Nitrgen Supplementatin f Grape Juie.. ffet n Amin Aid Utilizatin During Fermentatin F. FRRRA MNTR ~t and LNDA F. BSSN 2. The effet f supplementatin f grape juie with diammnium phsphate (DAP) and arginine n nsumptin f amin aids during grape juie fermentatin was examined in tw mmerial yeast strains, UCD 522 (Mntrahet) and Prise de Musse. Bth strains displayed similar patterns f amin aid uptake during fermentatin and seemed t be affeted similarly by nitrgen supplementatin. Nitrgen supplementatin inreased fermentatin rate f bth strains, and had a signifiant effet n bimass prdutin fr Prise de Musse but nt fr UCD 522. The rrelatin between juie nitrgen ntent and fermentatin rate was strnger in UCD 522 as mpared t Prise de Musse. Supplementatin with lw levels f DAP (.23 g/l, the legal allwable limit fr nitrgen supplementatin) had very little impat n amin aid utilizatin in this juie. Very high levels f DAP (2. g/l) greatly dereased bth the nsumptin and degradatin f arginine prvided in the medium. The presene f the plyamines putresine, spermine, and spermidine dereased utilizatin and degradatin f arginine signifiantly, but less strngly. The mbinatin f plyamines and DAP at high nentratin did nt mpletely prevent degradatin f arginine. KY WRDS: yeast, amin aids, nitrgen The nitrgen ntent f grape juie affets bth yeast ell grwth and fermentatin rate (1,9,19). Grape juie ntains a mplex mixture f amin aids and ther nitrgen sures, the nentratin f whih is influened by varietal and envirnmental fatrs (1,11,12). The utilizatin f individual nitrgen sures by Saharmyes has been investigated in detail (reviewed in 4); hwever, relatively little has been dne n the utilizatin f mplex mixtures f nitrgen mpunds suh as thse fund in the natural envirnment, grape juie. xtensive studies have appeared n the utilizatin f amin aids in wrt (reviewed in 25). Nitrgen-ntaining mpunds were lassified int fur distint grups in wrt, depending upn the time f disappearane during fermentatin (25). Castr (3) fund in examining 14 amin aids in grape juie, that the bulk f the amin aids were nsumed early in grape juie fermentatin, prir t mpletin f ell grwth phase. Utilizatin f a given amin aid may be affeted by the presene f ther nitrgen mpunds in the medium due t mpetitin fr uptake (5), trans-inhibitin f uptake (5), r nitrgen repressin (4). thanl inhibits amin aid transprt, with as little as 5% dereasing the ativity f the general amin aid permease by as muh as 8%, depending upn the mpsitin f the plasma membrane (2,6). Wdward and Cirill (29) 1Graduate Student and 2Assiate Prfessr, Department f Vitiulture and nlgy, University f Califrnia, Davis, CA 95616-7849. *Authr t whm rrespndene shuld be addressed. tpresent address: Department f Bilgial Sienes, Stanfrd University, Stanfrd, CA 9435. Aknwledgements: This researh was funded by a grant frm the Amerian Vineyard Fundatin. F. F. Mnteir was the reipient f Fulbright-Hays and Wine Spetatr Shlarships. We thank these generus dnrs fr these funds. The assistane f David M. Cns in figure preparatin is greatly appreiated. This researh was nduted at the University f Califrnia, Davis, CA. Manusript submitted fr publiatin 22 January 1991. Cpyright 1992 by the Amerian Siety fr nlgy and Vitiulture. All rights reserved. demnstrated that under lw nitrgen nditins, suh as present in the typial grape juie, the general amin aid permease wuld be in peratin. Thus, yeast ells may nt be apable f effiient amin aid uptake thrughut grape juie fermentatin. Nitrgen supplementatin may als affet the pattern f amin aid utilizatin. Ammnium is a preferred yeast nitrgen sure, and when plentiful, represses the expressin f atabli pathways fr the degradatin f ther nitrgen-ntaining mpunds (4). The nitrgen-rih mpunds knwn as the plyamines, putresine, spermine, and spermidine, are imprtant fr ell grwth and divisin, and their levels are high in all grwing ells (8,). The preursr fplyamine bisynthesis is rnithine, whih is derived frm arginine via arginase ativity. The pathway fr plyamine bisynthesis is regulated by the level f available plyamines (7,). Thus, the need fr plyamines uld drive the degradatin f arginine irrespetive f the need fr arginine as a nitrgen sure fr general bisynthesis. n this situatin, urea (the ther prdut f the arginase reatin) wuld beme an end prdut rather than an intermediate and pssibly be released frm the ells. Urea ntent f wine is f partiular imprtane, as this mpund is a preursr f ethyl arbamate (18,,21,22) and is a suspeted aringen (14). The redutin r eliminatin f its prdutin wuld be desirable. Radilabel traer experiments revealed that a majr sure f urea during vinifiatin fermentatins is the degradatin f arginine (18), an amin aid ften fund in high nentratins in grape juie (11,12). Reent wrk with a labratry yeast demnstrated that disruptin f the CAR1 gene, whih endes the enzyme arginase, essentially eliminated detetable urea prdutin by this rganism under the grwth nditins tested (27). This finding nfirms the entral rle f arginine metablism in the generatin f urea during Am. J. nl. Viti., Vl. 43, N. 1,1992
2~ MNTR and BSSN fermentative grwth. Disruptin f the CAR1 genes in a wine yeast might als prevent that yeast frm prduing urea. Hwever, arginine is relatively abundant in grape juie and is generally the majr yeast nitrgen sure, and industrial fermentatins are nt nduted under sterile nditins. The arginine wuld still be available fr degradatin by ther mirrganisms present during the fermentatin r in the finished wine, leading t ptential dramati mirbial stability prblems. Als, a strain unable t utilize the majr nitrgen sure might nt be able t mpete suessfully with ther wild strains f Saharmyes present in the juie and, thus, nt be able t dminate the fermentatin. Hwever, ells prvided with high levels f ammnium in as well as plyamines might nt need t degrade arginine. We undertk these studies t investigate the effet f nitrgen additin n utilizatin f amin aids in tw yeast strains, UCD 522 (als knwn as Mntrahet) and Prise de Musse, previusly shwn t differ dramatially in ability t prdue and/r release urea at the end f fermentatin (23,24). The impat f plyamine additin n utilizatin f arginine was als investigated. n a mpanin paper, we reprt the impat f supplementatin n release f amin aids and urea at the end f fermentatin. Materials and Mehtds Reagents: All hemials used were reagent grade. L-arginine (guanid-14c, 54.4 mcuries/mml) was btained frm New ngland Nulear (Dupnt, Wilmingtn, D). Medium mpnents were frm Dif (as supplied by Fisher Sientifi, Pittsburgh, PA). The plyamines were btained frm Sigma Chemial C. (St. Luis, M). Yeast strains and grwth nditins: UCD 522 was btained frm the ulture lletin f the Department f Vitiulture and nlgy; mmerial preparatins f this yeast were btained frm the Universal Fds Crpratin (Milwaukee, W). Prise de Musse was btained as a mmerial preparatin frm the Universal Fds Crpratin. Fr the studies n the effets fplyamine additin, the yeasts were grwn in YPD (yeast extrat, g/l; Bat-peptne, g/l; gluse, g/l) with agitatin n a rller drum t early statinary phase (24 hurs at 3 C). Fr inulatin,.2% (v/v) f these ultures were added t the must with the bjetive f starting the fermentatin with apprximately 1 5 viable ells/ ml. Viable ells per ml f inulated juie was determined by plating diluted samples pst-inulatin nt YPD plates t alulate lny frming units. n all ases, viable ell unts mathed thse predited frm the inulatin vlume. UCD 522 was the yeast strain utilized in these studies. Fr the experiments n the effet f diammnium phsphate and arginine supplementatin n amin aid utilizatin in Chenin blan,.25 g/l f dry ativated yeast was rehydrated arding t manufaturers instrutins, in ml deinized water fr 15 minutes at 35 C fr UCD 522 and at 4 C fr Prise de Musse. Grape juie fermentatins: Fr the mparisn f the effets f plyamine additin, a pink juie nentrate btained frm the & J Gall Winery was used. The amin aid mpsitin f this juie has been previusly reprted (18). Dupliate fermentatins were run at 3 C using 15-mL vlumes in test tubes, plaed within a glass ntainer nneted t traps t mnitr the release f labeled C 2 as previusly desribed (18). Spermine, spermidine, and putresine were all added, eah t a final nentratin f.1 mm. The fermentatins that were ammnium-supplemented reeived 2 g/ L f diammnium phsphate (an additin f 3.3 mm ammnium, fr a ttal f 35 mm ammnium in the juie). Chenin blan juie frm the 1988 harvest f the Davis Tyree Vineyard was used in the experiments mparing the effet f diammnium phsphate and arginine supplementatin. Tw liters fjuie were plaed in sterile 1-galln glass bttles, whih were immersed in a irulating water bath held at C. Degrees Brix were mnitred using hydrmetry and refratmetry. A standard urve f Brix versus refratmetry reading was generated fr eah juie. The refratmetry t Brix nversin was highly reprduible ver the range used in these studies. The -ml samples used fr the Brix measurements were nt returned t the samples, t prevent aeratin and rss-ntaminatin f the fermentatin. The juies were supplemented as given in the text. Yeast ell bimass was determined by absrbane at 58 nm. Absrbane values were rreted fr the initial absrbane reading btained fr juie. n rder t btain absrbane values in the linear range, sme samples required dilutin. n these ases, similarly diluted juie, after being filtered thrugh a.2 ~ filter, served as the blank value t be subtrated t btain a true absrbane reading. The white juies used did nt absrb signifiantly at 58 nm. Amin aid analysis: The amin aid analytial methds were desribed previusly (16). Tw amin aids (prline and ysteine) uld nt be analyzed, and sine tryptphan smetimes -eluted with rnithine, values f these tw mpunds als are nt reprted. Utilizatin f radiatively labeled arginine: n the ase f plyamine additin, degradatin f arginine was mnitred using radiatively labeled substrate as previusly desribed (18). Rate f fermentatin: The time between 5 t Brix fermented was averaged fr the repliate samples t be used in the statistial mparisn f fermentatin rates. These values fr fermentatin rate are independent f the length f the lag phase prir t the nset f fermentatin and f any variatin in the number f viable ells in the inulum. This regin f the Brix versus time urves displayed the steepest slpe, and is indiative f the maximal sugar utilizatin rate. The lines btained were defined by at least fur pints in all ases. T further analyze fatrs affeting fermentatin
NTRGN SUPPLMNTATN.. m 3 Table 1. Nitrgen mpund mpsitin f unsupplemented Chenin blan juie. Amin Aid Cnentratin (#mles/l) Aspartate 415 Threnine 382 Serine 332 Asparagine 7 Glutamate 63 Glutamine 212 Glyine 62 Alanine 561 Citrulline Valine 144 Methinine 2 sleuine 42 Leuine 28 Tyrsine 9 GABA a 642 Ammnium 437 Phenylalanine 122 Histidine 139 Lysine Arginine a GABA = y-aminbutyri aid. nd = nt deteted. nd and time t mpletin f fermentatin (Fig. 1). The juie reeiving bth DAP and arginine displayed the fastest fermentatin rate (Fig. 2) and shrtest time t mpletin f fermentatin. The juie reeiving arginine supplementatin nly fermented faster than the ther tw juies. The juie reeiving DAP supplementatin shwed the lngest time t mpletin f fermentatin. The r 2 value fr the rrelatin between ammnium equivalents and average rate f fermentatin was.785 fr UCD 522. The average fermentatin rates frm 5 Brix t Brix fermented were mpared using the Fisher's least signifiant differene (FLSD) test (Fig. 2) (StatView Statistial Prgram). The rates fr the ntrl and the DAP-supplemented juie were nt signifiantly different frm eah ther, but were signifiantly different frm the tw reeiving arginine, whih were als signifiantly different frm eah ther. Prise de Musse als displayed the fastest rate f fermentatin in the sample reeiving bth DAP and arginine, the ther three fermentatins being muh mre similar t eah ther than in the ase f UCD 522 (Fig. 1). The r 2 value fr the rrelatin between ammnium equivalents and fermentatin rate fr Prise de Musse was.496. Cmparisn f fermentatin rate frm 5 Brix t Brix fermented, revealed a small but rate and ell yield in different juies, the results f these studies n DAP and arginine supplementatin f Chenin blan were integrated with the results f several ther experiments using the pink juie nentrate (16,17,18), Sauvignn blan (16) and ther Chenin blan juies (Mnteir and Bissn, unpublished data) fr statistial analysis. Results and Disussin ffet f nitrgen supplementatin n fermentatin rates: The base Chenin blan juie initially had 1.2 mmle/l f arginine (Table 1), whih was nsidered t be defiient; arginine was added t raise this value t 2. mmle/ L. The arginine-supplemented Chenin blan juie used in these studies had a ttal nitrgen ntent f 6.3 mmle/l, with a ttal f 12.9 mmle/l f nitrgen equivalents. This juie was further supplemented with diammnium phsphate (.23 g/l; 3.5 mml/l f nitrgen equivalents), arginine (1.5 g/l; 34.4 mml/l nitrgen equivalents) r arginine plus diammnium phsphate (DAP) (a ttal f 37.9 mml/l f nitrgen equivalents). The ttal nitrgen ntent sf the fur juies in terms f nitrgen equivalents were: ntrl, 12.9 mm; DAP, 16.4 mm; arginine, 47.3 mm; DAP plus arginine, 5.8 mm. Tripliate fermentatins were run using tw mmerial yeasts, UCD 522 (Mntrahet) and Prise de Musse. n the ase f UCD 522, there was a strng rrelatin between amunt f nitrgen supplied and the fermentatin rate Mntrahet 5 15 25 Prise de Musse Time (hurs) 5 15 25 Time (hurs) v "~ Cntrl ~. DAP Arginine DAP + Arginine G, Cntrl DAP ":" Arginine DAP + Arginine Fig. 1. Fermentatin prfiles f tw yeast strains, UCD 522 (Mntrahet) and Prise de Musse, in Chenin blan juie with and withut nitrgen supplementatin. Pints shwn represent the average f the determinatins + standard errr.
4 m MNTR and BSSN.,. x.m L { L ---.m --.. { {{ - L C. L_.Q. 1!! < z z < z z z ~ ~ z ~ ~ ~ ~ + + Mntrahet Prise de Musse Fig. 2. Fisher's least signifiant differene analysis f fermentatin rates frm 5 Brix t Brix fermented as a funtin f yeast strain and nutrient supplementatin. Pints shwn represent the average f the determinatins + FLSD. signifiant differene between the sample reeiving arginine and the ntrl and DAP-supplemented juies (Fig. 2). The DAP plus arginine juie fermentatin rate was signifiantly different and muh faster than the ther treatments. n mparing Prise de Musse t UCD 522, UCD 522 fermented faster in the arginine supplemented juies, but there was n signifiant differene in rate mparing the ntrl and DAP nly juies between the tw strains. quatins based upn these experiments desribing the effet f milliequivalents f nitrgen n fermentatin rate were determined fr bth strains. Fr UCD 522, y (fermentatin rate frm 5 t Brix fermented in Brix/h) -.2178x (N milliequivalents) +.8234 (r 2 -.785, p =.1), and fr Prise de Musse, y =.144x +.9167 (r 2 -.496,p =.34). The differenes in the slpes suggest a muh strnger effet f nitrgen additin n fermentatin rate in UCD 522 than in Prise de Musse under these nditins. At a nitrgen value f 12.55 milliequivalents, the tw strains wuld be predited t have idential fermentatin rates. n juies with a nitrgen milliequivalents value belw 12, Prise de Musse wuld be expeted t have a faster fermentatin rate than UCD 522; at values abve 12, UCD 522 wuld be expeted t ferment mre quikly than Prise de Musse until nitrgen saturatin r maximal fermentatin rate was ahieved. n additin t ammnium, DAP als prvides phsphate; hwever, this juie was used in ther nutrient additin trials where it was determined nt t be phs-!! d G. L~J LU < z z z G ~ n," n," Mntrahet < 121! [.~ _ LJ W < z z < < Prise de Musse Fig. 3. Fisher's least signifiant differene analysis f final absrbane as a funtin f nitrgen supplementatin and yeast strain. Pints shwn represent the average + FLSD. phate-defiient. Mrever, in mparing the samples reeiving DAP nly, there was n stimulatin ffermentatin rate, again indiating that phsphate has n stimulatry effet under these nditins. Nitrgen supplementatin an als stimulate bimass prdutin, whih may affet fermentatin rate simply beause mre ells were available t nsume sugar. The final absrbane (58 nm) f eah fermentatin, a parameter whih is diretly rrelated with ellular bimass, was determined and analyzed using the FLSD test (Fig. 3). n the ase f UCD 522, nitrgen appeared t have a minimal effet n ell yield, with n signifiant differene in absrbane apparent between the ntrl and the tw arginine-supplemented treatments. The samples reeiving DAP nly displayed lwer final absrbanies. n ntrast, the higher nitrgen ntent f the tw juies supplemented with arginine yielded signifiantly higher absrbanies as mpared t the ntrl and the DAP-nly juies fr the Prise de Musse fermentatins. Thus, in this experiment, nitrgen additin in Prise de Musse appears t inrease bth fermentatin rate and bimass prdutin, while nly stimulating fermentatin rate signifiantly in UCD 522. Am. J. nl. Viti., Vl. 43, N. 1,1992
NTRGN SUPPLMNTATN..- 5 8 6 6 8 ~ 4 Fig. 4. Utilizatin f amin aids in UCD 522 (Mntrahet) with and withut supplementatin. Panel A: ntrl juie; Panel B: juie with DAP supplementatin; Panel C: juie with arginine supplementatin; Panel D:juie with bth DAP and arginine supplementatin. Amin aids: aspartate ( ), threnine (Q), asparagine (13), glutamate (ll), glutamine (A), glyine (,&), alanine (+), valine (X), methinine (), leuine (41,). 2 6 r 6 ~ 4 ~ = ~ 1 = A B. l -= 3 3 - t- -= ~ 4 Q,. v t.- 8 313 t- ( 3 - Fig. 5. Utilizatin f nitrgen mpunds in UCD 522 (Mntrahet) with and withut supplementatin. Panels are as given in the legend t Figure 4. Nitrgen mpunds: serine (), itrulline (), isleuine (Zl), tyrsine (ll), phenylalanine (A), histidine (A), lysine (+). ~ 13 ~ C Z " ~!
6- MNTR and BSSN = p. 6 4 6 / ~'-" < =_ 6 6 "~ C Fig. 6. Utilizatin f amin aids in Prise de Musse with and withut supplementatin. Panel A: ntrl juie; Panel B: juie with DAP supplementatin; Panel C:juie with arginine supplementatin; Panel D: juie with bth DAP and arginine supplementatin. Amin aids: aspartate (C)), threnine (e), asparagine (C), glutamate (B), glutamine (A), glyi ne ( ), alani ne ( + ), vali ne (X), methini ne ( ), leuine (4,). "P "~ l 3 3 Fig. 7. Utilizatin f nitrgen mpunds in Prise de Musse with and withut supplementatin. Panels are as given in the legend t Figure 4. Nitrgen mpunds: serine ( ), itrulline (e), isleuine ([3), tyrsine (B), phenylalanine (A), histidine ( ), lysine (+). ". )" ~- "~" ~" 3.-- Z._ ~ 3 8 ~ 1 ' 3 -~ ~ 8 = p. ~ 2,-- J/ " Z ~
. _ NTRGN SUPPLMNTATN.. m 7 18 " 36 -._ 2 " 1... 6 8 " Fig. 8. Utilizatin f ammnium, arginine and y- aminbutyri aid in UCD 522 (Mntrahet) as a funtin f nitrgen supplementatin. Panel A: ntrl juie; Panel B: juie with DAP supplementatin; Panel C: juie with arginine supplementatin; Panel D: juie with bth DAP and arginine supplementatin. Arginine (Q), NH 4 (), y- aminbutryi aid (). 33.C_. t e, ~_~. 1, v, v - - ' ~ v ' 8 ' 18 3 ~~ 2 41 ~- ~ 6 g Z w! v.'!v - l 1211 v v 1" u ~ ~v =.d! w v 2 Fig. 9. Utilizatin f ammnium, arginine and y- aminbutyri aid in Prise de Musse as a funtin f nitrgen supplementatin. Panel A: ntrl juie; Panel B:juie with DAP supplementatin; Panel C: juie with arginine supplementatin; Panel D: juie with bth DAP and arginine supplementatin. Arginine (C), NH 4 (), 7-aminbutyri aid (). " 8 8 6 1. ~ z l? -%. v. v v v, r-" v 1;
8- MNTR and BSSN ffet f supplementatin n utilizatin f amin aids: The effet f nitrgen supplementatin n the pattern f use f amin aids during the Chenin blan fermentatins was evaluated. n general, amin aids are nsumed quite quikly during grape juie fermentatin, disappearing well befre the terminatin f ell divisin (3,15). We had previusly shwn that the bulk f the amin aids are nsumed during the first few degree Brix drp (16), in agreement with the early wrk f Castr (3). t has been shwn in labratry strains under labratry nditins that amin aids and ther nitrgen sures mpete fr entry int the yeast ells (4,5) and that ammnium an redue the uptake f amin aids. t was, therefre, f interest t determine if supplementatin with arginine r ammnium wuld alter the pattern r timing f utilizatin f amin aids. There was very little differene in the pattern f utilizatin f nitrgen mpunds in the ntrl and DAP supplemented juies with UCD 522 (Fig. 4 and 5) r with Prise de Musse (Fig. 6 and 7). n the arginine-supplemented juies, initial uptake was still quite rapid; hwever, sme amin aids (glyine, alanine, and glutamine) either started being released very early (befre Brix had been fermented), perhaps as a nsequene f early ell lysis, r were nt mpletely nsumed, r bth. The biggest differenes bserved were in the amunts f amin aids released at the end f fermentatin. Utilizatin f arginine, ammnium in and 7-aminbutyri aid is pltted separately (Fig. 8 and 9). Supplementatin with DAP did nt seem t signifiantly affet the timing f arginine nsumptin in either strain. n the arginine-supplemented juies, the bulk f the available arginine (apprximately 75% t 8%) was nsumed prir t the first 5 Brix drp, the ttal time f nsumptin being extended nly slightly ver that bserved in the ntrl and DAP nly juie (Fig. 8 and 9, Panel C). By the time half f the sugar ( Brix) had been nsumed, uptake f arginine, as well as ther amin aids, had eased. Bth strains displayed similar behavir. n the juie reeiving bth DAP and arginine, arginine nsumptin was retarded fr bth strains, but the ttal amunt f arginine nsumed was idential t the amunt utilized in the trials reeiving arginine supplementatin nly (Fig. 8 and 9, Panel D). Less 7-aminbutyri aid was nsumed in the juies reeiving arginine supplementatin. 7-Aminbutyri aid utilizatin is influened by the presene f ther, mre preferred nitrgen sures in the medium (13,28). verall, bth strains shwed very similar patterns f amin aid utilizatin in respnse t nitrgen supplementatin. The terminatin f uptake f the amin aids uld be due t the ells exiting grwth phase and preparing fr statinary phase. Hwever, the transprt f amin aids is inhibited by ethanl (2,6,26). Ferreras et al. (6) reprted an 8% derease in the ativity f the general amin aid permease upn expsure t as little as 5% ethanl. Under nitrgen-limitatin nditins suh as the typial grape juie fermentatin, the general amin aid permease wuld be expeted t be in peratin (29). Depending upn the fatty aid mpsitin f the plasma membrane, the arginine-speifi permease is als inhibited apprximately 5% by ethanl nentratins as lw as 3% (26). Thus, amin aid nsumptin may ease beause f the aumulatin f ethanl in the medium. ffet f plyamine supplementatin n arginine utilizatin: Arginine an be degraded t serve as a nitrgen sure, r it an serve as a preursr fr plyamine bisynthesis. Plyamines are present in high nentratin in eukaryti ells during grwth phase (reviewed in 8,). The first step in plyamine bisynthesis is the nversin f rnithine t putresine atalyzed by rnithine dearbxylase. Arginase degradatin f arginine yields bth urea and rnithine. The rnithine generated an be either further metablized t prline r an be utilized fr plyamine bisynthesis. Degradatin fprline urs nly aerbially (4). ther rutes f rnithine degradatin in yeast have nt been reprted, althugh they annt be disunted. f arginine is being degraded primarily t prvide a sure f rnithine, and nt nitrgen, urea wuld nt need t be further metablized, thus beming an end prdut whih will be exreted frm the ells, as wuld be the ase in a juie high in bth arginine and nn-arginine nitrgen. Yeast an readily utilize plyamines present in the medium (), whih wuld negate the need t synthesize these mpunds. Thus, if bth ammnia and plyamines were prvided in the medium in suffiiently high nentratins, arginine might nt be degraded as a nitrgen sure but utilized simply as arginine fr prtein bisynthesis. The effet f supplementatin with plyamines r with a high nentratin f DAP (2 g/l) r bth n utilizatin f radiatively labeled arginine was investigated using the pink juie nentrate that has been utilized in many previus studies (16,17,18). This nentratin f DAP is nt exessively high in terms f ttal nitrgen equivalents fr a grape juie, yielding a level f ttal nitrgen n the high side but still within the range fr a typial Califrnia grape juie (23,24). This supplementatin des result in a juie unusually high in ammnium in nentratin relative t ther nitrgen sures. n this ase, high DAP had a strng inhibitry effet n arginine nsumptin (Fig. A). The amunt fradilabel stably assiated with the ells was highest in the DAP-supplemented juie, but was apprximately the same in the tw samples supplemented with plyamines (Figure B). Plyamine additin had an inhibitry impat n arginine utilizatin in the absene f added DAP and a slightly stimulatry effet n arginine nsumptin in the presene f high DAP under these nditins. n all ases, sme f the radilabel was revered as C 2, the urea derived frm arginine being apprximately 9% f the initial amunt f labeled arginine degraded in the ase f n supplementatin, apprximately 5% in the ase f supplementatin with plyamines nly, and abut 25% in the juies reeiving DAP r DAP plus plyamines (Fig. 11). While the
NTRGN SUPPLMNTATN..- 9!.8 Fig.. ffet f supplementatin with plyamines (putresine, spermine and spermidine) and with high nentratins f DAP n utilizatin f radilabeled arginine in UCD 522 (Mntrahet). Pints shwn represent the average f the determinatins + FLSD. Panel A: label remaining in the medium; Panel B: label assiated with ells. N supplementatin (), DAP supplementatin ( ), plyamine supplementatin (C), DAP plus plyamine supplementatin ( ). A 4._1 u~ -- 3 C > :3.. ' 2 1 L_... t~ m (n.. =,.. >.6 :3.4 " C,B 1 t_, " m..2,,j. d 8 " 6 t~!.-.,m C :,,.= 4 Cntrl DAP [] Plyamines DAP + Plyamines Fig. 11. Perent f arginine released frm the fermenting juie as C 2 as a funtin f DAP and plyamine supplementatin. The yeast strain used was UCD 522 (Mntrahet). Pints shwn represent the average f the determinatins + FLSD. -- <: 2 1' presene f DAP in very high nentratin greatly suppressed degradatin farginine, apprximately 25% f the available arginine was metablized t urea whih was further degraded t NH 3 and C 2, and an additinal 7% f the available arginine was ell-assiated), prbably inrprated as arginine int prtein. nterestingly, in the ase f supplementatin with plyamines nly, the inhibitry effet f plyamine additin n arginine degradatin was greater than predited n a simple nitrgen equivalents basis; i.e., the amunt f nitrgen in arginine that was nt degraded is signifiantly greater than the ttal amunt f nitrgen f the added plyamines. This finding suggests a rle f ply- amine ntent in the ntrl f arginine degradatin. Cnlusins Nitrgen supplementatin f Chenin blan juie affeted rate f fermentatin and the time required fr mpletin f fermentatin in bth yeast strains analyzed. Nitrgen supplementatin had a mre signifiant stimulatry effet n bimass prdutin in Prise de Musse as mpared t UCD 522 (Mntrahet), and a mre prnuned stimulatry effet n fermentatin rate in UCD 522. The nitrgen-indued stimulatin f sugar utilizatin in UCD 522 was independent f any inrease in bimass. The tw strains displayed similar
~ MNTR and BSSN patterns f amin aid utilizatin, and nitrgen supplementatin did nt appreiably affet the timing f nsumptin f amin aids during fermentatin. Supplementatin with a high diammnium phsphate nentratin did signifiantly redue nsumptin f arginine. Plyamine supplementatin mre mderately but signifiantly affeted arginine utilizatin. Juie plyamine ntent appeared t be invlved in the ntrl f arginine degradatin, sine there was a muh bigger redutin in arginine degradatin with plyamine supplementatin than wuld be predited based upn a strit preursr/metablite relatinship. Supplementatin with bth plyamines and high nentratins f DAP did nt mpletely eliminate arginine nsumptin and subsequent degradatin. Literature Cited 1. Bell, A. A., C. S. ugh, and W. M. Kliewer. ffets n must and wine mpsitin, rates f fermentatin, and wine quality f nitrgen fertilizatin f Vitis vinifera var. Thmpsn Seedless grapevine. Am. J. nl. Viti. 3:124-9(1979). 2. Calderbank, J., M. H. J. Keenan, and A. H. Rse. Plasma membrane phsphlipid unsaturatin affets expressin f the general amin aid permease in Saharmyes erevisiae Y185. J. Gen. Mir. 131:57-65 (1985). 3. Castr, J. G. B. The free amin aids f musts and wines.. The fate f amin aids f must during alhli fermentatin. Fd Res. 18:146-51 (1953). 4. Cper, T. G. Nitrgen metablism in Saharmyes erevisiae. n: The Mleular Bilgy f the Yeast Saharmyes:Metablism and Gene xpressin. J. N. Strathern,. W. Jnes and J. R. Brah (ds.) pp 39-99. Cld Spring Harbr Labratry, Cld Spring Harbr, New Yrk (1982). 5. Cper, T. G. Transprt in Saharmyes erevisiae. n: The Mleular Bilgy f the Yeast Saharmyes: Metablism and Gene xpressin. J. N. Strathern,. W. Jnes, and J. R. Brah, (ds.) pp. 399-461. Cld Spring Harbr Labratry, Cld Spring Harbr, New Yrk (1982). 6. Ferreras, J. M., R. glesias, and T. Girbes. ffet f the hrni ethanl atin n the ativity f the general amin-aid permease frm Saharmyes erevisiae var. ellipsideus. Bihim. Biphys. Ata 989:375-7 (1989). 7. Fnzi, W. A. Regulatin f Saharmyes erevisiae rnithine dearbxylase expressin in respnse t plyamine. J. Bil. Chem. 264:18-18(1989). 8. Heby,., and L. Perssn. Mleular genetis f plyamine synthesis in eukaryti ells. Trends Bihem. Si. 15:153-9 (199). 9. ngledew, W. M., and R.. Kunkee. Fatrs influening sluggish fermentatins f grape juie. Am. J. nl. Viti. 36:65-76 (1985).. Jnes,. W., and G. R. Fink. Regulatin f amin aid and nuletide bisynthesis in yeast. n:the Mleular Bilgy f the Yeast Saharmyes: Metablism and Gene xpressin. J. N. Strathern,. W. Jnes, and J. R. Brah, (ds.) pp.181-299. Cld Spring Harbr Labratry, Cld Spring Harbr, New Yrk (1982). 11. Kliewer, W. M. Free amin aids and ther nitrgenus fratins in wine grapes. J. Fd Si. 35:17-21 (197). 12. Kluba, R. M., L. R. Mattik, and L. R. Hakler. Changes in the free and ttal amin aid mpsitin f several Vitis labrusana grape varieties during maturatin. Am. J. nl. Viti. 29:2-11 (1978). 13. MKelvey, J., R. Rai, and T. G. Cper. GABA transprt in Saharmyes erevisiae. Yeast 6:263-7 (199). 14. Mirvish, S. S. The aringeni atin and metablism f urethan and N-hydrxyurethan. Adv. Caner Res. 11:1-42 (1968). 15. Mnk, P. R., D. Hk, and B. M. Freeman. Amin aid metablism by yeasts. n: Preedings f the Sixth Australian Wine ndustry Tehnial Cnferene. pp 129-33 (1986). 16. Mnteir, F. F., and L. F. Bissn. Amin aid utilizatin and urea frmatin during vinifiatin fermentatins. Am. J. nl. Viti. 42:199-8 (1991). 17. Mnteir, F. F., and L. F. Bissn. Bilgial assay f nitrgen ntent f grape juie and preditin f sluggish fermentatins. Am. J. nl. Viti. 42:47-57(1991). 18. Mnteir, F. F.,. K. Trusdale, and L. F. Bissn. thyl arbamate frmatin in wine: Use f radiatively labeled preursrs t demnstrate the invlvement f urea. Am. J. nl. Viti. 4:1-8 (1989). 19. ugh, C. S. Fermentatin rates f grape juie.. ffets f temperature and mpsitin n white juie fermentatin rates. Am. J. nl. Viti. 15:167-77(1964).. ugh, C. S. thyl arbamate in fermented beverages and fds.. Naturally urring ethyl arbamate. J. Agri. Fd Chem. 24:323-7 (1976). 21. ugh, C. S.,. A. Crwell, and B. R. Gutlve. Carbamyl mpund reatins with ethanl. Am. J. nl. Viti. 39:239-42 (1988). 22. ugh, C. S.,. A. Crwell, and L. A. Mney. Frmatin f ethyl arbamate preursrs during grape juie (Chardnnay) fermentatin.. Additin f amin aids, urea, and ammnia: effets f frtifiatin n intraellular and extraellular preursrs. Am. J. nl. Viti. 39:243-9 (1988). 23. ugh, C. S., D. Stevens, and J. Almy. Preliminary mments n effets f grape vineyard nitrgen fertilizatin n the subsequent ethyl arbamate frmatin in wines. Am. J. nl. Viti. 4:219- (1988). 24. ugh, C. S., D. Stevens, T. Sendvski, Z. Huang, and D. An. Fatrs ntributing t urea frmatin in mmerially fermented wines. Am. J. nl. Viti. 41:68-73 (199). 25. Piere, J. S. Amin aidsin malting and brewing. J. nst. Brew. 88:228-33(1982). 26. Rse, A. H. Saharmyes erevisiae as a mdel eukaryte. n: Current Develpments in Yeast Researh. G. G. Stewart and. Russell (ds.) pp. 645-52. Pergamn Press, New Yrk (198). 27. Suizu, T., Y. limura, K. Gmi, K. Takahashi, S. Hara, K. Yshizawa. and G. Tamura. Cnstrutin f urea nn-prduing yeast Saharmyes erevisiae by disruptin f the CAR1 gene. Agri. Bil. Chem. 54:537-9 (199). 28. Vissers, S., B. Andre, F. Muyldermans, and M. Grensn. ndutin f the 4-aminbutyrate and urea atabli pathways in Saharmyes erevisiae. Speifi and general transriptinal regulatrs. ur. J. Bihem. 187:611-6(199). 29. Wdward, J. R., and V. P. Cirill. Amin aid transprt and metablism in nitrgen-starved ells f Saharmyes erevisiae. J. Bateril. 13:714-23(1977).