeloHorizonte,MG,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Received20July2017 Accepted7January2018 Availableonline1March2018 AssociateEditor:AdalbertoPessoa

Keywords:

Saccharomycescerevisiae mtDNArestrictionpatterns Yeastpopulations

Chicha

Ecuadorianindigenousbeers

a

b

s

t

r

a

c

t

Chicha,atypeofbeermademainlywithmaizeorcassava,isatraditionalfermentedbeverage oftheAndeanregion.Therehaveonlybeenafewstudiesonyeastsassociatedwithchicha fermentation,andthespeciesdiversityoccurringduringtheproductionofthisbeverageis notknown.Theobjectiveofthisstudywastodeterminethebiodiversityofyeastsinchicha, andtocharacterizetheSaccharomycescerevisiaepopulationsassociatedwiththe produc-tionofchichadejora,seven-grainchicha,chichadeyuca,andchichademorochoinEcuador.The moleculardiversityofS.cerevisiaepopulationswasdeterminedbyrestrictionpolymorphism mitochondrialprofiles.Thebeverageswerecharacterizedbasedontheirphysicochemical parameters.Twenty-sixspecieswereidentified,andthemostprevalentspecieswereS. cere-visiaeandTorulasporadelbrueckii.Otheryeastspecieswereisolatedatlowfrequencies.Among 121isolatesofS.cerevisiae,68differentmtDNAmolecularprofileswereidentified.These resultsshowedthatchichasarefermentedbyahighnumberofdifferentstrainsofS. cere-visiae.Someotherspeciesprovidedaminorcontributiontothefermentationprocess.The chichapresentedgenerallysimilarphysicochemicalparameterstothoseobservedforother traditionalfermentedbeverages,andcanbeconsideredasanacidfermentedbeverage.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:[email protected](C.A.Rosa). https://doi.org/10.1016/j.bjm.2018.01.002

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Chichaormaizebeercouldberegardedastheoldestbeverage inLatinAmerica.Thenamechichapossiblyoriginatesfromthe wordchichab,fromthe originallanguagespoken inthe cur-rentterritoryofPanama,whichmeansmaize.Othertheories suggestthatthenameisderivedfromthewordChibcha,a civ-ilizationthatpopulatedColombiaandPanama,orrelatethe wordchichatoChichas,anethnicitypresentinsouthernBolivia beforetheestablishmentoftheIncas.1

Chicha isaclear, yellow, and frothybeverage present in theAndeanregionandinlow-lyingregionsofEcuador,Peru, Bolivia,Colombia,Brazil,andArgentina.2_{Thistraditional}

bev-erageispreparedmainlyfrommaize,butcurrently,thename isconsideredgenericandreferstoavarietyofbeverages, fer-mentedornot,preparedfromvariousothermaterials,suchas cassava,beans(suchasrice,oats,andquinoa)andfruits(such asbananas).InEcuador,thefirstreportsofchichaproduction datebackto200B.C.,beforetheestablishmentoftheIncas intheregion.1_{Thisbeveragewasofgreatimportancein}

tra-ditionalindigenouscultures,especiallyintheIncanculture, whereinitwasalsolinkedtofestiveceremonies.3

InEcuador,asintherestoftheAndeanregion,themost commonmaizechichaischichadejora(Fig.1).Thischicha is preparedfromtheyellowmaizegrain(maízamarillo),which ismalted(germinatedanddried).Forthepreparationofmalt, thegrainsareleftinwaterforaday.Thisstepisnecessary toachievetheoptimumgrainmoistureforgermination. Sub-sequently,the waterisdrainedand the maizeisplacedin basketsofstraytogerminateoveraperiodof13days.Once germinated,themaizeisputintostrawmatsorplastictarps underthesunfor2daystodrycompletely,whichstopsthe enzymaticactivitywithinthegrain. Afterdrying,thebeans are groundand theflourobtainedisused forthe prepara-tionofchicha.For this,the joraflourisaddedtocoldwater andthenthismixtureistransferredtovesselswithhotwater, andboiledforapproximately20min.Afterboiling,the mix-tureisstrainedand thenplacedinacontainer toferment. Theclayvessels,formerlyusedforboilingandfermentation, havebeenreplacedbyaluminiumpotsandplasticcontainers, respectively.Thespentgrainobtainedafterfiltrationistermed afrechoandservesasfoodforanimals.Thefermentation ves-selsareusuallyopen.Usuallyaftertwodaysofspontaneous fermentationbyindigenousmicroorganisms,thebeverageis readyforconsumption.Someproducerstypicallyboiljoraflour withotheringredients,includingpanela(brownsugarinsolid pieces).Othersmakeamixtureofpanelaandherbsandthen addthismixturetothejoraflourand water.Therearestill thosethataddpiecesoffruitandpanelatothebeverage,after filtering.

OtherchichabeveragesproducedinEcuadorincludechicha demorocho,madewithwhitemaize,andchichaproducedwith sevenvarietiesofmaizeincludingjora,maízamarillo(yellow maize),maízblanco(white maize),maíznegro (black maize), chulpi(chulpimaize),morocho(morochomaize)andcangil (pop-corn maize).Seven-grainchicha isproduced inthe townof Otavalo, in northern Ecuador, and is a very famous drink andappreciated throughoutthe country.Theyuca(cassava, Manihotesculenta)isalsoanimportantrawmaterialforthe

productionofchicha.4_{Thischichaisproducedbytheindigenous}

andmestizopopulationintheAmazonregionofEcuador. Few studies have been performed to identify the yeast speciesinchichas.Vallejoet al.5_{isolated Saccharomyces}

cere-visiaeasthesingleyeastspeciesattheendoffermentation from 10 samples ofchicha de jora collected from 10 differ-ent familiar traditional “chicherías”in the Cusco regionof Peru.Theseauthorssuggestedthatthisspecieswasmainly responsible foralcoholicfermentationinthese chicha sam-ples. Rodríguezet al.6 _{suggestthatSaccharomyces uvarumis}

responsible forthe traditionalfermentation ofapple chicha elaborated byaboriginal communitiesofAndeanPatagonia (Argentina and Chile). Mendonza et al.7 _{showed by}

high-throughput sequencing and culture-dependentapproaches thatS.cerevisiaewasthedominantspeciesinanArgentinian maize-basedchicha.Otherworksonchichafermentationlinked bacterialpopulationstothisprocess.4,8,9_{Despitetheworkof}

Vallejo et al.5 _{and Mendonzaet al.,}7 _{the yeastbiodiversity}

associatedwithmaizeandcassavachichaproductionisalmost unknown.Inthiswork,chichassoldinbulk(Fig.1),produced with differentsubstrates and differentfermentation times, were collected from markets,bars, restaurants, and in vil-lagesofEcuador.Theobjectivewastodetermineyeastspecies richnessandtocharacterizetheS.cerevisiaepopulations asso-ciated with the production of this beverage by restriction polymorphismmitochondrialDNA(mtDNA)analyses.In addi-tion,thephysicochemicalparametersofthebeverageswere determined.

Materials

and

methods

Forty-twochichasampleswerecollectedfromAugustto Octo-berof2010andApriltoSeptemberof2012intworegionsof Ecuador:theAmazonregion,withintheYasuníNationalPark (Orellana Province)and theAndeanregion,intheprovinces ofPichincha,ImbaburaandChimborazo.Thesamplesincluded twochichasdeyuca,34chichasdejora,threeseven-grainchichas, andtwochichasdemorocho.Inthesesamples,thefermentation wasconsideredfinishedbytheproducers,andthebeverage wasreadytodrink.Onesampleofchichadejorawassampled duringsuccessivefermentationtimes(0–5days).Thechichas werecollectedinsterilebottlesof100mL,transportedtothe laboratoryonice,andprocessedthesameday.

Yeastisolationandidentification

Aliquots of 25mL of each chicha were added to 225mL of sterile0.1%peptonewater.Foryeastisolation,0.1mLof appro-priate decimaldilutions,in triplicate,was spread on yeast extract-malt extract (YMA:1% glucose, 0.5%peptone, 0.3% malt extract, 0.3%yeast extract, 2%agar, and 0.02% chlor-amphenicol)andlysine(1.17% YCB,0.056%lysine,2%agar, and0.02%chloramphenicol)agars.TheYMAwasutilizedfor the isolationofSaccharomycesandnon-Saccharomycesyeasts whilethe lysineagar wasutilizedforthe isolationof non-Saccharomycesyeasts.Theplateswereincubatedat25◦Cfor 5daysand thedensity ofeachdifferentyeastmorphotype

Fig.1–Chichadejora(A);Morocho(whitemaize)(B);Chichademorochoreadytodrink(C);seven-grainchicha(D);cassavafor

chichadeyucaproduction(E);ChichadeyucareadytodrinkafteradditionofseedsoftheUngurahuapalm(F).

wasexpressedasthemeansofeachmorphotypeineach sam-pleincolony-formingunits(cfu/mL).Representativecolonies ofeachdifferentyeastmorphotypefrombothculturemedia werepurifiedon YMAplates and preservedat−80◦_{Cor in}

liquidnitrogenforsubsequentidentification.

Theyeast isolates were morphologically and physiolog-ically characterizedaccordingto Kurtzmanet al.10 _Isolates

withidenticalmorphological andphysiological characteris-ticsweregrouped together andsubjected toPCRusingthe coresequencesoftheprimer(GTG)5asdescribedbyGomes

et al.11 _{Isolateswith identical DNA banding patterns were}

groupedandtentativelyconsideredthesamespecies.Atleast 50%oftheyeastisolatesofeachmoleculargroupwere iden-tifiedbysequencing.Speciesidentificationwasperformedby sequenceanalysisoftheITS-5.8SregionandtheD1/D2 vari-abledomainsofthelargesubunitofrRNAgeneasdescribed previously.12–16_{Sequencingwasperformeddirectlyfrom}

puri-fiedPCRproductsusinganABI3130(LifeTechnologies,USA) automatedsequencingsystem.For yeastspecies identifica-tion,thesequencesobtainedwerecomparedtothoseincluded in the GenBank database using the Basic LocalAlignment SearchTool(BLASTathttp://www.ncbi.nlm.nih.gov).

RestrictionpolymorphismofmitochondrialDNA

ExtractionofmtDNAfrom121isolatesofS.cerevisiaewas per-formedaccording to the methodology described byQuerol andBarrio17 _{and modifiedbyFoschinoet al.}18 _ThemtDNA

was resuspended in 25␮Lof TE and stored at−20◦C. For

digestion,amixturecontaining10×buffer(Invitrogen,USA), 1␮LofRNAseA(Invitrogen,USA),1␮LofHinfI(Invitrogen, USA), and 10␮L ofDNA (approximately1500ng) was used. Thevolumewascompletedwithdeionizedwaterto20␮L.The tubeswerethenincubatedat37◦Cfor6h.ThemtDNA restric-tion fragmentswereseparatedbyelectrophoresisona1.5% agarosegel(Pronadisa,Spain)inTBEbufferat80Vfor2h.The gelswerestainedwithasolutionofGelRed(Biotium,USA)and visualizedandphotographedunderultraviolet(UV)light. Physicochemicalanalyses

The parameters ofpH, total reducing sugars,and ethanol contentweredetermined.Thedeterminationoftotal reduc-ing sugars was performed by the 3,5-dinitrosalicylic acid methodology.19 _{Theethanol, glycerol,and lactic and acetic}

acid contentswere determined byhighperformanceliquid chromatography(HPLC)inanAgilentchromatographermodel equippedwithacolumnRezexROA(300×7.8mm).

Results

Yeastidentification

Twohundredandfifty-fouryeastisolatesobtainedfromchicha sampleswereidentifiedasbelongingto26species.S.cerevisiae wasthe mostprevalent speciesoccurringin33of42 chicha samples(41samplesplusonesamplecollectedindifferent time),followedbyTorulasporadelbrueckiithatwasidentifiedin

Table1–Yeastspecies(cfu/mLandnumberofpositivesamplesforeachspecies)isolatedfromchichasamplescollected inEcuador.

Yeastspecies Chichadejora(n=35)a _{Seven-grainchicha(n=3) Chichademorocho(n=2) Chichadeyuca(n=2)}

Candidacalifornica 4.0×105₍₁₎b _{– – –} Candidahumilis 4.7×106_{(1) – – –} Candidasake 1.0×105_{(1) – 4.0×10}5_{(1) –} Candidasolani 3.3×105_{(1) – – –} Candidasorboxylosa 3.3×104_–4.6×105_{(3) – – –} Candidatropicalis – – – 3.3×104₍₁₎ Candidavinaria 3.3×104_{(1) – – –} Candidazeylanoides 8.6×105_{(1) – – –} Dekkeraanomala 3.3×105_{(1) – – –} Dekkerabruxellensis 8.0×105_–4.0×106_{(2) 1.6×10}5_{(1) – –} Galactomycescandidum 3.3×104_–6.6×104_{(2) – – –} Galactomycesgeotrichum 2.0×105_–1.0×106_{(5) – – –} Hanseniasporaopuntiae – – – 3.3×104₍₁₎ Hanseniasporaspp. 3.3×104_–5.2×107_{(9) – – –} Kazachstaniaexigua 3.3×105_–5.6×107_{(3) – – –} Kodamaeaohmeri – – – 3.3×104₍₁₎ Meyerozymaguilliermondii 3.33×104_{(1) – – –} Pichiafermentans 1.0×105_–1.6×106_{(5) – 3.3×10}5_{(1) –} Pichiakluyveri 6.6×105_{(1) – – –} Pichiakudriavzevii 3.3×105_–1.1×107_{(2) 2.6×10}5_–6.6×105_{(3) – 6.7×10}4₍₁₎ Pichiamanshurica – 6.6×104_{(1) – –} Rhodotorulamucilaginosa 3.3×104_{(1) – – –} Saccharomycescerevisiae 3.3×104_–3.8×107_{(28) 3.3×10}4_–5.6×107_{(3) – 1.5×10}7_–2.1×107₍₂₎ Saccharomycodesludwigii 2.0×105_–1.7×106_{(2) – 7.0×10}6_{(1) –} Torulasporadelbrueckii 3.3×104_–1.1×107_{(14) – 3.3×10}5_–7.0×106_{(2) 1.6×10}5_–4.3×105₍₂₎ Zygoascushellenicus 3.3×104_{(1) – – –}

a _{n=numberofsamplesanalysed.}

b _{Numberofsamplesthatthespecieswasisolated.Whentheyeastspecieswasisolatedinmorethantwosamples,theresultsshowincfu/mL}

thelowestandthehighestyeastcountsforeachspecies.

18samples.ThesetwospeciesaswellasPichiakudriavzevii, Candidasake, Dekkerabruxellensis, Pichiafermentans,and Sac-charomycodesludwigiiwereisolatedfromatleasttwotypesof chicha(Table1).Theotheryeastspecieswerepresentinonly onetypeofchicha.Thehighestyeastcountswereobtainedfor chichadejoraandseven-grainchicha.S.cerevisiaepopulations contributedtothehighestyeastcountsinmostsamplesof chichasdejora,seven-grainchichas,andchichasdeyuca.Inmost samplesthatthisspecieswasdetected,thepopulationcounts wereabove1.0×107cfu/mL.However,thisspecieswasnot iso-latedfromtwosamplesofchichademorocho,andT.delbrueckii andS.ludwigiipresentedthehighestcounts(7.0×106_cfu/mL)

inthisbeverage.

Onesampleofchichadejorawasstudiedduringfivedaysof fermentation(Table2).Atthestartoffermentation,S.cerevisiae waspresentataconcentrationof6.6×105_{cfu/mL,andthis}

speciesremainedinthebeverageuntilthefifthday,atwhich timecountswere4.5×106_{cfu/mL.Otheryeastspeciessuchas}

T.delbrueckiiandRhodotorulamucilaginosaappearedafterthe thirddayoffermentation.

The 121 isolates of S. cerevisiae studied represented 68 differentrestriction mtDNApatterns.For chicha de yuca,11 isolatesofS.cerevisiaewereanalysedrepresentingthree dif-ferentmtDNArestrictionprofilesintwosamplesstudied.For seven-grainchicha, 28 isolatesof S.cerevisiae were studied, representing19differentmtDNArestrictionprofilesinthree samplesstudied.Inchichadejorasamples,82isolatesofS. cere-visiaewereanalysed,and46mtDNArestrictionprofileswere

found(Table3).Two strains (mtDNAprofiles 1and 2)were foundinfourdifferentchichadejorasamples,butmostchicha samplesyieldedasetofstrainswithuniquemtDNArestriction profilesthatnotoccursinanothersample.

FourdifferentmtDNAprofilesofS.cerevisiaewereidentified inchichadejorastudiedduringthefivedaysoffermentation (Fig.2).

OnlytheS.cerevisiaestrainwithmtDNAprofile3occurred from the first to the fifth day of fermentation (Table 2). Strains with mtDNA profiles 2 and 3 showed increasing countsoverthecourseofthefermentation.Incontrast,the strain with mtDNA profile 1 decreased in number during the fermentationprocess, and after three daysit was not detected.

Physicochemicalanalyses

The highest pH value was 4.15, in sample 1 after 24h of fermentation, and thelowest valuewas2.62,insample 35 after 8daysoffermentation (Table 3).Thelowest valueof totalreducingsugarswas1.63g/Landthehighestvaluewas 36.24g/L, both foundin chicha de jorasamples. Thelargest amountofglycerolwas3.65g/L,foundinchichadejora. Lac-ticacidcontents rangedbetween1.59 and6.90g/L.Ethanol wasnotdetectedin10chichasamples.Thehighestvalueof ethanolwas5.97(%v/v)insample5ofchichadejoraafter24h offermentation.

Table2–Mitochondrialrestriction(mtDNA)profilesoftheSaccharomycescerevisiaestrainsisolatedfromchichadejora

during5daysoffermentationandidentifiedyeastspeciesateachtimepoint.

Daysoffermentation Yeastspecies/counts (cfu/mL)

Populationcounts (cfu/mL)ofeachS. cerevisiaemtDNAprofile

1 2 3 4 0 – – – – – 1 Saccharomycescerevisiae (6.9×105₎ 3.3×105 _3.3×104 _3.3×105 _– 2 S.cerevisiae(9.9×104_{) 6.6×10}4 _3.3×104 _{– –} 3 S.cerevisiae(5.3×105_), Torulasporadelbrueckii (3.3×104₎ – 9.9×104 _4.3×105 _– 5 S.cerevisiae(4.5×106_),T. delbrueckii(5.0×106_), Rhodotorulamucilaginosa (3.3×104₎ – – 2.2×106 _2.3×106

AnalysisofS.cerevisiaepopulationsbyrestrictionmtDNAprofiles.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

10000 bp

1500 bp 1000 bp

Fig.2–MitochondrialDNArestrictionpatternsobtainedfromachichadejorasampleatdifferenttimesoffermentation. Column:1:1kbDNAladder;2–7:profilesfoundwith1dayoffermentation(2–4:pattern1;5:pattern2;6–7:pattern3);8–10: profilesfoundwith2daysoffermentation(8–9:pattern1;10:pattern2);11–15:profilesfoundwith3daysoffermentation (11–13:pattern2;14–15:pattern3);16–17:profilesfoundwith5daysoffermentation(16:pattern3;17:pattern4).

Discussion

Ourresultsshowedthat inmostchichasamplesstudied, S. cerevisiae was the predominant species, followed byT. del-brueckii,P.kudriavzevii,C.sake,D.bruxellensis,P.fermentans,and

S.ludwigii,butgenerallyatmuchlowercounts.Alltheseyeast speciesmaycontributetothesensoryqualityofthebeverage.

S.cerevisiae wasalso the dominantspecies intwo produc-tionsamaizechichapreparedbylocalproducersfromMaimará and Tumbaya villages (Quebrada de Humahuacaregion) in NorthwestofArgentina.7 _{However, Candidaparapsilosisand}

anundescribedPichiaspecieswerethesecondandthirdmost abundantyeastsinthesefermentationsinArgentina.Vallero et al.5 _{isolated only S.cerevisiae from samplesof chicha de}

jora in 10 traditional “chicherías” in Cusco region in Peru. Theseresultsshowthatthefermentationprocessassociated withmaize-basedchichaiscarriedbyS.cerevisiaeandother non-Saccharomycesspeciesthat occurinminor frequencies. Thenon-Saccharomycesspeciesfoundinourstudycanbe iso-latedfromdifferentsubstratesworldwide,includingthoseof fermentationprocessesforbeverageproduction.1,5,20–22 _The

originofthenon-Saccharomycesspeciesassociatedwiththe chichasamplesstudied mightbeexplainedbythedifferent manufacturingprocessesforthesebeverages.Chichadejorais preparedindifferentways,employingawidevarietyofraw materialswithadditionalingredientssuch asfruits,herbs,

spices, brown sugar, and sugar. Seven-grain chicha is pro-ducedwithsevenvarietiesofmaizeflourandchichadeyuca withfewrawmaterialsincludingcassavaandtheseedofthe Ungurahuapalm.Chichademorocho,despitealsobeing man-ufacturedfrom awidevarietyofingredients,was collected at restaurants in LaMariscal, a district ofQuito (datanot shown), wherethe beverage was produced following Good ManufacturingPractice.Someingredientsusedinthechicha manufacturingprocess,asfruits,herbsandspices,areadded afterboilingthejoraflour.Basedonthisinformation,theyeast populations couldoriginatefrom theseingredientsusedin beveragepreparation,andfrom othersourcessuchas han-dlers, utensils, and vessels used during the fermentation process.

AhighnumberofdifferentmtDNAprofilesofS.cerevisiae werefoundassociatedwiththechichaproductionprocessin ourstudy.TheoccurrenceofthesedifferentmtDNAprofiles ofS. cerevisiaeassociatedwithtraditionalbeveragesis well documented.21,23,24 _{In addition, the occurrence ofdifferent}

strainsduringthefermentationprocess,asobservedinthe chichadejorastudiedduringthefivedaysoffermentationis verycommoninspontaneousfermentationduringtraditional beverageproduction.21,25,26_{AseachmtDNAprofilerepresents}

adifferentgeneticstrainofS.cerevisiae,thesestrainscouldbe contributingtodifferentsensoryproprietiesofthebeverage. TheselectionofthebestS.cerevisiaestrainsforchicha produc-tionisinteresting,andthesestudiescouldbebasedonstrain

Table3–PhysicochemicalparametersofthechichasamplesandtheoccurrenceofthedifferentmtDNAprofilesof

Saccharomycescerevisiaestrainsineachsample.

Chicha Daysof fermentation

pH Total reducing sugars(g/L)

Glycerol(g/L) Lacticacid(g/L) Ethanol(%v/v) mtDNAprofilesof theS.cerevisiae strains yuca 1 1 4.15±0.01a_{34.83±4.51 1.40±0.19 4.72±1.10 2.28 33,35} 2 4 3.94±0.00 7.93±0.03 1.34±0.19 4.58±2.44 3.15 33,34,35 jora 3 1 3.79±0.01 12.42±0.14 1.12±0.30 1.70±0.95 1.07 36,37,38 4 1 3.64±0.02 9.11±0.09 1.27±2.49 2.11±3.36 1.87 41 5 1 3.23±0.03 6.81±0.13 3.35±1.71 4.50±2.18 5.97 57,58 6 1 3.23±0.01 20.6±0.11 1.99±2.47 1.93±1.82 2.75 59 7 1 3.20±0.01 6.97±0.07 – 2.17±0.76 – 11,12,13,16,31 8 1 3.89±0.01 6.98±0.14 – – – 1,2,17 9 1 3.56±0.02 5.78±0.23 0.96±0.32 2.17±0.74 0.65 8 10 1 3.89±0.01 7.19±0.09 – 2.46±1.27 0.46 19,20,21 11 1 3.87±0.01 7.12±0.03 – 1.90±0.57 – 4,1,22 12 1 4.13±0.03 3.40±0.18 – – – – 13 1 3.73±0.02 7.26±0.03 NDb _{ND ND 1,5} 14 1 3.62±0.01 5.84±0.09 ND ND ND 24,25,26 15 2 3.45±0.00 36.24±0.10 2.04±1.65 3.77±2.92 2.76 40 16 2 3.30±0.00 10.08±0.03 0.83±0.21 3.79±0.42 1.21 60 17 2 3.34±0.02 1.63±0.02 – 2.23±1.23 0.20 10 18 2 3.88±0.01 7.17±0.12 – 1.80±1.79 – 1,17,22,23 19 2 3.47±0.01 4.77±0.04 ND ND ND – 20 3 3.55±0.09 34.20±1.87 2.51±2.91 1.94±1.95 2.40 55 21 3 3.24±0.01 6.94±0.06 – 2.27±0.06 – 13,14,16,32 22 3 3.68±0.00 6.92±0.06 – 1.83±2.00 0.37 5,18 23 3 3.93±0.01 6.64±0.06 ND ND ND 2,4,6 24 4 2.84±0.01 6.93±0.05 1.53±0.58 3.74±0.95 1.51 – 25 4 3.49±0.01 6.94±0.03 3.65±0.17 2.91±0.43 3.02 8 26 4 3.05±0.01 6.89±0.04 1.80±0.57 1.59±0.80 1.47 9 27 4 3.37±0.01 1.66±0.19 – 3.03±0.62 – – 28 4 3.37±0.01 6.97±0.05 – 1.95±0.77 – 13 29 4 3.56±0.02 3.31±0.09 – 1.95±1.64 – – 30 5 4.04±0.01 10.91±0.31 ND ND ND 7,27,28,29,30 31 6 4.14±0.00 6.56±0.06 – – – – 32 7 3.01±0.01 7.91±0.07 – 3.40±0.95 1.71 54,56,61 33 7 3.20±0.01 2.65±0.06 ND ND ND – 34 8 2.62±0.00 1.86±0.15 – 3.51±0.28 1.21 15 35 8 3.02±0.01 2.06±0.18 2.66±0.42 5.88±1.01 3.02 2,17 36 ND 3.39±0.00 16.70±0.59 3.38±1.10 3.79±1.09 4.22 2,39 sevengrain 37 ND 3.71±0.03 14.71±0.62 2.59±0.85 6.90±2.11 2.99 42,43,44,45,46,47 38 ND 3.30±0.01 7.48±0.21 – – 0.71 62,63,64,65,66,67, 68 39 ND 3.31±0.00 2.76±0.07 2.13±1.00 6.66±1.03 1.98 43,48,49,50,51,52, 53 morocho 40 1 3.79±0.01 9.17±0.10 ND ND ND – 41 2 3.84±0.02 7.82±0.23 ND ND ND – a _{Standarddeviation.} b _{ND=notdetermined.}

diversitywithdifferentmtDNArestrictionprofilesoccurring duringthefermentativeprocess.

ThelowpH valuesofthe chichastudied mightbe asso-ciatedwithacidcontent,specifically,lacticandaceticacids, producedduringfermentation.Theseorganicacidscan con-tributenegativelytothesensory qualityofthebeverage,as off-flavourscanresultwhentheseacidsarepresentinhigh amounts.23_{Thetotalreducingsugarsvariedwidely,possibly}

duetosamplingatdifferentfermentationtimesandthe diver-sityofrawmaterialsusedinchichapreparation.Thisvariation mightberelatedtothe efficiencyofthe microbial commu-nityinutilizingthesereducingsugars.Glycerolisanalcohol ofgreatimportanceinalcoholicbeverages,asit providesa sweetaromaandcontributestotheviscosityofthefinal prod-uct. The largest amount of glycerol was 3.65g/L, found in chicha de jora. Itis importantto note that glycerol in high

amountsisnotdesirablebecauseit givesastaleflavourto thebeverage.27

Altayetal.28_{notedphysicochemicalparametersvery}

sim-ilartothosefoundinthisstudywhenstudyingshalgamjuice, afermentednon-alcoholicbeverageproducedfromthe lac-ticfermentationofblackcarrot.Accordingtotheauthors,the pHofthisbeveragewasbetween3.15and4.25andthemajor fermentationproductsobtainedwerelacticacid(from5.18to 8.05g/L),aceticacid(0.57–0.83g/L),ethanol(0.79–6.41%)and volatilearomaticcompounds.Lowconcentrationsofethanol presentinsomesamplesandthepresenceoflacticacidmake thepossibilitythatfermentationbylacticacidbacteriainstead ofalcoholicfermentationhadoccurredinmajorscaleinthese samples.Withrespecttoboza,abeveragefermentedbyyeasts andlacticacidbacteria,thepHrangedfrom3.16to4.63and the ethanol content ranged from not detectable to 0.39%. Mendozaetal.7_{reportedethanolconcentrationaround1%in}

Argentineanmaize-basedchichas,resultsimilartothechichas analysed in our study.Therefore, the chicha samples stud-iedpresentedgenerallysimilarphysicochemicalparameters tothoseobservedforothertraditionalfermentedbeverages. Finally, the Ecuadorian chichas can be considered acid fer-mentedbeverages,andtheyeastspeciesassociatedwithits fermentativeprocessshouldcontributetothesingulararoma anduniqueflavourofthisbeverage.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Thiswork wasfundedbytheConselhoNacionalde Desen-volvimentoCientíficoeTecnológico(CNPq–Brazil),Fundac¸ão deAmparoàPesquisadoEstadodeMinasGerais(FAPEMIG –Brazil),theCoordenac¸ãodeAperfeic¸oamentodePessoalde NívelSuperior(CAPES–Brazil),andtheSecretaríaNacional de Educación Superior, Ciencia, Tecnología e Innovación (Ecuador).

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