The Vlatile Cmpsitin f Chardnnay Juices: A Study by Flavr Precursr Analysis M. A. SEFTON ~, I. L. FRANCIS 2, and P. J. WILLIAMS 3. The free vlatiles, as well as thse released frm their glycsidically bund frms by acid r enzyme hydrlysis, have been analyzed fr Chardnnay juices frm three successive vintages. One hundred eighty cmpunds were identified. Twenty-eight f these have nt been previusly reprted as grape cmpnents. Mre than seventy percent f the ttal cncentratin f vlatile secndary metablites cmprised thirteen-carbn nrisprenids, and these were mainly bserved in the acid- and the glycsidase enzyme-released fractins. Benzene derivatives accunted fr a further twenty percent f the ttal vlatile cncentratin, while mnterpenes made up nly five percent f the ttal. Lw cncentratins f vlatiles, apparently derived frm primary metablites r frm chlrphyll degradatin, were als bserved. Vlatiles with likely sensry significance were fund mainly in the acid hydrlysates. Pathways t these cmpunds are discussed. KEY WORDS: Chardnnay, cmpsitin, vlatiles, nrisprenids, mnterpenes, vlatile phenls, enzyme hydrlysis, acid hydrlysis, precursr, damascenne The analysis f vlatiles released frm flavrless glyccnjugates is a strategy being emplyed increasingly fr the investigatin f flavr cmpunds f fruits and wines (52). This strategy is particularly valuable fr flavr studies n fruits that cntain lw cncentratins f distinguishing free arma cmpunds, e.g., nn-flral grape varieties such as Chardnnay. Chardnnay juices seldm have a distinct arma, althugh the wines f this variety can usually be readily recgnized (1). Implicitly then, Chardnnay juices cntain cnstituents which, althugh drless, are capable f prducing characteristic arma cmpunds in the finished wine. Earlier wrk n Chardnnay juice used in Australian winemaking indicated that the variety was deficient in mnterpenes (11). This bservatin derived frm quantitative analyses f free and ptentially vlatile (mn)terpenes, i.e., FVT and PVT. Hwever, the PVT distillate cntained a high cncentratin f nrisprenid cmpunds, suggesting that these cmpnents might cntribute t the varietal character f wines made frm Chardnnay. The pssible invlvement fnrispren0ids in Chardnnay wine flavr was als suggested frm an earlier study f the headspace cmpsitin f Chardnnay wines. Mre than 150 cnstituents were identified in the wines frm six cnsecutive vintages, and the majr fermentatin alchls, esters, and acetic acid were recgnized as imprtant arma cmpunds. Hwever, the nly grape-derived vlatile with sensry significance that was bservable against the backgrund f fermen- 1,2.3The Australian Wine Research Institute, P.O. Bx 197, Glen Osmnd, SA 5064, Australia. *Authr t whm crrespndence shuld be addressed. Acknwledgements: Chardnnay juices were generusly dnated by G. Gramp and Sns Pty. Ltd. Samples frm the 1987 vintage were prepared by Mr. B. Wilsn and 9-hydrxymegastigma- 5,7-dien-3-ne was synthesized by Dr. P. Winterhalter in this labratry. We als thank Dr. C. R. Strauss f the CSIRO, Divisin f Chemicals and Plymers fr reference samples f sme benzene derivatives, and the Grape and Wine Research Cuncil fr financial supprt. Manuscript submitted fr publicatin 29 June 1992. Cpyright 1993 by the American Sciety fr Enlgy and Viticulture. All rights reserved. 359 tatin prducts was the nrisprenid cmpund damascenne (43). This paper reprts the results f a study f Chardnnay juices btained frm the same regin ver three cnsecutive vintages. The free vlatiles f the juices, as well as the acid- and glycsidase-released vlatile aglycnes f the precursr fractin, were identified and quantified. The data thus allw an assessment f the number and type f vlatile cnstituents that can be generated in Chardnnay wines n hydrlysis f the flavr precursrs. This hydrlytic release is respnsible, at least in part, fr the develpment f flavr in Chardnnay wines (15,54). Knwledge f the cmpsitin f the vlatiles released frm the grape glycsides culd, therefre, help t elucidate varietal armas f these wines. Materials and Methds General prcedures: All slvents were f high purity at purchase and were redistilled befre use. Details f analyses by GC-MS were as described previusly (38), except that the 1989 samples were analyzed with a Finnigan TSQ 70 mass spectrmeter cupled t a Varian 3400 gas chrmatgraph. Reference cmpunds were either available cmmercially, dnated, r prepared in ur labratry by standard synthetic methds. Preparatin f samples fr analysis: The Chardnnay juice samples were btained frm cmmercial batches f the 1987-1989 vintages frm the Suthern Vales regin f Suth Australia. These juices were prepared by the same winery and were sampled immediately prir t fermentatin. They were a mixture f free-run juice and light pressings and had been clarified by Pectinex 3XL enzyme (50 ml/tnne added at crushing and a further 15 ml/1000 L added in the tank). The 1989 juice was 22.8 Brix, and this typified the sluble slids cntent f the juices frm the tw earlier vintages.
360 SEFTON et al Table 1. Nrisprenid cmpnents f 1987, 1988, and 1989 vintage Chardnnay juices and f the glycside hydrlysates derived frm these juices. 1 2,6,6-Trimethylcyclhex-2-en-l-ne 2 3,5,5-Trimethylcyclhex-2-en-l-ne 3 2,6,6-Trimethylcyclhex-2-ene-l,4-dine 4 Vitispirane 5 2,6,6-Trimethylcyclhexane-l,4-dine 6 Riesling acetal 7 1,1,6-Trimethyl-l,2-dihydrnaphthalene 8 Damascenne 9 Actinidl- ismer 1 10 Actinidl - ismer 2 11 Actinidl - ismer 3 12 (5R*,9R*)-3,4-Dihydr-3-xedulan e 13 (5S*,9R*)-3,4-Dihydr-3-xedulan e 14 3,4-Dehydr-8-inne e 15 (7E)-Megastigma-4,7,9-trien-3-ne 16 8,9-Dehydrtheaspirne e 17 2-(3-Hydrxybut-l-enyl- 2,6,6-tri methylcyclhex-3-en- 1 -ne 18 3-Hydrxytheaspirane - ismer le 19 4-(2,3,6-Trimethylphenyl)-but-3-en-2-ne 20 3-Hydrxytheaspirane- ismer 2 e 21 3-Hydrxytheaspirane- ismer3 e 22 5,6-Epxy-3-hydrxymegastigm-7-en- 9-ne ismer 1 23 cis-3-ox-8-damascne 24 (6Z,8E)-Megastigma-4,6,8-trien-3-ne 25 (6Z,8Z)-Megastigma-4,6,8-trien-3-ne 26 Dihydractinidilide 27 tran-3-ox-8-damascne 28 3,4-dihydr-3-xactinidl ismer le 29 (6E,8E)-Megastigma-4,6,8-trien-3-ne 30 3,4-dihydr-3-xactinidl ismer 2 e 31 cis-3-ox-~-damascne 32 3,4-Dihydr-3-xactinidl ismer 3 e 33 (5S*,6R*)-9-Hydrxymegastigm-7-en-3-n 34 3-Hydrxydamascne 35 3,4-Dihydr-3-xactinidl, ismer4 e 36 9-Hydrxymegastigma-5,7-dien-3-ne 37 Megastigm-5-en-7-yne-3,9-dil 38 3,4-Dihydr-3-hydrxyactinidl e 39 Megastigm-5-ene-3,9-dil 40 trans-3-ox-~-damascne 41 3-Ox-~-inl ismer If 42 3-Ox-c~-inl ismer 2 f 43 3-Hydrxymegastigma-5,7-dien-9-ne 44 9-Hydrxymegastigma-4,6,7-trien-3-ne 45 Dehydrllilide 46 3-Ox-(z-inne 47 5,6-Epxy-3-hydrxymegastigm-7-en- 9-ne, ismer 2 48 9-Hydrxymegastigm-5-en-4-ne 49 9-Hydrxymegastigma-4,6-dien- 3-ne, ismer 1 cntinued References As As 1987 b 1988 1989 grape ak Kvats a Evid- r wine cmpindices Free Enz H* Free Enz H Free Enz H* ence ~ prduct nent n.d, 1274 1297 + + 1327 + + + 1355 1374 1423 1496 + 1510 1515 1564 1592 1614 1619 1632 1641 tr tr tr tr 7.4 20 4.4 21 + tr tr tr + 7 + 1.5 + 1.2 2.5 3.8 + + A (18) (55) 1 B (55) 2.7 3 C (38) (37) 5.5 22 C (38) (37) 1 C (38) 7.5 C (58) 11.5 C (38) 33 C (38) 15 tr 1.7 14 C (38) 23 tr 2 25 C (38) tr tr C (38) tr 1.5 1.5 tr 3.3 3.8 C (37) tr 3 8 1.8 10 11 C (37) 4 1.7 A (25) tr tr C (55) tr 1 C (59) 1654 + 1 45 17 C (38) 1657 + 14 11 tr C (59) 1658 + 1 A (25) 1668 5 tr 4.5 tr C (59) 1684 3 tr 3.2 tr C (59) 1694 + tr 5 tr tr 4 D 1748 1 D 1750 tr tr 8 tr 6.5 C (55) (37) 1796 tr tr C (55) 1802 + tr 4 2 5 C (38) (37) 1805 + 40 32 C (38) 1805 + + + tr 16 2 4.7 20 8 C (59) (37) 1810 tr 1 11 3 6 C (55) (37) 1815 + + + tr 48 7 6.6 37 11 C (59) (37) 1823 + 3.5 3 D 1830 + + + tr 20 1.5 1.7 18 2.7 C (59) (37 1848 + + + 3 117 180 31 108 90 C (38) (37) 1851 + + tr 75 3 bsc C (46) (37) 1853 + + + tr 5 2 8 1 C (59) (37) 1863 + 15 1 8 B 1871 + tr 2.4 1 4 C (38) 1884 + tr 2 tr 1 3 1 C (59) 1886 tr tr 2 C (39) 1893 + 82 2 50 1.5 C (38) (37) 1937 + + + 4 370 N80 71 260 ~60 C (46) (37) 1938 + ~86 ~60 C (46) (37) 1948 1 6 3 4.5 C (39) 1948 + 22 17 tr C (38) (37) 1959 + + + 1.2 12 6.5 10 C (38) (37) 1960 + + tr 1 3.3 4 C (38) (37) 1972 + + tr tr 2.5 6 6 C (39) 1988 + tr 6 tr 7 C (59) (37) 2001 + + + 1.8 53 18 23 40 20 C (38) (37)
VOLATILE COMPOSITION OF CHARDONNAY JUICE m 361 Table I cntinued 1987 b Kvats a indices Free Enz H 50 Blumenl C 2002 + 51 3-Hydrxymegastigma-5,7-diene-4,9-dine 2039 52 9-Hydrxymegastigma-4,6-dien-3-ne, ismer2 2081 + + + 53 An ismer f grasshpper ketne 546,9-Dihydrxymegastigm-7-en-3-ne 55 Grasshpper ketne 56 Llilide, ismerl 57 Vmiflil (BlumenlA) 58 Llilide, ismer2 59 Dehydrvmiflil 60 Blumenl B 1988 1989 c Evid- Free Enz H* Free Enz H ence tr 42 4.2 18 5 C tr 1 1 A (2) References As As grape ak r wine cmpprduct nent (38) (37) 1.7 57 21 21 46 21 C (38) (37) 2115 15 D 2121 + + + 1.7 545 185 60 105 64 C (40) (37) 2149 + 110 tr 23 C (38) 2170 tr 4 6 A (3) 2180 + + + tr 285 13 14 45 16 C (46) (37) 2200 + 1 A (3) 2212 + + + tr 44 10 3 17 6.5 C (38) (37) 2244 + 17 tr 1 8 2.5 C (38) (37) a Fr GC cnditins see Seftn et al, 1990; n.d., nt determined, b+, cnstituent bserved in this fractin. Free, uncnjugated juice vlatiles; Enz, vlatiles released by hydrlysis f the C18 islate with Rhapect C; H, vlatiles released by hydrlysis f the C18 R.P. islate at 50 C, ph 3.0 ver 28 days. c Cncentratin in #g/l f juice; tr, trace; bsc, bscured, da, mass spectrum the same as published spectrum; B, the mass spectrum was the same as that f the reference cmpund and the peak was enhanced by the reference when c-chrmatgraphed; C, previusly prven in this labratry; D, interpretatin f mass spectrum, ecartenid numbering, f Assignments interchangeable. A single juice sample frm each vintage was analyzed in duplicate. The acid hydrlysates were prepared as described by Seftn et al. (38). The islatin f the free juice vlatiles and acid hydrlysis prducts with Fren Fll, as well as the islatin f the grape glyccnjugate fractin with a C-18 reversed-phase (RP) adsrbent, were carried ut as described by Wilsn et al. (56). Enzyme hydrlysates were prepared as fllws. A slutin f Rhapect C (200 mg) in an aqueus ph 5 buffer [10 ml, prepared by mixing equal vlumes f aqueus citric acid slutin (0.1 M) and aqueus disdium hydrgen phsphate slutin (0.2 M)], was added t an aqueus slutin f the C-18 RP islate (5 ml, equivalent t 250 ml fjuice) in the same buffer (25 ml). The aqueus C-18 RP islate was stripped f the free vlatiles by liquid/liquid extractin with fren vernight, prir t the enzyme additin. The mixture was heated at 37 C fr 16 hurs, a slutin f 1-ctanl (20 ~g) in ethanl (100 ~L) then added, and the hydrlysate islated by cntinuus extractin with dichlrmethane ver 24 hurs. The rganic extract was dried (magnesium sulfate) and cncentrated by fractinal distillatin thrugh a Vigreux clumn packed with Fenskes helices prir t analysis by GC-MS. Results and Discussin The free vlatile cmpnents f the Chardnnay juices frm the three vintages and the vlatile hydrlysis prducts derived frm precursrs in these juices fall int fur brad categries: nrisprenids (Table 1), benzene derivatives (Table 2), mnterpenes (Table 3), and aliphatic cmpunds (Table 4). Sme additinal cmpnents f disparate bigenetic rigin are listed in Table 5. Quantitative data are given fr samples frm the 1988 and 1989 vintages, but nly qualitative data were available fr thse frm the 1987 vintage. Only thse cmpunds that have been identified are listed in the tables, and these accunt fr mre than 80% f the ttal cncentratin f the vlatiles bserved by GC-MS. Evidence fr the assignments, tgether with references t cmpunds as previusly bserved in grape r wine prducts, are given. Where n such reference is given, the cmpnent has nt, t the best f ur knwledge, been previusly reprted as a cnstituent f such prducts. Because many f the Chardnnay vlatiles have been earlier identified as ak wd cmpnents (37), these cmmn cnstituents are als indicated in Tables 1 and2. Mre than 100 minr r trace cnstituents, the identity f which culd nt be cnfidently assigned, were als bserved, but these are nt included in the tables. The ttal cncentratins f the free cmpunds as well as thse bserved after enzymatic and acid hydrlysis f the precursr fractins are presented in five categries fr 1988 and 1989 (Fig. 1). The relative prprtin f the acid- and enzyme-released cnstituents is similar fr each categry fr bth years, with nrisprenids dminating and the benzene derivatives ccurring at secnd highest cncentratin. The lw cncentratin f mnterpenes in these Chardnnay samples is als illustrated by the data. The free vlatiles in juices frm bth years were cmpsed largely f aliphatic cnstituents which can perhaps be regarded as primary rather than secndary metablites. It shuld be nted that thse cmpunds which dminated the free vlatile fractins, i.e. (Z)-3- hexenl 145, (E)-2-hexenl 146, n-hexanl 147, and 7- butyrlactne 150 (Table 4) culd have been prduced by unwanted partial fermentatin taking place during strage f the juices, with the three alchls 145-147 prduced by reductin f the crrespnding aldehydes which may be frmed frm lipxygenase xidatin f linleic acid. Whether these cnstituents were present ttally as grape metablites r were cntributed partly by pst-harvest cnditins is, therefre, pen t questin. Hwever, sme f these cmpunds were als
362- SEFTON et al Table 2. Vlatile phenls and ther benzene derivatives f 1987, 1988, and 1989 vintage Chardnnay juices and glycside hydrlysates derived frm these juices. 1987 b 1988 c 1989 c Kvats a Evidindices Free Enz H Free Enz H Free Enz H ence 61 Ethylbenzene n.d. + tr 1 tr E 62 1,3 and/r 1,4-Dimethylbenzene n.d. + tr tr tr E 63 1,2Dimethylbenzene n.d. + tr tr tr E 64 Benzaldehyde n.d. + + tr tr 1.2 tr tr C 65 Benzyl alchl n.d. + + + 10 80 7 175 22 8 C 66 Phenl n.d. + + + 1.2 tr 5 5 E 67 -Cresl n.d. + tr tr E 68 p-cresl n.d. + tr tr E 69 m-cresl n.d. + tr tr E 70 2-Phenylethanl 1283 + + + 4 70 10 95 38 16 C 71 Methyl salicylate 1306 + tr 3.8 1 2.5 tr E 72 Benzthiazle 1353 + + tr tr 1 tr E 73 Quinline 1376 tr tr E 73 3-Phenylprpanl 1403 + + tr tr tr 1 tr C 75 Benzic acid 1420 + + + 10 1.7 20 32 15 9 C 76 2,3-Dihydrxybenzic acid methyl ester 1469 + + tr tr tr 2 3.2 2 C 77 4-Vinylguaiacl 1483 + + + tr 100 25 1.4 16 10 C 78 4-Vinylphenl 1493 + + + tr 95 8 4 10 8.5 C 79 Eugenl 1505 + tr 5.6 1 7 C 80 Phenylacetic acid 1515 + + tr tr 2 1 C 81 Adimethylnaphthalene 1524 tr tr A (18) 82 Adimethylnaphthalene 1530 + tr tr A (18) 83 Indle 1537 + tr 1 tr 1.6 1 E 84 m-hydrxyacetphenne 1543 + tr 1 tr D 85 2-Hydrxymethylbenzicacidlactne 1589 + tr tr 1 1 tr A(18) 86 cis-cinnamic acid 1628 + D 87 Vanillin 1642 + + 1.6 1.6 2 9 2.5 4 C 88 Cumarin 1672 tr tr tr E 89 trans-cinnamic acid 1720 + + tr tr tr 4.3 6 B 90 Methyl vanillate 1728 + + + tr 14 6 2.7 6.5 5 C 91 Acetvanillne 1731 + tr 3 5 3 3 5 B 92 4-Hydrxybenzaldehyde 1776 + + + tr 1 1 1 C 93 Tyrsl 1781 + tr 1.4 1 2 C 94 3-Methxytyrsl 1799 + tr tr 10 3 D 95 Prpivanillne 1821 tr 4.4 tr D 96 4-Hydrxybenzic acid methyl ester 1833 + + tr 7 1 E 97 4-Hydrxyacetphenne 1851 + + tr 1 tr tr 1 C 98 Butyrvanillne 1860 + tr tr tr D 99 2,5-Dihydrxybenzic acid methyl ester 1863 + + + tr 3 2 5 1 4 C 100 4-Hydrxybenzic acid ethyl ester 1887 + + tr tr tr 4 1 E 101 Raspberry ketne 1901 tr tr tr C 102 Zingerne 1903 + tr 5 1 4 4 C 103 Vanillic acid 1911 + 7 4 B 104 Dihydrcniferyl alchl 1929 + + 17 1 11 2 C 105 Syringaldehyde 1950 + + + tr tr 3 2 3 C 106 cis-cniferyl alchl 1977 tr D 107 4-Hydrxybenzic acid 1979 + 6 5 B 108 Methyl syringate 2018 + + + tr 17 13 4 12 8 D 109 Acetsyringne 2021 + tr tr 1 tr 2 A (18) 110 Cniferyl alchl 2039 + tr 21 1 1 20 tr C 111 Prprisyringne 2100 + tr 10 5 1.3 6 4 E 112 Butyrsyringne 2107 tr 2 tr tr E 113 cis-ferulic acid 2109 + tr 3 D cntinued References As grape r wine prduct As ak cmpnent (26) (26) (26) (26) (29) (26) (26) (26) (29) (26) (29) (46) (26) (26) (26) (26) (29) (26) (29) (26) (29) (26) (26) (7) g (46) g (46) (26) (7) (16) (46) g (24) (46) g (26) (24) (7) g (7) (7)
VOLATILE COMPOSITION OF CHARDONNAY JUICE -- 363 Table 2 cntinued 114 Tryptphanl 115 cis-4-hydrxycinnamic acid 116 Syringic acid 117 Tryptphanl acetate 118 trans-ferulic acid 119 trans-4-hydrxycinnamic acid 1987 b 1988 c 1989 c Kvats a Evidindices Free Enz H Free Enz H Free Enz H ence 2119 + tr 1.5 tr 1 2.5 tr E 2170 + 1 D 2183 + tr E 2222 + 1.2 tr E 2264 + 1 10 8 C 2289 + 8 3 C References As grape r wine prduct As ak cmpnent (24) (16) Fr general details see Table 1. E, gas chrmatgraphic retentin time and mass spectrum identical t thse f a reference sample, g Observed in this labratry. Table 3. Mnterpene cmpnents f 1987, 1988, and 1989 vintage Chardnnay juices and glycside hydrlysates derived frm these juices. 1987 b 1988 c 1989 c As grape Kvats a Evid- r wine indices Free Enz H Free Enz H Free Enz H ence prduct 120 Limnene n.d. tr tr tr C (48) 121 5-(2-Buten-2-yl)-2,2-dimethyltetrahydrfuran n.d. tr 1 C (48) 122 Furan linall xide, ismer 1 n.d. + + 10 2.4 tr 5 2.5 C (48) 123 Furan linall xide, ismer 2 n.d. + + 6 tr tr 2.5 1.5 C (48) 124 Linall n.d. + + 5 1.6 3.5 C (48) 125 Htrienl n.d. tr 1.8 tr tr C (48) 126 Myrcenl n.d. 2.2 C (48) 127 cis-ociminl 1276 3 2 C (48) 128 trans-ociminl 1291 1.2 2.5 C (48) 129 Pyran linall xide, ismer 1 1302 + tr 1.3 tr 1.3 bsc C (48) 130 ~-Terpinel 1304 tr tr 1.6 5 1 1.5 2.5 C (48) 131 Pyran linall xide, ismer 2 1310 tr tr 1 1.8 1.5 C (48) 132 Nerl 1342 + 2.3 tr 6.5 C (48) 133 Z-2,6-Dimethylcta-3,7-diene-2,6-dil 1358 tr tr tr C (48) 134 2-Hydrxy-l,8-cinele 1370 tr 1.4 A (4) 135 E-2,6-Dimethylcta-3,7-diene-2,6-dil 1375 + + tr 5 1.5 bsc C (48) 136 Geranil 1375 + + 6 tr 18 C (48) 137 2,6-Dimethylct-7-ene-2,6-dil 1416 + 1 14 18 C (48) 138 cis-l,8-terpin 1497 + tr 20 8.5 C (48) 139 trans-1,8-terpin n.d. + C (48) 140 2,6-Dimethylct-7-ene-1,6-dil 1539 + + 2.5 1.5 1.5 6 A (50) 141 Geranic acid 1552 + + tr 3.5 1 1.5 8.5 1.5 C 142 Z-2,6-Dimethylcta-2,7-diene-l,6-dil 1554 + 18 tr 1 22 1 C (49) 143 E-2,6-Dimethylcta-2,7-diene-l,6-dil 1569 + + tr 60 1 6 40 4 C (49) Fr general details and references, see Table 1. Am. J. Enh Vitic., Vl. 44, N. 4, 1993
364- SEFTON et al Table 4. Aliphatic alchls, carbxylic acids, lactnes, and ethyl esters f 1987, 1988, and 1989 vintage Chardnnay juices and glycside hydrlysates derived frm these juices. 19875 1988 c 1989 c Kvats a Evidindices Free Enz H Free Enz H Free Enz H ence 144 Butanic acid n.d. + E 145 Z-3-Hexenl n.d. + + 4 10 tr 60 tr tr E 146 E-2-Hexenl n.d. + + --110 tr -150 --14 E 147 Hexanl n.d. + + -110 42-300 --5 E 148 Ethylhexanate n.d. + tr tr E 149 Pentanic acid n.d. + tr tr 1.4 1 E 150 y-butyrlactne n.d. + 90 tr tr 200 2.5 E 151 4-Hydrxypentanic acid lactne n.d. tr 2.2 E 152 Hexanic acid n.d.. + 25 3.3 60 20 15 E 153 4-Hydrxyhexanic acid lactne n.d. + tr tr 3 tr E 154 Ethyl ctanate n.d. + tr tr E 155 Heptanic acid 1279 tr tr 13 1 3 E 156 4-Hydrxyheptanic acid lactne 1360 tr tr 1 tr E 157 Octanic acid 1369 + + + 8 1.6 2 16 3 5 E 158 Ethyl decanate 1461 + + tr E 159 4-Hydrxyctanic acid lactne 1469 + + tr tr tr 2 tr tr E 160 Nnanic acid 1472 + + + 6 2 1 18 2 5 E 161 Decanic acid 1561 + + + 1.8 tr 1 3.6 1 E 162 4-Hydrxynnanic acid lactne 1573 + + tr tr 1 2.7 2 3.5 E 163 Undecanic acid 1654 + tr tr E 164 4-Hydrxydecanic acid lactne 1688 + + tr tr 1 E 165 Ddecanic acid 1760 + + 1 tr 2.4 2 2 E 166 Tridecanic acid 1851 + tr tr 1 2 E 167 Tetradecanic acid 1945 + + 1.2 2.5 2 bsc E 168 Pentadecanic acid 2037 + + tr 1 3 E 169 Hexadecanic acid 2146 + + 3 8 16 15 E 170 Heptadecanicacid n.d. + E 171 Linleicacid 2344 + + 12 2.5 A (18) 172 Octadecanic acid 2348 + tr tr 3 E As grape r wine prduct (29) Fr general details see Tables 1 and 2. Table 5. Miscellaneus vlatile cmpnents f 1987, 1988, and 1989 vintage Chardnnay juices and glycside hydrlysates derived frm these juices. 173 Maltl 174 2-Ethyl-3-methylmaleic anhydridl 315 175 2-Furicacid 176 2-Ethyl-3-methylmaleimide 177 2-Methyl-3-vinylmaleimide 178 5-(2-Hydrxyethyl)-4-methylthiazle 179 Diethyltartrate 180 2-Ethylidene-3-methylsuccinimide Fr general details see Tables 1 and 2. 1987 b 1988 c 1989 c Kvats a Evidindices Free Enz H Free Enz H* Free Enz H ence 1286 1.5 4 1 1 E 1315 tr tr 1 tr 1 A (18) 1334 + + 1.5 E 1462 + + tr 1.6 7 4 2 13 A (18) 1497 + + tr tr tr 1 10 A (18) 1507 + + tr tr 3.2 1 E 1556 tr tr tr E 1623 + + tr 1 A (18) As grape r wine prduct (47)
VOLATILE COMPOSITION OF CHARDONNAY JUICE- 365 1600-1400 - "5 1200- _1 ~ 1000 p, = 800 l-- t- O 600 - c 400-1,4-1 O P" 200 - _ BB nrisprenids D benzene derivatives I mnterpenes I--i aliphatics IB miscellaneus Fig. 1. Cncentratins f five categries f vlatiles, bserved as free cmpunds (free) r after release by either glycsidase enzyme (enz) r acid hydrlysis (acid) f precursr fractins frm the 1988 and 1989 Chardnnay juices. -0 88 free 88 enz 88 acid 89 free 89 enz 89 acid fund in the aglycne fractin, and thus in viv catablic pathways t 145-147 must be cnsidered [see als Humpf and Schreier, (19)]. The bservatin in the free fractin f less than 1 ~g/l quantities f the majr fermentatin esters, e.g., ethyl ctanate 154 and ethyl decanate 158 (Table 4), indicates that virtually n fermentatin f the juice had cmmenced prir t analysis. The extent f glyccnjugatin was in evidence fr all but the aliphatic cmpunds in the juices fr bth years (Fig. 1). The rati f the ttal cncentratin f the enzyme- r acid-released prducts t the free cmpnents was different fr the tw years and, in the 1988 juice, this rati indicated that glycsylatin f the secndary metablites was almst cmplete. Sme nrisprenids listed as enzyme hydrlysis prducts are presumably artifacts. These artifacts are derived frm tw surces. Firstly, extraneus acid hydrlysis f the aglycnes, presumably during sample preparatin can give, fr example, vitispirane 4, the actinidls 9-11 (Fig. 2), the xedulans 12 and 13, and the megastigmatriennes 24, 25, and 29 (Fig. 4). Facile frmatin f acid-generated prducts frm labile precursrs during enzyme hydrlysis and wrk-up has als OH 181 H+/FAS T PRE-HARVEST OH 4 6 7 HO I 37 8 34 HO 9-11 18, 20, 21 H+/SLOW POST- VINIFICA TION HO O 47 59 O GLYCOCONJUGATES GLYCOCONJUGATES OF 37 OF 34 I ROHAPECT C I ROHAPECT C 0 0 OH OH OH 33 41, 42 54 Fig. 2. Sme nrisprenids bserved in this study. Names f cmpunds are indicated in Table 1. + 44 27 40 Fig. 3. The generatin f damascenne 8 and related nrisprenids in the free vlatile fractin f Chardnnay juices and in the enzyme and acid hydrlysates f Chardnnay precursr fractins. Names f cmpunds are indicated in Table 1.
366 -- SEFTON et al 12 + + 24 49, 52 OH 13 25 29 Fig. 4. The frmatin f acid hydrlysis prducts frm the ismeric 9- hydrxymegastigma-4,6-dien-3-nes 49 and 52. Names f cmpunds are indicated in Table 1. been nted by Humpf and Schreier (19). The secnd surce f nrisprenid artifacts was thrugh the use f a high cncentratin f fungal enzyme which is knwn t generate sme xidative transfrmatin prducts frm genuine aglycnes. High cncentratins f these enzymes are necessary t effect near-cmplete hydrlysis f the glyccnjugate fractin (39). Cmpunds in this secnd grup f artifacts are the xdamascnes 27 and 40 which are frmed frm cnjugates f 3-hydrxydamascne 34, the hydrxyketne 44 which is an artifact f xidatin f the enyne di137 (Fig. 3), and dehydrvmifli159 which is derived, at least in part, frm xidatin and rearrangement f the epxide 47 (Fig. 2). Sme f the benzene derivatives listed in Table 2 may als be attributed t artifact frmatin. Thus, the relatively abundant 4-vinylguaiacl (77) and 4- vinylphenl (78) may be derived frm the crrespnding cinnamic acids thrugh decarbxylatin during gas chrmatgraphy (22). Sensry studies n the 1987 and 1989 (15)juice samples demnstrated that vlatiles released by enzyme hydrlysis had n sensry effect n the arma f wines t which the hydrlysates were added. Nevertheless, the enzyme prducts, thrugh their relative abundance, aided in the analysis, and helped in ratinalizing the frmatin f bth the acid generated prducts and the minr free vlatiles. In cntrast, the vlatile fractin released by mild acid hydrlysis, under cnditins simulating bttle ageing, had a highly significant sensry effect (15,54). Accrdingly, cmpunds present mainly in the acid hydrlysis fractin are thse mst likely t cntribute t the develped varietal character f Chardnnay wines. Such acid-generated fractins differ frm thse frmed by enzyme hydrlysis in that many acid-catalyzed cyclizatin, dehydratin and rearrangement prducts are + 2 given in the frmer (37,38). These acid-generated vlatiles are the fcus f discussin in this study. Nrisprenids (Table 1): Of the fur bigenetic classes int which these Chardnnay cmpnents can be gruped, the nrisprenids are dminant, bth in number and cncentratin, accunting fr abut 70% f the hydrlytically-released vlatile cncentratin (Fig. 1). Many f these cmpunds are key flavr cmpnents f fdstuffs and beverages and are als arma cnstituents f leaf prducts and perfumes (33). Sixty nrisprenids are listed in Table 1 and mre than 40 f the still unidentified vlatiles in the hydrlysates als appear t belng t this class. Plausible rutes fr the bigenesis f the nrisprenids listed in Table 1 frm grape cartenids have been suggested elsewhere (53). Key bigenetic steps that interrelate the majrity f the nrisprenid cmpnents are xidatin f a hydrxyl functin at C-3 and reductin f a ketne functin at C-9 (cartenid numbering, shwn fr 3-x-a-inl 41, Fig. 2). Als, frm the bserved accumulatin fketnes 33 and 54, it seems that reductin f the 4,5- duble bnd f a-inls is an imprtant bigenetic prcess in these Chardnnay samples. Reductin f the side chain 7,8- duble bnd is a significant prcess, but leads t the frmatin f less abundant nrisprenids than thse derived frm 4,5- duble bnd reductin. Cmpunds in which bth 4,5- and 7,8- duble bnds have been reduced, such as the recently reprted ak cmpnent 9- hydrxymegastigman-3-ne (37), were nt bserved in the Chardnnay juice samples. The fur 3-x cmpunds 33, 41, 42, and 54 were the majr cmpnents released by acid hydrlysis. Hwever, because three f these ketnes, 33, 41, and 54, were als abundant in the enzyme-liberated aglycne fractin, which had little sensry imprtance (15,54), their cntributin t Chardnnay varietal arma is presumably limited. The sensry impact f the secnd ismer f 3-x-a-in142, fund nly in the acid hydrlysates, needs further investigatin. Several nrisprenids with knwn flavr prperties were frmed at higher cncentratin during acid hydrlysis than during enzyme hydrlysis and these are discussed belw. The 3-Oxedulans 12 and 13 and 3- Oxmegastigmatrienes 24, 25, and 29: These five acid hydrlysis prducts are imprtant flavrants f tbacc (23,51) and have recently been bserved as cnstituents f mdel wine extracts f ak shavings (37). Wahlberg and Enzell (51) have suggested that these cmpunds may be frmed frm the ismeric ketnes 49 and 52 (Fig. 4), and this is supprted by recent data frm this labratry (37). The nexus between prducts 12, 13, 24, 25, 29 and their prgenitrs 49 and 52 was bserved in the 1988 and 1989 Chardnnay samples (Table 1) and is als reflected in the 1987 data in which the ketnes 49 and 52 were nly minr cmpnents and their prducts 12, 13, 24, 25, and 29 were nt detected. Vitispirane 4, Riesling acetal 6, and TDN 7: Recent
- 15% VOLATILE COMPOSITION OF CHARDONNAY JUICE- 367 studies (57) have shwn that the bicyclic nrisprenids vitispirane 4 and 1,1,6-trimethyl-l,2-dihydrnaphthalene (TDN) 7 are frmed in relatively high cncentratins during acid hydrlysis f Riesling wines. These tw vlatiles, tgether with the tricyclic cmpund Riesling acetal 6, were als frmed in high cncentratin when glycsidic fractins frm that variety were acid-hydrlyzed (57,58). The hydrcarbn TDN 7 has been cmmnly bserved at cncentratins in excess f its flavr threshld, and at such levels in wines, it is a cntributr t the kersene-like, bttle-aged character f many Riesling wines (42). Althugh the acid hydrlysates frm the Chardnnay precursr fractins studied here were particularly rich in nrisprenids, they were cmparatively pr in vitispirane 4, Riesling acetal 6 and TDN 7. Vitispirane 4 was prduced in the hydrlysates at cncentratins well belw its threshld in wine (41), while TDN 7 was prduced maximally in the 1989 sample at a cncentratin near t its detectin threshld in white wines (41). This presumably accunts fr the absence in aged Chardnnay wines f the type f bttle-aged develpment that characterizes Riesling. Vitispirane 4 was frmed in the acid hydrlysates at the cncentratin expected frm hydrlysis f its precursrs, i.e., cnjugates f the ismeric hydrxytheaspiranes 18, 20, and 21 which were bserved as aglycnes (57). The reasn fr the paucity f cmpunds 4, 6, and 7 in the Chardnnay hydrlysates has been attributed t a relative inactivity, in this variety, f enzymes reducing the 7,8- duble bnd f the megastigmane skeletn which appears t be a prerequisite t the frmatin f precursrs f these three cmpunds (53). Damascenne 8: The ptent flavrant damascenne 8 was bserved bth as a free vlatile in juice and as a cmpnent f the acid hydrlysates. Hwever, the cccurrence with 8 f varius side prducts frm its frmatin, suggests that the generatin fdamascenne in these tw fractins may cme abut thrugh different hydrlytic pathways. These pathways t 8 frm enyne dil 37 and allenic tril 181 are illustrated in Figure 3. Hydrlytic studies in ur labratry f synthetic substrates have shwn that the allenic tril 181 reacted rapidly at juice ph and at rm temperature t give damascenne as a minr prduct, alng with 3- hydrxydamascne 34 and the enyne dil 37 as the majr prducts (44). Furthermre, in the 1988 and 1989 enzyme hydrlysates, the bserved rati f 5:1 fr the artifact ketnes (27 + 40):44 [i.e., cmpunds frmed frm 34 and 37, (see Fig. 3) (39)] was apprximately the same as the rati f 3-hydrxydamascne 34:enyne dil 37, given by the hydrlysis f synthetic allenic tril 181. This supprts the prpsitin that hydrlysis f the tril 181 was respnsible fr the frmatin fthe damascenne that is present free in the pre-harvest juices, and simultaneusly generated the aglycnes 34 and 37. Althugh the allenic tril 181 has nt yet been identified as a grape cmpnent, its presumed prgenitr, the s-called Grasshpper ketne 55 (38), was a significant cmpnent f the enzyme hydrlysates. The hydrlytic studies f Skurumunis et al. (44) have als shwn that damascenne 8 was generated slwly at ph 3 as a minr prduct frm bth the enyne dil 37 and its C-9 glucside, tgether with 3- hydrxydamascne 34 as the majr prduct (Fig. 3). Imprtantly, at ph 3, neither 3-hydrxydamascne 34 nr its 3-glucside gave any damascenne 8, even after prlnged heating at 100 C. Bth damascenne 8 and 3- hydrxydamascne 34 were frmed in the Chardnnay acid hydrlysates (Table 1), and the quantity f damascenne 8 bserved in these hydrlysates (i.e., 10% f the cncentratin f 37 plus the artifact 44) was that expected frm hydrlysis f the free and cnjugated enyne dil. Thus, the frmatin fdamascenne 8 in the acid hydrlysates (analgus t pst-harvest frmatin) can be accunted fr entirely by hydrlysis f the free and cnjugated enyne dil 37. Benzene derivatives (Table 2): After the nrisprenids, benzene derivatives were the mst diverse and abundant grup f the hydrlytically released vlatiles seen in the samples, accunting fr between 10% and 20% f the ttal vlatile cncentratin in the fractins. The majrity f these cmpunds are presumably shikimate-derived, althugh several, such as the rth-hydrxymethylbenzates are prbably frmed frm plyacetate precursrs. Mre f the individual cmpunds in this grup were fund at higher cncentratin as acid- r enzymereleased aglycnes than as free cmpunds in the juices. Hwever, fr the 1989 sample, several cmpnents were present at a much higher cncentratin in the free fractin. These include benzyl alchl 65, 2- phenylethanl 70, vanillin 87, 3-methxytyrsl 94, and the phenlic acid esters 99 and 100. This bservatin emphasizes the variability f glycsidic binding f individual cmpunds, bth within this cnstituent categry and between years. Amng the cmpunds listed in Table 2 which have knwn sensry prperties, there were nne present at a cncentratin abve threshld (5,10,12). Furthermre, nly seven f these benzene derivatives 84, 85, 87, 91, 96, 97, and 109 were present at a greater cncentratin in the acid hydrlysates than in the enzyme-released fractins f bth the 1988 and 1989 juices; even fr these, the differences were nt great and the abslute cncentratin f each was lw. This indicates that the greater sensry impact f the acid hydrlysates cmpared t the enzyme hydrlysates (15,54) culd have nly been marginally cntributed t by the grup f cmpunds listed in Table 2. Hwever, several f these vlatiles such as vanillin 87, raspberry ketne 101, and zingerne 102, are imprtant arma cmpunds in fdstuffs and wuld be expected t influence the flavr f Chardnnay samples when they ccur at higher cncentratins than were recrded in this study. The cinnamic acids 118 and 119 are prgenitrs f the vinyl phenls 77 and 78, respectively, thrugh yeast fermentatin (8,9) r by the artifact frmatin f these vlatiles during GC analysis. The fermentative generatin f vlatiles frm 118 and 119 means that these
368 ~ SEFTON et al cinnamic acids can pssibly be imprtant fruit-derived flavr precursrs in wines. Hwever, GC is an inadequate analytical methd t quantify the cinnamic acids r t assess their likely cntributin t wine flavr thrugh 77 and 78. Mnterpenes (Table 3): The cncentratin f mnterpenes (ca 5% f the ttal vlatile cncentratin) in the Chardnnay samples is the lwest fund in any f the white grape varieties that we have examined s far, and this bservatin is cnsistent with the nnflral character f mst Chardnnay wines. The data fr the mnterpenes reprted in Table 3 are similar t thse given by Scienza et al. (36) fr neutral clnes f Chardnnay. The muscat clnes f Chardnnay studied by thse authrs were reprted t cntain higher cncentratins f mnterpenes. The majr mnterpenes bserved in the present study were the flavrless plyls (Z) and (E)-2,6-dimethylcta-2,7-diene-l,6-dils (142, 143), and the ccurrence f these as ubiquitus cnstituents f V. vinifera has been cmmented n previusly. Thse cmpunds fr which sensry threshld data are available (31) were present in bth the free and acidreleased vlatile fractins at cncentratins belw their threshld values. It is unlikely that the mnterpenes culd exert a significant arma influence individually, either n the juice r n matured wines made frm these juices. One further mnterpene, i.e., 2-hydrxy-l,8-cinele 134, a vlatile previusly identified in the variety Sauvignn blanc but nt in Chardnnay (4), was tentatively identified as a minr free and bund cnstituent in the 1988 Chardnnay juice. Aliphatic and miscellaneus cmpunds (Tables 4 and 5): The majrity faliphatic cnstituents reprted in Table 4 were fund as free cmpunds in the juices and attentin was fcused n the free fractin fr an assessment f these cnstituents in Chardnnay. A range f C-4 t C-18 unbranched aliphatic acids, tgether with n-hexanl (147), tw ismeric hexenls (145 and 146), and y-butyrlactne (150) accunt fr mst f the aliphatic fractin f the samples. Of the acids, thse with six t ten carbn chains were present at the highest cncentratin. Mst f the aliphatic cmpunds listed in Table 4 have been reprted befre as cnstituents f several grape varieties, and they cannt be cnsidered as vlatiles specific t Chardnnay. Furthermre, thse cmpunds fr which flavr threshld data are available (45) were present in the juice at a cncentratin belw their detectin threshlds and accrdingly their influence n juice r wine flavr wuld be limited. Mre imprtant are the C-4 t C-10 ~-lactnes, f which the C-8 t C-10 hmlgues are the mst ptent (13). Althugh in abslute terms the cncentratins f these cmpunds were lw, they were each near their detectin threshld, and cllectively, these lactnes may play a rle in Chardnnay flavr. Only the C-8 and C-9 lactnes were fund as enzyme hydrlysis prducts, and verall, higher cncentratins were released by acid hydrlysis. Presumably, glycsylatin plays a minr rle in cnjugating these cmpunds r their precursrs. Kinsella et al. (21) have prpsed that thermal hydrlysis f triglycerides cntaining a hydrxyacid residue leads t the frmatin f lactnes in milk. A similar mechanism may als accunt fr the lactnes fund in the acid hydrlysates. Amng the cmpunds f miscellaneus bigenetic rigin were several that may be cnsidered as chlrphyll degradatin prducts (6) and which were fund mainly in the acid hydrlysates. Three f these, 2-ethyl- 3-methylmaleic anhydride (174), 2-ethyl-3-methylmaleimide (176) and 2-methyl-3-ethylidine succinimide (180), have been islated frm varius leaf and fd prducts and their flavrs have been described (14,17,20,23,28,30,35). 2-Methyl-3-vinylmaleimide(177) has nt, t ur knwledge, been previusly reprted as a natural prduct. Cnclusins Acid hydrlysis f the glycsides gave many prducts different frm thse fund in the enzyme hydrlysates, bth in chemical structure and cncentratin. Acid hydrlysis f the bund fractin is implicit during cellar maturatin f wines, a prcess f particular imprtance t nn-flral varieties such as Chardnnay (1). The vlatiles f Chardnnay are apparently frmed thrugh several secndary metablic pathways. Cmpunds frm branches f the shikimic acid bisynthetic pathway that give armatic amin acids, benzic acids, cinnamic acids, and lignins are present tgether with mevalnic acid-derived vlatiles. The latter cmpunds arise either directly, i.e., as mnterpenes r, in the case f the nrisprenids, frm the apparent degradatin f cartenid pigments. Additinally, sme f the vlatiles are pssibly derived frm chlrphyll pigments while thers cme frm primary metablites f the vine. Cnsidering the disparate rigins f the many cmpunds fund, and the multiplicity f enzymes that are needed fr the synthesis and transfrmatin f these vlatiles, it is t be expected that great variatin culd ccur in the frmatin and accumulatin f individual cnstituents. The bservatin f individually imprtant fd flavrants, e.g., damascenne, 1,1,6-trimethyl-l,2 dihydrnaphthalene, several f the benzene derivatives and the ~-lactnes, suggests their ptential imprtance t Chardnnay flavr. In the samples studied, mst f these cmpunds were present at cncentratins near t r belw their individual sensry threshlds. Hwever, in abslute terms nly a small increase in cncentratin, resulting frm a stimulatin f ne f the bisynthetic pathways discussed abve, culd result in any ne f these ptential flavrants having a directing arma influence. It is imprtant t recgnize that nrisprenids and vlatile benzene cmpunds are als flavr cmpnents f ak wd (Tables 1 and 2), Thus, the cncentratins f many f these natural
VOLATILE COMPOSITION OF CHARDONNAY JUICE- 369 grape cmpunds culd be enhanced during barrel maturatin f Chardnnay wines. This culd accunt fr the harmny that apprpriate wd treatment can bring t Chardnnay wine flavr. In the case f the specific Chardnnayjuices studied here, acid hydrlysis f the glycsides f the 1989 juice gave vlatiles exhibiting sme f the key varietal armas that a wine prepared frm the juice als pssessed, i.e., tea, lime, and hney (15). Thus, the many cmpunds fund in the acid hydrlysate frm the 1989 juice (Tables 1 t 5) are, in the cmbinatin fund, capable f imparting these varietal wine attributes. Althugh the number f vlatiles bserved in the acid hydrlysates precludes sensry studies n individual cmpunds, it is ntewrthy that many f the nrisprenids have been fund in leaf prducts such as tbacc and tea (27). The assignment by the sensry panel f a significant tea arma t the Chardnnay hydrlysates (15) is an indicatin f the likely cntributin f the nrisprenids t this varietal wine flavr attribute. Future investigatins n this variety shuld invlve sensry studies n individual cnstituents within the grup f released flavrants, and thus lead t a mre cmplete understanding f flavr develpment in Chardnnay wines. Literature Cited 1. Amerine, M. A., and E. B. Ressler. Wines, Their Sensry Evaluatin. pp 25-27, W. H. Freeman and C., San Francisc (1976). 2. Becher, E., R. Albrecht, K. Bernhard, H. G. W. Leuenberger, H. Mayer, R. K. Muller, W. Schuep, and H. P.Wagner. Synthese vn Astaxanthin aus B-Jnn. 1. Erschliessung der enantimeren Cls-Wittigsalz dutch chemische und mikrbilgische Racematspaltung vn (+)-3-Acetxy-4-x-B-jnn. Helv. Chim. Acta 64:2419-35 (1981) 3. Behr, D., I. Wahlberg, T. Nishida, and C. R. Enzell. Tbacc Chemistry 50, (3S,5R,8S,9~)-5,8-Epxy-6-megastigmene-3,9-dil and (3S*,5R*,6R*,7E,9~)-3,6-Epxy-7-megastigmene-5,9-dil. Tw new nrcartenids f Greek tbacc. Acta Chem. Scand. 33B:701-4 (1979). 4. Bitteur, S. M., R. L. Baumes, C. L. Baynve, G. Versini, C. A. Martin, and A. Dalla Serra. 2-ex-Hydrxy-l,8-cinele: A New cmpnent frm grape var. Sauvignn. J. Agric. Fd Chem. 38:1210-13 (1990). 5. Bidrn, J.-N., P. Chatnnet, and M. Pns. Influence du bis sur certaines substances derantes des vins. Cnnaiss. Vigne Vin 22:275-94 (1988). 6. Brckman, H., and D. MUller-Ench. Substituted succinic acids. III Abslute cnfiguratin f the 2-methyl-3-ethylsuccinic acids. Chem. Ber. 104:3704-10 (1971 ). 7. Chatnnet, P., J.-N. Bidrn, and M. Pns. Incidence du traitment thermique du bis de ch6ne sur sa cmpsitin chimique. 2 e Partie" Evlutin de certains cmps6s en functin de I'intensit6 de brulage. Cnnaiss. Vigne Vin 23:223-50 (1989). 8. Chatnnet, P., J.-N. Bidrn, and M. Pns. 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