research article effects of fermentation temperature and...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 870802 6 pageshttpdxdoiorg1011552013870802

Research ArticleEffects of Fermentation Temperature andAeration on Production of Natural Isoamyl Acetate byWilliopsis saturnus var saturnus

Murat Yilmaztekin1 Turgut Cabaroglu2 and Huseyin Erten2

1 Inonu University Faculty of Engineering Department of Food Engineering 44280 Malatya Turkey2 Cukurova University Faculty of Agriculture Department of Food Engineering 01330 Adana Turkey

Correspondence should be addressed to Murat Yilmaztekin muratyilmaztekininonuedutr

Received 2 April 2013 Revised 7 June 2013 Accepted 9 June 2013

Academic Editor Isabel Sa-Correia

Copyright copy 2013 Murat Yilmaztekin et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Isoamyl acetate is a natural flavour ester widely used as a source of banana flavour by the food industry Williopsis saturnus varsaturnus is a yeastwhich can produce isoamyl acetate by esterification of amyl alcoholswith acetyl coenzymeAvia fermentationTheevaluation of this kind of production as an alternative way to obtain natural banana flavour could be possible if the levels producedwere high enough to make a commercial product In this study the effects of temperature (15∘C and 25∘C) and aeration (aerobicsemiaerobic and anaerobic) on the production of isoamyl acetate byWilliopsis saturnus var saturnus from sugar beetmolasses wereexamined According to the results obtained isoamyl acetate production rate and specific productivity were higher at 25∘C than at15∘C and at semiaerobic condition than aerobic and anaerobic conditions Williopsis saturnus var saturnus showed a productionrate of 0703mg Lminus1 hminus1 and a specific productivity of 00297mg Lminus1 cellminus1 hminus1 isoamyl acetate with semiaerobic condition at 25∘CThe maximum amount of isoamyl acetate reached with these conditions was 118mgL

1 IntroductionFlavour compounds found widespread application in foodbeverages cosmetics detergents and pharmaceutical prod-ucts with a world-wide industrial size estimated at US $ 22billion in 2012 [1] Most of the available flavour compounds(84) are now produced via chemical synthesis althoughextraction from natural material continues [2] Howeverrecent market surveys have shown that consumers preferfoodstuff that can be labelled as ldquonaturalrdquo Although flavoursmay be produced by chemical transformation of naturalsubstances the resulting products cannot legally be labelledas natural [3] An alternative route for flavour synthesiswhich is based on employment of new biotechnologicalprocesses de novo microbial processes (fermentation) orbioconversions of natural precursors using microbial cells orenzymes (biocatalysis) has increased considerably in the pasttwo decades [4ndash6]

Esters are commonly used flavouring agents very appre-ciated for the fruity aromas they provide They are employed

in fruit-flavoured products baked goods wines and dairyproducts Acetate esters such as ethyl acetate hexyl acetateisoamyl acetate and 2-phenylethyl acetate are recognised asimportant flavour compounds [3] Especially isoamyl acetatethe characteristic compound of banana flavour is producedwith an amount of 74 tonnes per annum [7] Several yeasts arecapable of producing large amounts of fruity ester flavoursThe genus Williopsis synthesizes high amounts of volatileesters for example isoamyl acetate [8ndash10]

During fermentation yeasts synthesize a vast numberof aroma and flavour compounds The production of thesecompounds is due to yeast metabolism since significantdifferences in their production have been demonstratedby the use of different yeast genera species and strainsApart from the choice of yeast several factors contribute toflavour production These include changes in fermentationconditions such as temperature pH aeration agitation andthe nature and concentration of the substrate utilized [11] It isreported that fermentation temperature aeration and culture

2 BioMed Research International

medium affect the lipid metabolism which is related to celldevelopment membrane integrity and the production ofseveral byproducts especially those directly related to flavourcompounds [12] In this study influence of fermentationtemperature and aeration on the production of isoamylacetate byWilliopsis saturnus var saturnus (W saturnus) fromsugar beet molasses was investigated

2 Materials and Methods

21 Microorganism andMedium W saturnus HUT 7087 wasobtained from the HUT (Japan) culture collection Yeastwas maintained on Malt Extract Agar (015 Malt ExtractAgar (by mass) obtained from Merck Germany) slants andreculturedmonthly Sugar beet molasses which was obtainedfrom the Ozmaya Co (Adana Turkey) was diluted withdeionized water to obtain a 10 Brix molasses solution Thesolution was then adjusted to pH 30 with 1NH

2SO4(Merck

Darmstadt Germany) to remove heavy metals that wouldaffect cultivationThemolasses solution was allowed to standfor 24 h at room temperature and then centrifuged at 5000timesgfor 15min The supernatant was adjusted to pH 50 with10NNaOH (Merck Darmstadt Germany)Themediumwassterilized at 121∘C for 15min in shake flasks and bioreactorsand used as preculture and cultivationmediums respectively[10]

22 Culture Conditions Batch cultivations were carried outin duplicate 3 L laboratory bioreactors (New BrunswickBioFlo 110 USA) containing 2 L of cultivation medium Theyeast was harvested from a 48 h preculture by centrifugationand used to inoculate the bioreactors at a level of 1 times107 cells mLminus1 The agitation system consisted of two 6-

bladed Rushton-type impellers (52mm) operating at thestirrer speed of 100 rpm The culture pH was left uncon-trolled (pH at inoculation time was 50) Temperature wasmaintained at 15 and 25∘C by a heat blanket and a chillerattached to the bioreactors for temperature experiments Acondenserwas used on the top of the bioreactor to prevent theloss volatile flavour compoundsThe experiments performedto examine the effect of temperature were conducted underanaerobic conditions Aeration experiments were carried outunder fully aerobic (12 L air hminus1 = 02 vvm) fully anaerobic(12 L nitrogen hminus1 = 02 vvm) and semiaerobic (3 L air hminus1= 005 vvm) conditions at the temperature value which wasselected as best in temperature trials Air and nitrogen weregiven into the bioreactors through a sterile filter during thecultivations [10] Samples were taken at interval times fromliquid medium for analytical determinations

23 Analytical Procedures Parameters monitored through-out fermentations included density and viable yeast cellnumber The number of viable cells was determined undera light microscope with a Thoma chamber using methyleneblue staining The supernatant of the cultivation mediumwas used to determine the density by a digital density meter(Mettler Toledo Columbus OH) [10]

The determination of products (2-methylbutanol 3-methylbutanol and isoamyl acetate obtained from Merck

Germany) was carried out by direct injection of samplesinto a gas chromatograph (Shimadzu GC-14B Kyoto Japan)equipped with a splitsplit less injector and a flame ionizationdetector as described by Yilmaztekin et al [10] Estersand higher alcohols were separated using a CP-WAX-57CBcapillary column (025mm id times 60m length times 04 120583m filmthickness) GC settings were as follow injection temperature160∘C oven temperature 4min at 40∘C then increased by18∘C per minute up to 94∘C and 40∘C per minute up to180∘C and finally 4min at 180∘C detector temperature180∘C carrier gas He (13mLminminus1) split rate 1 50 Thequantification was performed by internal standard method3-Pentanol (Merck Darmstadt Germany) was added as aninternal standard Standard solutions containing all com-pounds were prepared and analysed in duplicate RelativeResponse Factors (RRFs) were calculated from peak areas foreach compound using

RRF = (119860 is119860119888

) times (

119862is119862119888

) (1)

where 119860 is is area of internal standard 119860119888is area of com-

pound 119862is is concentration of internal standard and 119862119888is

concentration of compound A linear plot was obtained witha correlation coefficient of at least 0999 for all compoundsThe results represent the means of two determinations withtheir standard deviations

24 Statistical Analysis All experiments were carried out induplicate independent cultures and all results were reportedas mean plusmn standard deviation Data analyses were performedby using SPSS 90 forWindows (SPSS Inc Chicago IL USA)An analysis of variance (ANOVA)was performed A Student-Newman-Keuls test was applied on the individual variablesto compare means and assess their significant differences (atsignificant levels of 119875 le 0001 and 119875 le 005)

3 Results and Discussion

The production of isoamyl acetate was influenced by thefermentative conditions which affected the physiologicalactivity and growth of the yeast The amount of viableyeast cells at different fermentation conditions was givenin Table 1 While low temperature and anaerobic conditionsinhibited yeast growth and decreased sugar consumptionelevated temperature and aerobic fermentation conditionsallow a faster and extensive yeast growth According toBeltran et al [12] temperature clearly affects yeast growth andfermentation kinetics They found that fermentation ratesand maximal yeast population were higher at 25∘C than at13∘C in wine fermentations but cell viability was higher at13∘C than at 25∘C This better viability of the yeasts at lowtemperature may be related with a different compositionof the cell membrane Furthermore wort aeration resultsin increased yeast growth since oxygen is essential for keyreactions in the biosynthesis of sterols and unsaturated fattyacids which are needed for yeast growth [13] Mauricio et al[14] also suggested that anaerobic conditions inhibited yeastgrowth and decreased fructose consumption and ethanolproduction in wine

BioMed Research International 3

Table 1 Viable yeast cells (times107 cells mLminus1) at different fermentation conditions

Fermentation conditions Time (h)0 36 72 108 144 180 216

15∘C 10 plusmn 012 29 plusmn 014 465 plusmn 010 625 plusmn 017 665 plusmn 020 725 plusmn 023 58 plusmn 01125∘C 10 plusmn 022 718 plusmn 004 103 plusmn 035 895 plusmn 010 102 plusmn 010 935 plusmn 021 558 plusmn 017Anaerobic 10 plusmn 024 735 plusmn 031 103 plusmn 044 102 plusmn 034 1045 plusmn 010 858 plusmn 022 NTlowast

Semi-aerobic 10 plusmn 018 175 plusmn 054 284 plusmn 049 246 plusmn 044 258 plusmn 051 246 plusmn 033 236 plusmn 021Aerobic 10 plusmn 016 2175 plusmn 035 20 plusmn 015 221 plusmn 013 20 plusmn 022 NTlowast NTlowastlowastNT not tested

Table 2 Effect of temperature on isoamyl acetate concentration production rate specific productivity and esterhigher alcohol ratio

Temperature Isoamyl acetate(mg Lminus1)

Isoamyl acetate productionratex

(mg Lminus1 hminus1)

Isoamyl acetate specificproductivityx

(mg Lminus1 cellminus1 hminus1)

Isoamyl acetatetotal amylalcohol ratiox

15∘C 2943 plusmn 010 0136 plusmn 00005 00235 plusmn 00002 175 plusmn 02325∘C 2072 plusmn 002 0432 plusmn 00004 00436 plusmn 00003 069 plusmn 012Level of significance lowastlowast lowastlowast lowastlowast lowastlowast

xIsoamyl acetate production rate specific productivity and esterhigher alcohol ratio were calculated at times when isoamyl acetate concentration reach themaximum lowastlowast 119875 le 0001

31 Effect of Temperature on Amyl Alcohols and IsoamylAcetate Production Isoamyl acetate synthesis by yeast is per-formed by the action of alcohol acetyltransferase (AATFase)which catalyses the reaction between amyl alcohols (2-methylbutanol and 3-methylbutanol) and acetyl-CoA Theavailability of the substrates affects the amount of isoamylacetate produced It has been reported that the amount ofisoamyl alcohol in sake mash is important on the isoamylacetate production in sake brewing [15] Calderbank andHammond [16] also reported that the availability of 2-methylbutanol and 3-methylbutanol is very important incontrolling the amount of isoamyl acetate Therefore theamount of isoamyl acetate produced is influenced by theexisting concentration of amyl alcohols in the medium

The amount of 2-methylbutanol and 3-methylbutanolshowed an increase throughout the fermentation at both15∘C and 25∘C The maximum amounts of 2-methylbutanoland 3-methylbutanol produced at 15∘C were 192 and124mg Lminus1 respectively The maximum concentrations of 2-methylbutanol and 3-methylbutanol at 25∘Cwere higher thanthat of 15∘C (248 and 249mg Lminus1 resp) The results demon-strated that the production of amyl alcohols was dependenton the temperature and the higher alcohol formation wassuppressed at low fermentation temperatures (15∘C) Higheralcohols are produced by yeast via the catabolic (Ehrlich)and anabolic pathways (amino acid metabolism) duringfermentation [17] Yeast strain fermentation conditions andwort composition all have significant effects on levels ofhigher alcohols that are formed [18 19] Conditions whichpromote yeast growth such as high levels of nutrients (aminoacids oxygen lipids and zinc) increased temperature andagitation increase the production of higher alcohols [20]The synthesis of amyl alcohols is especially sensitive totemperature changes Since fusel alcohols are the productof amino acid and sugar metabolism it is recognized that

110051011015102102510310351041045

05

101520253035

0 36 72 108 144 180 216 264 300 372 444 516

Isoa

myl

acet

ate (

mg

L)

Time (h)

Den

sity

(gc

m3)

Figure 1 Effect of temperature on isoamyl acetate production at15∘C (⧫) and 25∘C (998771) and on density of the medium at 15∘C (loz) and25∘C (Δ)The bars indicate standard deviations for two independentcultivations

parameters such as temperature affecting the assimilation ofthe latter also control fusel alcohol production [20] On theother hand conditions which restrict yeast growthmdashsuchas lower temperature and higher CO

2pressuremdashreduce the

extent of higher alcohol production [21]The effect of temperature on isoamyl acetate production

patterns by Williopsis saturnus var saturnus is shown inFigure 1 and Table 2 respectively As can be seen in Figure 1there was a slow decrease in the density of themedium at 15∘Ccompared to 25∘C However the amount of isoamyl acetateproduced against the consumed substrate was higher at 15∘CThe concentration production rate and specific productivityof isoamyl acetate and esterhigher alcohol ratio at 15∘C and25∘C were significantly different (119875 le 005) While the maxi-mum isoamyl acetate producedwas higher at experiment per-formed 15∘C (294mg Lminus1) than at 25∘C (207mg Lminus1) on thecontrary isoamyl acetate production rate was higher at 25∘C(043mg Lminus1 hminus1) than at 15∘C (014mg Lminus1 hminus1) The specific


productivity was also higher at 25∘C (0044mg Lminus1 cellminus1 hminus1)compared to 15∘C (0024mg Lminus1 cellminus1 hminus1) The ratio of theisoamyl acetate to total amyl alcohols which is a parameterknown as to influence the sensory properties of the mediumwas also influenced by temperature An increased content ofesters gives an enhanced fruity flavour and this can improvewhen higher alcohol content decreases [10]

Esters are the product of an enzyme-catalyzed conden-sation reaction between acyl-CoA and a higher alcohol [22]Fundamentally two factors are important for the rate of esterformation during fermentation the availability of the twosubstrates (acetylacyl-CoA and alcohols) and the activityof enzymes (AATFase) Hence all parameters that affectsubstrate concentrations or enzyme activity will influenceester production [13 19 23 24] Dufour et al [23] indicatedthat acetate esters increasedwith temperature during fermen-tation and this increase is related to the stimulation of theAATases Furthermore it is well known that the formationof higher alcohols is also temperature dependent so thatchanges in temperature may cause changes in the availabilityof fusel alcohols that are necessary for ester formation [16]Generally increased fermentation temperatures in the rangeof 10ndash25∘C lead to increased ester production Accordingto Peddie [13] when temperature is elevated the concen-tration of esters produced during the fermentation is alsoincreased due to increasingmembrane fluidity An increase inmembrane fluidity may allow more esters to diffuse into themedium Verstrepen et al [22] reported that up to 75 moreesters are produced at 12∘C than at 10∘C However differentesters may show different temperature dependencies Somestudies show that ethyl acetate and phenyl ethyl acetate areproduced maximally at 20∘C whereas maximal productionof isoamyl acetate and ethyl caprylate occurs at 15∘C [18]Thistemperature dependency is not valid for all yeast strains andsome strains may show different temperature dependenciesfor the production of certain volatile esters [25] In this studythe production rate and specific productivity parameterswere taken into account for the performance of temperaturetrials Hence the subsequent trials to investigate the effect ofaeration on isoamyl acetate production were carried out at25∘C

32 Effect of Aeration on Amyl Alcohols and Isoamyl AcetateProduction In the course of anaerobic fermentation the pro-duction of amyl alcohols was inhibited compared to semiaer-obic and aerobic conditions The maximum concentrationsof 2-methylbutanol and 3-methylbutanol were obtained withsemiaerobic conditions (928mg Lminus1 and 438mg Lminus1 resp)followed by aerobic and anaerobic fermentations Undersemiaerobic conditions the concentrations of amyl alcoholspeaked at exponential phase and then decreased at stationaryphase probably as a result of their utilization for isoamylacetate production Results showed that aeration stimulatesthe production of amyl alcohols

Branyik et al [26] stated that control of higher alco-hol formation can be well balanced by the choice of anappropriate yeast strain wort composition (amino acidslipids zinc) fermentation conditions (temperature aerationand residence time) immobilization method and reactor

110051011015102102510310351041045

0

20

40

60

80

100

120

140

0 24 48 78 102 126 150 174 198

Isoa

myl

acet

ate (

mg

L)

Time (h)

Den

sity

(gc

m3)

Figure 2 Effect of aeration on isoamyl acetate production at aerobic(⧫) anaerobic (◼) and semiaerobic (998771) conditions and on densityof the medium at aerobic (loz) anaerobic (◻) and semiaerobic(Δ) conditions The bars indicate standard deviations for twoindependent cultivations

design The majority of these interventions are based on thestimulation of the growth intensity for example dissolvedoxygen concentration and temperature enhancing the higheralcohol formation According to Crowel and Guymon [27]aerated fermentations formed as much as five times theamount formed under anaerobic conditions and the amylalcohols accounted for most of the increase resulting fromaerobic conditions Some authors also demonstrated thataeration stimulated the production of higher alcohols [19 2428ndash30]

The effect of aeration on substrate consumption andisoamyl acetate production by Williopsis saturnus var sat-urnus is shown in Figure 2 There was a sharp decreasewith the density of the medium at semiaerobic experimentcompared to aerobic and unaerobic experiments which isrelated to yeast growth The isoamyl acetate production ratespecific productivity and esterhigher alcohol ratio weregiven in Table 3 The concentration production rate andspecific productivity of isoamyl acetate were significantlyaffected with aeration conditions (119875 le 005) The maximumisoamyl acetate concentrationwas observedwith semiaerobiccondition (1181mg Lminus1) followed by anaerobic and aerobicfermentations (207 and 88mg Lminus1 resp)

Mauricio et al [14] reported that the addition of oxygento the must increases ethyl acetate and fatty acid ethyl estersconcentrations decreasing those of higher alcohols 23-butandiol acetic acid and higher alcohol acetates Contraryto the mentioned results Inoue et al [15] demonstratedthat Hansenula mrakii could produce a large amount ofisoamyl acetate when the cells were cultured under aerobicconditions According to Peddie [13] wort aeration resultsin increased yeast growth since oxygen is essential for keyinteractions in the biosynthesis of sterols and unsaturatedfatty acids which are needed for yeast growth Increasedyeast growth creates additional demand for acetyl CoA forthat purpose and so the availability of acetyl CoA for estersynthesis is reduced even a low rate of aeration duringfermentations can strongly inhibit ester formation [13 18 1924 31] Generally anaerobic conditions decreased the pro-duction of butyl acetate isoamyl acetate phenethyl acetate


Table 3 Effect of aeration on isoamyl acetate concentration production rate and specific productivity

Aeration Isoamyl acetate(mg Lminus1)






Aerobic 884 plusmn 008c 0082 plusmn 00005c 00037 plusmn 00003c 171 plusmn 032cy

Anaerobic 2072 plusmn 002b 0432 plusmn 00004b 00276 plusmn 00011b 077 plusmn 002b

Semiaerobic 11805 plusmn 559a 0703 plusmn 00333a 00297 plusmn 00009a 137 plusmn 060a

Level of significance lowast lowast lowast lowast

xIsoamyl acetate production rate specific productivity and esterhigher alcohol ratio were at times when isoamyl acetate concentration reach the maximumyDifferent letters in the same column indicate significant difference (119875 le 005) lowast 119875 le 0001

and hexyl acetate compared to semiaerobic conditions [14]Under semiaerobic conditions the concentrations of theacetates peaked and then decreased probably as a result oftheir hydrolysis under the action of cellular esterases theactivity of which increases at the end of fermentation [32]Confirming these results in our study the concentration ofisoamyl acetate peaked at the end of cellular growth undersemiaerobic conditions and then decreased at the end offermentation

4 Conclusions

Temperatures studied had a significant effect on the produc-tion of isoamyl acetate by Williopsis saturnus var saturnususing sugar beet molasses The maximum isoamyl acetateconcentration was reached at 15∘C while the maximumisoamyl acetate production rate was at 25∘C Aeration wasan important factor for the production of isoamyl acetateIsoamyl acetate concentration showed a decrease at aerobicand anaerobic conditions At certain levels of aeration (semi-aerobic) isoamyl acetate concentration was at maximumThese results showed that the production of isoamyl acetateby Williopsis saturnus var saturnus appears to be a potentialway for isoamyl acetate production However an amount of118mgL is still not enough to make it commercial Furtherstudies are required regarding genetic modifications on yeaststrain as well as suitable bioprocessing strategies for anefficient isoamyl acetate production process

Acknowledgments

This work was financially supported by the Scientific andTechnological Research Council of Turkey (Project noTUBITAK-TOVAG-3393) and the Academic Research Proje-cts Unit of Cukurova University (Project no ZF2004D34)The authors also thank OzmayaCo (Adana Turkey) for sugarbeet molasses

References

[1] Leffingwell and Associates ldquoFlavor amp Fragrance Industry Lead-ersrdquo httpwwwleffingwellcomtop 10htm 2012

[2] M Soares P Christen A Pandey M Raimbault and C RSoccol ldquoA novel approach for the production of natural aromacompounds using agro-industrial residuerdquo Bioprocess Engineer-ing vol 23 no 6 pp 695ndash699 2000

[3] M A Longo and M A Sanroman ldquoProduction of food aromacompounds microbial and enzymatic methodologiesrdquo FoodTechnology and Biotechnology vol 44 no 3 pp 335ndash353 2006

[4] L JanssensH LDePooterNM Schamp andE J VandammeldquoProduction of flavours by microorganismsrdquo Process Biochem-istry vol 27 no 4 pp 195ndash215 1992

[5] U Krings and R G Berger ldquoBiotechnological production offlavours and fragrancesrdquo Applied Microbiology and Biotechnol-ogy vol 49 no 1 pp 1ndash8 1998

[6] G Reineccius Flavor Chemistry and Technology CRC PressBoca Raton Fla USA 2006

[7] M Yilmaztekin H Erten and T Cabaroglu ldquoEnhanced pro-duction of isoamyl acetate from beet molasses with addition offusel oil by Williopsis saturnus var saturnusrdquo Food Chemistryvol 112 no 2 pp 290ndash294 2009

[8] T Iwase T Morikawa H Fukuda K Sasaki and M YoshitakeldquoProduction of fruity odor by genus williopsisrdquo Journal of theBrewing Society of Japan vol 90 no 5 pp 394ndash396 1995

[9] H Erten and I Campbell ldquoTheproduction of low-alcohol winesby aerobic yeastsrdquo Journal of the Institute of Brewing vol 107 no4 pp 207ndash215 2001

[10] M Yilmaztekin H Erten and T Cabaroglu ldquoProduction ofisoamyl acetate from sugar beet molasses byWilliopsis saturnusvar saturnusrdquo Journal of the Institute of Brewing vol 114 no 1pp 34ndash38 2008

[11] C Abbas ldquoProduction of antioxidants aromas colours fla-vours and vitamins by yeastsrdquo in Yeasts in Food and BeverageA Querol and G H Fleet Eds pp 285ndash335 Springer BerlinGermany 2006

[12] G Beltran M Novo J M Guillamon A Mas and N RozesldquoEffect of fermentation temperature and culture media onthe yeast lipid composition and wine volatile compoundsrdquoInternational Journal of Food Microbiology vol 121 no 2 pp169ndash177 2008

[13] A B H Peddie ldquoEster formation in brewery fermentationsrdquoJournal of the Institute of Brewing vol 96 pp 327ndash331 1990

[14] J C Mauricio J Moreno L Zea J M Ortega and M MedinaldquoThe effect of grape must fermentation conditions on volatilealcohols and esters formed by Saccharomyces cerevisiaerdquo Journalof the Science of Food and Agriculture vol 75 pp 155ndash160 1997

[15] Y Inoue S Trevanichi K Fukuda S Izawa Y Wakai andA Kimura ldquoRoles of esterase and alcohol acetyltransferase onproduction of isoamyl acetate in Hansenula mrakiirdquo Journal ofAgricultural and Food Chemistry vol 45 no 3 pp 644ndash6491997

[16] J Calderbank and J R M Hammond ldquoInfluence of higheralcohol availability on ester formation by yeastrdquo Journal of


the American Society of Brewing Chemists vol 52 no 2 pp 84ndash90 1994

[17] L A Hazelwood J Daran A J A Van Maris J T Pronk andJ R Dickinson ldquoThe Ehrlich pathway for fusel alcohol pro-duction a century of research on Saccharomyces cerevisiaemetabolismrdquo Applied and Environmental Microbiology vol 74no 8 pp 2259ndash2266 2008

[18] J R M Hammond ldquoBrewerrsquos yeastrdquo in The Yeasts H A Roseand J S Harrison Eds pp 7ndash67 Academic Press London UK1993

[19] C Boulton and D Quain Brewing Yeast and FermentationBlackwell Science Great Britain UK 2001

[20] S Landaud E Latrille and G Corrieu ldquoTop pressure and tem-perature control the fusel alcoholester ratio through yeastgrowth in beer fermentationrdquo Journal of the Institute of Brewingvol 107 no 2 pp 107ndash117 2001

[21] R S Renger S H Vanhateren and K Luyben ldquoThe formationof esters and higher alcohols during brewery fermentation-theeffect of carbon-dioxide pressurerdquo Journal of the Institute ofBrewing vol 98 no 6 pp 509ndash513 1992

[22] K J Verstrepen G Derdelinckx J Dufour et al ldquoFlavor-act-ive esters adding fruitiness to beerrdquo Journal of Bioscience andBioengineering vol 96 no 2 pp 110ndash118 2003

[23] J PDufour PMalcorps andP Silcock ldquoControl of ester synthe-sis during brewery fermentationrdquo in Brewing Yeast Fermenta-tion Performance K Smart Ed pp 213ndash233 Blackwell ScienceOxford UK 2003

[24] K J Verstrepen G Derdelinckx and F R Delvaux ldquoEsters inbeer-part 2 controlling ester levels during beer fermentation abiochemical approachrdquo Cerevisiae vol 28 pp 22ndash30 2003

[25] C Ramos-Jeunehomme R Laub and C A Masschelein ldquoWhyis ester formation in brewery fermentations yeast strain depen-dentrdquo in Proceedings of the 23rd European Brewery Conventionpp 257ndash264 Oxford University Press London UK 1991

[26] T Branyik A A Vicente P Dostalek and J A Teixeira ldquoAreview of flavour formation in continuous beer fermentationsrdquoJournal of the Institute of Brewing vol 114 no 1 pp 3ndash13 2008

[27] E A Crowel and J F Guymon ldquoInfluence of aeration andsuspended material on higher alcohols acetoin and diacetylduring fermentationrdquo American Journal of Enology and Viticul-ture vol 14 pp 214ndash222 1963

[28] R B Boulton V L Singleton L F Bisson and R E KunkeePrinciples and Practices of Winemaking Chapman amp Hall NewYork NY USA 1998

[29] D R Berry and J C Slaughter ldquoAlcoholic beverage fermenta-tionsrdquo in Fermented Beverage Production A G H Lea and J RPiggott Eds pp 25ndash41 Kluwer Academic NewYork NYUSA2003

[30] P Ribereau-Gayon D Dubourdieu B Doneche and A Lon-vaud Handbook of Enology the Microbiology of Wine andVinifications Wiley West Sussex UK 2006

[31] M J Lewis and T W Young Brewing Kluwer Academic NewYork NY USA 2002

[32] J C Mauricio J J Moreno E M Valero L Zea M Medinaand J M Ortega ldquoEster formation and specific activities of invitro alcohol acetyltransferase and esterase by Saccharomycescerevisiae during grape must fermentationrdquo Journal of Agricul-tural and Food Chemistry vol 41 no 11 pp 2086ndash2091 1993

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medium affect the lipid metabolism which is related to celldevelopment membrane integrity and the production ofseveral byproducts especially those directly related to flavourcompounds [12] In this study influence of fermentationtemperature and aeration on the production of isoamylacetate byWilliopsis saturnus var saturnus (W saturnus) fromsugar beet molasses was investigated

2 Materials and Methods

21 Microorganism andMedium W saturnus HUT 7087 wasobtained from the HUT (Japan) culture collection Yeastwas maintained on Malt Extract Agar (015 Malt ExtractAgar (by mass) obtained from Merck Germany) slants andreculturedmonthly Sugar beet molasses which was obtainedfrom the Ozmaya Co (Adana Turkey) was diluted withdeionized water to obtain a 10 Brix molasses solution Thesolution was then adjusted to pH 30 with 1NH

2SO4(Merck

Darmstadt Germany) to remove heavy metals that wouldaffect cultivationThemolasses solution was allowed to standfor 24 h at room temperature and then centrifuged at 5000timesgfor 15min The supernatant was adjusted to pH 50 with10NNaOH (Merck Darmstadt Germany)Themediumwassterilized at 121∘C for 15min in shake flasks and bioreactorsand used as preculture and cultivationmediums respectively[10]

22 Culture Conditions Batch cultivations were carried outin duplicate 3 L laboratory bioreactors (New BrunswickBioFlo 110 USA) containing 2 L of cultivation medium Theyeast was harvested from a 48 h preculture by centrifugationand used to inoculate the bioreactors at a level of 1 times107 cells mLminus1 The agitation system consisted of two 6-

bladed Rushton-type impellers (52mm) operating at thestirrer speed of 100 rpm The culture pH was left uncon-trolled (pH at inoculation time was 50) Temperature wasmaintained at 15 and 25∘C by a heat blanket and a chillerattached to the bioreactors for temperature experiments Acondenserwas used on the top of the bioreactor to prevent theloss volatile flavour compoundsThe experiments performedto examine the effect of temperature were conducted underanaerobic conditions Aeration experiments were carried outunder fully aerobic (12 L air hminus1 = 02 vvm) fully anaerobic(12 L nitrogen hminus1 = 02 vvm) and semiaerobic (3 L air hminus1= 005 vvm) conditions at the temperature value which wasselected as best in temperature trials Air and nitrogen weregiven into the bioreactors through a sterile filter during thecultivations [10] Samples were taken at interval times fromliquid medium for analytical determinations

23 Analytical Procedures Parameters monitored through-out fermentations included density and viable yeast cellnumber The number of viable cells was determined undera light microscope with a Thoma chamber using methyleneblue staining The supernatant of the cultivation mediumwas used to determine the density by a digital density meter(Mettler Toledo Columbus OH) [10]

The determination of products (2-methylbutanol 3-methylbutanol and isoamyl acetate obtained from Merck

Germany) was carried out by direct injection of samplesinto a gas chromatograph (Shimadzu GC-14B Kyoto Japan)equipped with a splitsplit less injector and a flame ionizationdetector as described by Yilmaztekin et al [10] Estersand higher alcohols were separated using a CP-WAX-57CBcapillary column (025mm id times 60m length times 04 120583m filmthickness) GC settings were as follow injection temperature160∘C oven temperature 4min at 40∘C then increased by18∘C per minute up to 94∘C and 40∘C per minute up to180∘C and finally 4min at 180∘C detector temperature180∘C carrier gas He (13mLminminus1) split rate 1 50 Thequantification was performed by internal standard method3-Pentanol (Merck Darmstadt Germany) was added as aninternal standard Standard solutions containing all com-pounds were prepared and analysed in duplicate RelativeResponse Factors (RRFs) were calculated from peak areas foreach compound using

RRF = (119860 is119860119888

) times (

119862is119862119888

) (1)

where 119860 is is area of internal standard 119860119888is area of com-

pound 119862is is concentration of internal standard and 119862119888is

concentration of compound A linear plot was obtained witha correlation coefficient of at least 0999 for all compoundsThe results represent the means of two determinations withtheir standard deviations

24 Statistical Analysis All experiments were carried out induplicate independent cultures and all results were reportedas mean plusmn standard deviation Data analyses were performedby using SPSS 90 forWindows (SPSS Inc Chicago IL USA)An analysis of variance (ANOVA)was performed A Student-Newman-Keuls test was applied on the individual variablesto compare means and assess their significant differences (atsignificant levels of 119875 le 0001 and 119875 le 005)

3 Results and Discussion

The production of isoamyl acetate was influenced by thefermentative conditions which affected the physiologicalactivity and growth of the yeast The amount of viableyeast cells at different fermentation conditions was givenin Table 1 While low temperature and anaerobic conditionsinhibited yeast growth and decreased sugar consumptionelevated temperature and aerobic fermentation conditionsallow a faster and extensive yeast growth According toBeltran et al [12] temperature clearly affects yeast growth andfermentation kinetics They found that fermentation ratesand maximal yeast population were higher at 25∘C than at13∘C in wine fermentations but cell viability was higher at13∘C than at 25∘C This better viability of the yeasts at lowtemperature may be related with a different compositionof the cell membrane Furthermore wort aeration resultsin increased yeast growth since oxygen is essential for keyreactions in the biosynthesis of sterols and unsaturated fattyacids which are needed for yeast growth [13] Mauricio et al[14] also suggested that anaerobic conditions inhibited yeastgrowth and decreased fructose consumption and ethanolproduction in wine

















110051011015102102510310351041045

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0 36 72 108 144 180 216 264 300 372 444 516

Isoa

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ate (

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L)

Time (h)

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(gc

m3)











110051011015102102510310351041045

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0 24 48 78 102 126 150 174 198

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4 Conclusions


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















110051011015102102510310351041045

05

101520253035

0 36 72 108 144 180 216 264 300 372 444 516

Isoa

myl

acet

ate (

mg

L)

Time (h)

Den

sity

(gc

m3)











110051011015102102510310351041045

0

20

40

60

80

100

120

140

0 24 48 78 102 126 150 174 198

Isoa

myl

acet

ate (

mg

L)

Time (h)

Den

sity

(gc

m3)



















4 Conclusions


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






110051011015102102510310351041045

0

20

40

60

80

100

120

140

0 24 48 78 102 126 150 174 198

Isoa

myl

acet

ate (

mg

L)

Time (h)

Den

sity

(gc

m3)



















4 Conclusions


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















4 Conclusions


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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