Effets of Aing Unsturte Ftty Ais on Ftty Ai Composition of Shromyes erevisie n Mjor Voltile Compouns in Wine L.L. Dun, Y. Shi, R. Jing, Q. Yng, Y.Q. Wng, P.T. Liu, C.Q. Dun, G.L. Yn* Centre for Vitiulture n Oenology, College of Foo Siene n Nutritionl Engineering, Chin Agriulturl University, Beijing 183, P.R. Chin Sumitte for pulition: Ferury 215 Aepte for pulition: My 215 Key wors: Wine yest, unsturte ftty is, ftty i omposition, rom ompouns The synergisti influenes of three unsturte ftty is (UFAs, nmely linolei i, olei i n linoleni i) on the ftty i omposition of Shromyes erevisie n mjor voltile ompouns were investigte in syntheti grpe juie. The ition of UFAs le to orresponing inrese in UFAs in the ellulr lipi, whih ws ompnie y reverse reution in the ontent of meium-hin sturte ftty is (C6: to C14:) n little vrition in tht of long-hin sturte ftty is (C16: to C24:). The supplementtion of UFAs onsierly improve yest growth n fermenttion tivity n, in prtiulr, inrese the onentrtions of most voltile ompouns in wine, inluing higher lohols (2-phenylethnol, 2-methyl-1-propnol n 3-(methylthio)-1-propnol), meium-hin ftty is (utnoi i, hexnoi i n otnoi i), ette esters (isomyl ette n 2-phenylethyl ette) n ll ethyl esters. Remrkle liner reltionships were further foun etween ethyl esters n the onentrtion of the e UFAs (R 2 from.99 to.996), whih signifintly intensifie the fruity, flowery n sweet ttriutes of the finl wine, s ssesse y lulting the oour tivity vlues. Our results suggest tht rtionlly inresing the onentrtion of UFAs is not only prtil metho to improve yest fermenttion tivity, ut lso potentil pproh to mnipulting wine rom. INTRODUCTION Unsturte ftty is (UFAs) re require for Shromyes erevisie to grow uner neroi onitions. They re essentil not only for mintining memrne integrity n funtion, ut lso for pting to fermenttion stresses, suh s high sugr n ethnol toxiity (Holerg & Mrglith, 1981; You et l., 23; Rupčić & Jurešić, 21). A higher egree of unsturtion of the ell memrne n mintin higher memrne fluiity n gurntee the tivity of memrne-ssoite enzymes n trnsporters, suh s ATPse n generl mino i permese (GAP) (Clernk et l., 1985; Ros & S- Correi, 1992). The min UFAs of S. erevisie re olei i (18:1) n plmitolei i (16:1), whih re erive from the esturtion of the sturte ftty is (SFAs) plmiti i (16:) n steri i (18:) y the oxygenepenent enzyme 9 esturse enoe y the OLE1 gene respetively (Ym et l., 25). Beuse it lks 12 -ftty i esturse n ω3-ftty i esturse, S. erevisie nnot proue polyunsturte ftty is (PUFAs), suh s linolei i (18:2n6) n α-linoleni i (18:3n3). The ility of PUFAs to mintin memrne fluiity is higher thn tht of monounsturte ftty is (MUFAs), n they thus offer etter protetion to ells uner hrsh onitions (Yzw et l., 29). The lipi n ftty i omposition of yest ell memrnes n e moifie y fermenttion temperture, oxygen vilility, growth rte n the presene of sterols, whih in turn influene ell growth n fermenttion tivity (Fujii et l., 1997; Reon et l., 29) n strongly ffet the formtion of voltile metolites, inluing esters, ette n ethyl esters, higher lohols n meium-hin ftty is (MCFAs), ll of whih re relevnt to wine rom (Thurston et l., 1982; Muriio et l., 1997; Beltrn et l., 28; Vrel et l., 212). During wine fermenttion, the sene of oxygen suppresses the ftty i esturtion of yest. An lterntive to the iosynthesis is the iret uptke of UFAs from the grpe juie, whih n e use to voi yest stuk fermenttion (Vrel et l., 212). Grpe erries ontin.15% to.24% (wet weight sis) lipis (Gllner & Peng, 198). UFAs represent the mjor omponents of the totl lipis. Linolei i (C18:2n6) is the most unnt, followe y olei i (C18:1n9) n α-linoleni i (C18:3n3) (Anín et l., 1998; Sntos et l., 211). The onentrtions of UFAs in grpe must epen lrgely on the grpe ultivr (Anín et l., 1998) n *Corresponing uthor: E-mil: glyn@u.eu.n Aknowlegements: This work ws supporte finnilly y the Chin Agriulture Reserh System (CARS-3) S. Afr. J. Enol. Viti., Vol. 36, No. 2, 215 285
286 Effet of UFAs on Yest Ftty Ais n Arom Compouns tehnologil fermenttion proeures, suh s grpe must lrifition (Vrel et l., 1999) n grpe-skin mertion (Vlero et l., 1998). Severl stuies hve investigte the effet of exogenous UFAs on intrellulr ftty i omposition or wine rom iosynthesis uring wine fermenttion. Reon et l. (29) foun tht supplementtion with plmitolei i (C16:1) n shorten the hin length n reue totl ftty is (inluing UFAs n SFAs) ompre with the ontrol, whih inreses the viility of the yest n reues the urtion of wine fermenttion. The profile of rom ompouns ws not ville in this stuy. The influene of exogenous UFAs on rom ompouns is ssoite with the type n onentrtion of UFAs. The ition of olei i (31 mg/l) n ergosterol (25 mg/l) n inrese the proution of higher lohols n ette esters, exept for 1-utnol n 1-pentnol, whih exhiit the opposite tren (Muriio et l., 1997). Thurston et l. (1982) foun tht the presene of linolei i (5 mg/l) n suppress the proution of ethyl esters n ette esters. In one stuy of synergisti effet, Tween 8, ontining 7% of olei i n 3% of plmi i n steri i, ws e n improve the esters, higher lohols n voltile ftty is of wine (Vrel et l., 212). Beuse the three UFAs oexist in grpe must n the onentrtions vry with grpe ultivr n fermenttion proeure, it is essentil from the wine proution perspetive to investigte their synergisti effet on the yest ftty i omposition of yest ells n yest-erive rom ompouns uring wine fermenttion. To te, however, no omplete informtion is ville. Therefore, in this stuy we investigte the synergisti effets of UFAs, inluing linolei i, olei i n linoleni i, on yest fermenttion tivity n the ftty i ompositions of ells n voltile rom ompouns. To hieve this purpose, four series onentrtions of UFA mixtures were set oring to their onentrtion n rtio in rel grpe must, whih were initilly e to the meium (Gllner & Peng, 198; Delfini et l., 1992; Anín et l., 1998; Vlero et l., 1998; Vrel et l., 1999; Sntos et l., 211). To filitte this investigtion, simplifie, hemilly efine meium (MS3) tht resemles the nutrient omposition of grpe juie ws use. MATERIALS AND METHODS Yest strins n ulture mei The S. erevisie strin EC1118 (Lllemn In. Denmrk) ws use in this stuy. The omposition of the syntheti grpe must MS3 ws similr to tht use in previous reserh (Bely et l., 199), with little moifition. Beuse the purpose of the stuy ws to investigte the effet of UFAs on yest ell metolism, Tween 8 n soium olete were not e. The omponents in the MS3 meium were s follows: min ompouns (in g/l): gluose (24), itri i (6), mli i (6), KH 2 PO 4 (.75), K 2 SO 4 (.5), MgSO 4-7H 2 O (.25), CCl 2-2H 2 O (.155), NCl (.2); minerl slts (mg/l): MnSO 4 -H 2 O (4), ZnSO 4 (4), CuSO 4-5H 2 O (1), KI (1), CoCl 2-6H 2 O (.4), H 3 BO 3 (1), NMoO 4 -H 2 O (1); vitmins (in mg/l): myo-inositol (2), niotini i (2), lium pntothente (1.5), thimine hyrohlorie (.25), pyrioxine hyrohlorie (.25), iotin (.3); nitrogen ompouns (in mg/l): proline (612.6), glutmine (55.3), rginine (374.4), tryptophn (179.3), lnine (145.3), glutmi i (12.4), serine (78.5), threonine (759.2), leuine (48.4), sprti (44.5), vline (44.5), phenyllnine (37.9), isoleuine (32.7), histiine (32.7), methionine (31.4), tyrosine (18.3), glyine (18.3), lysine (17), ysteine (13.1), mmonium hlorie (46); lipis (in mg/l): ergosterol (15). The UFA mixture (inluing linolei i, olei i n α-linoleni i, purhse from Sigm, purity 98%) ws e to the meium in the onentrtions shown in Tle 1. The minimum UFAs were require to ensure norml ell growth n fermenttion uner neroi onitions (Aeituno et l., 212), whih serve s the ontrol (Tle 1, meium A). The onentrtion of UFAs in mei B, C n D were four-, eight- n 12-fol higher thn tht in meium A respetively. The proportions of the three UFAs were etermine oring to the results of previous stuies (Anín et l., 1998; Sntos et l., 211). Fermenttion onitions Yest strins were pre-ulture eroilly in YEPD (1% yest extrt, 2% peptone, 2% gluose) t 12 rpm n 28 C for pproximtely 15 h to the lte exponentil phse. Before the inoulums, nitrogen ws sprge to eliminte the oxygen from the meium. The inoultions were in 5 ml flsks equippe with glss pillry stoppers ontining 3 ml of MS3 meium. The initil yest inoulums (1 1 6 CFU/mL) were ultivte sttilly t 25 C. Fermenttion ws rrie out in three iologil repets. The progress of fermenttion ws monitore ily y mesuring ell ensity (OD 6 ) n sugr onsumption. Smples tken from the fermenttion flsks were immeitely entrifuge to ollet the ell-free superntnts for the nlysis of the min fermenttion prouts n rom ompouns n the yest ells for the nlysis of intrellulr ftty is. UFA hnges in the mei n the intrellulr ftty is uring fermenttion were nlyse t the pre-fermenttion ( h), miexponentil (5 h), lte-exponentil (7 h), en-exponentil (94 h) n mi-sttionry phses (166 h), n t the en of fermenttion (266 h for mei A, B n C n 238 h for TABLE 1 Conentrtions of olei i, linolei i n α-linoleni i in MS3 mei (A, B, C n D) Vrile Conentrtions of UFAs in ifferent mei (mg/l) meium A meium B meium C meium D olei i 1 5 9 13 linolei i 3 15 27 39 α-linoleni i 8 4 72 14 S. Afr. J. Enol. Viti., Vol. 36, No. 2, 215
Effet of UFAs on Yest Ftty Ais n Arom Compouns 287 Sugr Sugr onentrtion (g/l) (g/l) 3 2 1 2 3 4 5 meium A meium B meium C meium D 6 9 6 3 OD OD 6 6 1 5 1 15 2 25 3 Time (h) Time (h) FIGURE 1 Cell growth (OD6) n resiul sugr onentrtion uring loholi fermenttion in ifferent mei. Fermenttion phse (fermenttion time): mi-exponentil (5 h), lte-exponentil (7 h), en-exponentil (94 h) n mi-sttionry phses (166 h) n t the en of fermenttion (266 h for mei A, B n C n 238 h for meium D). meium D) (Fig. 1). The nlysis of the min fermenttion prouts n rom ompouns were onute t the en of fermenttion. All fermenttion smples were store t -2 C until nlysis. Eh nlysis ws repete in triplite for eh iologil repet. Anlysis of min fermenttion prouts The onentrtions of the min fermenttion prouts, gluose, frutose, ethnol, glyerol, mli i, itrte i n eti i, were etermine y high-performne liqui hromtogrphy (HPLC, 12 series, Agilent Tehnologies, In., Plo Alto, CA, USA) using n Aminex HPX-87H olumn (3 mm 7.8 mm, Bio-R, CA, USA), s esrie in previous stuy ut with little moifition (López & Gómez, 1996). The moile phse flow rte of.6 ml/min ws hieve with n elution grient ompose of 5 nmol/l H 2 SO 4 in wter: (1) The injetion volume ws 2 μl for the gluose, frutose, glyerol n lohol nlysis. The olumn temperture ws 45 C with refrtive inex etetor (RID). The totl nlysis time ws 3 min. (2) The injetion volume ws 1 μl for the mli i, itri i n eti i nlysis. The olumn temperture ws 6 C, with ioe rry etetor (21 nm, DAD). The totl nlysis time ws 3 min. Anlysis of ftty is The nlysis of the ftty is in the yest ells ws onute oring to the proeure of Reon et l. (29). Yest ell smples were ple in sele tues n sponifie with 1 ml of 5% NOH in 5% methnol/wter. The sponifition ws onute t 1 C for 3 min. The sponifie prt then ws oole to room temperture, n 2 ml HCl 6 M ws e to the tues. Free ftty is were extrte y ing.5 ml of hexne:methyl-tert-utyl ether (1:1, v/v). The tues were vortexe twie for 3 se eh. The orgni phse ws ollete fter entrifugtion (3 rpm, 3 min). Ftty is were quntifie with 789NGC gs hromtogrph equippe with 5975BMS mss spetrum system (GC-MS, Agilent, USA) on n HP-INNOWAX olumn (6 m.25 i..,.25 μm film thikness, J&W Sientifi, USA) uner helium rrier gs (1 ml/min). The temperture ws progrmme from 8 C (hel for 1 min) to 22 C t 25 C/min n from 22 C to 25 C (hel for 2 min) t 5 C/min. One mirolitre of extrt ws injete in splitless moe. The temperture of the trnsfer line n the injetor were 28 C n 25 C respetively. The mss spetrometer ws operte in the eletron impt (EI) moe t 7 ev, n in the seletive ion moe (SIM) rnge of 2 to 35 m/z. The lirtion ws previously performe with pure stnr hemils. Anlysis of rom ompouns The nlysis of the finl wine oour roms ws onute oring to previous reserh (Zhng et l., 27), s follows. Five millilitres of the finl fermenttion smple, 1. g of NCl n 1 μl of 4-methyl-2-pentnol (1.39 mg/ml wter, internl stnr) were lene in 15 ml smple vil tht ws tightly ppe with PTFE silion septum n ontine mgneti stirrer. Susequently, the vil ontining the smple ws hete t 4 C for 3 min on heting pltform y gittion (8 rpm). A 789NGC gs hromtogrph equippe with 5975BMS mss spetrum system (GC-MS, Agilent, USA) on n HP-INNOWAX olumn (6 m.25 i..,.25 μm film thikness, J&W S. Afr. J. Enol. Viti., Vol. 36, No. 2, 215
288 Effet of UFAs on Yest Ftty Ais n Arom Compouns Sientifi, USA) ws pplie for the nlysis of the rom ompouns. The pre-trete (onitione t 27 C for 1 h) SPME fire (5/3 μm DVB/Croxen/PDMS, Supelo, Bellefonte, PA, USA) ws then inserte into the hespe n extrte for 3 min with ontinue heting (4 C) n gittion (8 rpm). The fire ws instntly esore in the GC injetor for 8 min t 25 C. The GC inlet ws set in the splitless moe. The strting temperture of the oven ws hel t 5 C for 1 min n then inrese to 22 C t rte of 3 C/min n hel there for 5 min. The mss spetrometer ws operte in the eletron impt (EI) moe t 7 ev, n in the sn n seletive ion moe (SIM) rnge of 35 to 35 m/z. The lirtion ws previously performe with pure stnr hemils se on previous stuies (Howr et l., 25). Estlishment of oour tivity vlue n romti series To evlute the ontriution of eh rom ompoun to the totl oour profile, the oour tivity vlue (OAV) ws lulte y iviing the onentrtion of ompoun y its oour threshol (OT, etermine from the wine mtrix) tht ws otine from the literture ite in Tle 4. To otin the whole rom profile of the wine, n romti series ws estlishe y grouping the OAVs of the voltiles exhiiting similr oour esriptions (Peino et l., 26; Wu et l., 211). Sttistil nlysis Anlysis of vrine (ANOVA, lest signifint ifferene metho t signifine level of P.5) ws use to evlute ifferenes etween the voltile n non-voltile ompouns in the fermenttion smples y the SPSS sttistil pkge version 19. (SPSS In., USA). RESULTS AND DISCUSSION Yest growth n sugr onsumption Fig. 1 shows the progress of fermenttion with ifferent UFA onentrtions. The ition of UFAs gretly improve yest growth n sugr onsumption. Cells rehe the sttionry phse fter 1 h of fermenttion in ll ses. The highest iomss (OD 6 vlues) ws oserve in meium D, followe y mei C, B n A. The sugr onentrtion in meium D ws reue elow 2 g/l fter 238 h of fermenttion n ws shortene y 28 h ompre with tht of the three other ses. Corresponingly, positive reltionship etween initil UFA onentrtion n the mximum iomss (OD 6 vlues, R² =.9514) n mximum gluose onsumption rtes (R² =.9567) ws estlishe. These results onfirme the onlusion tht the initil ition of UFAs n improve yest growth n inrese yest fermenttion tivity (Reon et l., 29). Min fermenttion prouts The finl onentrtions of ethnol, glyerol, eti i, itri i n mli i were further etermine, n the results show tht there ws no signifint ifferene mong the ifferent smples (Tle 2). This fining suggests tht the vrition in the UFA onentrtions i not hve onsierle influene on the primry metolites of the sugr. Ftty i omposition of yest ells It generlly is elieve tht the improvement in ell growth n fermenttion performne tht is use y UFAs results from the moifition of the omposition of intrellulr ftty is. We thus etermine the ftty i ontent of the ells. First, the profiles of the supplemente UFA onentrtions in the mei uring fermenttion were investigte. The results showe tht the three UFAs were rpily tken up y yest ells n tht the UFA levels erese rpily. The highest UFA uptke rtes were oserve in meium D uring fermenttion, followe y mei C, B n A (Fig. 2). In four ses, the UFAs were lmost ompletely onsume fter 94 h of fermenttion when the yest ells rehe the en-exponentil phse (t not shown). Corresponingly, the intrellulr UFA ontent inrese signifintly n the level of prtiulr UFAs ws positive following the supplemente UFA onentrtion (Fig. 3). The inorportion of UFAs le to n inrese in UFAs in the totl ftty i ontent from 74.35% in meium A to 8.55% in meium C n 83.55% in meium D when the ells rehe the miexponentil phse; the highest UFA levels were otine t this time. After the mi-exponentil phse, the UFA ontent grully erese n finlly ws mintine t onstnt level fter the en-exponentil phse in ll smples, ut the vlues with UFAs ition were ll higher thn those of the ontrol (meium A). Our results were onsistent with previous stuy, whih showe tht the UFA onentrtion of yest ells eline s the fermenttion progresse ue to ell growth uner neroi fermenttion (Venter et l., 1989). The inorportion of UFAs resulte in the highest totl onentrtion of FAs oserve in meium D, followe y yest in mei C, B n A (Fig. 3, ANOVA, P <.5). It shoul e note tht plmitolei i (C16:1n-9), whih is onsiere one of the min ftty is in the omposition of yest ells, ws not etete in our reserh (t not shown). This n e srie to the ft tht ftty i esturtion nnot our in yest ells uner neroi onitions (Snoek & Steensm, 27). Our results emonstrte tht the enrihment of UFAs in ells is essentil for yest pttion to environmentl stress uring loholi fermenttion (You et l., 23; Rupčić & Jurešić, 21). Long-hin sturte ftty is (LSFAs, C16: to TABLE 2 Min fermenttion prouts of the finl wines Meium ethnol (%, v/v) glyerol (g/l) eti i (g/l) itrte i (g/l) mli i (g/l) A 11.46 ±.22 6.8 ±.8.65 ±.4 4.73 ±.12 6.7 ±.1 B 1.93 ±.37 5.99 ±.7.66 ±.3 4.62 ±.1 5.9 ±.19 C 11.45 ±.16 6.31 ±.4.73 ±.4 4.78 ±.2 6.8 ±.3 D 11.18 ±.13 6.17 ±.3.72 ±.2 4.68 ±.8 6.8 ±.12 S. Afr. J. Enol. Viti., Vol. 36, No. 2, 215
Effet of UFAs on Yest Ftty Ais n Arom Compouns 289 Uptke rte of unsturte ftty is y yest ells (mg/l/h) 8 6 4 2 mi-exponentil-phse lte-exponentil-phse meium A meium B meium C meium D FIGURE 2 Uptke rtes of unsturte ftty is (UFAs) y yest ells in ifferent mei uring loholi fermenttion. Signifint ifferenes in UFA onentrtions mong mei in the sme fermenttion phse re lelle y letters,, n (ANOVA, P <.5). en-exponentil-phse mi-sttionry-phse en-fermenttion Ftty is in yest ells in ifferent mei (mg/g ry wine yest) Ftty is in yest ells in ifferent mei (mg/g ry wine yest) 18 12 6 4 3 2 1 Long-hin sturte ftty is Unsturte ftty is meium A meium B meium C meium D 6 3 24 16 8 Meium-hin ftty is Totl ftty is pre-fermenttion pre-fermenttion mi-exponentil-phse mi-exponentil-phse lte-exponentil-phse lte-exponentil-phse en-exponentil-phse en-exponentil-phse mi-sttionry-phse mi-sttionry-phse en-fermenttion en-fermenttion pre-fermenttion mi-exponentil-phse lte-exponentil-phse en-exponentil-phse mi-sttionry-phse en-fermenttion FIGURE 3 Conentrtions of unsturte ftty is (UFAs), meium-hin ftty is (MCFAs), long-hin sturte ftty is (LCSFAs) n totl ftty is (TFAs) in wine yest uring wine fermenttion. Signifint ifferenes in min ftty i onentrtions mong yest in ifferent wines in the sme fermenttion phse re lelle y letters,, n (ANOVA, P <.5). pre-fermenttion mi-exponentil-phse lte-exponentil-phse en-exponentil-phse mi-sttionry-phse en-fermenttion S. Afr. J. Enol. Viti., Vol. 36, No. 2, 215
29 Effet of UFAs on Yest Ftty Ais n Arom Compouns C24:, 15.54% to 39.38%) were the seon lrgest group of ftty is in the totl ftty is, followe y meiumhin ftty is (MCFAs C6: to C14:,.74% to 9.32%). Surprisingly, the vrition in intrellulr UFA onentrtion h no signifint effet on LSFA synthesis, ut it i hve onsierle impt on the ontent of MCFAs. The highest onentrtion of MCFAs ws oserve in yest ultivte in meium A, followe y mei B, C n D (Fig. 3, ANOVA, P <.5), whih suggests tht there is negtive reltionship etween the intrellulr MCFAs n UFAs. At present we nnot fully explin the phenomenon; however, two eutions were propose oring to previous stuies. First, Dufour et l. (28) suggeste tht the inrese in UFAs in yest ells n le to the relese of MCFAs from the ftty i synthse omplex (FAS1 n FAS2) n further serete them into the meium. This phenomenon ws lso oserve in our stuy, s shown elow. The seretion might e prtilly responsile for the reution in MCFA ontent in the ells. An lterntive explntion, propose y Reon et l. (211), sttes tht, in ition to UFAs, MCFAs re funtionl ompouns tht help yest ells pt to stress onitions y inresing the memrne fluiity. The inorportion of exogenous UFAs into the ell memrne might e effiient for ells to hnle stress onitions n my thus weken the synthesis of MCFAs. The etile mehnism nees to e investigte further. Arom ompouns Although they h no onsierle impt on the primry metoli ompouns of sugr, the vritions in UFAs signifintly ffete the synthesis of wine rom ompouns. Generlly, the voltile ompouns were promote y the ition of UFAs, n the totl onentrtions in the finl wines of C n D were 1.21- n 1.25-fol tht oserve in wine A respetively (Tle 3, ANOVA, P <.5). Moreover, the onentrtions of ethyl esters, whih re the min romontriuting ompouns, in wines C n D were 1.84- n 2.82-fol higher thn tht in wine A respetively. Twentyeight rom ompouns were etete in our stuy six MCFAs, seven lohols, five lehyes, seven ethyl esters n three ette esters. Of these, 22 ompouns oul e ientifie, n six ompouns oul e ientifie tenttively (Tle 3). With the exeption of uneyl lehye in wine D, ll of the rom ompouns oul e quntifie in ll the smples. Meium-hin ftty is MCFAs (C6 to C12) re proue primrily y yests s intermeites in the iosynthesis of long-hin ftty is n re premturely relese from the ftty i synthse (FAS) omplex. These voltile ftty is n iretly ontriute to the flvour of wine, or serve s sustrtes tht prtiipte in the formtion of ethyl ettes. In ontrst to the intrellulr MCFAs, the ition of UFAs resulte in greter umultion of MCFAs in the meium. This result n e srie to the repression of the gene enoing etyl- CoA roxylse (ACC1) y exogenous UFAs, whih uses the relese of MCFAs from the ftty i synthse omplex (FAS1 n FAS2) n their seretion into the environment (Dufour et l., 28). Among the six ientifie MCFAs, otnoi i ws the highest, followe y hexnoi i, enoi i, oenoi i, utnoi i n heptnoi i. The highest onentrtions of otnoi i, hexnoi i n utnoi i were foun in wine C (14.59, 3.26 n.35 mg/l) n wine D (12.44, 3.23 n.36 mg/l), ompre with those of wine A (1.22, 2.21 n.27 mg/l) (Tle 3, ANOVA, P <.5). Ethyl esters The C6, C8 n C1 meium-hin ftty i ethyl esters re importnt flvour-tive metolites tht re responsile for the highly esire fruit rom hrters in wine. They re generte y n lohol n the yl group erive from tivte MCFAs. Among the seven ientifie ethyl esters, ethyl enote ws quntittively the highest, followe y ethyl otnote, ethyl hexnote n ethyl oenote. Higher ontents of ethyl esters were forme in wines C n D, n the totl onentrtions were 1.84- n 2.82-fol higher thn tht in wine A (Tle 3, ANOVA, P <.5) respetively. In ition, signifint positive reltionships etween ethyl esters, prtiulrly ethyl hexnote ( fruity oour) n ethyl otnote ( fruity, florl n sweet oour), n the e UFA onentrtions were estlishe (R 2 from.99 to.996). Suffiient MCFAs re ruil for the higher iosynthesis of plesnt ethyl esters (Serens et l., 28); therefore, the improvement oserve for the ethyl esters following ftty i tretment y UFAs n e srie to the umultion of the sustrte MCFAs in the meium. This onlusion ws supporte further y the oservtion tht liner orreltions existe etween MCFAs n the orresponing ethyl esters (t not shown). Alohols Seven lohols were ientifie in our reserh, inluing three ftty lohols (1-hexnol, 1-heptnol n 1-otnol) n four higher lohols (2-phenylethnol, 3-methyl-1-utnol, 2-methyl-1-propnol n 3-(methylthio)-1-propnol). The etile mehnism of ftty lohol iosynthesis y wine yest is not fully unerstoo. Our results inite tht tretment with UFAs n inrese the proution of ftty lohols. The highest onentrtions of 1-hexnol n 1-otnol were foun in wine D (444.6 μg/l n 18.24 μg/l vs 35.93 n 12.93 μg/l in wine A respetively), wheres the highest onentrtion of 1-heptnol ws in wine C (42.82 μg/l vs 28.14 μg/l in wine A) (Tle 3, ANOVA, P <.5). The higher lohols re preominntly forme y yest uring fermenttion from α-keto is, proess tht involves the egrtion of n mino i (i.e., vline, leuine, isoleuine, threonine n phenyllnine) vi the so-lle Ehrlih pthwy (Hzelwoo et l., 28). High levels of 2-phenylethnol, 2-methyl-1-propnol n 3-(methylthio)-1-propnol were oserve in wines C n D. Higher lohols n hve oth positive n negtive impt on the rom n flvour of wine, epening on their onentrtions. Exessive onentrtions (exeeing 4 mg/l) n result in strong, pungent smell, wheres n optiml level (elow 3 mg/l) imprts plesnt hrter (Brtowsky & Pretorius, 29). In this stuy, totl onentrtions of 295.9 mg/l n 36.3 mg/l were foun S. Afr. J. Enol. Viti., Vol. 36, No. 2, 215
Effet of UFAs on Yest Ftty Ais n Arom Compouns 291 TABLE 3 Conentrtions of rom ompouns in wines Arom ompouns Oour threshol Retention inex ID Fermenttion meium A B C D utnoi i (mg/l) 1 () 163 I.27 ±.1.27 ±.3.35 ±.3.36 ±.4 hexnoi i (mg/l).42 () 1855 I 2.21 ±.15 2.66 ±.13 3.26 ±.29 3.23 ±.33 heptnoi i (μg/l) 199 I 62.44 ± 5.77 75.9 ± 7.59 82.78 ± 1.62 77.85 ± 5.68 otnoi i (mg/l).5 () 275 I 1.22 ±.6 13.16 ± 1.58 14.54 ± 1.59 12.44 ± 1.45 enoi i (mg/l) 1 () 2292 I 2.37 ±.38 2.41 ±.48 2.8 ±.36 2.29 ±.47 oenoi i (mg/l) 1 () 25 I 1.29 ±.17 1.37 ±.17 1.55 ±.14 1.71 ±.15 Totl meium-hin ftty is (mg/l) 16.42 ± 1.43 19.94 ± 1.2 22.58 ± 2.13 2.11 ± 1.87 ethyl hexnote (mg/l).5 () 1232 I.22 ±.2.25 ±.5.98 ±.1 1.57 ±.1 ethyl otnote (mg/l).2 () 1429 I.69 ±.5.71 ±.2.88 ±.12 1.1 ±.16 ethyl nonnote (μg/l) 1535 I.65 ±.18 1.1 ±.9 1.88 ±.14 2.86 ±.18 ethyl enote (mg/l).2 () 1637 I.9 ±.2 1.64 ±.1 3.14 ±.33 4.13 ±.43 ethyl oenote 2 () 1848 I.11 ±.1.25 ±.3.44 ±.5.61 ±.1 (mg/l) ethyl myristte (μg/l) 2 () 255 TI 4.92 ±.52 11.74 ± 1.55 21 ± 2.18 27.65 ± 4.88 ethyl plmitte (μg/l) > 2 () 2263 TI 6.52 ±.61 11.74 ± 1.7 17.81 ± 1.88 18.56 ± 1.17 Totl ethyl esters(mg/l) 1.93 ±.16 2.88 ±.21 5.48 ±.37 7.37 ±.61 1-hexnol (μg/l) 8 () 1344 I 35.93 ± 1.7 142.99 ± 17.16 231.4 ± 15.6 444.6 ± 45.23 1-heptnol (μg/l) 3 () 1457 I 28.14 ± 2.75 27.76 ±.72 42.82 ± 2.75 37.52 ± 3.55 1-otnol (μg/l) 11 () 1562 I 12.93 ± 1.7 24.92 ± 1.17 41.36 ± 4.8 18.24 ± 16.58 2-phenylethnol (mg/l) 1 () 1928 TI 42.98 ± 4.2 58.5 ± 5.4 76.77 ± 5.86 71.33 ± 4.49 3-methyl-1-utnol 3 () 126 I 184.21 ± 13.45 157.36 ± 22.61 186.51 ± 13.62 25.84 ± 24.18 (mg/l) 2-methyl-1-propnol 4 () 1111 I 18.22 ± 1.32 22.17 ± 3.3 32.24 ± 3.8 28.55 ± 4.4 (mg/l) 3-(methylthio)-1- propnol (μg/l) 5 () 1726 I 23.78 ± 1.9 26.88 ± 2.83 31.74 ± 3.6 29.26 ± 2.52 Totl lohols (mg/l) 245.5 ± 1.1 238.26 ± 12.64 295.87 ± 14.57 36.34 ± 33.7 3-methyl-1-utnol.3 () 122 TI 1.9 ±.6 1.25 ±.15 1.22 ±.14 1.69 ±.16 ette (mg/l) ethyl ette (mg/l) 7.5 () 877 I 36.54 ± 3.43 41.8 ± 2.24 39.19 ± 5.83 42.56 ± 6.41 2-phenylethyl ette (μg/l) 25 () 183 I 1.2 ±.12 1.75 ±.15 1.91 ±.11 1.98 ±.13 Totl ette esters (mg/l) 37.64 ± 3.91 42.33 ± 2.27 4.41 ± 4.24 44.25 ± 6.21 otnl (μg/l).7 (e) 129 I 7.53 ±.7 8.11 ±.83 8.71 ±.88 7.53 ±.1 nonnl (μg/l) 1 (e) 1394 I 8.35 ± 1.58 3.51 ± 2.43 34.76 ± 4.4 19.93 ± 1.75 enl (μg/l).1 (e) 1495 I 1.7 ±.17 4.99 ±.25 5.68 ±.21 2.84 ±.11 uneyl lehye (μg/l) 1596 TI 1.43 ±.14 1.71 ±.17 1.61 ±.16 Tr oenl (μg/l) 2 (f) 1693 TI 2.51 ±.27 4.3 ±.7 5.44 ±.3 3.95 ±.3 Totl lehyes (μg/l) 2.89 ±.94 94.57 ± 6.53 17.39 ± 9.43 59.87 ± 5.21 Totl rom ompouns onentrtions (mg/l) 31.51 ± 15.63 33.5 ± 24.81 364.45 ± 2.79 378.13 ± 22.91 Oour threshols of rom ompouns were oring to previous referenes: () Guth, 1997; () Li et l., 28; () Leffingwell & Assoites, 1999; () Fzzlri, 1978; (e),ferreir et l., 24; (f) Boelens & Vn Gemert, 1987. ID, reliility of the ientifition proposl: I, ientifie, mss spetrum n retention inex gree with stnrs; TI, tenttively ientifie, mss spetrum grees with retention inex (Wu et l., 211) n/or the mss spetrl tse. Letter,,, n in the sme row show signifint ifferenes oring to the nlysis of vrine t P <.5. Bolfe inites onentrtion of rom ompoun ws over its oour threshol. Tr: tre. S. Afr. J. Enol. Viti., Vol. 36, No. 2, 215
292 Effet of UFAs on Yest Ftty Ais n Arom Compouns TABLE 4 Oour tive vlues, oour esription n romti series of ll rom ompouns in finl wines Arom ompouns Oour tive vlues(oavs) Oour esription Aromti A B C D series Ftty rni () Ftty hrsh, heese () hexnoi i 5.26 ±.36 6.33 ±.31 7.76 ±.69 7.69 ±.79 ftty, heese, otnoi i 2.44 ± 1.2 26.32 ± 3.16 29.8 ± 3.18 24.88 ± 2.9 ftty, rni, enoi i 2.37 ±.38 2.41 ±.48 2.8 ±.36 2.29 ±.47 rni, heese () Ftty oenoi i 1.29 ±.17 1.37 ±.17 1.55 ±.14 1.71 ±.15 lurel oil () Ftty ethyl hexnote 44.1 ± 4 5.2 ± 1 196.56 ± 9 314.38 ± 2 fruity, green pple, Fruity nn () ethyl otnote 346.8 ± 17.5 355.19 ± 1 44.95 ± 11 56.48 ± 1.5 sweet, florl, fruity, nn, per () Sweet, florl, fruity ethyl enote 4.48 ± 1 8.2 ±.5 15.72 ± 1.65 2.67 ± 2.15 fruity, plesnt () Fruity otnl 1.76 ± 1 11.58 ± 1.19 12.45 ± 1.26 1.76 ±.14 itrus () Fruity nonnl 8.35 ± 1.58 3.51 ± 2.43 34.76 ± 4.4 19.93 ± 1.75 itrus () Fruity enl 1.7 ± 1.7 49.9 ± 2.5 56.8 ± 1.1 28.4 ± 1.1 green, fresh () Hereous oenl 1.26 ±.14 2.15 ±.4 2.72 ±.15 1.98 ±.15 green oriner () Hereous 1-heptnol 9.38 ±.92 9.25 ±.24 14.27 ±.92 12.51 ±.18 green () Hereous 3-methyl-1-6.14 ±.4 5.25 ±.54 6.22 ±.59 6.86 ±.45 lohol, Chemil utnol nil polish () 2-phenylethnol 4.3 ±.35 5.85 ±.25 7.68 ±.25 7.13 ±.41 pollen, roses (e) Florl 3-methyl-1- utnol ette 36.47 ± 2 41.77 ± 5 4.74 ± 4.67 56.25 ± 5.33 nn, fruity, sweet () Fruity, sweet ethyl ette 4.87 ±.46 5.48 ±.3 5.23 ±.78 5.67 ±.85 pinepple, vrnish, lsmi (e) Fruity, hemil Oour esription ws oring to the previous referenes: () Peino et l., 26; () Li et l., 28; () Buettner et l., 23; () Yng et l., 28; (e) Aznr et l., 21. Signifint ifferenes in OAVs in the sme row re lelle y letters,, n (ANOVA, P <.5). in wines C n D respetively, whih suggests tht higher lohols ontriute positively to the rom of the wine. At present, we nnot fully explin why the ition of exogenous UFAs promotes higher lohol synthesis, ut we inferre tht it might e ssoite with the improvement of mino i trnsporttion into ells euse it hs een emonstrte tht S. erevisie growing in meium enrihe with UFAs (linolei i n olei i) n inue generl mino i permese (GAP) tivity n inrese the trns-shipment of mino is into yest ells (Clernk et l., 1984, 1985), whih is enefiil for the synthesis of higher lohols (Hernánez-Orte et l., 26). The etile mehnism nees to e investigte further. Aette esters Aette esters re nother importnt rom ompoun tht re generte y the retion of etyl-coa with higher lohols tht re forme from the egrtion of mino is or rohyrtes n hve signifint effet on the fruity flvour of wine. The ette esters etete in our stuy inlue ethyl ette ( pinepple, vrnish n lsmi oour, Tle 3), isomyl ette ( nn n sweet oour, Tle 3) n 2-phenylethyl ette ( honey, roses n flowery oour). No signifint ifferene ws foun in ethyl ette in ll the smples, lthough higher onentrtions of 3-methyl-1-utnol ette (isomyl ette) n 2-phenylethyl ette umulte in wines C n D (Tle 3, ANOVA, p <.5). This ws in ontrst to the results of previous stuy, whih showe tht ette esters were inhiite y exogenous UFAs ue to the repression of ATF1 trnsription, the gene enoing the enzyme (lohol etyltrnsferse) tht tlyses the synthesis of ette esters (Fujii et l., 1997). Our results inite tht, espite the negtive regultion, inrese UFA onentrtions n enhne the proution of ette esters y inresing yest growth n sustrte proution (higher lohol). Alehyes High levels of enl n oenl n use n unplesnt, intense, green oour in wine, wheres lower ontent of C8 to C11 lehyes n ontriute to itrus fruit rom (Culleré et l., 211). The effets of UFAs on the proution of lehyes were ifferent from other rom ompouns, whih were lrgely epenent on the UFA onentrtion. Reltively high UFA ontents (wines B n C) resulte in higher onentrtions of nonnl, enl n oenl ompre with wine A, wheres further inresing the UFA ontent (wine D) le to gret reution in the lehye level (Tle 3, ANOVA, P <.5). The OAVs erese from 34.8-, 56.8- n 2.7-fol in wine C to 19.9-, 28.4- n 2.-fol in wine D respetively, s shown elow. This fining implies tht mnipulting the initil UFA onentrtion potentilly n regulte the influene of lehyes on wine rom. S. Afr. J. Enol. Viti., Vol. 36, No. 2, 215
Effet of UFAs on Yest Ftty Ais n Arom Compouns 293 Influene of ing UFAs on oour profile of finl wine The OAV n ssess the extent of the potentil sensory rom ontriution for eh voltile ompoun. An rom ompoun with n OAV over one is onsiere to mke flvour ontriution to wine (Guth, 1997). As shown in Tle 4, 16 rom ompouns h n OAV ove 1 in our stuy. Aoring to previous reserh (Aznr et l., 21; Buettner et l., 23; Peino et l., 26; Li et l., 28; Yng et l., 28), these ompouns re minly ssoite with the ttriutes nn, pple, per, strwerry, itrus sweet, roses, fruity, ftty, heese, rni, nil polish, lsmi n green (Tle 4). The OVAs of ethyl hexnote (fruity, green pple, nn ttriutes) n ethyl enote (fruity ttriute) in wine D were 6.15- n 3.62-fol higher thn those of wine A. Other importnt voltile rom ompouns, inluing ethyl otnote, 2-phenylethnol n 3-methyl-1-utnol ette, lso h higher OAVs in wines C n D thn in wine A. They ertinly re responsile for the inrement of fruity n flowery ttriutes in wines C n D, s shown elow. The oour of ompoun is esrie in terms of severl esriptors tht re gree upon y experts, n n romti series n e efine s group of voltile ompouns with similr oour esriptors (Wu et l., 211). To etter unerstn the influene of UFAs on the wine oour profile n the ontriution of vrious voltile ompouns to the olftory impression of wine, n romti series ws further estlishe in our stuy y omining the OAVs of group of voltiles with similr oour esriptions (Peino et l., 26; Wu et l., 211). Six romti series of voltile ompouns were estlishe: fruity, florl, sweet, hereous, hemil n ftty (Fig. 4). Of these, the fruity series ws prominent, followe y the flowery, sweet, hereous, ftty n hemil series. The higher OAVs of ethyl esters, higher lohol n ette esters (suh s ethyl otnote, ethyl hexnote, 2-phenylethnol n 3-methyl-1-utnol ette) le to wines C n D feturing the fruity, flowery n sweet series (Fig. 4, ANOVA, P <.5). Although wines C n D lso h reltively higher OAVs in the hereous n ftty series, their influene on the totl oour profile ws limite ue to the low onentrtion or high oour threshol of prtiulr ompoun, suh s oenl, 1-heptnol n 1-hexnol (Tle 3). As result, there ws no onsierle isrepny etween the hereous n ftty series mong the wines. Similrly, no signifint ifferene ws oserve mong the smples for the hemil series. Bse on these results, it n e onlue tht n inrese initil UFA onentrtion in the meium n intensify the fruity, florl n sweet ttriutes of wine n thus improve the oour profile of the wine. The purpose of the present stuy ws to evlute the synergisti impt of three UFAs on voltile ompouns from the point of view of prtil proution. Clerly, to fully unerstn the mehnism of how UFAs regulte rom ompouns it is essentil to investigte the seprte influene of iniviul UFAs. In ition, the influene of yest strin speifiity shoul e onsiere. These relte experiments re eing onute in our lortory. In this stuy, we use the syntheti grpe juie MS3 meium to eliminte the influene of grpe ultivr rom ompouns n rom preursors on the proution of yest-erive rom ompouns; however, the utilistion of nturl grpe must my hnge the effet of the vrition in UFA 12 8 4 wine A wine B wine C wine D OAVs 5 Fruity Florl Sweet Hereous Ftty Chemil FIGURE 4 Arom series in finl wine A, wine B, wine C, n wine D. Arom ompouns re lulte for florl series: ethyl otnote n 2-phenylethnol; for fruity series: ethyl otnote, ethyl hexnote, ethyl enote, otnl, nonnl n 3-methyl-1- utnol ette; for sweet series: ethyl otnote n 3-methyl-1-utnol ette; for hereous series: enl, oenl n 1-heptnol; for hemil series: 3-methyl-1-utnol n ethyl ette; for ftty series: hexnoi i n otnoi i. Signifint ifferene in rom series mong wines re lelle y letters,,, n (ANOVA, P <.5). S. Afr. J. Enol. Viti., Vol. 36, No. 2, 215
294 Effet of UFAs on Yest Ftty Ais n Arom Compouns onentrtion on the types n levels of voltile ompouns, euse omplex flvour preursors (suh s glyosie rom moleules) (Losos et l., 27) n other enzymetlyse retions (suh s 1ipoxygense-tlyse retions) (Delu et l., 29) my our. Therefore, further work is neee to evlute the extent to whih hnging the initil UFA onentrtion ffets the proution of wine rom ompouns in rel grpe must. CONCLUSIONS The present results inite tht supplementtion with mixe UFAs (inluing linolei i, olei i n linoleni i) n orresponingly inrese the intrellulr UFA ontent, whih simultneously les to the reverse reution of MCFA ontent ut oes not influene LSFA synthesis. In ition to improving yest ell growth n fermenttion tivity, the itions of mixe UFAs promote the most voltile rom ompouns, inluing MCFAs, higher lohols, ette esters n ll ethyl esters. 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