Impliatin f Aeti Aid in the Indutin f Slw/Stuk Grape Juie Fermentatins and Inhibitin f Yeast by Latbaillus sp. C. G. EDWARDS ~*, A. G. REYNLDS 2, A. V. RDRIGUEZ 3, M. J. SEMN 4, and J. M. MILLS ~ The impat f aeti aid and its suggested rle in induing slw/stuk alhli fermentatins was studied. The inhibitry effet f aeti aid against Saharmyes erevisiae was dependent n the strain as evidened by the wide range f minimum inhibitry nentratins bserved (4.5 t 7.5 g/l). Latbaillus kunkeei, a spilage baterium previusly determined t ause slw/stuk alhli fermentatins, prdued 4 t 5 g/l aeti aid when grwn in a Chardnnay grape juie. Daily additin f sterile-filtered slutins f aeti aid t Riesling and Chardnnay fermentatins at the same rate prdued by L. kunkeei delayed mpletin f the fermentatins. Althugh the Riesling fermentatins with aeti aid eventually beame dry (<2 g/l fermentable arbhydrate), Chardnnay fermentatins beame stuk. Yeast ppulatins in the Chardnnay grape juies with r withut aeti aid exeeded 1 7 fu/ml and were nt different during fermentatin. This finding was in ntrast t previus inhibitin studies between S. erevisiae and L. kunkeei where the yeasts studied did nt enter lgarithmi grwth in juies inulated with the baterium. Therefre, exessive prdutin f aeti aid an slw alhli fermentatins but des nt slely aunt fr the baterial inhibitin f yeast. KEY WRDS: Latbaillus kunkeei, Saharmyes erevisiae, alhli fermentatin, slw/stuk fermentatins, vlatile aidity, aeti aid In reent years, sme winemakers have bserved rapid wine spilage by rganisms dubbed "ferius" latbailli [2]. Bultn et al. [2] haraterized this spilage as being very swift with abundant baterial grwth during the early stages fvinifiatin. Huang et al. [2] islated three lati aid bateria, designated YH-15, YH-24, and YH-37, frm mmerial wines underging slw/stuk fermentatins. These strains slwed labratry-sale Chardnnay fermentatins atalyzed by Saharmyes erevisiae Epernay 2 while inulatin fyh-15 als slwed fermentatins inulated with S. erevisiae EClll8 [11]. The rapid grwth f YH-15 in unsulfited grape juie bserved by Huang et al. [11] fit the desriptin f "ferius" latbailli given by Bultn et al. [2]. The rganism ahieved a peak ppulatin f > 19 fu/ml tw days after inulatin. YH-15 was subsequently identified as a nvel speies f Latbaillus while YH-24 and YH-37 were wild types f enus eni [7,8], an rganism frmerly knwn as Leunst ens [5]. Regarding pssible mehanisms fr yeast inhibitin, Bultn et al. [2] indiated that enugh aeti aid uld be prdued by "ferius" latbailli in tw r three days t inhibit yeast metablism. Aeti aid is well knwn t be inhibitry t yeasts [6], influening 1,3,4,~Department f Fd Siene and Human Nutritin, Washingtn State University, Pullman, WA 99163-6376; and 2Cl Climate enlgy/vitiulture Institute, Brk University, St. Catharines, ntari, Canada. *Crrespnding authr [FaxL 59-335-4815; e-mail: <edwards@wsu.edu>]. Aknwledgements: The authrs thank the Washingtn Wine Advisry Bard and the Nrthwest Center fr Small Fruit Researh fr finanial supprt f this prjet, The Hgue Cellars (Prsser, WA) fr dnatin f Chardnnay grape juie, and Lallemand In. (Mntreal, Quebe, Canada) fr the dnatin f seleted yeasts. The authrs wuld als like t thank Dr. T. Henik- Kling (NYSAES, Crnell University, Geneva, NY) fr advie related t HPLC analyses f musts and wines. Manusript submitted fr publiatin 11 May 1998. Cpyright 1999 by the Amerian Siety fr Enlgy and Vitiulture. All rights reserved. 24 Am. J. Enl. Viti., Vl. 5, N. 2, 1999 bth grwth and fermentative abilities [13,19,21]. In agreement, Rasmussen et al. [22] reprted that additin f 4 g/l midway thrugh grape juie fermentatins slwed the fermentatins. Hwever, it is nt knwn whether the amunt f aeti aid prdued by spilage latbailli wuld inhibit yeast grwth and indue slw/ stuk fermentatins t the extent nted by Huang et al. (11) wrking with YH-15. Furthermre, while Huang et al. (11) reprted high amunts f vlatile aidity present in wines inulated with YH-15 (3 g/l), muh lwer levels were present in the slw/stuk fermentatins inulated with YH-24 r YH-37 (.37 and.38 g/ L, respetively). This led t the speulatin that aeti aid may nt be the sle mehanism fr the bateriaindued inhibitin f yeast. The bjetives f this study were t (a) mpare the suseptibility f different strains f wine yeast t aeti aid and t (b) investigate the inhibitry atin f the aid n Saharmyes during vinifiatin. Materials and Methds Media and rganisms: The medium used fr ultivatin f lati aid bateria was mdified Rgsa (MR) brth and agar [1]. Wrt agar was used fr the prpagatin f yeasts [14] and was prepared using diastati diamalt (Premier Malt Cmpany, Grsse Pint, MI). Yeasts were btained frm Lallemand In. (Mntreal, Quebe, Canada) and inluded Saharmyes erevisiae var. erevisiae T73, CY379 (Burgblan), 71B, UCD 522 (Mntrahet), D254, Epernay 2, W~idenswil 27, and V-1116 (K1) and S. erevisiae var. bayanus EClll8 (Prise de Musse), UCD 595 (Champagne), and SB1 in the ative dry frm. Baterial strain YH-15, riginally islated by Huang et al. [11], has been
SLW/STUCK FERMENTATINS- 25 identified as a nvel speies f Latbaillus and prpsed t be named Latbaillus kunkeei [7]. Initial fermentatins with and withut L. kunkeei: Chardnnay grape juie was prepared frm grape juie nentrate btained frm the Califrnia Cnentrate Cmpany (Aamp, CA). Enugh water, gluse and frutse (5:5 w/w), and KHC 3 were added t the nentrate t yield a juie f ph 3.8 and 2.9 Brix. Diammnium phsphate (J. T. Baker In., Phillipsburg, NJ)was added t ahieve a nentratin f.35 g/l. The juies were treated with 3% H22 until the ttal S 2 nentratin was redued apprximately t < 3 ppm as determined by the aeratin-xidatin methd [18]. The juie was stred a3 C prir t use. Thawed juie was sterile-filtered thrugh a Durapre.45-pm filter (Millipre C., Bedfrd, MA) int sterile 3.8-L glass arbys s that eah arby ntained apprximately 2 kg. Pwdered ellulse (Sigmaell TM Type 2, Sigma Chemial C., St. Luis, M) was sterilized at 121 C fr 15 minutes as an aqueus suspensin and was added t the filtered juie at a rate f.1% (w/v) t simulate partiulate matter nrmally present in grape juie t be fermented [14]. S. erevisiae, L. kunkeei, r bth S. erevisiae and L. kunkeei were inulated int the Chardnnay juies. Inulatin fs. erevisiae int the apprpriate arbys lagged tw days behind that f inulatin f L. kunkeei. T prepare inulum, L. kunkeei was inulated int 1 ml MR brth and inubated fr three days at 3 C. After inubatin, the ells were transferred int 5 ml MR brth and inubated fr an additinal three days. Cells were harvested by entrifugatin (5 ~ g fr 2 min), washed with 25 ml sterile phsphate buffer (ph 7,.23 M NaH2P 4 /.3 M Na2HP4.7H2), and suspended in varying amunts f buffer t yield an initial ppulatin in the grape juie f a 15 fu/ml thrugh inulatin f 1 ml f the ell suspensin. The ative-dry frm f S. erevisiae ECl118 was rehydrated in sterile phsphate buffer at 4 C (1.6 g/2 ml buffer). After 15 minutes, 1 ml f the suspensins were aseptially inulated int glass arbys at initial ppulatins f a 15 fu/ml. Fermentatins were arried ut at 21 C in tripliate. Carbys were anaerbially sampled using a C 2 siphn system. During fermentatin, the nentratins f arbhydrates (gluse and frutse), ethanl and rgani aids (aeti, lati, and mali aids) were mnitred daily using HPLC (methd 1, see belw). MIC values f aeti aid: Minimum inhibitry nentratin (MIC) values f aeti aid were determined fr different yeasts using a syntheti mirbilgial medium. Strains were inulated int 1 ml MR brth and inubated 48 hurs at 25 C. Cells were harvested by entrifugatin, washed (2 ), and suspended in phsphate buffer. Cells were inulated int the brth desribed by Phwhinda et al. [2] ntaining nentratins f aeti aid that ranged frm 4. t 8. g/l in inrements f.5 g/l. All media were adjusted t ph 3.8 using 5% H3P 4. After inulatin and inuba- tin at 25 C fr 36 hurs, grwth was evaluated using a KletSummersn phteletri lrimeter (Klett Manufaturing C. In., New Yrk) and defined as grwth (> 3 units) r n grwth (< 3 units). Determinatin f the MIC values was repliated twie. S. erevisiae T73 and V-1116 were grwn and harvested as previusly desribed. The yeasts were inulated t yield ppulatins f 15 fu/ml int the brth f Phwhinda et al. [2] whih ntained 4 g/l aeti aid (ph 3.8). Cultures were inubated at 25 C during whih viability was mnitred using wrt agar as spread plates and a spiral plating system (Autplate 4, Spiral Biteh, Bethesda, MD). Additin f aeti aid t Riesling/Chardnnay fermentatins: Riesling grape juie (ph 3.8 and 17 Brix) was prepared in a similar fashin as the Chardnnay juie by diluting grape juie nentrate btained frm the Califrnia Cnentrate Cmpany (Aamp, CA) using water and gluse/frutse. Inrpratin f diammnium phsphate, redutin in ttal S2, sterile filtratin, and additin f pwdered ellulse t the juie was amplished as previusly desribed. Sterile-filtered slutins f aeti aid were added daily t sme f the fermentatins (1 ml slutin/arby) t yield the same nentratin f aeti aid as that fund in the earlier Chardnnay fermentatins inulated with L. kunkeei. Sterile phsphate buffer was added t ntrl fermentatins (withut added aeti aid) t aunt fr hanges in vlume due t the additin f aeti aid t the ther fermentatins. The ative-dry frm f S. erevisiae ECl118 was prepared as previusly desribed and inulated at a ppulatin f 15 fu/ml, 14 hurs fllwing the first additin f aeti aid. Fermentatins were repliated in tripliate and were sampled 1 t 15 hurs after additin f aeti aid fr HPLC analysis (methd 1, see belw). A send Chardnnayjuie (ph 3.6, 21.5 Brix) was btained frm a mmerial winery. Juie filtratin, ellulse additin, and daily additin f aeti aid were perfrmed as desribed fr the Riesling juie. A ulture f S. erevisiae EClll8 was prepared by inulating an islated lny frm wrt agar int MR brth. After inubatin fr three days at 25 C, ells were harvested by entrifugatin and suspended in phsphate buffer fr inulatin int juies at 15 fu/ml. Fermentatins were sampled 12 hurs after additin f aeti aid fr analysis by HPLC (methd 2, see belw), ttal sluble slids using the methd f Ingledew and Kunkee [12], and yeast viability using wrt agar and the spiral plating system. ttplc analysis. Methd 1: A VaElut 1-tube system equipped with 3-mL CH lumns (Varian Assiates In., Harbr City, CA) was used with 12-mL glass sample reeiver tubes. Clumns were initially generated with ne lumn vlume f methanl fllwed by an equal vlume f deinized water. Reuse was pssible if ne lumn vlume f 1 N HC1 in methanl fllwed by an equal vlume f deinized water were applied prir t generatin. Am. J. Enl. Viti., Vl. 5, N. 2, 1999
~ 26 medwards et al Samples t be analyzed were prepared by adding 1. ml internal standard mixture (5. g/l ribitl + 5. g/l prpini aid) t 2. ml sample/alibratin standard. The samples were vrtexed, added t the CH lumn, and lleted in the reeiver tube. An additinal 3. ml deinized water was added t eah sample tube, vrtexed, and passed thrugh the CH lumn. The pled extrats (6 ml) were vrtexed, pured int 12-mL plasti syringes, and filtered thrugh 2.5-m hydrphili BD disk filters (Millipre C., Bedfrd, MA) int vials fr HPLC analysis. The HPLC system nsisted f a HewletPakard (Ls Angeles, CA) ChemStatin, autsampler (mdel 15), and dide array detetr (mdel 14A) upled t an Is (Linln, NE, USA) single pistn pump (mdel 235) and gradient prgrammer (mdel 236), and a Waters (Franklin, MA) refrative index detetr (mdel R41). Separatin was amplished using tw analytial lumns (HPX-87H, Bi-Rad Labratries, Herules, CA) plumbed in series. Clumn temperature 12 1 8 e~. m " 6 L. r- e - 4 g 2 1 8 6 1 2 3 Fermentatin time (hr) l=frutsel was maintained at 65 C using in a lumn heater. The mbile phase nsisted f.65% H3P 4 maintained at a flw rate f.7 ml/min. Ttal sample vlume injeted was 1 ~tl. Gluse, frutse, aeti aid, mali aid, lati aid, and ethanl were identified using retentin times f knwn standards. Methd 2: Samples were prepared and analyzed as remmended by T. Henik-Kling (persnal mmuniatin, 1997). Samples were filtered thrugh.45-ktm filters (Gelman Nyln Ardis 4, Ann Arbr, MI) prir t diret injetin nt a Rainin/Varian HPLC system (Wburn, MA) nsisting f a pump (Dynamax SD-2), autsampler, UV detetr (Dynamax UV-1) mnitring at 214 nm, and refrative index detetr (Dynamax RI- 1). Separatin was perfrmed using a 4 X 125 mm DS- 5S reverse phase lumn (HewletPakard, Ls Angeles, CA) fllwed by a 1 3 mm HPX-87H lumn (Bi-Rad Labratries, Herules, CA). Clumn temperatures were maintained at 6 C using a lumn heater. The mbile phase nsisted f 1 mn H2S 4 with 2% w/v aetnitrile and maintained at a flw rate f.6 ml/min. Ttal sample vlume injeted was 1 ~tl with quantitatin by peak area using an external standard alibratin. Results and Disussin The ability fl. kunkeei t inhibit alhli fermentatins atalyzed by S. erevisiae is illustrated in Figure 1. The deline in nentratins f gluse and frutse was slwer in fermentatins inulated with L. kunkeei than in ntrl fermentatins withut the rganism. Here, Chardnnay fermentatins inulated with S. erevisiae EClll8 alne beame dry after 2 hurs, while the nentratins f gluse and frutse in juie inulated with S. erevisiae and L. kunkeei were greater than 2 g/l and 4 g/l, respetively, after 25 hurs. Yields f ethanl in fermentatins with S. erevisiae and L. kunkeei were half that f ntrl fer- 12" 1 8 := 6 L_ 8 4 2 4 e~ 2 I i ~ i l 2 3 Fermentatin time (hr) Fig. 1. Deline f gluse and frutse in a Chardnnay juie inulated with (@) Saharmyes erevisiae EC1118, (G) Latbaillus kunkeei YH-15, r (A) S. erevisiae and L. kunkeei. 1 2 3 Fermentatin time (hr) Fig. 2. Prdutin f ethanl during fermentatin f a Chardnnay juie inulated with () Saharmyes erevisiae ECl118 r (A) S. erevisiae and L. kunkeei. Am. J. Enl. Viti., Vl. 5, N. 2, 1999
SLW/STUCK FERMENTATINS m 27 mentatins inulated with yeast alne (Fig. 2), a trend nsistent with redued arbhydrate fermentatin (Fig. 1). The ability f L. kunkeei t slw alhli fermentatins was first bserved by Huang et al. [11]. Huang et al. [11] reprted that slw/stuk fermentatins inulated with L. kunkeei ntained high amunts f vlatile aidity, apprximately 3 g/l. Hwever, it was unlear whether these vlatile aidities were by-prduts f baterial r yeast metablism. While it is well knwn that lati aid bateria prdue aeti aid ([9], S. erevisiae an als prdue the aid during fermentatin depending n many fatrs [3,4,1,16,17,22,23]. Grwth f S. erevisiae alne in the Chardnnay grape juie yielded aeti aid in nentratins nt exeeding.5 g/l (Fig. 3), an amunt well within the nrmal range fund in wines [9]. Hwever, nentra- tins f aeti aid in juies inulated with L. kunkeei r with bth S. erevisiae and L. kunkeei apprahed 5 g/l, a nentratin in exess f legal limits [25]. Thus, the high vlatile aidities present in wines inulated with L. kunkeei bserved by Huang et al. [11] were prbably a result f baterial metablism rather than being synthesized by yeast. In additin t aeti aid, L. kunkeei prdued lati aid and metablized mali aid (Fig. 3), in agreement with knwn harateristis f this rganism [8]. Rasmussen et al. [22] suggested that ne ausative agent fr slw/stuk alhli fermentatins was prdutin f aeti aid by ther mirrganisms. In fat, Phwhinda et al. [2] estimated that a nentratin f 1 g/l was required fr mplete inhibitin f S. l 9-1 8- A _1 E I.I. 1 7- e~ im, 1-1 6-8 ~- 6 4!._. dk~ 2 Lati aid...!i i t 5 2 4 6 8 1 9 18 _1 il_ 17 Mali aid ~!i,== 1 6 1! L-, 1 2 3 Fermentatin time (hr) Fig. 3. Changes in nentratin f aeti, lati, and mali aids in a Chardnnay juie inulated with () Saharmyes erevisiae EC1118, ([3) Latbaillus kunkeei YH-15, r (A) S. erevisiae and L. kunkeel t5 2 4 6 8 Time (hr) Fig. 4. Grwth f Saharmyes erevisiae V l 116 and T73 in a mirbilgial medium withut () r with (121) 4 g/l aeti aid added. Am. J. Enl. Viti., Vl. 5, N. 2, 1999
~ 28 --EDWARDS et al. Table 1. MIC values f aeti aid in a syntheti medium (ph 3.8) against different yeasts. Yeast MIC value (g aeti aid/l) S. erevisiae var. erevisiae T73 7.5 CY 379 7.5 71B 7. - 725 UCD 522 7. - 7.5 D254 6.5-7. Epernay 2 6.5-7. W&denswil 27 6. - 6.5 V 1116 (kl) 5.5-6. S. erevisiae var. bayanus EC 1118 5.5-6. UCD 595 5. - 5.5 SB1 4.5-5. Lureir [19] mmented that yeast fermentatins may be nsiderably slwed by aeti aid during the latter stages f fermentatin when the nentratin f ethanl is high. ne study that did use grape juie was that f Rasmussen et al. [22] wh bserved prgressively slwer fermentatins by additin f 1 t 4 g/l aeti aid midway thrugh fermentatin f Chardnnay and Merlt musts. Unfrtunately, a "ne-time" additin f aeti aid des nt neessarily simulate baterial prdutin f the aid during vinifiatin. As an example, L. kunkeei required apprximately 2 hurs in rder t prdue nentratins f 4 t 5 g/l in a Chardnnay juie (Fig. 3). 9 erevisiae. While Pampulha and Lureir [19] nted that the fermentatin rate f ne strain f S. erevisiae drpped t 1% at a nentratin f 6 g/l aeti aid (ph 3.5), Mairella et al. [15] fund that a nentratin f 7.5 g/l resulted in an 8% redutin in ell density f S. erevisiae var. anamensis. The inhibitin f yeast by aeti aid als depends n the presene f ther inhibitry mpunds suh as ethanl [13,19,21]. In rder t mpare sensitivities f different yeast strains t the aid, minimal inhibitry nentratin (MIC) values were determined. Table 1 illustrates that the strains differed in their tleranes t aeti aid with T73, CY379, 71B, and UCD 522 being mre tlerant evidened by higher MIC values (7 t 7.5 g/l) than EClll8, V-1116, UCD 595, and SB1 (MIC = 4.5 t 6. g/ L). With the exeptin f SB1, these MIC values were higher than the amunt f aeti aid prdued by L. kunkeei grwn in grape juie (Fig. 3). Yeasts with lw (V-1116) and high (T73) MIC values were als inulated int a medium ntaining 4 g/l aeti aid. As shwn in Figure 4, bth strains exhibited lnger lag times in the presene f the aid, with a smewhat lnger time fr V-1116. Hwever, V-1116 and T73 ahieved similar peak ppulatins f > 17 fu/ml in the presene r absene f aeti aid. Data presented in Figure 4 and Table 1 demnstrate that mst wine yeasts an grw in the presene f 3 t 5 g/l aeti aid, nentratins similar t the amunt f vlatile aidity fund in sme slw/sluggish fermentatins by Huang et al. [11] and the amunt f aeti aid prdued by L. kunkeei (Fig. 3). Beause data presented in Figure 4 and Table 1 were determined using syntheti media, it remained unknwn as t whether additin f aeti aid at these nentratins uld slw alhli fermentatins f grape musts. Mst studies regarding aeti aid inhibitin f yeast have relied n syntheti media ntaining lwer nentratins f fermentable arbhydrates than grape juie and wuld, therefre, have lwer amunts f ethanl present [13,19,21]. In supprt, Pampulha and L. 6.,- 45 ~ L. 3 e,, (9 15 t 9 t._ 6 = 45 L. = 3 (9 t 15 5 1 15 2 25 I... W i thaeti:ai d...!!! 5 1 15 2 25 Fermentatin time (hr) v ".,,,. 3= (9 N~ 2.m L_.i-i (9 1 " t Fig. 5. Deline f gluse (e) and frutse () in Riesling fermentatins inulated with Saharmyes erevisiae ECl118 withut r with inreasing amunts f aeti aid (CI) added t the fermentatins. Am. J. Enl. Viti., Vl. 5, N. 2, 1999
SLW/STUCK FERMENTATINS- 29 T better evaluate the influene f baterial-synthesized aeti aid during fermentatin, the aid was sequentially added t a Riesling fermentatin at a rate similar t that prdued by L. kunkeei when grwn in a Chardnnay grape juie (Fig. 3). Firstly, gluse was utilized faster than frutse by the yeast in all fermentatins (Fig. 5), a arbhydrate preferene that is well knwn [2]. Sendly, the nentratins f gluse and frutse were <2 g/l 125 hurs after inulatin in fermentatins withut aeti aid. Hwever, fermentatins with aeti aid required a lnger time t ahieve the same nentratin, apprximately 1 hurs and 1 hurs fr gluse and frutse, respetively. These bservatins are in agreement with Rasmussen et al. [22] regarding the slwing f fermentatins in the presene f aeti aid. The Riesling juie used fr these fermentatins ntained 16 g/l gluse + frutse (Fig. 5), an amunt that was lwer than the 2 g/l in the Chardnnay juie in whih aeti aid prdutin by L. kunkeei was initially evaluated (Fig. 3). Thus, fermentatin was indued in a send Chardnnay juie t determine whether exessive nentratins f aeti aid an slw the fermentatin f a juie ntaining a higher amunt f fermentable arbhydrate (22 g/l). Similar t the Riesling fermentatin, the fermentatin rate was slwed thrugh additin f aeti aid as evidened by the delines in nentratins f ttal sluble slids (Fig. 6) and gluse and frutse (Fig. 7). Hwever, unlike the Riesling fermentatins, the Chardnnay fermentatins ntained apprximately 8 g/l residual frutse 9 hurs after inulatin and did nt mplete fermentatin (Fig. 7). Thus, the presene f very high nentratins f aeti aid, 4 t 5 g/l, an yield a slw r stuk alhli fermentatin. Even thugh the nentratins f aeti aid added t the Riesling and Chardnnay fermentatins were far abve the legal limits permitted in wine, the presene f the aid at these levels des nt fully ex- 21 18 15 ~ 12 "13 9 6 3 i - i i i 4 8 12 16 Fermentatin time (hr) Fig. 6. Deline f sluble slids in Chardnnay fermentatins inulated with Saharmyes erevisiae EC1118 withut () r with (13) inreasing amunts f aeti aid added t the fermentatins. v 125 1 5 u 25 u '}!._. e- C 125 1 5 25 I WRhut aet!~aid!,, 2 4 6 8 1., r6 [ Wit. haetiaid!,!. 2 4 6 8 1 Fermentatin time (hr) 1 "D =m 4= (9 U 3 t._ (9 2 Fig. 7. Deline f gluse () and frutse ( ) in Riesling fermentatins inulated with Saharmyes erevisiae ECl118 withut r with inreasing amunts f aeti aid (13) added t the fermentatins. plain the inhibitin f yeast bserved by Huang et al. (11). In their study, Huang et al. [11] nted that S. erevisiae did nt enter lgarithmi grwth in a Chardnnay juie when L. kunkeei was inulated tw days befre the yeast. If aeti aid prdutin by L. kunkeei was the sle mehanism fr yeast inhibitin, the viability f S. erevisiae in a must with added aeti aid shuld be similar t that reprted by Huang et al. [11]. In fat, the ppulatins f yeast were nt different in fermentatins with r withut the aid (Fig. 8). Here, yeast entered lgarithmi grwth and ahieved peak ppulatins in exess f 17 fu/ml. As suh, the primary influene f the aid appears t be t redue fermentative ability rather than ell viability. This finding was in ntrast t the inhibitin f yeast grwth by L. kunkeei as reprted by Huang et al. [11]. Am. J. Enl. Viti., Vl. 5, N. 2, 1999
~ 21 --EDWARDS et al.._i LI.. v. m m. m,,q = 1 8 "1 2 4 6 8 I 12 Fermentatin time (hr) Fig. 8. Yeast viability in Chardnnay fermentatin inulated with Saharmyes erevisiae ECl118 withut () r with (Q) inreasing amunts f aeti aid added t the fermentatins. Cnlusins Minimum inhibitry nentratins f aeti aid against S. erevisiae ranged frm 4.5 t 7.5 g/l in a syntheti medium. Riesling and Chardnnay juie fermentatins t whih aeti aid was added at the same rate prdued by L. kunkeei all slwed. While the Riesling fermentatins eventually beame dry, the Chardnnay fermentatins with aeti aid ntained higher amunts f ttal sluble slids and frutse than ntrl fermentatins. Yeast grwth in fermentatins withut r with aeti aid were similar and ppulatins exeeded 1 7 fu/ml. This was in ntrast t the previusly bserved antagnism f yeast by L. kunkeei where S. erevisiae did nt enter lgarithmi grwth. These results imply that aeti aid may be partially invlved in yeast inhibitin by L. kunkeei but that additinal inhibitry mehanisms are prbable. Given these bservatins, additinal researh is required t eluidate the mehanism f inhibitin f yeast by lati aid bateria inluding investigatin f any synergism between aeti aid and ther yeast r baterial metablites suh as ethanl and/r lati aid. Literature Cited 1. Beelman, R. B. Develpment and utilizatin f starter ultures t indue mallati fermentatin in red table wines. In: Preedings frm the University f Califrnia, Davis, Grape and Wine Centennial Sympsium. pp 19-117 (1982). 2. Bultn, R. B., V. L. Singletn, et al Priniples and Praties f Winemaking. 64 pp. Chapman and Hall, New Yrk (1996). 3. Delfini, C., and F. Cervetti. Metabli and tehnlgial fatrs affeting aeti aid prdutin by yeasts during alhli fermentatin. Viti. Enl. Si. 46:142-15 (1991). 4. Delfini, C., and A. Csta. Effets f the grape must lees and insluble materials n the alhli fermentatin rate and the prdutin f aeti aid, pyruvi aid, and aetaldehyde. Am. J. Enl. Viti. 44:86-92 (1993). 5. Diks, L. M. T., F. Dellagli, and M. D. Cllins. Prpsal t relassify Leunst ens as enus eni [rrig.] gen. nv., mb. nv. Inter. J. Syst. Bateril. 45:395-397 (1995). 6. Dres, S. rgani aids. In: Antimirbials in Fds. pp 95-136. Marel Dekker, In., New Yrk (1993). 7. Edwards, C. G., K. M. Haag, and M. D. Cllins. Identifiatin f sme lati aid bateria assiated with sluggish/stuk fermentatins. Am. J. Enl. Viti. 49:445-448 (1998). 8. Edwards, C. G., K. M. Haag, et al Latbaillus kunkeei sp. nv., a spilage rganism assiated with grape juie fermentatins. J. Appl. Mirbil. 84:698-72 (1998). 9. Fugelsang, K. C. Wine Mirbilgy. 245 pp. Chapman and Hall, New Yrk (1997). 1. Giudii, P., and C. Zambnelli. Bimetri and geneti study n aeti aid prdutin fr breeding f wine yeast. Am. J. Enl. Viti. 43:37-373 (1992). 11. Huang, Y.-C., C. G. Edwards, et al Relatinship between sluggish fermentatins and antagnism f yeast by lati aid bateria. Am. J. Enl. Viti. 47:1-1 (1996). 12. Ingledew, W. M., and R. E. Kunkee. Fatrs influening sluggish fermentatin f grape juie. Am. J. Enl. Viti. 36:65-76 (1985). 13. Kalathens, P., J. P. Sutherland, and T. A. Rberts. Resistane f sme wine spilage yeasts t mbinatins f ethanl and aids present in wine. J. Appl. Bateril. 78:245-25 (1995). 14. King, S. W., and R. B. Beelman. Metabli interatins between Saharmyes erevisiae and Leunst ens in a mdel grape juie/wine system. Am. J. Enl. Viti. 37:53-6 (1986). 15. Mairella, B., H. W. Blanh, and C. R. Wilke. By-prdut inhibitin effets n ethanli fermentatin by Saharmyes erevisiae. Biteh. Bieng. 25:13-121 (1983). 16. Mill&n, C., and J. M. rtega. Prdutin f ethanl, aetaldehyde, and aeti aid in wine by varius yeast raes: rle f alhl and aldehyde dehydrgenase Am. J. Enl. Viti. 39:17-112 (1988). 17. Mrun, E. G., C. Delfini, et al Fatrs affeting aeti aid prdutin by yeasts in strngly larified grape musts. Mirbis. 74:249-256 (1993). 18. ugh, C. S., and M. A. Amerine. Methds fr Analysis f Musts and Wines. 377 pp. Jhn Wiley and Sns, New Yrk (1988). 19. Pampulha, M. E., and V. Lureir. Interatin f the effets f aeti aid and ethanl n inhibitin f fermentatin in Saharmyes erevisiae. Biteh. Lett. 11:269-274 (1989). 2. Phwhinda,., M. L. D61ia-Dupuy, and P. Strehaian. Effets f aeti aid n grwth and fermentative ativity f Saharmyes erevisiae. Bitehnl. Lett. 17:237-242 (1995). 21. Rams, M. T., and A. Madeira-Lpes. Effets f aeti aid n the temperature prfile f ethanl tlerane in Saharmyes erevisiae. Biteh. Lett. 12:229-234 (199). 22. Rasmussen, J. E., E. Shultz, et al Aeti aid as a ausative agent in prduing stuk fermentatins. Am. J. Enl. Viti. 46:278-28 (1995). 23. Shimazu, Y., and M. Watanabe. Effets f yeast strains and envirnmental nditins n frmatin f rgani aids in must during fermentatin. J. Ferm. Tehnl. 59:27-32 (1981). 24. Trmp, A. The effet f yeast strain, grape slids, nitrgen and temperature n fermentatin rate and wine quality. S. Afr. J. Enl. Viti. 5:1-6 (1984). 25. Zeklein, B. W., K. C. Fugelsang, et al Wine Analysis and Prdutin. 621 pp. Chapman and Hall Publishers, New Yrk (1995). Am. J. Enl. Viti., Vl. 5, N. 2, 1999