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Food Sci Nutr. 20 18 ;1– 8.
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1
www.foodscience-nutrition.com
Received:21June2018
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Revised:18A ugust2018
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Accepted:22Augus t2018
DOI: 10.1002/f sn3.80 3
ORIGINAL RESEARCH
Composition and biochemical properties of l- carnitine fortified
Makgeolli brewed by using fermented buckwheat
Namhyeon Park1 | Thi Thanh Hanh Nguyen2 | Gang-Hee Lee1 | Shi-Na Jin1 |
So-Hyung Kwak1 | Tae-Kyung Lee1 | Yeong-Hwan Choi3 | Seong-Bo Kim4 |
Atsuo Kimura5 | Doman Kim1,2
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,
providedtheoriginalworkisproperlycited.
©2018TheAuthors.Food Scien ce & NutritionpublishedbyWileyPeriodicals,Inc.
1GraduateSchoolofInternational
AgriculturalTechnolog y,SeoulNational
University,Pyeongchang-gun,Gangwon-do,
Korea
2InstituteofFoodIndustrialization,Institutes
ofGreenBioScienceandTechnology,Center
forFoodandBioconvergence,Seoul
NationalUniversity,Pyeongchang-gun,
Gangwon-do,Korea
3KooksoondangBreweryCo.,LTD.,
Hoengseong-gun,Gangwon-do,Korea
4CJCheilJedang,LifeIngredientandMaterial
ResearchInstitute,Suwon,SouthKorea
5ResearchFacultyofAgriculture,Hokkaido
University,Sapporo,Japan
Correspondence
DomanKim,GraduateSchoolof
InternationalA griculturalTechnology,Seoul
NationalUniversity,Pyeongchang-gun,
Gangwon-do,Korea.
Email:kimdm@snu.ac.kr
Funding information
KoreaInstituteofPlanningandEvaluation
forTechnologyinFood,Agriculture,
Forestry(IPE T)throughAgriculture,Food
andRuralAffairsResearchCenterSupport
Program,MinistryofA griculture,Food
andRuralAffairs(MAFRA);Grant/Award
Number:710012-03-1-HD220;Korea
InstituteofPlanningandEvaluationfor
TechnologyinFood,Agriculture,Forestry
andFisheries(IPET)throughHighValue-
addedFoodTechnologyDevelopment
ProgramMinistryofAgriculture,Foodand
RuralAffairs,RepublicofKorea,Grant /
AwardNumber:116013032HD020;
NationalResearchFoundationof
Korea(NRF),Grant/AwardNumber:
2016K1A3A1A19945059
Abstract
MakgeolliisatraditionalKoreanalcoholicricebeverage.Itisbrewedofingredients
containingstarch,Nuruk,andwater.Inordertoimprovethequalityandfunctionality
ofMakgeolli,theRhizopus oligosporusfermentedbuckwheatcontaining18.7mg/kg
of l-carnitinewereutilizedtobrewl-carnitinefortifiedMakgeolliwithrice.Makgeolli
was prepare d in two-stage ferm entation method an d total rutin and quer cetin in
eachfermented buckwheat Makgeolliwereincreased 1.8-fold greater than buck-
wheatMakgeolli.DPPHantioxidantactivitywasenhancedinfermentedbuckwheat
MakgeollithanbuckwheatMakgeolli(21.9%–65.7%).Theamountsofl- carnitine in
riceMakgeolli,buckwheatMakgeolli,andfermentedbuckwheatMakgeolliwere0.9,
0.8–1.0, and1.0–1.9mg/L, respectively.The fermented buckwheat Makgeolli not
onlypromotedhealthbenefitbyincreasingl-carnitineandflavonols,butalsomade
effective alcohol production (2.8%–8.4%) compared to common buckwheat
Makgeolli,indicatingthepotentialindustrialapplicationwithhealthbenefits.
KEYWORDS
antioxidantactivity,buckwheat,l-carnitine,quercetin,Rhizopus oligosporus,rutin
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PARK et Al .
1 | INTRODUCTION
MakgeolliisatraditionalKoreanalcoholicbeverage.Itisbrewedby
fermentationofingredientscontainingstarch, Nuruk (a traditional
fermentation starter composing ofvariousmicroorganisms such as
fungi,yeasts,andlacticacidbacteria),andwater(Baeketal.,2010).
MakgeollicontainsvitaminBcomplex,organicacids,andbioactive
substances with yeasts,resultinginhigh nutritionalandfunctional
values,includingantioxidantproperty(Kim,Park,&Sung,2012).The
sizeandtypeofmicroorganismsinNurukarecontinuouslychanged
atdifferentstagesofMakgeollifermentation.Theysignificantlyre-
main in the finalproduct (Kimetal., 2015).Thesemicroorganisms
notonlyparticipateinsaccharificationand alcoholicfermentation
(Nile,2015),butalsocontributetoMakgeolli’suniquewhitecreamy
textureandflavor(Lee&Choi,2005).However,becauseofalackof
uniquecharacteristics,inferioracceptability,andfunctionality,pop-
ularit y of Makgeolli h as been decli ning (Kim, Cha ng, Ko, & Jeong,
2013).ToimprovethequalityofMakgeolliwithmicrobialactivities
andfunctionalcharacteristics,utilizationofrawmaterialsandman-
ufactureprocesses have beenstudied (Kim,Lee, Lee,Choi, &Lee,
2004;Kim,Chang,etal.,2013).
Buckwheat(Fagopyrum sp p.)isaregionalspecialtyinBongpyeong
ofKorea .I tisagoodso urceofnutritional lyvaluabl ep rote in,lipid,di-
etaryfiber,minerals,flavonoids,fagopyrin,tocopherols,andphenolic
substancessuchas3-flavanols,rutin,phenolicacids,andtheirderiv-
atives (Holasova etal., 2002; Jiang etal., 2007;Oomah, Campbell,
& Mazza, 1996). Among buck wheat species, common buck wheat
(Fagopyrum esculentum) and Tartary b uckwheat (Fagopyrum tatari-
cum)arecultivatedashumanfoodsources.Commonbuckwheathas
asweettastewithalargeseedsizewhereasTartarybuckwheathasa
bittertastewithasmallseedsize.Tartarybuckwheatseedscontain
100-foldhigheramountsofrutincompared tocommonbuckwheat
seeds (Fabjanetal., 2003; Jiang etal.,2007). Common buckwheat
fermentationbyusingRhizopus oligosporushasbeenreportedinour
previous study (Park etal.,2017).Duringbuckwheat fermentation
by R. oligosporus, macr omolecule s are hydrolyzed by e nzymes an d
corresponding hydrolytic products are coupled with metabolism
which can change biochemical compositions of food substrates
(Handoyo & Morita, 2006; de Reu, Linssen, Rombouts, & Nout,
1997 ). l-carnitin e can be synthesi zed from lysine and m ethionine
(Bremer,1983). Buckwheat waschosen due to its higher content
ofprecursorslysineandmethioninefor l-carnitinesynthesiswhen
comparedwiththose ofother crops(Park etal.,2017).Buckwheat
without additionalnutrients hasbeen fermented using R. oligospo-
rus,producingfour timeshigheramountofl-carnitinethanoriginal
buckwheat(Parketal.,2017).l-carnitineisaquaternaryammonium
compoundnaturallyfound inmeat (Walter&Schaffhauser,2000).
Itsmajorrole isacarrierof long-chainfattyacidintomitochondria
forbeta-oxidation.Ithasgoodinfluenceonischemic heartdisease
andrecoveryafterexercise(Walter&Schaffhauser,2000).
Makgeolli using buckwheat has been studied (Cho, Seo, Lee,
& Cho, 2012; Kang, Choi, Choi, Yeo, & Jeong, 2014). Cho etal.
(2012) have exploited several cereals, including buckwheat, to
brewMakgeolliandcompare differencesof pHandalcoholin final
Makgeolli. In addition, Kang etal. (2014) have analyzed various
commercial Makgeolli, including buckwheat Makgeolli. However,
thesepreviousresearchesonlyindicatedgeneralcharacteristicsof
buckw heat Makgeol li such as pH, acid ity, alcohol, red ucing sugar,
aminoacidicity, color, and volatile acidity (Cho etal., 2012; Kang
etal., 2014).Therefore, in this study, we focus on using combina-
tion of common and Tartary buckwheat or mix of common and
Tartary fermented buckwheat by R. oligosporus as ingredients to
brew functionally-enhanced Makgeolli. Their chemical composi-
tion, antioxidant properties,andgeneralqualities(pH,alcohol,and
acidity)weretheninvestigated.Especially,functionalcompounds(l-
carnitine, rutin, quercetin)in these Makgeolli were analyzed using
LC/MS.
2 | MATERIALS AND METHODS
2.1 | Preparation of fermented buckwheat
Rhizopus oligosporus was identified using Biolog (Biolog Inc., CA,
USA)and16SrRNAasdescribedinourpreviousreport(Parketal.,
2017).R. oligosporuswasincubatedonPotatoDextroseAgar(Difco,
Detroit , MI, USA) plat es at 28°C for spor ulation. Fer mentation of
buckwheat was carried out as described in our previous report
(Park etal., 2017) with some modifications. Briefly, 250gof com-
monbuckwheat seed(Bongpyeong, Korea) weremixedwith 250g
of Tartary buckwheat seed (Bongpyeong) and soaked in distilled
water for 12hr.After draining,soaked buckwheat seedswere au-
toclaved at 121°C for 15m in. These auto claved buck wheat seeds
werethencooledtoambienttemperature(20–22°C)andmovedinto
atetragonalstainless-steelcontainerandinoculatedwith 5%(v/w)
R. oligosporus (1.6×106 spores/ml). After covering the container
withwrappingpaper,buckwheat seedswereincubatedat28°Cfor
3days. These fermented buckwheat seeds were then lyophilized
(EyelaFD-550; Rikakikai Co.,Tokyo,Japan)at0°C under10Pafor
5daysandstoredat−20°Cforfurtherstudy.Commonbuckwheat,
Tartarybuckwheat,andmixtureofcommonbuckwheatandTartary
buckwheat seed (1:1) without fermentation as control were pre-
paredasdescribedabove.
2.2 | Brewing of l- carnitine enhanced Makgeolli
with fermented buckwheat seed
Fortygramsof rice(Nonghyup,Naju,Korea)weresoakedfor12hr
and autoclaved at 121°C for 15min (Jinju Gokja, Seoul, Korea).
Steamed rice was thencompletely cooledandmovedintoaplastic
bottle with 8g of Nuruk and80ml of distilledwater.The mixture
wasincubatedat22°Candstirredtwiceadaytoprepareseedmash.
After2days,thesameamountofsteamedrice,Nuruk,anddistilled
waterwere addedintothe seedmashforfirst-stage fermentation.
Duringthefirststage,themixturewasstirredoncedaily.After1day
of fermentation, different cereals were added for second-stage
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PARK et Al.
fermentation. For rice Makgeolli, twice the amount of the same
material used atthepreviousstagewasadded.Forbuckwheatand
fermentedbuckwheatMakgeolli,differentratiosofricetosteamed
buckwheatseed(10,20,or40gin80goftotalsecond-stagecere-
als)or fermented buckwheat seed powder were added with addi-
tionalNurukandwater.Thesemixtureswereincubatedat22°Cfor
5days. Thefinalproducts were roughly filtered with twofolds of
cottonandstoredat−20°Cforfurtheranalyses.
2.3 | Analyses of ethanol, pH, and acidity
Makgeolliwascentrifugedat12,600×gfor10minandthesuperna-
tant was usedto determine pH (Suntex, Taipei, Taiwan).Makgeolli
samples were titrated using 0.1M NaOH solution (Lee, Haq,
Saravana,Cho,&Chun,2017).Aciditywascalculatedasaceticacid
based onthe volumeofNaOHused for titration to pH 8.2.Tode-
terminealcoholcontent,100mlofMakgeolliwasdistilledfirstwith
arotaryevaporator(Hei-VAP,Heidolph,Germany).Thefinalvolume
wasthenadjustedto100ml.AlcoholcontentofMakgeolliwasthen
measuredusinganalcoholhydrometer(Joylab,Seoul,Korea).
2.4 | Analysis of l- carnitine content
Forsamplepreparation, one gramofmilled Makgeolli ingredients
(rice, buckwheat seed,or fermented buckwheat seed) was added
into 10ml distilled water and extracted for 1hr.After centrifuga-
tion at 8, 000×g for 10min at 4°C , 100μl supernat ant was mixed
with 900μl acetonitrile and centrifuged at 12,600×g for 10 min.
Afterfiltrationwitha0.2μmmembranesyringefilter(SartoriusAG,
Germany),1μlsample wasinjectedintothe LC/MS.ForMakgeolli
samplepreparation,100μlsamplewasmixedwiththesamevolume
ofdistilledwateranddilutedwithacetonitrilefollowedbycentrifu-
gation,filtration,andinjectionasdescribedabove.Theusedsystem
wasWatersAcquityH-classwithWatersQDadetector(Waters,MA,
USA) with Wate rs Acquity UPLC Beh Hilic 1.7μm, 2.1×100mm
column. l-carnitine contents in different kinds of ingredients and
Makgeolliwere determined usinga LC/MS system (Waters)asde-
scribedinourpreviousreport(Parketal.,2017).Acalibrationcurve
waspreparedwithanexternalstandardmethod(0.01,0.05,0.1,0.5,
and 1 μg/ml).Thelinearitybetweenconcentrationofstandardsand
areawasevaluated(r2>0.99).Recoverywasconfirmedbystandard
additiontechnique(0.0125,0.025,0.05,and0.1μg/ml)inorder to
determinethematrix effect of ingredientsandMakgeollionquan-
tification.Eachrecoverywasanalyzedbyone-samplet-testinSPSS
version 23.0 forWindows(SPSSInc., Chicago,IL,USA) toevaluate
significantdifference from 100%(p < 0.05;Supportinginformation
TableS1).
2.5 | Analyses of rutin and quercetin contents
Onegramofrice, buckwheatseed, and fermented buckwheat
seed were e xtracte d with 10ml of 70% (v/v) ethanol for 1hr
and serially diluted (10 and 100 times) by acetonitrile. Final
Makgeollisamplesweremixedwiththesamevolumeofdistilled
water and deproteinized using acetonitrile. All samples were
filtered using a 0.2μm membrane syringe filter. Then 1μl of
eachsamplewasinjectedintoLC/MS(WatersH-classequipped
with QDa detector, MA, USA). Kromasyl C18 column (1.8μm,
2.1×100mm)wasusedtoanalyzerutinandquercetincontents
with solventA(100%tripledistilledwater with0.1%(v/v) for-
micacid)andsolventB(10 0%acetonitrilewith0.1%(v/v)formic
acid). Con ditions for the m ass detector we re as follows: ele c-
trospr ay ionization (ESI) n egative, 609.5m/z, capill ary energy
(CE)of0.8kV,conevoltage(CV)of25Vforrutin;ESIpositive,
303m/z,CEof1.5kV,andCVof 10Vforquercetin.Asblank,
90% (v/v) acetonitrile was used. Calibration curve linearity
rangedfrom0.05to5μg/mlforquercetin.Itrangedfrom0.1to
5 μg/mlfor rutin(r2 >0.99).Recoverytestsforbuckwheat and
Makgeolliwereperformedbystandardadditionmethodwitht-
test(p < 0.05)toevaluate significantdifference(Supportingin-
formationTableS1).
2.6 | Analysis of reducing sugar content
Reducing sugar content in Makge olli ingredients was determined
using DNS me thod (Gonçalve s, Rodriguez-Jasso, Gom es, Teixeira ,
&Belo,2010; Park,Kim, & Jeong,2018)with glucoseasstandard.
Sample s were extra cted by the sam e process as for an alysis of l-
carnitine.Theyweremeasuredwithadifferentdilutionrate.
2.7 | Analysis of antioxidant activity
Antioxidant activities of Makgeolli were evaluated by
2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging method
(Kimetal.,2012).Briefly,10μlofeachMakgeollisampleandwater
ascontrolweremixedwith100μMDPPHat37°Cfor30minintotal
darkness.Aftercentrifugingthemixtureat12,600×gfor10min,the
absorbance of each supernatantwas measured using a microplate
reader (M olecular Devi ce, Sunnyvale , CA, USA) at 517nm. DPPH
radic al scavenging ac tivity (SC) w as converted into p ercentage of
antioxidantactivityasfollows(Choi,Kang,&Mok,2018):
2.8 | Statistical analysis
Each analysis of general and functional compounds was repeated
threetimes.Resultsoftestwerepresentedasmean±standarddevia-
tion.Analysisofvariance(AN OVA)wasconductedusin gSPSSversion
23.0forWindows(SPSSInc.).Levene’stest forequalityofvariances
was conducted first. Significant difference was evaluated using
Scheffe’smethodbetweendifferentMakgeollisamples.Significantly
differentMakgeolliwasindicatedbydifferentsuperscriptlowercase
alphabetintablesandfigures.Statisticalsignificancewasconsidered
at p < 0.05.
SC(%) =(Abs of control - Abs of sample)∕(Abs of control) ×100
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PARK et Al .
3 | RESULTS AND DISCUSSION
3.1 | Analyses of rutin, quercetin, l- carnitine, and
reducing sugar contents in Makgeolli ingredients
Amountsofrutinandquercetininrawmixed buckwheat seed
were 7.3 and 1.1g/kg, respectively. In soaked and steamed
buckwheat,theiramountswere10.8and0.6g/kg,respectively.
Infermentedbuckwheat,amountsofrutinandquercetinwere
7.1and1g/kg,respectively(Table1).Rutinandquercetinwere
not detected in rice ( Table1). By soaking and steaming, the
content of rutin in buckwheat was increased47.9%whilethe
contentofquercetininbuckwheatwasdecreased45.5%.These
contents were signifi cantly (p < 0.05 ) different from t hose in
raw ones . Our results wer e consistent with pr evious report s
(Li,Li,Ding, & Park,2008;Qin,Wu,Yao,&Ren, 2013),show-
ingthatrutin contentwasincreased bysteam-treating soaked
buckwheat materials. Rutininbuckwheat was hydrolyzed ina
fewminutesuponadditionofwaterbecauseofthepresenceof
rutin-degradingenzymesinTartarybuckwheatseed(Yasuda&
Nakagaw a, 1994)an d flavonol 3-gluco sidase in Tartar y buck-
wheat testa (Suzuki, Honda, Funatsuki, & Nakatsuka, 2002).
However,activitiesoftheseenzymesarecompletelyinhibited
at 80–85°C (Suz uki etal., 2002; Yasuda & Na kagawa, 1994).
Steamingmightinhibitrutindegradationandacatalyticreverse
shift re action of rut in-sy nthesizing coul d take place, thus i n-
creasingtheextractionofflavonoidsfromthematerial(Barber
& Behrma n, 1991).Fur ther stud y is needed to obt ain clearer
explanation.
Rutinandquercetincontentsinfermentedbuckwheatweresim-
ilarwith thoseof rawbuckwheat(Table1).However,rutin content
in fermented buckwheat was decreased by 34.3% while querce-
tin contentwas increasedby 66.7% compared to those of soaked
and steamed buckwheat. Various molds such as Penicillium and
Aspergilluscan userutinby rutin catabolic pathway (Tranchimand,
Brouant,&Iacazio,2010).Therefore,R. oligosporus, which is one of
moldsmightusethispathwayorasimilarpathway,thusalteringrutin
andquercetincontents.
l-carnitinecontentsin raw riceand buckwheatseed were7and
5.7mg/kg, respectively (Table1). After soaking and steaming, l-
carnitinecontent wasdecreasedto3.6mg/kg in riceand 4.6mg/kg
inbuckwheat(Table1).Leachingofl-carnitineduringheattreatment
with water might account for the decrease in l-carnitine (Knüttel-
Gustavsen&Harmeyer,2011).Fermentedbuckwheatcontained4.1-
fold increase in theamountof l-carnitine (18.7mg/kg) compared to
soaked ands teamedbuck wheatseed with significant difference. It
showed a 3. 3-fold incre ase over the orig inal one (p < 0.05; Table1).
Ourpreviousstudyhasrevealedthatl-carnitinecontentinbuckwheat
extractpowderafterfermentationusingR. oligosporus was increased
fourfoldcomparedtothatinnon-fermentedbuckwheatextractpow-
der (Park etal., 2017). Thus, although whole buckwheat seed was
fermented with R. oligosporus,fermentedbuckwheatseedcontained
similar augmentation of l-carnitine, resulting in improved potential
functionalityduetoenhanced l-carnitine withabundant flavonolsof
buckwheathulls.
Reducing sugar contents of rice, soaked and steamed rice,
buckwheat,soakedandsteamedbuckwheat,andfermentedbuck-
wheat with R. oligosporus are sh own in Table1. Reduc ing sugar
contents i n raw rice and buck wheat seed wer e 5.1a nd 21.3g/
kg,respectively.Soakingandsteamingprocessincreasedreducing
sugar contents (to23.1g/kg inrice and22.9g/kg in buckwheat
seed). Reducing sugar contents in fermented buckwheat seed
was 5.2-fold higher (110.7g/kg) th an that in the origina l buck-
wheat. Theseincreasesin reducingsugarscouldbeattributedto
hydrothermalandcatalytichydrolysisbyR. oligosporus(Nagamori
&Funazukuri2004;Sarrette, Nout,Gervais, & Rombouts,1992)
which could affect the brewing of Makgeolli. Higher reducing
sugarcontentmightbeoneofthereasonswhyethanolcontentin
finalfermentedbuckwheatMakgeolliishigherthanthatinunfer-
mentedbuckwheatMakgeolli.
3.2 | Characterization of different
kinds of Makgeolli
The pH of rice M akgeolli, 6. 25% or 12.5% buckwh eat Makgeolli,
or fermented buckwheat Makgeolli was 4.0 without significant
Ingredient Rutin (g/kg) Quercetin (g/kg) l- carnitine (mg/kg)
Ruducing
sugars (g/kg)
Rice(noprocess) ND ND 7.0±1.7b5.1±0.6c
Rice(soak&
steam)
ND ND 3.6±0.4d23.1±1.4b
Buckwheat(no
process)
7.3±0.2b1.1a5.7±0.8bc 21.3±1.1b
Buckwheat(soak
&steam)
10.8±0.2a0.6c4.6±0.6cd 22.9±1.3b
Buckwheat
(Fermentation)
7.1±0.2b1.0b18.7±0.4a110.7±0.7a
Notes.ND:notdetected.
a,b,c,dDifferentsuperscriptslower-casealphabetsaftervaluesmeandifferentgroups(p < 0.05).
TABLE1 Functionalcompoundsin
rice,buckwheat,andfermented
buckwheat
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PARK et Al.
difference(p > 0.05;Table2).However,the pH of25%buckwheat
Makgeolliwas4.1andthatoffermentedbuckwheatMakgeolliwas
4.2, which was significantly (p < 0.05) different from that of rice
Makgeolli(Table2).Buckwheatcontainsmoreessentialaminoacids
thanrice(Motaetal.,2016)andfermentationbyR. ligosporus further
increas ed these amino a cid contents in bu ckwheat ( Wronkowska,
Christ a, Ciska, & Sora l-Śmiet ana, 2015). The amin o acid contents
of Makgeo lli were shown to be asso ciated with the raw m aterial
(Kangetal.,2014).Asaresult,theincreasedpHin25%buckwheat
Makgeolli or fermented buckwheat Makgeolli may be due to in-
creased aminoacidcontentsandtheirbuffering capacity (Thomas,
Hynes,&Ingledew,2002).
Results of acidit y of Makgeolli samp les are shown in Table2.
The acidity of fermented buckwheat Makgeolli was significantly
(p < 0.05) higher (40%–60%) than that of rice or buckwheat
Makgeolli(Table2).Theincreaseinacidityoffermentedbuckwheat
Makgeolli could be explained by the characteristic of fermented
TABLE2 GeneralcharacteristicsofdifferentMakgeolli
Typ e pH Alcohol (%) Acidity (%) Sugar (%)
RM 4.0b15.6±0.5a0.5d9.8±0.8a
BM_6.25 4.0b14.3±0.6ab 0.5d9.4±0.2ab
BM_12.5 4.0b14.5±1.1ab 0.6cd 9.6±0.4ab
BM_25 4.1ab 12.5±1.1b0.6d8.1±0.4b
FBM_6.25 4.0b15.5±0.5a0.7bc 10.2±0.7a
FBM_12.5 4.0b14.9±0.4ab 0.7ab 10.1±0.8a
FBM_25 4.2a13.2±1.6ab 0.8a10.1±0.4a
Notes.BM:buckwheatMakgeolli;FBM:fermentedbuckwheatMakgeolli;
RM:riceMakgeolli.
Makgeolli and the numbers withtypes of Makgeollimean the ratioof
buckwheatorfermentedbuckwheatinfinalMakgeolli.
a,b,cDifferentsuperscriptsoflower-caselettersaftervaluesmeandiffer-
entgroups(p < 0.05).
FIGURE1 Analysisofrutin,quercetin,l-carnitineandDPPHantioxidantactivity.(a)rutin,(b)quercetin,(c)l-carnitine,and(d)DPPH
antioxidantactivityindifferentMakgeollisamples.RM:riceMakgeolli;BM:buckwheatMakgeolli;FBM:fermentedbuckwheatMakgeolli.
a,b,cDifferentlower-caselettersonbargraphsmeansignificantdifferenceateachsupplementlevel(p < 0.05).*Superscriptstarmark
indicatestheratioofsupplementsinBMorFBM
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PARK et Al .
buckw heat itself s ince it could cont ain organic a cids produce d by
Rhizopus(Magnuson&Lasure,2004).
Alcohol contentwas15.6%forriceMakgeolli,14.3%for6.25%
buckwheatMakgeolli,14.5%for12.5%buck wheatmak geolli,12.5%
for 25% buck wheat Makgeo lli, 15.5% for 6.25% fe rmented buck-
wheatMakgeolli,14.9%for12.5%fermentedbuckwheatMakgeolli,
and 13.2% for 25% fermented buckwheat Makgeolli (Table2).
Theloweralcohol contentinbuckwheatorfermentedbuckwheat
Makgeo lli compared to that i n rice Makgeolli mig ht occur by the
factthatbuckwheatseedcontainshigherdietaryfiberandasmaller
amount of starch (Bonafaccia, Marocchini, & Kreft, 2003) which
canaffecttotal glucoseamountsforyeasttouseduringMakgeolli
brewing. Also, the reason for higher alcohol content in fermented
buckwheat Makgeolli than buckwheat Makgeolli could be pre-
fermentationpolysaccharidasewhich canbe producedbyR. oligos-
porus(Sarretteetal.,1992).Itcandegradeunavailablecarbohydrate
ofseedsintoadditional availablesugars.These additional glucoses
byfermentationmightbeexploitedbyyeasts(whichproduceetha-
nol)laterintheMakgeollibrewingprocess.
TheprimarypurposeofMakgeollibrewingistoproduceethanol,
animportantcomponentinfluencingflavorand preservation(Kim,
Park, etal., 2013). From these result s, pre-ferment ation of buck-
wheat see d seems to be useful fo r brewing Makgeo lli, especially
whenwholeseedswithhullsareused.
Sugar contents in different kinds of Makgeolli are shown in
Table2. Among three kinds of Makgeolli, fermented buckwheat
Makgeollihasslightly highersugar contentthanriceorbuckwheat
Makgeolli.
3.3 | Analyses of rutin, quercetin, l- carnitine, and
antioxidant effects in different Makgeolli
Amountsofrutinin6.25%,12.5%,and 25% buckwheatMakgeolli
were 63.0, 109.3, and 173.3mg/L , respectively. They were 19.6,
22.1, and 45.6mg/L in 6.25%, 12.5% , and 25% fermented buck-
wheat Makgeolli, respectively. However, rutin was not detected
inrice Makgeolli (Figure1a). Amountsofquercetinwere 7.6, 11.5,
and18.3mg/Lin6.25%,12.5%,and25%buckwheatMakgeolli,re-
spectively.Theywere52.5,104.1,and187.0mg/Lin6.25%,12.5%,
and25% fermentedbuckwheatMakgeolli,respectively.Quercetin
was not detected in rice Makgeolli (Figure1b). The sum of rutin
and quercetin in fermented buckwheat Makgeolli was increased
(by2.1%in6.25%fermentedbuckwheatadded,4.5%in12.5%fer-
mentedbuckwheatadded,and21.4%in25%fermentedbuckwheat
added) co mpared to that in no n-fe rmented buck wheat Makge olli.
l-carnitinewasdetectedinriceMakgeolli(0.7mg/L).Therewas no
significant difference in l-carnitine content between buckwheat
Makgeolli samples (6.25% and 12.5%) except 25% of buckwheat
Makgeolli (p<0.05) (Figure1c). Amounts of l- carnitine were in-
creasedsignificantly(p < 0.05)inallfermentedbuckwheatMakgeolli
comparedtothoseinriceMakgeolliandbuckwheatMakgeolliwith
samesupplementratio.In addition,thecalculatedconversion ratio
of l-carnitine fromingredientstofinalMakgeolliwasalmost stable
for all Makgeolli samples (51.4%±3.1). This result indicates that
l-c arnitine in fin al Makgeolli is de pendent on l-c arnitine levelsin
Makgeolliingredients.Tothebestofourknowledge,l- carnitine con-
tentinMakgeolliandl-carnitinefortifiedMakgeolliarereportedfor
thefirsttimeinthispaper.
Allbuckwheat andfermentedbuckwheat Makgeollishowed
significantly increased antioxidant activities compared to rice
Makgeolli (p < 0.05; Figure1d). Antioxidant activity in individ-
ual fermented buckwheat Makgeolli was higher than that of
each buck wheat Makgeolli using the s ame percent of supple-
ment. An tioxidant act ivities of 6. 25% and 12.5% of ferme nted
buckwheatMakgeolli were significantlydifferent from thoseof
6.25% and 12.5%ofbuckwheat Makgeolli (p < 0.05; Figure1d).
The higher antioxidant activity of buckwheat and fermented
buckwheatMakgeollicomparedtoriceMakgeolli mightbedue
tothe presence ofl-carnitine,tocopherols,phenolicsubstance
such as 3-flav anols, rutin , phenolic aci ds, and their de rivatives
in buck wheat which are k nown to possess a ntioxidant ac tivity
(Holasovaetal.,2002;Oomahetal.,1996).Especially,thehigher
antioxidantactivityoffermentedbuckwheatMakgeollithanthat
of buckwheat Makgeolli might result from R. oligosporus that
can prod uce α-a mylase and endo genous carb ohydrate-cleav ing
enzymes, since these enzymes can produce polyphenols from
carbohydrates-conjugated phenolic compounds during fermen-
tationofbuckwheat(McCue&Shetty,2003).In addition,R. oli-
gosporus is known to produce β-glucosidase, β-glucuronidase,
or xylanase to degrade cell wall matrix (Huynh, Van Camp,
Smagghe,& Raes,2014).Thus,duringfermentation,R . oligospo-
rusmightbio-convertbound-phenoliccompoundsintounbound
phenolics as aglycone forms (Huynh etal.,2014). Furthermore,
during the process of brewing Makgeooli, different kinds of
fungi ( Aspergillus,Rhizopus), yeast, and va rious lactic ac id bac-
teria(Nile, 2015)canproducetannase,phenolicaciddecarbox-
ylase, benzyl alcohol dehydrogenase, and β-glucosidase that
can degr ade some phenolic compoun ds (Landete etal., 2010).
Therefore,fermentationprocessesmightreleasephenoliccom-
pounds fromplant matrixes followedbymetabolic pathwaysof
flavonoids, including glycosylation, deglycosylation, ring cleav-
age, methylation, glucuronidation, and sulfate conjunction in
waystoproducenewbioactivecompounds(Huynhetal.,2014).
Increased contents of flavonoids might influence on DPPH-
radicalSC, resulting in higherantioxidant activityoffermented
buckwheat Makgeolli thanbuckwheat Makgeollias well as rice
Makgeolli. Quercetin has higherantioxidant activity compared
torutin (SC50 of quercetin=37.4μM,SC50 of rutin ≥1,000μM)
(Kong,Mat-Junit,Aminudin,Ismail,&Abdul-Aziz,2012;Nguyen
etal., 2015). In addition,anincreaseofl- carnitine contained in
fermentedbuckwheat Makgeollicouldincreasethe antioxidant
activitybecausel-carnitineisanantioxidantcompoundthatcan
preventoxidativestressandregulatecellularrespirationbynitric
oxide(Brown,1999).Theseresultssuggestthatpre-fermentation
techniqueofbuckwheatseed by R. oligosporuspriortobrewing
Makgeo lli could incre ase antioxidant a ctivitie s of final produ ct
|
7
PARK et Al.
with enhanced functional compounds (l-carnitine, rutin, and
quercetin).
4 | CONCLUSION
In this study, forthe first time, l-carnitinefortified Makgeolli was
successfully brewed using rice and fermented whole buckwheat
seedpreparedwithR. oligosporus.Fermentedbuckwheatseedcon-
tained increased l- carnitine content and maintained its richness
in rutin an d quercetin as m ajor functi onal compoun ds. This result
indicatesthatitispossible to enhancethefunctionalityandwiden
the appl ication of buck wheat as a food ingr edient. In addit ion, l-
carnitinefortifiedMakgeolliusingfermentedbuckwheatseedwith
natural fortificationofl-carnitine andflavonolsis expected toen-
hancehealth effects.Asa result, thisbrewingtechniquewith pre-
fermentationseemstobeeffectivewhenwholebuckwheatseeds
are used be cause it enhan ces functio nal compounds wi th alcohol
productionandantioxidantactivitycomparedtooriginalbuckwheat
Makgeolli.
ACKNOWLEDGMENTS
The authors acknowledge the technical advice and assistance of
JenahParkfromWatersKoreaforLC/MSoperation.Thisworkwas
partiallysupportedbyKoreaInstituteofPlanningandEvaluationfor
TechnologyinFood,Agriculture,Forestry(IPET)throughAgriculture,
Food and Rural Affairs Research Center SupportProgram, funded
by Ministr y of Agriculture, Food and Rural Affairs (MAFRA)(D.
Kim, 710012-03-1-HD220),andbyKoreaInstituteofPlanningand
EvaluationforTechnologyinFood,Agriculture,ForestryandFisheries
(IPET) through High Value-added Food Technology Development
Program (116013032HD020) funded by Ministry of Agriculture,
Food and Rural Affairs, Republic of Korea, by the Basic Science
Research Program through the National Research Foundation of
Korea(NRF)fundedbytheframeworkofInternationalCooperation
Program m anaged by the NRF (2016K1 A3A1A19945059), and by
OTTOGICorporationthroughResearchandPublicationProject.
CONFLICT OF INTEREST
Nonedeclared.
ETHICAL STATEMENT
Allauthorswereactivelyinvolvedintheworkleadingtothemanu-
script andwill hold themselves jointly andindividually responsible
for its con tent. This stu dy does not involve any h uman or animal
testing.
ORCID
Doman Kim http://orcid.org/0000-0003-0389-3441
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
SupportingInformationsectionattheendofthearticle.
How to cite this article:ParkN,NguyenTTH,LeeG-H,etal.
Compositionandbiochemicalpropertiesofl- carnitine
fortifiedMakgeollibrewedbyusingfermentedbuckwheat.
Food Sci Nutr. 2018;00:1–8. https://doi.org/10.1002/
fsn3.803
