Bacteria isolated from Korean black raspberry vinegar with low biogenic amine production in wine

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

Industrial

Microbiology

Bacteria

isolated

from

Korean

black

raspberry

vinegar

with

low

biogenic

amine

production

in

wine

Nho-Eul

Song

a

_,

_Hyoun-Suk

_Cho

b

_,

_Sang-Ho

_Baik

a,∗

a_Department_of_Food_Science_and_Human_Nutrition,_and_Fermented_Food_Research_Center,_Chonbuk_National_University,_Jeonju,_Jeonbuk,

RepublicofKorea

b_Gucheondong_Bokbunja_Co.,_Muju,_Jeonbuk,_Republic_of_Korea

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6October2014 Accepted15September2015 Availableonline2March2016 AssociateEditor:SolangeInes Mussatto

Keywords:

Biogenicamines Histamine Aceticacidbacteria Vinegar

Wine

a

b

s

t

r

a

c

t

Ahighconcentrationofhistamine,oneofthebiogenicamines(BAs)usuallyfoundin fer-mentedfoods,cancauseundesirablephysiologicalsideeffectsinsensitivehumans.The objectiveofthisstudyistoisolateindigenousAcetobacterstrainsfromnaturallyfermented BokbunjavinegarinKoreawithreducedhistamineproductionduringstarterfermentation. Further,weexamineditsphysiologicalandbiochemicalproperties,includingBA synthe-sis.TheobtainedstrainMBA-77,identiﬁedasAcetobacteracetiby16SrDNAhomologyand biochemicalanalysisandnamedA.acetiMBA-77.A.acetiMBA-77showedoptimalacidity% productionatpH5;theoptimaltemperaturewas25◦C.Whenwepreparedandexamined theBAssynthesisspectrumduringthefermentationprocess,Bokbunjawinefermented withSaccharomycescerevisiaeshowedthatthehistamineconcentrationincreasedfrom2.72 ofBokbunjaextractto5.29mg/Landcadaverineanddopaminewasdecreasedto2.6and 10.12mg/L,respectively.BokbunjavinegarpreparedbyA.acetiMBA-77asthestarter,the his-tamineconcentrationofthevinegarpreparationstepwasdecreasedupto3.66mg/Lfrom 5.29mg/Linthewinepreparationstep.Toourknowledge,thisistheﬁrstreportto demon-strateaceticacidbacteriaisolatedfromBokbunjaseedvinegarwithlowspectrumBAand wouldbeusefulforwellbeingvinegarpreparation.

Introduction

Biogenicamines(BAs)areorganicbasesthatprimarilyoccur infermentedfoodsduetothedecarboxylationofaminoacids bycertainmicroorganismswithbiologicalactivity.Duetothe

∗ _{Corresponding}_author.

E-mail:baiksh@jbnu.ac.kr(S.-H.Baik).

undesirable physiologicaleffects causedby ahigh concen-trationofBAsinsensitivehumans,manyeffortshavebeen performedinordertoreducetheirconcentrationinvarious foods.Inparticular,theconsumptionoffoodswithhigh-level concentration of histamine can have vasodilation effects1 in humans aswell ascause headaches, heartpalpitations,

http://dx.doi.org/10.1016/j.bjm.2016.01.016

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edema,vomiting,diarrheaandothersymptoms2_;_thus,_there aregreatdemandstoreducethehistamineconcentrationin thepreparationoffermentedfoods.

Infermentedfoods,severalspeciesofhistamine-producing lactic acid bacteria (LAB) and yeast belonging to the Lac-tobacillus, Leuconostoc, Pediococcus genera,3 _Oenococcus _oeni,4

Staphylococcus spp., Enterobacter cloacae, and Candida spp.5 have been determined in a wide range of food products, not only fermented from ﬁsh, meat, vegetables and dairy products,but also from other typesoffoods, suchas beer and wine. Although many other BAs, except histamines such as putrescine, spermidine, methylamine, ethylamine, phenylethylamine,isoamylamineandcadaverine,areusually derived from the raw materials ofgrapes and grapemust themselves,mostoftheBAscanbesynthesizedordegraded duringfermentation.6_With_regard_to_wine_{fermentation,}_BAs, especiallythroughfermentationprocesses, are wellrelated withthe malolacticfermentationstep.Inthis step,a large amountofBAsareformedinwinebythedecarboxylationof

Lactobacilli.7 _LABs_isolated_from_wine,_such_as_P. _parvulus,_L.

mali,Leu.Mesenteroids,L.brevis,O.oeniand L.hilgardii,were showntoproducevariousBAswithdifferentBAspectrum.8_It hasbeenalsodemonstratedthatmanyyeaststrainspresent inwines(Saccharomyces cerevisiae,Brettanomyces bruxellensis, Kloeckera apiculata, among others) can produce histamine, phenylethylamine and cadaverine.9,10 _These _results_clearly indicatethatmicroorganisms,suchasfermentationfactors, arecriticalforcontrollingBAsynthesisduringfermentation.

Vinegarfermentationusually occurred afteralcohol fer-mentationunderabsolutelyaerobicconditionduringlittlebit longtimefermentationthroughtheoxidationoftheethanol by AAB. When the total BAs contents in various vinegars wereexamined,interestingly,Balsamicand“PedroXiménez” Sherryvinegarsshowedthe highestamounts ofBAs; how-ever,apple,whiteand Sherrywinevinegarshadthelowest concentrations,therebyindicatingthatbiogeniccontents sig-nificantlyremainedaftervinegarfermentation,althoughthe amountofBAswere differentdependingonthe vinegars.11 AlthoughtheproducedBAsaftervinegarfermentation usu-allyoriginatefromyeastsorLABsduringwinefermentation, the finalproductsofvarious vinegarsstill contains signifi-cantamounts ofBAs, whichmightcausesignificant health problemsbecauseBAslevels areincreasedduring malolac-ticfermentation.12_Hence,_there_are_numerous_LABs_that_are isolatedinordertocontrolBAsduringfermentation; further-more,theyareexaminedfortheirBAreductionability,aswell asconveyapotentialpossibilityforBAsreductioninthefinal wineproduct.13_However,_there_has_been_no_study_yet_on_AAB forBAreductionabilityduringthefermentationprocess.8_Only minimalknowledgeon the BAsynthesis abilityofAABs is present.

Black raspberry (Rubus coreanus Miquel), which is well known as ‘Bokbunja’ in Korea, is mainly cultivated in the southern parts of Korea, China and Japan, and has been increasedincultivationareaduetotheexpandedusageas atraditionalherbalmedicine.14 _Because_Bokbunja_contains rich ingredients such as ﬂavonoids, tannins, triterpeno-sidesand phenoliccompounds, variousfunctional aspects, suchascell proliferation inhibitionand apoptosis stimula-tion inHT-29 humancoloncancer cells15 _and_{anti-fatigue,}

anti-gastropathic, anti-inﬂammatory, anti-rheumatic and antioxidantactivities,havebeenreported.Duetoits extraor-dinaryfunctionality,atthepresent,varioustypesofprocessed products, such as sugar extracts, wine and vinegars, have beenactivelyproducedtocreatehigh-valueaddedBokbunja foodproducts.ThetraditionalBokbunjavinegarismadefrom the fermentationoffully ripenedfruitsthroughatwo-step fermentationofalcoholandvinegarfermentationstepswith the sugar-soakingsteppriortofermentation.Many studies indicated that the chemical constituents change the raw Bokbunjafruitandthefermentedwineandbeveragechange enhance the functional aspects, such as the antioxidant activity. Nevertheless, a high concentration of BAs might alsobeformedduringtheethanolandvinegarfermentation throughthedecarboxylationofaminoacids.

Inthisstudy,ourpurposewastoisolateindigenousAAB strains whichpossess the biogenic aminereductionability especiallyforhistamineproductionfromnaturallyfermented Bokbunjavinegar,andexaminedtheirusefulnessfor prepa-ration offermentedBokbunja vinegarshowingreduced BA concentrationwithespeciallyhistamine.

Materials

and

methods

Bacteriaandcultureconditions

AABwereisolatedfromnaturallyfermentedBokbunjavinegar intheMujuarea.Sampleswereserialdilutedandcultivated inaGYECagarmedium(5%glucose,1%yeastextract,ethanol 3%, 1.5%calcium carbonate,0.8%agar)for3 daysat29◦C. IsolatedAABcoloniesshowedaclearzoneontheGYECagar plate,whichwascultivatedinGYEbroth(5%glucose,1%yeast extract,3%ethanol)for3daysat29◦C,160rpm.Culturebroth wascollectedeverydayforanalyzingthepH,◦Brix,acidity(%) andcellgrowth.

Identiﬁcationby16SrDNAsequencing

Bacterial cells were grown aerobically in GYE broth at 26◦C. The bacterial sets of two primers 27f and 1490r were applied inorder to amplifythe V3 variable regionof the eubacterial 16S rDNA fragments. The DNA sequences of primers 27f (5-AGTTTGATCCTGGCTCAG-3) and 1490r (5-GTTACCTTGTTACGACTTC-3)were used. PCRswere per-formed in a Bio-Rad thermocycler (MyCycler, Bio-Rad Lab-oratories, Hercules, CA, USA) as described by Kopermsub andYunchalard.16_The_PCR_products_were_separated_in_1.0% agarosegelina1×TBEbuffer.Afterelectrophoresis,thegels werestainedwithethidiumbromideanddocumentedbythe GelDocXR+usingtheImageLabSoftware(Bio-Rad Laborato-ries,USA)imagingSystem2000(Bio-RadLaboratories,USA). DNAsequencingwasperformedwiththeABI-PrismBigDye DeterminatorCycleSequencingReadyReactionkitand ABI-Prism377Sequencer(AppliedBiosystemsJapan,Tokyo,Japan). Sequence similarity searches were carried out using the Basiclocalalignmentsearch(BLAST)ontheEMBL/GenBank databases; phylogenetic trees were constructed using the neighbor-joiningmethod.Finally,API20NEKit(bioMerieux,

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France)was also used forthe identiﬁcation of non-enteric Gramnegativerods.

SEManalysis

Cell morphology was observed by FE-SEM (Field Emission ScanningElectronMicroscope).Thebacterialcellsgrowingon GYEbrothfor3daysat26◦Cwereﬁxedin2.5% glutaralde-hydeindistilledwaterfor30min,post-ﬁxedin1%osmium tetroxide indistilled waterfor 20min, dehydratedthrough gradedethanol;thepelletwasdriedfor24hbeforethe obser-vationandgold-coatedbysputtering.Thecellswerecoated withgoldbyacoater(SC502,Polaron,WestSussex,UK)and observed under a scanning electron microscope (SEM 515, Philips,Netherlands).

Phenotypicandtechnologicalfeaturesofstrains

CellshapewasobservedafterincubatingtheculturesinGYE brothat28◦Cfor3days.Oxidationofethanoltoaceticacid wastested inthe GYE brothcontainingglucose 10g, yeast extract10g,ethanol30mL(perliter)after3daysof incuba-tion at28◦C.Growth on 30% d-glucose; 0.5%yeast extract and 30% glucose incubation 7 days 28◦C and tolerance to ethanol(5,10,15and20%,v/v)weretested; agradient vol-umeofabsoluteethanolwasaddedtoeachflaskcontaining thesamemedium.17_Growth_on_single_carbon_sources_was car-riedout withfructose,glycerol,raffinose,sucrose,mannose andsorbitol.Thecarbonsourcesweresterilizedbyfiltration (0.2␮m)andaddedtothesterilebasalmedium(yeastextract, 0.5g; vitamin-free casaminoacid, 3g) to afinal concentra-tionof0.3%by2%pre-cultureinoculation.18_The_control_was usedasaculturebrothwithoutthecarbonsource.The opti-caldensitywasmeasuredat600nmusingspectrophotometer (BeckmanSpectrophotometer,USA).Catalaseactivitywas per-formedbyaddingyoungcellstoadropofa10%H2O2solution andobservingtheproductionofO2,asdescribedpreviously.19 Catalaseoptimumcultureconditionwasalsodemonstrated. FivefactorsofculturebrothpHwereconsideredforthe cul-tureconditions.Fivehundred␮Lofpre-culturewasinoculated into50mLofeachoftheGYEbroth.Thebrothcultureswere thenincubatedat29◦Cat160rpm.Theacidityoftheculture mediawasmeasured bytitrating10mLsampleswith0.1N NaOHusingphenolphthaleinasapHindicator.

WineandvinegarfermentationwithBokbunja

OrganicallygrownblackraspberrywasharvestedfromMuju provinceinSouthKoreaandquicklycooledinthefreezerat −20◦_C_before_use._The_cooled_black_raspberry_was_mixed_with organicsugar(GoiasaGoiatubaAlcoolLtda,Brazil)ataratioof 6:4,andthenripenedfor40daysat20◦C.Thepressedcrude extractwasﬁltered,sterilizedat85◦Cfor15–20mindiluted to20–25◦Brixwithdistilledwater.Ethanolfermentationwas carried out bystarter yeast, S.cerevisiae no.7013(Fermivin, DSMFoodSpecialties,France).Dryyeastwasre-hydratedto 10%(w/v)bydistilledwaterat37◦Cand swelledfor30min before inoculation in the Bokbunja crude extract; ethanol fermentationwasstartedwiththeinoculationof0.02%(v/v) re-hydratedstarteryeastandfermentedat23◦Cfor5days.

Static vinegar fermentationby the traditionalmethod was performeddirectlyinoculate10%ofstartercultureinto the obtainedBokbunja wine.Theacetic acid fermentationwas performedinacrockat23◦Cwithoutagitationfor14days.

AnalysisofBAsproducedbymicroorganisminculture

brothbyHPLC

ThedeterminationofBAwascarriedoutbyhighpressure liq-uidchromatography.AnalysiswasperformedwithHPLC1200 series(AgilentTechnologies,SantaClara,CA,USA).The5mL samplesand5mLof0.1NHClweremixedand1mLofmixed solutionwerederivatizedwith0.5mLinternalstandard (1,7-diaminoheptane, 100mg/L) using saturated Na2CO3 0.5mL and1%dansylchlorideacetonesolution1mLat45◦Cfor1h. Afterderivatization,10%prolinesolution1mLwasaddedin ordertoremovetheremainedderivatizationreagents.5mLof etherwasmixedwithshakingfor3minandasupernatantwas driedusingN2gas.Sampleswereinjectedintriplicateontothe columnafteradding2mLofacetonitriletothedriedsamples, followed bybeingfilteredthrougha0.45␮mfilter(Minisart NY 25,Sartorius-stedimbiotech). All separations were per-formedonaCapcellPakC18Column(4.6mm×250mm,5␮m, ShiseidoCo.,Tokyo,Japan);thefluorescencewavelengthfor detectionwas254nm.Thecolumnwassetat40◦C,flowrate 1mL/min.Asthemobilephases,acetonitrilewasused.The programusedwasasfollows:lineargradientelutionfrom55% for10min;65%for5min;80%for10minfollowedby90%for 15min;thetotaltimewas40min.Elevenamines(tryptamine, 2-phenylethylamine,putescine,cadaverine,histamine, sero-tonin,trymine, spermidine,spermine,l-noradrenaline,and dopamine)wereinjectedasthestandardsolution.

Statistics

All analyses were performed in triplicate. The data were analyzed by a one-way ANOVA using the SPSS version 16.0 program. The results on the analyses are expressed as mean±standard deviation (SD). The differences among groupswereassessedbyusingDuncan’smultiplerangetests. Statisticalsigniﬁcancewasconsideredatp<0.05.

Results

and

discussion

Isolation,screeningandidentiﬁcationofAABfrom

naturallyfermentedKoreanblackraspberryvinegar

Bokbunjavinegars,asthescreeningsource,wasobtainedfrom variousareasinMuju,Korea(127◦44,35◦54),whichfermented BokbunjanaturallybyusingorganicallygrownBokbunja. Dur-ing theacetic acidfermentationover acourseof144days, AABcontinuouslyincreaseduptolog5.8CFU/mL.Themost high-level ethanolconcentrationwas12% attheinitial fer-mentationstageof3dayswithaconcomitantgrowthofyeast. Then,theaciditygraduallyincreasedfrom1.06%(w/v)to2% (w/v)after45daysoffermentation,andthenﬁnallyachieved 4.4%(w/v)acidityaftervinegarfermentationfor144days.

From a total of147 AAB-like strains isolated from nat-urally fermented Bokbunja vinegar on the GYEC medium,

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0 1 2

MBA-41MBA-42MBA-55MBA-59MBA-60MBA-73MBA-75MBA-77MBA-78MBA-79MBA-81MBA-82_MBA-103_MBA-105_MBA-110

3 4 5 6 7 His ta m in e (pp m, mg /L) Strains a ab abc bcd bcd bcd bcde

cde cde cde cde

de ef

fg g

Fig.1–Productionofhistamine(ppm,mg/L)byMBA-77 isolatedfromnaturallyfermentedBokbunjavinegar.The determinationofbiogenicaminewascarriedoutbyHPLC. Elevenamines:tryptamine,2-phenylethylamine,

putescine,cadaverine,histamine,serotonin,trymine, spermidine,spermine,l-noradrenaline,dopaminewere derivatizedwithdansylchloridewithinternalstandard (1,7-diaminoheptane)andinjectedasstandardsolution. Valuesrepresentmeansoftriplicate

determination±standarddeviation.Differentletters(e.g. a–g)wereassignedtosigniﬁcantlydifferentgroupsandthe sameletterswereassignedtosigniﬁcantlysimilargroups (p<0.05).

fifteenstrains,displaying bothahigh-level aceticacid pro-ducingactivitiesaswellasahighgrowthrate,wereselected. Then,thefifteenstrainswereexaminedforBAspectrumat amodifiedconditionaftercultivationontheGYECmedium. Asshown in Fig. 1, mostof the examined strains showed anidenticalBAprofile,showingonlythehistaminedetected. Cadaverin, spermidine, spermine, tyramine, serotonin and dopaminewerenotdetectedinouranalysis.Whenwe com-paredthe histamineconcentration, strain MBA-77 showed

Gluconacetobacter liquefaciens IFO 12388 Acetobacter syzygii strain G4-4

Acetobacter ghanensis 384

Acetobacter estunensis Acetobacter oeni B13

Acetobacter aceti

Acetobacter indonesiensis Acetobacter tropicalis A77

Acetobacter persicus Acetobacter farinalis G360-1 Acetobacter orleanensis Acetobacter cerevisiae

Acetobacter malorum EW-m

Acetobacter pasteurianus NBRC 3225

0.01

Acetobacter aceti NCIB

MBA-77

Acetobacter fabarum LMG Acetobacter lovaniensis NBRC 103497

Fig.3–Phylogenetictreeof16SrDNAsequencesofMBA-77 isolatedfromnaturalfermentedBokbunjavinegar.

SequencesimilaritysearcheswerecarriedoutusingBasic localalignmentsearch(BLAST)ontheEMBL/GenBank databasesandthephylogenetictreeswereconstructed usingtheneighbor-joiningmethod.

thesigniﬁcantlylowestconcentration(3.05mg/L)ofhistamine (p<0.05)withthehighestaceticacidinthemedium,asshown inFig.1.However,strainMBA-59showedasigniﬁcantlyhigher histamineproduction(p<0.05).

IdentiﬁcationoftheselectedAABfromnaturally

fermentedKoreanblackraspberryvinegar

AsshowninFig.2, theisolatewasrod-shapedandregular short lengthsizeofabout 4␮mlength. When the selected ﬁfteen strains were identiﬁed by 16S rDNA sequence, all thestrainsshowed highsimilaritywithA.acetistrainNCBI 8621with99–100%homology.Inparticular,thestrainMBA-77 showingthelowesthistamineproductionshowedhigh sim-ilaritywiththeA.acetistrainNCBI8621with99%homology (Fig.3).Moreover,inthebiochemicaltestresultsusingAPI20 NEKit,theselectedstrainMBA-77showedatypical charac-teristicofAAB,therebyindicatingalmostidenticalchemical metabolicpropertieswithA.aceti(datanotshown).Asshown inTable1,theselectedstrainMBA-77wasrevealedasGram negative,catalasepositiveandoxidizedethanoltoaceticacid.

200 nmº EHT=15.00 kV 200 nmº EHT=15.00 kV WD=2.7 mm Mag=50.00 K X WD=2.7 mm JBNU CURF EM Lub. JBNU CURF EM Lub. Signal A=InLens Mag=50.00 K X Signal A=InLens

Fig.2–Fieldemission-scanningelectronmicroscope(FE-SEM)ofcellsofaceticacidbacteriaisolate(MBA-77)growninGYE broth(50,000×).

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Table1–PhenotypicandtechnologicalfeaturesofaceticacidbacteriaisolatedfromtraditionalKoreanfermentedvinegar.

Strain Ethanol(%) d-Glucose(%) Growthonsinglecarbonsource(%) Catalase

activity 5 10 15 20 30 Fructose Glycerol Rafﬁnose Sucrose Mannose Methanol

A.acetiMBA-77 + − − − − + ++ Wa ₊ ₊₊ _W ₊

a _w:_weak.

Alltogether,wewereabletoidentifytheselectedstrain MBA-77asAcetobacteraceti,andnamingitA.acetiMBA-77.

Physiological,biochemicalandgrowthproperties

As shown in Table 1, A. aceti MBA-77 were tolerantat 5% ethanolconcentrationandshowedbettergrowthand avail-abilityat5%ethanol. Thisabilitytogrow from 5%(v/v)of ethanol is one ofthe mainphenotypic traitsfor selecting theAABstarterforvinegarproduction.20_When_the fermen-tationproﬁlewas examinedbyusingvarioussinglecarbon sources, the A. aceti MBA-77 showed weak growth in the presentmethanolasacarbonsourceaswellasalowergrowth rateintherafﬁnosecontainingmedium.However,theA.aceti

MBA-77 was able to ferment rafﬁnose and mannose more efﬁciently.AccordingtoWuet al.,18 _Acetobacter_pasteurianus speciesshowedahighdegreeofphenotypicvariability,such asethanoltolerance,growthon30%ofd-glucoseandsingle carbon sources, which are in accordance with the pheno-typicvariabilityofA.acetiandA.acetiMBA-77.Someofthese featureswerepreviouslyusedasthediscriminativetoolsfor taxonomicpurposesinordertodifferentiateAcetobacterfrom

GluconobacterandGluconacetobactergenera,becausethegrowth ond-mannitoland30%ofd-glucoseweredifferent.18_However, inourresult,thesameA.acetispeciesshowedsimilar pheno-typicvariability,exceptfortheslightdifferencerafﬁnoseand mannosefermentability(datanotshown).Theoptimalacetic acidproductionofA.acetiMBA-77wasshownatpH5after 192hfermentation,asshowninFig.4.ComparedtoA.aceti

MBA-77,theoptimalgrowthofAABwasgenerallyobserved atpH4–5foroptimalaceticacidproductionduetothefast declineofviablecellsofA.acetibyalowoxygen concentra-tionwhenthepHvaluewaslowerthan3.4.21_AAB_is_also_able togrowatlowerpHvalueswherebacterialactivityhasbeen detectedforpHvaluesunder3,eventhoughtheoptimalpH growthofAABisbetween5.0and6.5.17_Thus,_the_observed optimalconditionoftheA.acetiMBA-77forgrowthandacetic acidproductionappearstobeataveryunusuallyhighlevel. Theseresultsmightbeexplainedbythepropertiesofthe orig-inalsourceweusedinthisstudy forscreeningbecausethe pHofBokbunja seedvinegarmaintained avery low pHof 2.4–3.0duringvinegarfermentation.Thus,theA.aceti MBA-77mightbeadaptedtosuchlowpHenvironment.Moreover, theA.acetiMBA-77showedthehighestaceticacid produc-tion atpH5,which correspondstomostoftheAABs have beenknownforoptimalaceticacidproductionatpH5during fermentation.

Whenweexaminedthetemperatureforoptimalaceticacid fermentation,theA.acetiMBA-77showedahigheracidity(%) at25◦Cafter192hfermentation,asshowninFig.5,whichis

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 4 3 5 6 7 Acidity % pH

Fig.4–OptimalpHofA.acetiMBA-77isolatedfrom traditionalKoreanfermentedvinegar.Theacidity(%)was measuredundervariousconditions.ThepHexperiments wereperformedunderﬁvefactorsofculturebrothpH(3,4, 5,6and7)incubatedat160rpm.Eachexperimentwas performedintriplicate. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 20 25 29 37 Acidity% Temperature(ºC)

Fig.5–OptimaltemperatureofA.acetiMBA-77isolated fromtraditionalKoreanfermentedvinegar.Theacidity(%) wasmeasuredundervariousconditions.Thetemperature experimentswereperformed20,25,29and37◦C,incubated at160rpm.Eachexperimentwasperformedintriplicate.

relativelylowerthanthegenerallyknownAABs.Intheabove optimumtemperature(25–30◦C),AABs weredeactivatedby thedenatureofenzymes,membranedamageandsensitivity tothetoxiceffectofaceticacid.22

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Table2–Productionofbiogenicamines(mg/L)inBokbunjaextract,wineandvinegarfermentedbyaceticacidbacteria isolatedinBokbunjavinegar.

Bokbunjaextract Bokbunjawine Bokbunjavinegara

Tryptamine nd nd nd 2-Phenylethylamine nd nd nd Putescine nd nd nd Cadaverine 5.09±0.08a _2.6_±_0.16b _nd Histamine 2.72±0.03c _5.29_±_0.05a _3.66_±_0.11b Serotonin 70.25±1.24b _80.98_±_1.74a _nd Tyramine nd nd nd Spermidine 1.21±0.03b _4.77_±_0.09a _nd l-Noradrenaine nd nd nd Dopamine 11.43±0.35a _10.12_±_0.07b _nd Spermine nd nd nd Totalamines 90.7±1.73b _104.06_±_2.11a _3.66_±_0.11c

a _Bokbunja_vinegar_fermented_with_A._aceti_MBA-77.

nd:notdetected.

Dataarethemean±SDofthesamplesanalyzedintriplicate.

Differentlowerletters(e.g.a–c)wereassignedtosigniﬁcantlydifferentbetweensamplesforeachbiogenicamine(p<0.05).

BAanalysisforBokbunjaextract

ToanalyzetheBAsproductionduringBokbunjavinegar fer-mentation,vinegarfermentationinalabscalewasperformed and sampling was carried out at each fermentation step ofthe Bokbunjaextract,winefermentationand aceticacid fermentation with a single culture inoculation of A. aceti

MBA-77.AsshowninTable2,BAspectrumofcadaverine, his-tamineandspermidinewasalreadyobservedintheBokbunja extract;5.09,2.72and1.21mg/L,respectively.García-Marino etal.23_reported_that_grape_must_before_winemaking_contains BAs(8.35mg/L); histamine3.26mg/L, ethylamine1.03mg/L, putrescine 3.21mg/L and cadaverine 0.85mg/L. Their BA spectrums were slightly different but consistent with our resultthattotalBAcontenttendstoincreasethroughout wine-making (13.17mg/L). Surprisingly, serotonin and dopamine weredetectedas majorBAsinBokbunja extract.Wecould notdetectotherBAsoftyramineandputrescineinthe Bok-bunja extract, which already has been identiﬁed as being dominantinrawmaterials.6_It_has_been_known_that_the_BAs of putrescine, spermidine and histamine are normal con-stituentsoftherawmaterialsinwinefermentation;further, adiversehighconcentrationofBAsmightoccurduringthe fermentationprocessesofthewine-makingprocessthrough thedecarboxylationofaminoacids.24

ChangeofBAsduringethanolfermentationbyS.

cerevisiae

DuringBokbunjawinefermentationprocessesbyS.cerevisiae

asthestarter,20◦Brixofinitialsugarwasreducedtoaﬁnal 10◦Brix, accumulating approximately 12% ethanol after 5 dayswithcontinuousincreasingyeastuntillog7.0CFU/mL. WhenweanalyzedBokbunjawineafter5daysofalcoholic fermentation, the BAs ofcadaverine, histamine, serotonin, spermidine and dopamine were detected. The histamine concentration increased from 2.72 of Bokbunja extract to 5.29mg/LofwinebyS.cerevisiaeduringwinefermentation.

Similarobservationshavebeenreportedbyotherauthors9,10 thatthisresultgenerallyduetotheyeastsmetabolitevarious amino acidsduring alcoholfermentation. Wedidnot con-trol the winefermentation process byS.cerevisiaein order to understand the effect of AABs we used in this study. When we compared the obtained BAs proﬁle with several commercialBokbunjawinespurchasedfromdifferentregions in SouthKorea,25 _our _results_were _slightly _different. Com-paredtoallthesamplesinwhichputrescineandtyramine exist as the most dominant amines detected100% in the samples, and tryptamine,cadaverine and spermidinewere foundin83%,78%and83%ofthesamples;theleastcommon amineswere2-phenylethylamineandhistamine,whichwere detectedin44%and50%ofthesamples,respectively. How-ever,inourresult,putrescineandtyraminewerenotdetected, and the most prevalent BAs of spermidine and dopamine were detectedin88%;cadaverine,histamineandspermine were found in 2.6, 5.3 and 4.8%, respectively, in Bokbunja winefermentedintheMujuprovince.Theseresultsclearly indicatedthatBAspectrawere signiﬁcantlyexpandedafter the Bokbunja wine fermentationstepby using S.cerevisiae

no.7013 as astarter culture. As shown inTable 2, ethanol fermentation byS. cerevisiaeno.7013 resultedin signiﬁcant changesonBAs.Duringfermentation,cadaverine(5.09mg/L) anddopamine(11.43mg/L),whichwereoriginallypresented intheBokbunjaextract,wasdecreasedto2.6and10.12mg/L. However,otherBAs,suchashistamine,serotoninand sper-midine,wereproduced2.57,10.73and3.56mg/L,respectively, during alcoholfermentation.It isnotunusualforthe new synthesisofotherBAsduringethanolfermentationbecause generally,fourBAs,suchashistamine,tyramine,putrescine and cadaverine,havebeen foundinahigherconcentration inwine.Furthermore,mostoftheBAsynthesisisresponsible forthemicrobialfermentationofyeastandLABduringwine fermentation.Ithasbeendemonstratedthatmanystrainsof yeastspresent inwines(S.cerevisiae,B. bruxellensis,K. apic-ulata,etc.)canproducehistamine,ethanolamine,agmatine, phenylethylamine and cadaverine. Caruso et al.9 _reported

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thatB.bruxellensisproducedthehighesttotalBAs(15mg/L), followedbyS.cerevisiae(12.14mg/L)andhistamine; cadaver-ine,putrescineandtryptaminewereallproducedbystrains less than 4mg/L. Also, theyreported that the highest fer-mentative yeast (S. cerevisiae) showed the highest amount ofagmatine, andmiddlefermentativeyeastCandidastellata

also showed a capacity to produce cadaverine. Generally, in alcoholic beverages, the toxic dose is considered to be between 8 and 20mg/L for histamine, 25 and 40mg/L for tyramine, whereas as little as 3mg/L ofphenylethylamine cancausenegativephysiologicaleffects.26 _Although_the_BA contentsinBokbunjawineinthisstudydidnotexceedthe toxic levels (especially for histamine of 5.29mg/L), which arethehighesthistamineconcentrationallthroughthe fer-mented wine at present, it strongly indicates that there must be a novelmethod to decrease those BAs, including histamine.

ChangeofBAsduringaceticacidfermentationbyA.aceti

MBA-77afterethanolfermentation

Aceticacid fermentationwasstarted byinoculatingstarter culture after 5days ofalcohol fermentation with increas-ingAABincreaseduptolog5.8CFU/mLduring14daysand ﬁnallyconverted to acetic acid up to6% during fermenta-tion (data not shown). Lots of AAB from various vinegars and wine based on different raw materialssuch as grape, strawberryor persimmon hasbeen isolated and examined fortheiracetiﬁcationasastarter.27–30_When_Acetobacter

mal-orum recovered from strawberry wine were used as native startersforacetification,theaceticacidconcentrationreached was5.5–6.6%(w/v)whichalmosttwotimeshigherthan spon-taneousacidificationonstrawberry.30Similarly,ourselected strainA.acetiMBA-77asthenaturalstarterwasalsoshowed goodfermentationprofile;onBokbunjaasrawmaterials, indi-catingthattheuseofindigenousstrainforacetificationmust beapplicableforefficientfermentationacetificationprocess. Moreover,whenweexaminedthe BAsofBokbunja vinegar afteracetic acidfermentationbyusingourselectedA.aceti

MBA-77 as the starter during the 14 days, we found that the histamineconcentrationwasdetectedin theBokbunja vinegarsample as 3.66mg/L, which isa 0.7-times reduced level compared toBokbunja wine (5.29mg/L), as shown in

Table 2. Moreover, in addition to a significant decrease of histamineconcentrationafteracetic acidfermentation,the expanded BA synthesis spectrum after ethanol fermenta-tionsignificantlydecreasedbyA.acetiMBA-77aswell.Even thoughthereducingrateofhistamineconcentrationdidnot increase ashigh aswe expected inthis experiment, how-ever, otherBAs occurred inBokbunjawine canbereduced significantly, which clearly indicates that the introduction ofA. aceti MBA-77 as the starter for acetic acid fermenta-tionwasaveryeffectivetoolforreducingtheBAscontents, includinghistamine,inthepreparationofBokbunjavinegar. Generally,a high concentrationofBAs canbe formed dur-ing the ethanol fermentation through the decarboxylation ofvariousaminoacids.23,31 _In_addition, _BAs_are physiolog-icallydegradedthroughoxidativedeaminationcatalyzedby aminesoxidase.Someaminesdegradingbacteriahavebeen studied.32,33However,duetosignificantinhibitionactivityby

ethanol forspecificenzymes,suchasmonoamine oxidase, diamineoxidaseandhistamine-N-methyltransferase,which areresponsiblefortheneutralizationofBAstonon-toxic prod-uctsunder normalconditionsafterwinefermentation, the fermentedwinecontainingBAsmightcauseunfavorableside effectseveniftheconcentrationofeachindividualBAsdidnot reachuptothehightoxiclevel.6_Namely,_it_would_be desir-abletoreduceethanolconcentrationorBAsconcentrationin the fermentedfoods inorder toprevent those unfavorable sideeffects. Interestingly,vinification itselfmaynotreduce thealreadyformedBAs,includinghistamine.Rather,amore highcontentofBAconcentrationisfoundaftervinification.34 Thisresultmightbecausedbytediousandlong-term vine-garfermentationinunstableenvironments.ComparedtoBAs inbalsamicvinegarresultingin51.5–525␮g/Lofputrescine, 23.1–60.4␮g/Lofcadaverineand101–223.3␮g/Lofspermidine byOrdó ñezetal.,11_however,_those_BAs_and_as_well_other_BAs, suchascadaverine,serotonin,spermidineanddopaminein Bokbunjawine,werenotdetectedafteraceticacid fermen-tation inBokbunjavinegar. Previously, itwas alsoreported thatthemostabundantlyremainedBAsaftervinegar fermen-tationwereputrescineandhistamine,whichwereproduced afterwinefermentationandtransferredtovinegar.11Thetoxic effectsofhistamineamongtheBAsareenhancedinthe pres-enceofcadaverineandputrescinebyinhibitingthehistamine metabolizing enzymes of diamine oxidase and histamine methyltransferase.1_When_we_examined_the_putrescine con-centration of Bokbunja vinegar, we found that putrescine wasnotdetectedallthroughtheBokbunjavinegar fermen-tation,therebyindicatingthatvinegarfermentationbyusing Bokbunja mustbeusefultocontroltheBAs, although Bok-bunjaalsocontainsnumerousfunctionalcomponents,such as phenolic compounds, like other materials. During vine-garfermentation,theactualtotalcontentofBAsinvinegars are very diversedependingon theused lawmaterials ran-gingfrom23.35to1445.2␮g/L;moreoveritscontentsgenerally decreasedafterwinefermentation(130mg/L)perhapsdueto thedegradationbysomebacterialstrainspresentinwineor vinegar.11However,mostofthestudiesonbacterialreduction invinegarfermentationhavebeenfocusedmainlyonLAB. Pre-viously,Landeteetal.8_reported_that_none_of_the_AABs_among the 40 strains and 36 ofyeast strains which isolated from wineorgrapemustproduceBAswhereasLABisolatedfrom wine, Pediococcussp.,Lactobacillus sp.,L.mesenteroidsandO. ovnicanproducehistamine,tyramine,phenylethylamineand putrescine.Also,P.parbulusandL.hilgardiihavebeendetected as high histamine-producing bacteria.7 _In _our _studies, _the total BA concentration significantly decreased after vine-garfermentation(3.66±0.11mg/L,onlyhistamine)compared to wine (104.06±2.11mg/L) and extract (90.7±1.73mg/L), eventhoughwetriedvinegarfermentationbyusingA.aceti

MBA-77. Because A. aceti MBA-77 was never identiﬁed as BA-degrading AAB,this istheﬁrstreporteverpublishedto indicate that an acetic acid bacterium is able to decrease histamine.

Inthis study,wewere abletoisolatetheMBA-77 strain, whichshowedhigheraceticacidsynthesisandlowBA syn-thesis abilityaswell. Theselectedstrain was identiﬁedas

A. aceti by a biochemical and 16S rRNA sequencing anal-ysis. Theoptimal condition for A. aceti MBA-77 was pH 7

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and25◦C.Moreover,A.acetiMBA-77wastolerantatthe5% ethanolconcentrationalongwithalowergrowthrateina raf-ﬁnosecontainingmedium.When theselectedwasused as thestarter foracetic acid fermentation,BAs spectrumand concentration present in Bokbunja extract were increased duringalcoholicfermentation.However,thoseincreasedBAs spectrumandconcentrationweredecreasedaftervinegar fer-mentationbyA.acetiMBA-77.TheAABisolatedhereinisthe ﬁrstapplicationasastartercultureforBAanalysisinvinegar; furthermore,itmightbehelpfultomanufacturevinegarwith alowBAsynthesis.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

This research was supported by High value-added Food Technology Development Program, of the Korea Institute of Planning and Evaluation for Technology in Food, Agri-culture, Forestry and Fisheries (IPET),and the Ministry for Food,Agriculture,Forestry,andFisheriesofRepublicofKorea (313037-03).

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