, STUDYTITLE DATA REVPEW IN SUPPORT OF THE REQUEST FOR TEE E? UUWE" PPON OF METHYL ANTHRANILATE FROM THE REQUWEMENTS OF .A TOLERANCE ON ALL RAW AGRICULTUERAL COMMODITIES DATA REQUIREMENTS Food Quality Protection Act.

AUTBOR Leonard R. Askham, PbD.

REVIEW COMPLETED ON April 2, 1992 SUBMITTOR Bird Shield Repellent Corporation P. O. Box 785 Pullman, WA 99163 , I' STATEMENT OF DATA CONFIDENTIALITY CLAMS No claim of confidentiality is made for any information contained in this study on the basis of its falling within the scope of FIFRA lO( d)( l)( A), @) or (C)

Submitter: ' Bird Shield Repellent Corporation Submitting Agent:& eonard R Askham, PhD. Date 6 3 GOOD LABORATORY PRACTICE STATEMENT Bird Shield Repellent  EPA Reg. No. 66550 1 L This submission does not fall within the requirements of 40 CFR Part 160 /

.. .. .. TITLE Product Identity and Composition 1. Product Identity and Composition 2. MerckIndex 3. Registry of Toxic Effects of Chemical Substances Biological Derivation of Methyl Anthranilate 1. 2.

3. 4. Methyl Anthranilate Content of Ohio Concord Grapes Concord Wine Composition as Affected by hditurity and Processing Technique The Odor Quality of Labrusca Grapes An Investigation of the Volatile Flavor Composition of Vitus Labrusca Grape Musts and Wines. I. Methyl Anthranilate  Its Role in the Total Aroma'Picture ofLabrusca Varieties Isolation and Identification of Volatiles from Catawba Wine Methyl Anthranilate as an Aroma constituent of American Wine. Inheritance of Methyl Anthranilate and Total Volatile Esters in yi& SPP Red Wine Aroma: Identification of Headspace Constituents SECTION Tab. A'

Tab. B US/ FDA Registration of Methyl Anthranilate asaGenerallyRecognized as Safe( GRAS) CompoundTab. C 1. 21 CFR 182.60  Synthetic Flavoring Substances and Adjuvants;

2. 2 1 CFR 184.102 1 Benzoic Acid Methyl Anthranilate Methyl Anthranilate as a Flavor and Perfume Ingredient 1 .. Fenaroli's Handbook Qf Flavor Ingredients, Second Edition, Volume 2 2. Perfume Synthetics and Isolates U. S. Department of Commerce Scientific Literature Review of Anthanilates in Flavor Usage. Vol. 1. Introduction and Summary Tables of Data, Bibliography Tab. D Tab. E.

1. Toxicity to Mouse, Rate and Guinea Pig 2. Chemical Identity and Physical Properties 3. Pharmacological and Toxicological Effects 4. NaturalOccurrence 5. Flavor and Extract Manufacturers' Association (FEMA) and 5. Addendum to Table IV 2 7. Bibliography 8. DataGuide National Academy of Science WAS) Use Levels.

AcuteToxicityforMice, Hamsters, Birdsand Fish Tab., F 1  Food Fiavoring and Compounds of Related Structure. I.

2. Acute Oral Toxicity and Repellency of 933 Chemicals to 3. Studies of Drug Adsorption from Oral Cavity: Physio Acute Oral Toxicity House and Deer Mice.

chemical Factors AEecting Absorption from Hamster Cheek Pouch.

998 Chemicals to One or More Species of Wild and Domestic Birds. 5 . Comparison of Fish Toxicity Screening Data and QSAR Predictions for 48 Aniline Derivatives. 4. The Acute Oral Toxicity, Repellency and Hazard Potential of (@ T TAB A. PRODUCT IDENTITY AND COMPOSITION TABLE OF CONTENTS \.%/ '

1 . Product Identity, and Composition 2. The MerckIndex 3. Material SafetyData Sheet 4, Registry of Toxic Effects of ChemicalSubstances, Volume 1 (c) Additional. informtion on iqredient~ (i) a~ chanical name is methyl arrthranilate othersvnorrvms: Anthranilic acid, methyl ester Mew1 2 amhdIemoate Methyl o aminobenzoa~ 2 AminabenZoic acid m y 1 ester (ii) CPS Resistry NLmJser: . 134 20 3 (1985 1986) {iii) StmAud. Forrnrla (i) Methyl an ate is mined synthetically by . . present# of sulfuric acid (see pspenaix 1).

(ii) Fonnilatim of BIRD SKIELD (Refer to i0nfidmtia. l state esterifying anthmdlic add w i t h methyl alcdbol in the ment of fonuila, fonn 8570 4, Confidmtial Attadrment, CnxS F& fer? mce 1). \ !

i.

i I THE MERCK INDEX AN ENCYCLOPEDIA OF CHEMICALS, DRUGS, AND BIOLOGICALS ELEVENTH EDITION Susan Budavari, Editor Maryadele J. O'Neil, Associate Editor Ann Smith, Assistant Ediror Patricia E. Heckelman, Editorial Assistant Published by MERCK. & CO., INC.

R A H W A Y , N .J .. U .S .A .

1989 I 17.96% H 12.08% iN 18.65%. 0 21.30%. CH, NHCH, >H, OH. f h e in vivo precursor of choline. Has been isolated 'rom a mutant of Neurospora c m s n which has lost its ability o synthesize choline: Horowitz. J. Bioi Chem. 162, 413 '1946). Prepd by mixing ethylene oxide with concd methyl mine soh with external cooling: Knorr. Matthes. Ber. 31, 1069 (1898); Lowe ct 02.. Brit. pat. 763,434 (1956 to Oxirane Ltd.); Nikolaev et al., Zh. qbshch. Khim. 33, 391 (1963). Viscous liquid. Fishy odor. dm 0.937. bp, 60 155 156"; >p12 64 659 n$ 1.4385. Miscible with water. alcohol, ether. Zorrosive to skin, cork, metals. Strong base. Forms a deli xuescent salt with HCI. LD, orally in rats: 2.34 glkg, Toxic Substances Lisr. H. E. Christensen, Ed. (1974) p 339. Picrate, yellow crystals, mp 148 150". Caurion: Irritating to skin, eyes, mucous membranes.

minophenol sulfate; p hydroxymethylaniline sulfate; Pho 5940. p Methylaminophenol Sulfate. Monomethyl p

:ol; Verol; Rhodol; Armol; Elon: Genol; Graphol; Photo Rex: Pictol; Planetol; Metol. C,, H, N, O, S: mol wt344.38.

:HOC6H, NHCHJ2. H2SO,.

Sol in 20 parts cold, 6 parts boiling water; slightly sol in alc; Crystals. Discolors in air. mp about 260' with decompn.

nsol in ether. Keep well closed and protected from light. C 48.82%, H 5.85%. N 8.14%. 0 27.87%, S 9.31%.

USE: Photographic developer, dyeing Furs.

vlaniline. C, H, N: mol wt107.15. C 78.46%. H 8.47%. N 5941. Methylaniline. ~~ e~ hglbenze~ ramine: rnonomerh

13.07%. C, H, NHCHJ. Made by heating aniline chloride and methyl alcohol under pressure.

exposure to air. d p 0.989. mp 5579 bp 194 196'. nZ1J Colorless or slightly yellow liquid; becomes brown on 1.5702. Slightly sol in water; sol in alc, ether. LD orally% rabbits: 280 mglkg.

esrer; methyl 2 aminobenzoate; neroli oil, artificial. C, H, 5942. Methyl Anthranilate. 2 Aminobenzoic acid mefhyl NO,; mol wt 151.16. C 63.56%. H 6.00% N 9.27%, 0 other essential oils and in grape juice: also obtained syn 21.17%. Occurs in neroli. ylang ylang. bergamot, jasmine,

thetically by esterifying anthranilic acid with CH, OH in presence of HCI.

water: freely sol in alcohol or ether. LD, orally in rats. Crystals. d 1.168, mp 24 259 bpIs 135.59 Slightly sol in mice: 2910. 3900 mglkg, P. M. Jenner et al.. Fwd Cosmer. . Toxicol. 2, 327 (1964). CSEl As perfume for ointments: manuf synthetic perfumes.

cenediono; 8 methylanthraquinone. C,, H,, O$ mol wt 5943. 2 h lethylanthraquinone. 2. MelhpI 9, f 0 ahthro

222.23. C 81.06%. H 4.54% 0 14.40%. Occurs in teak wood. Prepd by oxidation of 2 methylanthracene; by for

Syn 4, 43 (1925); from 6 and 7 methylanthraquinone l mation from phthalic anhydride and toluene: Fieser. 0%.

carboxylic acids with powdered copper in quinoline: Fiescr. Martin. J. Am. Chem. Soc. 58. 1443 (1436); from 1.4 naph thoquinone and isoprene: Carothers. Berchet, ibid. 55. 2813 (1933).

Needles from alcohol, mp 177". Sublimes. Very sol in benzene, toluene. xylene; sol in alcohol, ether. glacial acetic acid. concd H, SO,; insol in water.

noside; methylarbutoside. C, H, O,; mol wt286.28. C 5944. Methytarbutin. 4 Melhox~ phengl~ D~ lucopyra

54.54%, H 6.347' 0 39.12%. Occurs together with arbutin I~ " HANUFACNREB WHEATEC ' I : ' mD& ss: 2 S. 076 0rchard" Rd. ,,

Wheaton, IL 60187 EMERGENCY TELEPHONE: 708 682 3024 I _. Hethyl Anthranilate CBEHICdL NAXE AND SYNONYMS: Methyl 2  Aminobenzoate *

TRADE NAME AND SYNONYMS: HETHYL ANTHRANILATE, HA  FCC CHpfIcILL FAKILY: Aromatic Acid Ester FOWLA: C8H9N02' 6 4 2 2 . C H NH CO H CAS REGISTRY NUHBER: 134 20 3 DOT SHIPPZHC CLASSIFICATION: Cherni. lc , NO1 PBODUCT NUMBER: .. *

..

S E C T I O N 11 . HAZARDOUS INGREDIENTS M T E R U L : .*

Methyl 99+ TLV (Units)

SECTION 111 . PHYSICAL DATA  r"

ik." d  ,i \? " ..

,: BOILING POINT, 76 e Xg: 206OC 6 FREEZING POINT : 24 *c SPECIFIC CBAVITY: 1.165 VAPOR PRESSURE AT .2OoC. : < lmm Hg VAPOR DENSITY (Air  1): No Data Found SOLUBILITY IN WATER: Approximately. O. 1% X VOLATI& ES BY VOLUME: 0.5% max. a s w a t e r ]E; VAPORILTION RATE : No Data Found * *

APPEARANCE AND ODOR: Colorless t o pale y e l l o w l i q u i d vith a b l u i s h fluorescence, o r w h i t e c r y s t a l s a t room temp.

. I .I".. . . . ...*.    . *. Crape like' odor '. .. .. . . "

..

CTION XV.  FIRE AND EXPLOSION WARD DATA FLASH POINT: . 212'F (CC)

FLAHHABLE LIMITS I N AIR: No Data Found EXTINGUISHING' HEDIA: Carbon Dioxide Dry Chemical, Foam. SPECIAL FIRE FICXTING PROCEDURES:, F u l l p r o t e c t i v e e q u i p m e n t i n c l u d i n g s e l f c o n t a i n e d breaching apparatus should be used. Uater spray'may be i n e f f e c t i v e . If water 1s used, fog nozzels are;,$. referable. Water may be used t o c o o l c l o s e d c o a t a i n e t r t o p r e v e n t p r e s s u r e b u i l d u p .a n a p o s s i b l e a u t o i g n i t i o n or explosion when exposed to 0 .

AUTOIGNITION TEMPERATURE: No. Data Found .. extreme heat. E' .:+ n. uN" suaL FIRE AND \$

\ \+ J EXPLOSION w s ; +Closed containers may explode (due t o the build up of '& ,4 ., '\ I Pressure) wh? n'@ XpOSed t o extreme heat. >. I 1 .. THRESHOLD LIMIT VALUE: None Assigned EFFECTS OF OVEREwosu~~: E x c e s s i v e e x p o s u r e m y c a u s e s k i n a n d eye i r r a t a t i o n . Other t o x i c e f f e c t s a r e unknown. Methyl a n t h r a n i l a t e is included i n t h e GKAS (Generally recognized as s a f e ) l i s t ..

E m ~G m c y AND FIRST AXD PROCEDURES: I F INHALED: If a f f e c t e d , remove. from e x p o s u r e . R e s t o r e b r e a t h i n g . Keep warm and q u i e t . I F ON SKIN: Wash affected a r e a t h o r o u g h l y w i t h s o a p a, nd water. I F IN EYES: F l u s h e y e s w f t h large amounts of water for 15 minutes. Get medical a t t e n t i o n . IF SWALLOWED: Never give anything by mouth t o a n u n c o n s c i o u s p e r s o n . G i v e s e v e r a l g l a s s e s o f water. If vomiting is not spontaneous, induce vomiting by g e n t l y t o u c h i n g t h e back of t h e p a t i e n t s t h r o a t w i t h a f i n g e r . Keep a i r w a y c l e a n . S e e k q e d i c a l a t t e n t i o n .

TOXICITY Oral LDSu (. Rat) 3,000  5,000 mg/ kg.

c SECTION .VI. REACTIVITY DATA. . I

STABILITY: Stable WCOHPATIBILITY: O x i d i z i n g .,

HAZARDOUS DECOHPOSITION PRODUCTS: BY FIKE: Carbon Dioxide, Carbon Monoxide ,. and Oxides of Nitrogen. HAZARDOUS POLYMERIZATION: Will Not Occur. _. . ..

~

. SECTION VI1 SPILL OR LEAK PROCEDURES ..: "

STEPS TO BETAKEN IN CASE THEMATERIAL IS SPILLED OR RELEBEP: ' S p i l l e d material can be c o l l e c t e d a n d h e l d for reclaim or placed in a cov'ered waste disposal c o n t a i n e r .

WASTE DISPOSAL .METHOD: S a n i t a t y l a n d f i l l or i n c i n e r a t e Cr, a p p r o v e d f a c i l i t y . Do not i n c i n e r a t e c l o s e d c o n t a i n e r . Dispose of In accordance with Federal, State , .a n d L o c a l r e g u l a t i o n s r e g a r d i n g p o l l u t i o n . '

~ I"". rrPT SECTION "". V I I I a E C T A L .. PR! XE NJSS% Z$ SN RESPIRATORY PROTECTION: Use a r e s p i r a t o r a p p r o v e d for organic vapors, fumes and mists i n areas laden wtth vapor. VENTILATION: L o c a l e x h a u s t p r e f e r a b l e ,. g e n e r a l e x h a u s t a c c e p t a b l e . . .

PROTECTIVE GLOVES: R e q u i r e d f o r l o n g or repeated contact. EYE PROTECTION: Wear s a f e t y s p e c t a c l e s w i t h unperforated. sideshield6. OTHERPROTECTIVEEQUIPMENT: Eye wash, Safety shower, \

..

. SECTION IX . SPECIAL PRECAUTIONS 1 PRECAUTIONS TO BETAKEN IN HANDLING AND STORAGE: .Do not store neer open flames or o x i d i z i n g a g e n t s . Avoid stor€ ng i n high t e m p e r a t u r e a r e a s r OTHERPRECAUTIONS: Avoid excessive exposure: Do n o t t a k e i n t e r n a l l y .

REVISED: June 17, 1988 SUPEBCEDES: July 20, 1984 ..

,_

.. ., .. ~MEIHYLANIHRANIIATE"

safety data for =thy1 anthranilab summarized in the follwjng reference bo&: in mishy of Toxic Eff O f chemical substances R e g i s t r y of Toxic Effects of chdm substances edited by R. L. Tatken and R. J. Lewis, Sr. 1981 82 edition. U. S. Dept. of Health and Human Services, Public Health service Centers for Disease Control, National Institute for Occupational Safety and fiealth, Cincinnati, OH 45226 me above ref lists the following toxicity data:

mth~ l late CAS RN: 134 20 3 Skin Irritation  it 500 mz/ 24 hours: Merate Q t Edited by Rodger L. Tatken and Richard J. Lewis, Sr.

US. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service Centers for Disease Control National Institute for Occupational Safety and Health Cincinnati, Ohio 45226 June 1983 .. Cnnu b34.71 " " RATAW hU# ".

WECS U6UOGRAPHIC REFERENCES 1981 82 M?? w 1. 2.

3. 4.

5. 6 . 7.

8. Methyl anthranilate Content of Ohio Concord Grapes. Concord Wine Composition as Affected by Maturity and Processing The Odm Quality of Labrusca Grapes. An Investigation of the Volatile Flavor Composition of Vitus Technique.

Labrusca Grape Musts and Wines. I. Methyl Anthranilate  Its Role in the Total Aroma Picture of Labrusca. Varieties. Isolation and Identification of Volatiles &om Catawba Wine Methyl anthranilate as an Aroma Constituent of American Wine. Inheritance of Methyl anthranilate and Total Volatile Esters in Red Wine Aroma: Identification of Headspace Constituents.  vitis spp. a" . ..

' A Research Note Methyl Anthranilate Content of Ohio Concord Grapes JIM WEN R. LIU and JAMES F. GALLANDER ABSTRACT Concordgrapesgrown at five locations in Ohiowerecollected in 1982 and 1983 for analysis of methyl anthranilate (MA) in frearun and heat extracted juice samples. The hiA content of freerun juice ranged from 0.33 2.05 mg/ L in 1982 and 0.14 0.91 mg/ L in 1983. The MA content was higher in heat extracted juice than in frearun juice. Heat extracted juice contained 0.19 2.60 mdL of MA in 1982 and 0.22 3.50 mg/ L in 1983. In general, the MA content in creased with maturity and was highest in grapes grown in the cool est regions.

INTRODUCTION METHYL ANTHRANILATE (MA), the methyl ester of o aminobenzoic acid, is one of the several compounds which contribute to the characteristic aroma of Concord grapes, This compound was used in synthetic grape flavored products before it was found in grapes (Acree, 1981). The measurement of MA. provides basic information for the con trol of flavor quality of Concord grapes and its products. There are several methods available for the determination of MA. These include a colorimetric procedure (AOAC, 1980), a g a s chromatographic procedure (Mattick et al., 1963), and a fluorometric method (Casimir et al., 1976). Robinson et aL (1949) reported that MA of New York Concords developed mainly ih the last stage of maturation and. declined slightly when fruits became overmature. Clore et al. (1965) and Fuleki (1972) obtained similar results with Washington and Ontario Concords, respectively. Clore e t al. (1965) also reported that season, pruning, vineyard location, and type of soil could affect MA content of Corn cord grapes. In Ohio, about 72% of the present acreage is planted t o the Concord cultivar (Cahoon, 1984). Approximately 97% of the Concord crop in Ohio is used for processing. The purpose of this study was to determine the MA content of Ohio Concord grapes as influenced by season, vineyard location, and maturation.

MATERIALS & METHODS Sample collection Fruit samples were collected in the 1982 and 1983 seasons from five different vineyard locations in Ohio. Ai each location 10 vines were selected for harvest a t three different levels of maturity. me fust level of maturity was chosen as the time when all berries had completedcolorchange( post veraison). Thesecondandthird maturity wereabout 1 2 and 2 4 wk, respectively, afterthe first sampling. The grapes were harvested a t increments of approximately 2'Brix when possible. For each maturity. two berries were randomly taken from each of the middle 20 clusters, located in the upper portion of the fine. The berries from the 10 vines were pooled, and a total of 400 benies Were collected for each replicate. Duplicated samples were kept in plastic containers and stored overnight at 4° C.

Authors Liu and Gallander are affiliated with the Dept o f Horticul ture. Ohio Agricultural Research & Development Center, The Ohio state Univ., Wooster. OH 446993.

280 JOURNAL OF FOOD SCIENCE Volume 50 (1985) Sample preparation Berries (100) from eachreplicatewerepassedthrough, press (Squeezo strainer) to obtain free run juice. Approha mL free run juice was centiifuged on an IEC 140 rotor at mately 900 x g for 10 min to remove gross particles. natant was analyzed for soluble solids (" Brix) with a refrac Thejuice was then stored in glass containers at 17° C u analysis could be performed. Heat extracted juice was prepared by macerating appr 200g of berries in a Waring Blendor at low speed for 1% macerate was placed in a 250 mLbeaker, covered with glass, andheated in a water bath at 85° Cfor 1 hr. macerate was cooled to 40° C and drained through cheesecl heat extracted juice was then centrifuged as described for run juice and the supernatant was decanted and stored at glass containers The frozen juice samples of both freerun and heat e x n a thawed and brought to room temperature prior to analyk fa Analysis of MA The fluorometric procedure of Casimir et d (1976) was the determination of MA. A 10 mL sample of grape juice wa distilled in a Cash distillation assembly (Research and Devel Products, Berkeley, CA) rather than in a micro Kjeldahl app as described by Casimir et aL (1976). Preliminary experiments indicated that in order to use the assembly for steam distillation, a slight modification on the p dure of Casimir et aL (1976) wasnecessary to obtain more 96.0% recovery of MA. Approximately 76 mL of distillate lected in 8 min in a 100 mL volumetric flask containing 20 m pH 7 buffer. Thebufferwaspreparedaccording to Carimj (1976). The collected distillate was brought to vo1um. e wilh distilled water. The temperature of the distillate was kept at 1° C. Thefluorescence of the distillate was then measured Model J4 7439 Fluoro colorimeter( AmericanInstrumen SilverSprings, MD) equippedwith a primaryfilter (pea mission at 360 nm) and a. secondary filter (at 415 nmand The fluorometer was calibrated with standard solutio anthranilate. The standard eqor of the mean of three M A ?O. OlQ mg/ L for a grape juice samplecontaining 1 with a tom1 of 300 mLglass distilled water. The Cash assemblywasflushedbetweensamples three RESULTS & DISCUSSION RESULTS indicated that there was a large variation in content between two replicated juice samples. For exa the MA content of heat extracted juice collected a on 9/ 9/ 82 was 0.75 2 0.40 mg/ L, and the MA c free run from Wooster on 9/ 14/ 83 was 0.23 k (Tables 1 and 2). Fuieki (1972) reported that cons1 variability was observed when juice t o be analyzed was tained by pressing the grapes. To reduce variability, he ommended the use of whole grapes instead ofjuice steam distillation.

2.05 mg! L in 1982 and O714 O. 9i mg/ L in 1983. For extracted juice, MA content varied from 0.19 2.60 mgi 1982 and 0.22 to 3.50 mg/ L in 1983. In general, heat traction increased MA coritent in the juice. Rice (1975) ported that hot pressed ,,(= oncord juice contained mu higher MA than did cold:$ ressed juice. Clore et d (196 analyzed the MA content rjf Washington Concords. fhe M The MA content of free run juice ranged from 0.3 .

8 . z* 2753 9t7 13.9 ! S IA 2948 911 5 15.7 0.77 2 0.09 1.16 t 0.06 OB5 t 0.02 0.72 t 0.03 23 18 a m 2424 9/ 7 2593 9/ 15 12.4 0.67 2 0.05 1.00 i 0.12 14.5 1.13 f 0.06 15.2 1.34 i 0.16 0.93 t 0.08 1.02 i 0.20 0.33 t 0.04 0.19 3 0.02 0.69 t 0.02 0.75 3 0.4Q . 15.6 1.76 t 0.04 2.22 2 0.60 0.91 2 0.05 0.00 f 0.12 1.87 t 0.12 0.54 t. o. oa 1891 8130 11.7 201 2 919 21 92 9/ 28 2183 911 14.2 2372 911 3 2479 9/ 23 15.1 1.66 t 0.04 15.9 2.05 2 0.20 2087 9t9 14.0 0.70 + 0.12 13.4 1.52 t 0.18 .~ 11BC 0.42 3 O. 0Zd 0.55 * 0.01 2272 9123 15.0 "  1.55 i 0.33 2.60 t 0.05 2414 1015 17.4 1.34 2 0.01 1.63 3 0.03 . of degrees above 50° F within the state from

April to the date of harvest; 1. c.. X: t( dally ~~

maxlmum daily mlnlmum)/ 2 50].

*~y f duplicate F #* , dup{ icates t standard error of mean f " Degree Date Soluble s { of solids MA content (rng/ L) wm'vd #: ion daysb harvest P ~r i x ) Free run Heat extract 2836 911 12.6' . 0.17 f 0. Old 3133 0.28 i 0.00 3299 9/ 21 .16.4. 0.18 t 0.06 0.48 f 0.00 0.22 t 0.01 2552 916 271 9 911 4 13.7 0.14 2 0.02 0.42 i 0.1 3 15.1 0.22 + 0.04 912 1 0.62 i 0.06 16.3 0.30 : 0.02 0.70 3 0.02 2214 916 14.6 0.23.2 0.08 911 4 089 t 0 01 17.0 2448 912 1 18.1 0.41 t 0.13 1.69 t 0.05 0.63 k 0.1 81.90 t 0.1 0 2507 917 14.1 2668 0.28 t 0.02 *I 911 5 0.74 i 0.02 15.7 279 1 9/ 22 OS1 i 0.06 1.97 t 0.03 034 t 0.09 0.44 f 0.04 056 t 0.01 1.70 t 0.14 0.91 t 0.06 350 i 0.1 0 ,I 9/ 12, 14.9 0.16 i 0.02 .. cpcs3 2840 antral) 2352 "

1 16.1 0.70 i 0.05 232 + 0.12 E 2377 9/ 8 129 2555 9/ 19 155 2624 9/ 27 16.6 r of degrees above 50'F region wltnin the state from April to the date of harvest; Le., Z[ (aally cIU of duplicate & O! duplicate f standard error of mean c 5 m e a t for the whole grape, skin, pulp. juice drained from central Ohio vineyards (Morrow and Columbus) tended to i. w. and juice drained from skins was 6.0, 5.8, 1.8, 2.2 be lower than those from northern Ohio (Wooster, San @ Zd ppm, respectively. They concluded that most of the dusky, and Madison) at comparable levels of maturity. :. w h y 1 anthranilate was, located in the skin fraction. Clore et a1 (1965) reported the effect. of vineyard location fr. gcneral, the MA content increased with maturity. This on MA content, but noticed that soil type, pruning severity, P m weement with the findings of other researchers (Rob and solar radiation could also influence MA content. Based t t at.. 1949; Clore et al., 1965; Flueki, 1972). Robin an the total number of degree days, the MA content seemed a t t al. (1949) reported that MA content of New York to be related to the regional climate. Grapes from colder PrRn .. Concords increased during ripening and decreased regions contained higher concentrations of MA. The MA . ftlt when the fruit became overmature. However, Clore content in 1983 was generally lower than that of 19S2.

4 @ d (1965) found that MA content did not necessarily This was apparently due to a warmerseason in 1983. ; k

bQnc when grapes became ' overmature. In this study, were some decreases in MA contrni in 1982 from the c m a t u r i t f t o t h e t h i r d m a t u r i t y f o r grapes from the REFERENCES '* 4mk and Madison vineyards. Whether this Kas due to . A ~~~, T. E. 1981. The odor quty ofi& rusca =apes. ACS s Y m p ~

m i n a t i o n of MA beyond the third maturity. No de AOAC. 1980. "Official Methods of Andy&" 13th e a Association 1  in MA content was observed for samples collected in ' Cahoon. G. A. 1984. Private communication. Horticulture Dept. i.: m 3 *ason. Ohio Agriculturd Research 6 Development Center. Ohio State 'k MA content of Concord grapes from southern and ', ... . casimir. D. 3.. Moyer, J. c, md Mattick. L. R. 1976. Fluorometric % Univ.. Wooster. OH.

Volume 50 IISBSI JOURNAL OF .FOOD SCIENCE 281 Pa &coming overmature is not clear, since there was no sium Series 170: 11.

of Official Analytical Chemists. U'ashington, DC.

.. determination of methyl anthranilate In Concord grape juict. Daw varietier Ch. 4 In "Chemistry of Winem" JAOAC 69: 269. Clore, W. J., Neubert. A. M., Carter: G. H.. Ingalsbe. D. W.. and Bmm Robinson. W. B.. Shaulis. N. J.. and Pederson. C. S. 19 mund. V. P. 1965. Composition of U'ashington uro& aced Concord studies of grapes grown for juice manufactum. F m pap .nd juices. Wash. Agr. ESP. Sta. Tech. Bull. 482 96.

papes grown io Ontdo during ripening in the 1970 season. Can. J. Plant ScL 52: 863. Matti4 L. R.. Robinson. W. B.. Weirs, L. D., and Barry. D. L. 1963. Determination of methyl anthranilate in grape juice by electron Rim. A. C. 1975. Chemistry of a inemaking from native American dfinfty gas Chromatography. 1. Amic. Food Chem. 11: 334. Webb. P. 88. Am. C h c a SOC. Adv. Chem, Sex. 197. . a F d e a T. 1972. Changes in the chemical composition of Concord Ma received 7/ 13/ 84; accepted 8[ 27/ 84. . :*

KINETICS OF WATER UPTAKE BY FOOD PRODUCTS.. . From page 279 REFERENCES Baumann. €I. 1966. Apparatur nach Baumann Zur bestimmung der Anstrichm. 68( 9): 741. flussigkeit soufnahme von pulnigen substanzen. Fette, Seifen,

Gerschenson. L. N.. Boquet. R.. and Bartholomai. G. B. 1983. Ef fect of thermal treatments on the moisture sorption isotherms tinurn L.). Lebensm. Wiss. u. Technol. 16: 43. of protein isolate. starch and flour from cheackpea (Cicer arie

Hermansson, A. M. 1972. Functional properties of proteins for foods. Swelling. Lebensm. Wiss. u. Technol. 5( 1): 24. Kuntz, D. A., Nelson, A. 1 ... Steinberg, M. P.. and Wei. L. S. 1970. Control of chalkmess in soymilk. J. Food Sci. 43: 1279. Pilooof. A. M. R., Bartholomai. G. B. and Chirife. J. 1982. Kinetics of nitrogen solubility loss in heated flour and protein isolates Rdston. M. L. and Jennrich, 1 1978. Dud. a derivative free algo from bean, Phaseolus vulgaris. J. Food Sci. 47( 1): 4.

Romo. C. R. 1980. The extraction characterization together with rithm for nonlinear least squares. Technometrics 20( 1).

nutritional and technological properties of protein contained in chilean bean and rapeseed meal. Thesis. National CoUepc ol,

Torgarsen, H. and Toledo, R. T. 1977. Physical propenies Technology. X'eybridge.

preparations related to their functional characteristics g' "4\ : nuted meat systems. J. Food Sei. 42( 6): 1615. cpcc :f Urbanski. G. E.. Wei. L. S'. Nelson. A'I.. and Steinberg. M p . Flow characteristics of soybean constituents contro& ed'b, '%

Ms received 3/ 14/ 84; reevked 7/ 21/ 84; accepted 9/ 24/ 84. of total to imbibed water. J. Food Sei. 48: 691. %.& a*

assistance, and Dra. 0. Kith i s heartily thanked for her & The authors ais0 acknowledge Millipore Co. for their est in this work and for valuatile comments. I i I . l$ V*

Richard R. Nelson and Terry E. Acree Respectively Graduate Research Assistant and Associate Professor of Biochemistry, Department of FoodScience, New York State Agricultural Experiment Station, Cornell University, Geneva, New York 14456.

The authors gratefully acknowledge the gift of Concord pigment from G. Hrazdina, assistancein the sensory analysisfrom R. M. Butts and 1. D. Tyler, andstatisticalas sistance from J. Barnard.

This manuscript approved by the Director of the New York State Agricultural Experi

Presented at the Annual Meeting of the American Society of Enologists, June 23,

Received June 23,1977.

Accepted for publication January 17,1978. ment Station for publication as Journal Paper No. 3123.

1977, Coronado, California.

2 ? ABSTRACT I Concord grapes were harvested from the vine trifluoroethane). Compounds in the solvent extract vards of the New York State Agricultural Experi mere separated by gas chromatography and their f kent .Station, Geneva, during the 19'76 vintage. odors evaluated using a sniffing device attached I \vines \vere prepared by three techniques commonly to the gas chromatographic effluent port. Nineteen . for producing Concordmines of various styles. compounds were identified by combined gas chroma

.. A ,vhite \vine was prepared from 12" Brix fruit that tographyjmass  spectrometry. The wines showed pressed immediately after crushing, a red wine vastly different varietal characters that cannot be , '.. as prepared from 16" Brix fruit that was crushed explained by variation in methyl anthranilate con fermented on the skins, and another red wine , centration alone. Sensory analyses were conducted was prepared from 16" Brix fruit processed by in order to examine the correlation of odor intensity ' thelmal vinification. All musts were ameliorated in with methyl anthranilate concentration and total to produce finished wines containing 12:; volatile concentration. This report. demonstrates that v ,' ~ ethanol. The flavor components of each wine the volatile composition of Concord wine differs sig . r \\ ere extracted with Freon 113 (1,1,2 trichloro 1,2,2 nificantly with maturity and processing technique. t i REVIEW OF LITERATURE pounds have been identified by Holley et al. (6), Literature is extensive on the flavor composition Stevens e t a]. (18) Neudoerffer et a]. (lo), and. \ of \f ine and other a] coho] icbeverages. The corn Stern et d. (16). Particular attention has been given pounds identified have been thoroughly reviewed bs to the role of methyl anthranilate in the varietal i. Kahn (7) and by Webb and Muller (19). There has Chal. aCter of native h e r i c a n Varieties in work b; v l been little investigation, however, into the effects of Scott (131, Sale and U'ilson (12), Fufeki (41,

. ' the \volatile composition of table wines. Shaulis and This report examines the volatile composition of ; Robinson ( 14) noted the importance of seasonal Concord wine by instrumental and sensory means as by fruit maturity and by enological tech

' ' season, maturity, and fermentation technique on l% xIman (3), and Nelson et a]. (9).

t 'hariation on methyl anthranilate concentration in affected ,' Concord and Fredonia grapes. Stevens et al. (17) nique '. compared volatiles isolated. from Grenache juice and ' 1 , from Grenache Rose wine. Hardy (5 ) examined MATERIALS AND METHODS :'flavor development in Muscat of Alexandria grapes . Wine preparation: The experimental wines were ' ,,, during ripening, and Stern et al. (15) followed cow . prepared from 40 kg lots of Concord grapes har k$? sitional changes of Zinfandel \Tine during aging. vested during the 19T6 \ intage. The fruit was har

\A,.; The volatile composition ofConcord juice has vested by hand, separated from the steqs and leaves 1 studied by several workers. A total of 85 com in a modified Healdsburg stemmel (S ), :and crushed 83 .' ..

..

. . Am. J. Enol. Vitic., VoL 23, No. 2,1978 ..

'! I  ~ ~ ~~~ ". ~ ""

b ~u I Y L V ~~U WINE COMPOSITiON : 4 in a fluted roller type crusher. Sulfur dioxide was added as potassium metabisulfite to a level of 100 ppm. All musts were ameliorated with sucrose to 21" Brix and then fermented with a Montrachet 522 ... dry yeast inoculum. Enologicai variables were as follows: a) A white wine was prepared from Con cord grapes harvested at 11.9" Brix. Immediately Lw, ' after crushing, the juice was expressed with a hy draulic rack and cloth press. The must was settled for 12 hours, and the clear portion was then racked into glass fermentors and inoculated. b) A red wine was prepared by fermenting crushed 16" Brix Con cord grapes in contact with the skin for five days. The skins were then removed, ameliorant was added, and the fermentation was completed in glass fer mentors. c) The third sample was prepared by bringing crushed 16" Brix grapes to 60° C for 15 minutes in a steam kettle. The juice was then drawn off and settled for 12 hours, and the clear portion was racked into glass fermentors, ameliorated, and inoculated. \

Sofventextraction: The volatile components .of the ,finished wines were isolated by solvent extrac tion with Freon 113 (1,1,2 trichloro 1,2,2 trifluoro ethane). Equal parts (3000 ml) wine and solvent were stirred for 30 minutes, and the solvent layer was removed, dried over anhydrous .MgSO,, and finally concentrated 12,000 fold in a rotary evapo rator at 20° C. instrumentalanalysis: Organoleptic analysis of the GC effluent was done on a Packard 800 gas 1::; chromatograph with a 4 m x 2 mm glass column 2 packed with 10% SP 1000 on. Chromosorb W. The c, sensory character as each compound emerged was as sessed .with a sniffing device attached to the effluent port of the GC (1). Volatile components were identi fied with a Varian 1400 gas chromatograph equipped with a similar column and interfaced to a Bendix 12 time of flight mass spectrometer. The eompo nents were quantified with a Hewlett Packard 5830 A gas chromatograph with a 4 m x 2 mm stainless: steel column and the same packing material. The internal standard was dodecanol. . .

Sensory analysis: Sensory analyses were made by the seven member wine variety eyaluation taste panel of the New York State Agricultural Experi ment Station. Two questions were asked: 1) are there detectable differences between samples ; and 2) what is the magnitude of any differences? Com pletely randomized triangle tests were used for the first analysis, and scoring on an unstructured scale was used for the second. Several other samples were included for reference : White Riesling, Delaware, Catawba, and. ,two synthetic samples prepared by adding the 19 compounds identified by GC/ MS in amounts found in the thermally vinified sample to a solution of 12% v/ v ethanol and 0.75oJo w/ w ,._". tartaric acid in distilled water. Methyl anthranilate i was omitted from one of the synthetic samples. To (\< exclude bias due to color differences, pigment iso . .I iated from Concord grape juice was added to an samples until the color matched the thermally pipi. f ied sample.

RESULTS AND DISCUSSION Fig. 1 shows typical chromatograms of the &re, Concord extracts. A total of 64 compounds were de. tected in the cold pressed white wine (coded Cpw), 64 compounds in the red wine fermented on the skins 21 Fig. 1. Chromatograms of 12,000 fold essenceextracted from cold pressed white, fermented on skins red, and thermally vinified red Concord wine.

L y ,

Am. J. Enol. Vitic., Voi. 29, No. 2, '1978 .,

.. .~ , "..

Table 1. Volatile composition ofConcord wine. R "

i Cornpound Cold Fermented Thermally Retention pressed on skins vinified time (W ) (w l l ) (W l ) (mid "

trt;, 4 acetate a Is.? butyl acetate f: hyf butyrate 1 tsjamyl acetate . b~ amyt alcohol Ethyl hexanoate i Hexyl acetate Ethyl lactate ' i : .:`: '::?. Hexanot ..

Diethylsuccinate Phenethyl acetate [ Hexanoic acid '. Phenethyl alcohol ' Octanoic acid 250 29 140 1400 61 00 730 ndb 280 20 370 50 1 20 1800 9000 41 0 36 340 140 5 49 1300 470 460 120 280 340 130 ' 96 1900 1200 5200 4300 6300 2400 51 0 200 170 6200 8900 630 57 230 170 41 1100 640 240 860 1600 71 00 5600 4.3 7.4 8.0 10.9 13.8 14.7 16.0 18.4 18.6 19.7 21.8 28.7 29.8 34.6 34.8 37.6 43.1  Methyl anthranilate 660 380 560 553  Decanoic acid 2200 640 2300 57.7 *Concentration of compounds calculated ona single strength wine basis as determined with dodecanol used as the internal ? standard.

* nd = not detected.

Table 2. Classification of .volatile components of Concord wine.

.. .. ,

i Processing techniques Class CPW OSR TVR 7.8 Acids 10.4 4.2 ' 9.5 Alcohols 11.3 13.5 ?.. : . .. 16.2 .. . Esters 3.9 2.2 3.6 &:. d Total essence 27.4 22.3 37.1 i Acetates 1.8 (ppml 2.4 i .' .

CpW, `cold pressed white; OSR, on the skinsred; N R , ther mally vinified red. .. .,

.. Am. 3. Enol. Vitic., CONCORD WINE COMPOSlTlON 85 triangle tests. The results are given in Table 3. Six '

of the seven panelists could successfully distinguish CPW from OSR, all seven panelists could distinguish OSR from TVR, and five panelists could distin guish CPW from TVR. The respective results are significant at p = 0.01, p = 0.001, and p = 0.05 (2). The intensity of typical "American" or "la brusca" character was investigated by scoring on an unstructured scale (Fig. 2) from 0, no detectable "labrusca" character, to 20, extremely intense "la brusca" character. The compiled data were evalu ated by a two way analysis of variance followed by Duncan's multiple range test. The results are shown in Table 4. It is apparent that the white wine made from low Brix grapes is significantly lower in "la brusca" character than the other Concord samples. Although it is not surprising that the Catawba wine was scored relatively high in "labrusca" char acter, it is interesting that the methyl anthranilate concentration of that sample was less than 0.1 ppm. It can also be seen that the synthetic wines were poor imitations of "1abrusca" flavored wines, re gardless of the presence or absence of methyl anthranilate.. Statistically, the CPW wine has no more "labrusca". character than the Delaware or the White Riesling wines. That appears to confirm the thought that the compounds responsible for Concord varietal character are formed during the latter stages of maturation. Although Robinson and Shaulis (11) showed that to be true for methyl anthranilate formation, this work indicates that it is not responsible for varietal character. Although both red wines have distinctive. `` la brusca" character, they are quite different from each other. The compounds responsible for the difference have not yet been positively identified. It seems, however, that the extremely high level of acetates in the thermally vinified wine, particularly isobutyl, isoamxl, and phenethyl, may play a role. Table 5 lists the acetates detected and the relative amounts found in the two red Concord wines. Each com pound has a strong fruity or spicy aroma that could contribute to the character of the thermally vinified wine.

Table 3. Varietal character: difference test.

(Randomizedtriangletests) Comparison % correctresponse Level of significance CPWa vs. OSRb 86 .01 CPW vs. TVRc 71 .05 OSR vs. TVR loo . .001 a Cold pressed white wine.

Red wine fermented in contact with skins.

e Thermally vinified red wine.

I Vol. 29, No. 2,1978 .. ~~ ~~

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.. 86 CONCORDWINECOMPOSITION

Table 4. Analysis of variance and Duncan's multiple range test.

Sample Treatment Mean Concord Therm. vinif. 14.43 Concord Ferm. on skins 12.36 Catawba Cold press 11.36 Concord Cold press 7.07 Delaware Cold press 6.21 Riesling Cold press 5.64 Synthetic No met. anth. 5.07 Synthetic With met. anth. 1.93 ____

Error df = 42, Error ms = 30.1477, F = 4.2000, LSD = 6.0397.

In conclusion, the thermally vinified wine had a higher odor intensity than the other wines. It also had a higher level of total extractable volatiles. Even so, the white wine was significantly lower in odor intensity yet had a higher level of extractable volatiles than the wine fermented on the skins. The lower level of total volatiles in the sample fermented on the skins did not appear to affect odor intensity significantly. Thermal vinification, through the production of high levels of acetates or other compounds, yields a wine with maximunl intensity of Concord aroma. It can be concluded, however, that the Concord varietal character is composed of one 01 more trace compo nents that have yet to be identified and that these compounds are formed during the latter stages of maturation. . The nineteen compounds identified, including methyl anthranilate, constituted over 90% of the total extractable essence. These compounds certainly contribute to the "\* inous" character of Concord wine but make little 01 no contribution to Concord varietal character. Identification of the compounds responsible for the varietal character in Concord and other wine varieties will rest yith the identifica tion of trace components present in pg/ 1 quantities.

ODOR EVALUATION OF ''AMERICAN CHARACTER Taster Date Directions  Examine the odor of each wipe: Rate the intensity of typical "American" character according to your own defini tion and experience by placing a vertical line at the appropriate position on the scale provided..

Sample "American" Low High 71 23 "

44 60 Fig. 2. Unstructured scaleused in scoring intensity of "labrusca" odor in experimental wines. Responses were con verted for statistical analysis using a20 pt grid. 1 1 Table 5. Relative acetate concentration in red Concord wine.

CompoundFerrn. on skins Therm. vinification X in,; ~

Ethyl acetate ". 38 Isobutyl acetate 50 200 300 Isoamyl acetate 1 E? 6200 244 Hexyl acetate 36 57 96 58 Phenethyl acetate 860 796 "

370 510 \

LITERATURE CITED 1. Acree, T. E., R. M. Butts, R. R. Nelson, and C. y. L ~~, ,Sniffer to determine the odor of gas chromatographic efil,,. ents. Anal. Chem. 48: 12,1821 (1976). 2. Amerine, M. A., and E. B. Roessler. Wines: Their SensorV Evaluation. W. H. Freeman and Co., San Francisco. Appendix D, p. 181 (1976). 3. Friedman, t. E. Concord grape quality. N. Y. S. Nortic. sot. Proc. 121: 132 6 (1976). 4. Futeki, T. Methyl anthranilate and total volatile estero content of grape cultivars grown in Ontario. hpubl. Paper presented at the, Annual. Meeting of the American Society of Enologists, EasternSection, Erie, Pennsytvania, August, 1976. 5. Hardy, P. J. Changes in volatiles of Muscat grapes dur ing ripening. Phytochemistry 9: 709 75 (1970). 6. Holley, R. W., B. Stoyla, and A. D. Holley. The identifica. tion of some volatile constituents of Concord grape juice. Food Res. 20: 326 30 (1955). 7. Kahn, J. H. Compounds identified in whiskey, wine, and . beer: a tabuiation. J. Assoc. Off. Anal. Chem. 52: 1766 78( 1969). 8. Moyer, J C. An experimental grape stemmer. Farm Res. 23: 1,15 (1957). 9. Nelson, R. R., T. E. Acree, C. Y. Lee, and R. M. Butts., Methyl anthranilate as an aroma constituent of American wine. J. Food Sci. 42: 57 9 (1977). IO. Neudoerifer, T. S., S. Sandler, E. Zubeckis, and M. D. Smith. Detection of an undesirable anomaly in Concord grape i by gaschromatography. J. Agric. FoodChem, 13( 6): 584 8 (1965). 11. Robinson, W. B., and N. J. Shaulis. Ripening studies of grapes grown in 1948 for juice manufacture. Fruit Prod. J. Am. . Food Manuf. 29: 2.36 7,54,62 (1949). 12. Sale, J. W., and J. 8. Wilson. Distribution of volatile flavor in grapes and grapejuices. J. Agric. Res. 33: 307 10 13. Scott, R. D. Methyl anthranilate in grape beverages and flavors. Ind. Eng. Chem. 15: 732 3 (1923). 14. Shaulis, N. J., and W. B. Robinson. The effect of season, i pruning severity, and trellising on some chemical character : istics of ConcordandFredonia grape juice. Proc. Am. Hortic. .

15. Stern, 0. J., G. Guadagni, and K. L. Stevens. Agingof wine: changes in Zinfandel volatiles. Am. J. Enol. Vitic. 26:

16. Stern, 0. J., A. Lee, W. H. McFadden, and K. L. Stevens. Volatiles from grapes: identification of volatiles from Concord essence. J. Agric. Food Chem. 15: llOO 3 (1967). 17. Stevens, K. L., R. A. Flath, L. Alson, and D. J. Stern. Volatiles from grapes: comparison of Grenache juice and Grenache Rosewine. J. Agric. Food Chem. 17: 1102 6( 1969). 18. Stevens, K. L. A. Lee, W. H. McFadden, and R. Teranishi. Volatiles .from grapes: some volatiles from Concord essence. J Food Sei. 30: 1106 7 (1965). 19. Webb, A D., and C. J. Muller. Volatile aromacompo nents of wines and' other fermented beverages. Adv. &PI. Microbiol. 15: 75 146,( 1972). (1926):

SOC. 62: 214 20 (1953).

208 13 (1975). 1 Am. J. Enol. Vitic., Vol. 29, No. 2,1978 ..

.. :.. . _... . . .. . x .. .. . . .. . . .  . . ..,_ 2 The Odor Quality o f Labrusca Grapes TERRY E. ACREE New York StateAgricultural Experiment Station, Cornell University, Geneva. NY 14456 C u l t i v a r s o f t h e s p e c i e s vinifera are the most p r e v a l e n t g r a p e s p l a n t e d i n t h e w o r l d . O r i g i n a l l y grown i n Europe and Asia . v i n i f e r a g r a p e s . o f w h i c h ,t h e r e a r e s e v e r a l thousand named c u l t i v a r s (i ). a l s o d o m i n a t e t h e v i t i c u l t u r e of the New Vorld. In North &erica. however, two o t h e r s p e c i e s of (h b r u s u , Bailey and L p t u n d i f o u . Hicha'ux) are growo i n s i g n i f i c a n t q u a n t i t i e s (?). The s e v e r e c l i r c a t e a n d v i r u l e n t d i s e a s e i n c e n t r a l and e a s t e r n N o r t h America made early c u l t i v a t i o n o f v i n i f e r a g r a p e s l a r g e l y m s u c c e s s f u l , a t l e a s t i n t h e E n g l i s h C o l o n i e s . T h i s s t i m l a t e d t h e h y b r i d i z a t i o n and c u l t i v a t i o n o f t h e c o r e t o l e r a n t n a t i v e g r a p e s p e c i e s f o r many years. With t h e d e v e l o p e c t of n e t h o d s f o r d i s e a s e c o n t r o l acd the expansion of v i t i c c l t u r e i n t o c l i m a t e s more amenable t o .v i n i f e r a g r a p e s , t h e p e r c e n t o f n a t i v e s p e c i e s h a s d e c r e a s e d  t o l e s s t h a n f i v e p e r c e n t of the total Korth American .grape production (2 ). Alth6uL. h dwarfed by t h e s i z e of t h e v i n i f e r a g r a p e c r o p , i n excess of 4.000.00@ t o n s , t h e p r o d u c t i o n af labrusca grapes has increased i n the last 30 y e a r s . T h i s c o n t i n u e d d e n a c d f o r l a b r u s c a g r a p e s is due t o t h e i r s u p e r i o r q u a l i t y for the production of grape juice and j e l l y . I t is t h e u n i q u e f l a v o r o f labrusca grapes. and i n p a r t i c u l a r t h e c u l t i v a r Concord; that i s r e s p o n s i b l e f o r t h e i r s u p e r i o r i t y . I n f a c t . t h e . f l a v o r o f labrusca grapes has . become t h e s t a n d a r d of i d e n t i t y €o r g r a p e juice and j e l l y i n n o r t h America. TtiE m O R OF LaBBllscBsBhEEs The major flavor differences among grape prcducts nade with d i f f e r e n t s p e c i e s o f g r a p e s a r e f o u n d i n t h e o l o r . It is e a s y t o d i s t i n g u i s h t h e o d o r s of g r a p e j u i c e s made from vinifera. l a b r u s c a . a n $ m u s c a d i n e s p e c i e s . I n c o n t r a s t , t h e t a s t e of g r a p e s , t h a t is t h e i r s w e e t n e s s . . sourness, and b i t t e r n e s s . is frequently manipulated by p r o c e s s o r s t o t h e e x t e n t t h a t a:; y t z s t e difference among grapes of l i f f e r e n t s p e c i e s a r e o b l i t e r a t e d . The one word which has been used €o r c e n t u r i e s t o ' d e s c r i b e t h e o d o r c h a r a c t e r o f n a t i v e g r a p e s is foxy". In f a c t e a r l y ..

009~ 6~ 5~/~ 1/ 0170~ 01 lSOS. OO/ O 0 1981 American Chemical Society 12 QUALITY OF SELECTED FRUITS AND VEGETABLES 2. ACREt European v i s i t o r s t o t h e New World named t h e n a t i v e l a b r u s c a g r a p e s g r o w i n g i n New England the Northern Fox G r a p e , a n d t h e grapes growing i n t h e S o u t h t h e S o u t h e r n Fox Grape. The o r i g i n a l meaning of t h e word "foxy" was e x p l i c i t l y s t a t e d i n 1722 by Robert Beverly when h e d e s c r i b e d the smell o f .t h e s e g r a p e s a s resembling t h a t o f a fox(&). This musky a n i m a l l i k e a r o n a is s t i l l c o n s i d e r e d a n u n d e s i r a b l e a t t r i b u t e i n &r a p e s . b u t "f o x y " m u s t not b e c o n f u s e d w i t h t h e p o w e r f u l f r u i t y I f l o r a l , o r candy o d o r s e s s e n t i a l t o t h e q u a l i t y o f g r a p e j u i c e a n d j e l l y . Even t h o u g h t h e s e o d o r s may n o t b e a p p r e c i a t e d i n most wine types, they are n o t t h e same as f o x i n e s s . T h e c u l t i v a r s of w r u s c m (Concord, Catawba. Delaware, etc.) were appreciated by e a r l y A m e r i c a n g r a p e b r e e d e r s b e c a u s e o f t h e i r g e n e r a l l a c k of foxiness. However, Niagara i s t h e o n e r e m a i n i n g . counercial c u l t i v a r w h i c h h a s a l v a y s b e e n c o n s i d e r e d f o x y . T h e r e f o r e , f o x i n e s s s h o u l d n o t b e c o n s i d e r e d t h e d o m i n a n t o d o r q u a l i t y o f L n b r u s c a g r a p e s . I n t h i s p a p e r , t h a t o d o r q u e l i t y c o m o n t o 611 f a b r u s c a g r a p e s will be r e f e r r e d t o a s "l a b r u s c a c h a r a c t e r . D u r i n g t h e s e n s o r y s t u d i e s o f h u n d r e d s of d i f f e r e n t g r a p e c u l t i v a r s c o n d u c t e d a t t h e E x p e r i m e n t S t a t i o n i n Geneva, New `iurk. p a n e l i s t s h a v e , w i t h g r e a t r e g u l a r i t y , i d e n t i f i e d t h e presence of l a b r u s c a c h a r a c t e r i n t h e o d o r of l a b r u s c a g r a p e s and e [ t e n d o t h e s a n e w i t h h y b r i d c r o s s e s b e t w e e c y. b b r u s u and cther g r a p e s p e c i e s . F u r t h e m o r e , t h e y c a n d e t e c t t h e p r e s e c c e o f s p e c i f i c o d o r  c o n p o n e n t s , w h i c h i n v a r y i n g d e g r e e s , seem t o make L I ~ ' t h e t o t a l s e n s o r y e f f e c t . F o u r d e s c r i p t o r s w h i c h are irequently used by t h e s e p a p e l i s t s a r e f o x y ,"f l o r a l ". n e t h y l ~n t h r a n i l a t e l i k e ' *, and "cotton candy". It is c e r t a i n l y u n w i s e cia a s s u m e t h a t t h e s e o d o r c o m p o n e n t s a r e . i n every c a s e . r e l a t e d t o s i n g l e c h e m i c a l s p e c i e s . b u t t h e y a r e p r o b a b l y less c o m p l i c a t e d i n t h e i r c t m i s t r y t h a n t h e conpound mix t h a t p r o d u c e s t h e t o t a l p e r c e p t i o n o f l a b r u s c a c h a r a c t e r . Such d e s c r i p t o r s are u s e f u l i n o u r a t t e m p t s t o s o r t o u t t h e s,, alI number of odor active compounds present i n n a t u r o l products. However, they have l i t t l e m e a n i n g f r o n o n e l a b o r a t o r y t o t h e n e x t . F o r e x a m p l e . t h e a r o m a d e s c r i b e d i n o u r v o r k as cotton candy" appears, for r e a s o n s w h i c h w i l l b e e x p l a i n e d l a t e r , t o b e d u e t o t h e same compoufid responsible f o r t h e s t r a w b e r r y o d o r d e t e c t e d i n t h e l a b o r a t o r y of Rapp( j) in Gemany. Tne c o n f u s i o n t h e s e n o n c h e m i c a l d e s c r i p t o r s c r e a t e , w i l l be ninimized once WE know t h e c a u s a t i v e a g e n t s for o d o r p e r c e p t i o n . Then we can use a chemical name t o d e s c r i b e t h a t p e r c e p t i o n . In t i t & meantime, we must t o l e r a t e t o some e x t e n t t h e u s e o f t h e s e v o r d s i n o u r d a y t o d a y r e s e a r c h . ,IETHYL AHTHRAFI~ Studies Gf t h e v o l a t i l e c o m p o s i t i o n of grapes have, through the y e a r s , r e v e a l e d t h e p r e s e c c e of so many odor active coapocnds t h a t i t is v e r y u n l i k e l y t h a t a s i n g l e compound i s responsible for more than a f e w p e r c e n t o f t h e t o t a l o d o r c h a r a c t e r o f g r a p e s ,* f any kind( h). Furthermore, among t h e h u n d r e d s of v o l a t i l e s ..

.. ..

.. present o d o r a c t present together c u l t i v a r a specif One e v e r a s s ) (1). h i produce i n g r a p e anthrani 3 abrus ca i n o n l y nethyl a t h i s co t h e l a b r labrusca Ha n t o have o d o r t b r to v i n i labrusca There a propyl, i n l a b r u contribu placed For exarn i n d i c a t e t h e q u a l t h e p e r t h e anou e s t e r c breeders odor cha One preseccc products f l a v o r s of flavc unintent FOXIKES$

f r o n 1 2 been ur "2  has an grapes. I n so abrusca s n d t h e e. The in 1722 ipeS a s ma i s candy . Even types . ruscana e a r l y ack of r e r c i a l r e f ore, i t y o f , t o a l l grape Jar New zd t h e pes and and x c e o f 3 make c11 are .. foxy

0.

t h a t u t . t h e :la t urz 1 s r a t o r y .roGk as plained i o r t h e .en;; any. . i. 11 be epdion. n. ' I n f t h e s e 2. ACREE Odor Quality of Lahrrtsca Grapes 13 p r e s e n t i n g r a p e s (F i g u r e I.)* only a very feK probably have any o d o r a c t i v i t y . T h e r e m u s t e x i s t a number of c h m i c a l compounds p r e s e n t i n g r a p e s a t p a r t i c u l a r c o n c e n t r a t i o n s t h a t when taken together produce t h e c h a r a c t e r i s t i c o d o r q c a l i t y o f a p a r t i c u l a r c u l t i v a r . A s s o c i a t e d w i t h e a c h of t h e s e o d o r a c t i v e c h e m i c a l s is a s p e c i f i c . though n o t n e c e s s a r i l y u n i q u e , o& or q u a l i t y . One such compound i s m e t h y l a n t h r a n i l a t e , t h e f i r s t conpound ever associated w i t h o d o r c h a r a c t e r of a p a r t i c c l a r g r a p e s p e c i e s (2 ). O r i g i n a l l y i d e n t i f i e d i n n e r o l i o i l (8) it vss u s e d t o produce synthetic grape flavored products before i t vas observed i n g r a p e s . In 1923 S a l e a n d W i l s o n (2 ) a n a l y z e d t h e m e t h y l a n t h r a n i l a t e c o n t e n t i n 55 c u l t i v a r s of g r a p e s . i n c l u d i n g b o t h labrusca and v i n i f e r a c u l t i v a r s . They f o u n d m e t h y l a n t h r a n i l a t e i n , o n l y 14 of t h e s e . I n 1976 using gas chromatography. we found m e t h y l a n t h r a n i l a t e i n only 8 of 4 5 c u l t i v a r s (m). Certainly t h i s compound p l a y s a n i m p o r t a n t r o l e i n t h e f l a v o r of sone of t h e l a b r u s c a g r a p e s b u t i t i s n o t  s o l e l y r e s p o n s i b l e f o r t h .e l a b r u s c a c h a r a c t e r . Nany v i n e s w i t h a s t r o n g l a b r u s c a c h a r a c t e r h a v e b e e n f o u n d t o have a methyl a n t h r a n i l a t e c o n t e n t vel1 below t h e a p p a r e n t odor threshold (m). F u r t h e r n o r e , t h e a d d i t i o n o f t h i s compound t o v i n i f e r a v i n e s a t v e r y h i g h c o n c e n t r a t i o n s d o e s n o t p r o d u c e a labrusca aroma n o r d o e s i t produce a v i n e w i t h any foxiness. There are small amounts of o t h e r a n t h r a n i l a t e e s t e r s . e t h y l . propyl, =. butyl and the analogous compound p aminoacetophenone i n l a b r u s c a g r a p e s b u t t h e r e are n o d a t a e x p l a i n i n g t h e i r precise contribution, i f any, t o t h e =e t h y l a n t h r a n i l a t e l i k e aroma. I n t h e l a s t 60 y e a r s , p e r h a p s t o o much e q h a s i s has beer. placed on t h e m e t h y l a n t h r a n i l a t e c o n t e n t of l a b r u s c a g r a p e s . For example. ueasurements of t h i s cornpour. 6 heve beer. used t o i n d i c a t e g r a p e n a t u r i t y (u ); it has been used as a n i n d i c z t o r of t h e q u a l i t y o f g r a p e p r o d u c t s (u ), and as z means of nonitorinL t h e p e r f o r m a n c e o f e s s e n c e r e c o v e r y e q u i p z e n t (u ). F u r t h e r m o r e , t h e amount of methyl . a n t h r a n i l a t e c o m b i n e d . w i t h t h e v o l a t i l e e s t e r c o n t e c t of grapes is p r e s e n t l y b e i n g used t o a i d t h e g r a p e b r e e d e r s i n t h e i r a t t e m p t t o p r o d u c e new c u l t i v a r s w i t h c e r t a i n o d o r c h a r a c t e r i s t i c s (&). One of t h e d a n g e r s o f p l a c i n g t o o much inportance on the p r e s e n c e o f m e t h y l a n t h r a n i l a t e i n g r a p e p r o t u c t s i s t h a t s u c h products may tend t o snell more l i k e t h e s i m p l e i m i t z t i o n g r a p e f l a v o r s r a t h e r t h a n t h e n a t u r a l p r o d u c t . I t i s one of t h e g o a l s of f l a v o r r e s e a r c h t o p r e v e n t s u c h i r o n i e s f r o e o c c b r r i n g unintentionally. FOXINESS So f a r o u r a t t e m p t s to i s o l a t e L? pure chemical component f r o m l a b r u s c a o r a u s c a d i n e g r a p e s . w i t h a c l e a r f o x y odor have b e e n u n s u c e s s f u l . T h e r e is, h o w e v e r r o n e c c n p o n e n t , grans 2 texen 1 01. apparently present in a l l g r a p e s p e c i e s . t h a t h a s a n o d o r . v e r y siniilar t o t h e f o x y oZor cooponect of Kiagara g r a p e s . P o s s i b l y . t h i s c o m p o u n d . i s p r e s e n t i n f o x y s n e l l i n g 14 < .. " ." 7 I 31VLllN33NO3 A 000'tl NO113VUJ *% E 2. ACRE1 grapes B t h e i r odo concentra 10 times breeders commercia grapes t c o m e r c i a foxy f l a v x€ uLu& w I n a present d e a c t i v a t p o l a r i t y and 100% p o l a r i t y e x t r a c t s was t h e c h a r a c t e r presence reported n a t u r e 1 p The labrusca anouhts o v i n i f e r a products Concord t h a t foun chardonna Bece products t h e o d o r c o n t r i b u t s u b s t a n t i threshold t h e n e t h grapes is Besi o t h e r goo such con r e n i n i s c c COTTOA Q!

Experimer q u a l i t y c f o r t h e has been p o l l u t i o r t h e o d o r 1.3 cycl0 For 16 QUALITY 01; SELECTED FRUITS AND VEGETABLES 2: ACRI 1:

40 20 100%

40 20 100%

80 60 40 20 100%

80 60 40 20 20 40 60 80 100 120 140 M /e i I 20 40 80 100 120 140 M /e QUALITY OF SELECTED FRUITS AND VEGETABLES COMPOUND ODOR QUALITY 0 DAMASCENONC FOXY ANIMAL . FlORAl FRUITY COTTON CANDY STRAWBERRY BURNT SUGAR ". "

2.

90 _ Anhvestigation of the Volatile Flavor Composition of V'tus Labrusca Grape Musts and Wines. 1. Methyl Anthranilate  Its Role in the Total Aroma Picture of Labrusca Varieties.*

Richard H. Tomlinson and Joe boison* I'

Contribution from Ckemistq Dqartment, McNaster LTniversitzj,. Canada U S &Ill and the Chemistry Department, University of Saskatchewan; Saskatoon, Canada S7N OW0 . L'anthranilate de mifhyle u 4tZ considhi comme 6tant le. compos4 volatile le pfas &terminant dans l'ar6me des vins et des moicts de misinq uConcozrl" et f u t longtemps considirC conrme Ctant l'agent r& Cla tmr du go5t dissimzd& associ4 ti l'ardmedes uins 'Concord" et des autres variltts Vitis labrusca. Des risultats exphimentaux notweaux uiennent demon trerqzte des compos& colatiles aromatiques azttres qtre l'anthmnilate de d t h y l e peurent dtre des agents ri vdlateurs del'ar6me camcteristique de ces variit4s nard amh'caines.

Abstract Methyi anthranilate has been proposed as the single most important volatileflavor component in Concord grape musts and wines and has long been held respon sible for imparting the typical "ffoxiness" associated with the flavor of Concord and other Vitis labrusca Varieties. New experimental evidence is presented to demonstrate that volatileflavor compounds other than methyl anthranilate m y be respomible for imparting the typical flavor associated with these native North American varieties.

$ Presented as part of a session on uAnalysis of Trace Components in Alcoholic Beverages", Cas& 86 Xeeting, Toronto, Ontario, Canada, October 64,1986. * Author to whom correspondence m y be directed. e n t address: Agricuiture Canada. Food ProductionandInspectionBranch, Animal Pathology Laboratory, 116 Veterinary Road, University of Saskatchewan Campus, Saskatoon, Saskatchewan,  Canada C717 9Dr) Methyl anthranilate has been considered the single,  "

.most important volatile flavor component in Concord grape musts and wines since its presence was fwt detected in Concord grapes by Power and Chestnut in 1921 (1). Subsequently, its presence in several Vitis .

labmica varieties has been confirmed by Scott (21, Sale and Wson (31, Robinson et al. '( 4 7), and by. HoIIey' et d. (8). This compound has been held responsible for imparting the "foxiness" associated with the flavor of the native North American varieties Witis Zabntsca) and their hybrids. This viewpoint has been so preva lent in the minds of vintners and viticulturalists in the eastern United States and Canada, where the grapes of the pative vines form the backbone of the wine industry, that viticultural and vinification practices in this region have hitherto been Focussed on the reduc tion or total removal of this compound from the prod ucts they make for consumers. These practices have unfortunately not yielded the anticipated superior wines. Could it be that vintners and viticulturalists in the eastern, United States and Canada have geared their efforts to the removal of a cornpound which is not sole13 responsible for imparting the "distasteful" characteristics associated with these varieties? More recent investigations into the volatiie aroma composition of the native North American grapes and wines have ,paid particular attention t o the role of methyl anbhfanilate in the overall aroma of these vari . '

 eties (9 15). iri? lile it has been established that the vola tile aroma eomposition of Concord depends signifi cantly on p e processing technique (141, it is becoming increasingly $ear that the flavor character is ti^^ of these native &iieties cannot be explained by the presence Investigation of Volatile Flmor Composition in Wines 7 or absence of methyl anthranilate alone. Some native I. D.) glass column packed with Superpak 20 M (Anal

. . varieties such as Baco Noir, Elvira, Delaware (161, abs, Connecticut, U. S. A.) held in a Varian 1800 Gas Aurore (16,18), and Catawba (16,17,21) show no .Chromatograph. The injection port temperature was detectable concentrations of methyl anthranilate and set at 250% and the oven temperature was programmed yet possess the distinctive Labrusca flavor. Various  ". from 20 200° C at G" C/ min and held at the final temper authors have also concluded that methyl anthranilate ature for 10 min. A glass lined microsample splitter done cannot be the most significant odorous compo fitted at the end of the colUmn provided an approxi nent in Vitk labrusca varieties (12,14,1921): Although mately 1: lO split ratio of the chromatographic effluent previous investigators have speculated that the possi to oneof two flame ionization detectors (FID set at ble presence of one or more compounds, other than 250° C) and an exit port. Th'e exit port effluent was methyl anthranilate, may rather be responsible for either directed to a fraction collector or a heated recep imparting the typical hbrusca flavor to these vari tacle (SNIFF PORT) where the nose of the experi eties (14), there is no evidence in the literature that menter could evaluate its significance. such compourids have been detected and identified. This paper presents new experimental evidence to clarify the role of methyl anthranilate in the total aroma picture Packed Column GClMS Analysis of the native North American varieties in a continuing research effort to identify the compound( s) that To provide qualititiie. 'identification of the compo . '

adequately portray the flavor characteristics of the nents separated by gas chromatography., GC'separa . Yitis labrusca varieties. tion was conducted on an identicalcolumnused for the packed column GC analysis held in a Varian 3700 Gas p,:? Experimental focussing, magnetic sector mass spedrometer (> IX70 . .

. ~ .. .. Chromatograph coupled to a high resolution, double

Ld 70F, VG Analytical Ltd. Altrincham, England) Aiamtus and Materials . a jet separator.

r . A newdesign of solvent extractor was developed for this analysis. It was used in conjunction with a new design of concentrator apparatus to successfully isolate and enrich the volatile flavor fraction from 95 mL of Cabernet Sauvignon wine using 250 mL of purified Freon 11 (trickdomfluoromethane) as extracting solvent (E)., Wine samples for this analysis were obtained from Chateau Gai Wineries, St. Catharines, Canada, An& Wineries, Canada, and the private collection of one of the authors (RHT).

Isolation and Enrichment of the Volatile Flavor Extract Volatile flavor components in Pichon Lalande (Vini fm), Vidal (Labncscu), Concord (Lubrusca), MouIin Rouge (Labrusca), Delaware (Labrusca), Elvira (L a b c a ), and Similkameen Red (Labmsca) were . isolated and concentrated 2500 fold, using the previ ously descriied method (22). The concentrated extract i "' was stored in a screw capped vial for further experi

I' mentation, GC andtor GCNS analysis. GC Experimekts to Evaluate Regions of Organoleptic Significance (SNIFF PORT)

Aliquots (5 10 pL) of the concentrated volatile flavor extract were injected into the packed column GC and a sensory description of the separated and eluting components was obtained (Figure 1).

Experiments to Evaluate the Amma Contribution of the Total Extract to the Wine In order to determine if the nature of the solutions produced by mixing the flavor extract with the wine base depended on the method of mixing, a total of 20 pL of the concentrated volatile flavor extract was injected into the GC and trapped into 150 mL of cooled wine base via thelfraction collector. Samples were saved for sensory evaluation (Table I). Another 20 JLL aliquot of the same flavor extract was added diiectly to a second 150 mL portion of cooled wine base. The resulting mixtures were saved for sensofy evaluation (Table 11). ,. I ' I GC ~qeriments to Reconstitute Vitis ,. , . Gas chromatographic separation of the concentrated The .. failure of our attempts to produce homogeneous .. I ' , . PackedColumn GC Analysis Labrusca Wines . , 1,; . iz '1 1 )I ;i , ..

 . , ,,   "

,,,: r ;   4  : . .. c A  a +I... ..:" " . .

P c c Richard H. Tomlinson & 3w Boisrm 2 .I t i I! .. '

P E N t I C . TIlocri=>

Figure 1. Sensory description of the eluting components in the chromatographic profile of a concentrated volatile flavor extract of Concord wine on a 6 h. X 0.4 in. O. D. (2 mm LD.) Superpak 21, Jf glass column. (Peaks represetit FID recorder responses while the descriptors represent the analyst's perception at the sniff port of the components eluting from the CC.)

bases for the Labrusca varieties using the previously described isolation concentration procedures used successfilIy for Pichon Lalande (22) led us to conduct these procedures with a modified version of the solvent extractor and concentrator operated under carefully controlled inert atmospheric conditions (22). The isolated and enriched volatile flavor extracts obtained under these new operating conditions were used to repeat the previously described experiments .to eval uate the aroma contribution of the total extract to the wine. The results of the sensory analysis performed on the resulting homogeneous solutions are show. in Table 111.

GC Experiments to Evaluate the Contribution of Sections of the Chromatographic Profile to the Wine In order to assess the aroma contribution of various fractions (especially methyl anthranilate) of the elution profile to the wine, a total of 20 pL of the concentrated volatile flavor extract obtained under the modsed conditions was injected into the column and the k c  tions eluting from the Superpak 20 M column were trapped jnto 150 mL of cooled \vine bases as pictorially """  "_ L  1 :. T" .. Figure 2. Partial Reconstructed Ion Chromatogram of a Standard Mixture of volatile flavor compounds depicting the experiments conducted to evaluate the flavor contribu tion of sections of the chromatographic profile on the wine. X. Ethyl hexanoate 2. Amyl butyrate 3. Ethyl Iactate 4. cis 3 hexen 1st 5. .Ethyl octanoate 6. Zmethylbutyi hexanoate 7. Benzaidehyde. 8. I Octanol9. y Butyrolac tone 10. Ethyl benzoate 11. Ethyl decanoate 12. Diethyl succinate 13.2 Phenethyl acetate 1.1. Hexanoic acid 15. 2 Phenethyl alcohol 16. t cinnamaidehyde 17. Octanoic acid IS. Methyl anthranilate 19. Decanoic acid 20. Dh+ Llli, l O? 0 7%"  I..* 1 Investigation of Volalile Flavor Composition in Wina Table 1. Experiments t o Evaluate the Aroma Contribution of the Total .Extract to the Wine.

20 pL of Flavor Extract (a) 150 mL, Wme base* (b)

Concord Xoulin Rouge Vidal Elvira Delaware S i e e n RH Pichon Lalande Observation aftera + b Concord

f u r f a c e Film Forme Concord Moulin Rouge Vidal Elvira Delaware Moulin Rouge .. . .. S i e e n Rei Pichon Lalande I ~ ~~

Concord Xoulin Rouge Vidal Elvim Delaware Pichon L'aIapd. Sirnilkameen Rec Von Homogeneous m . "med . Table 111. GC E xperiments to Reconstitute \'itis Labnuar Wines using the lfodiied Isolation Concentration Method. " Vidai Strong Burned Odor leveloped Concord Jfoufin Rouge Vidd Elvira Delaware S i e e n Red Pichon Lalande Elvira Delaware Concord Xoulin Rouge Vidal Elvira Delaware S i e e n Red Pichon Lalande Table IV. GC Experiments to Evaluate the Flavor Contribution of Sections of the Chromatographic Profile to the Wine.

Concord Moulin Rouge Vi& Elvira Similkameen Red f Delaware Sirnilkameen Red Pichon Lalande Flavor of PI Regenerated Homogeneous Soin. Formed No Detectable hbrusca Flavor Perceived ?oncord Houlin Rouge Vidal Zlvira Pichon Lalande leiaware S i e e n Red ?ichon Lalande Wine base is the bland product ,remaining after the volatile flavor La:" L  * L 94 Richard H. Tonrlinson & Joe Boison (a) all components eluting up to and including 2 sitions of Vitis l a h s c a varieties have been investi phenethyl alcohol were trapped into wine base A; gated using techniques essentially developed for Vi?& (b) all components eluting up to but excluding methyl fm varieties without any major modifications. After anthranilate were trapped into wine base B; ,the apparatus developed for this analysis was modified (c) all components eluting from 2 phenethyl alcohol " to enable the isolation concentration procedures to be (inclusive) up to but excluding methyl anthranilate were trapped into wine base C; (d) all components eluting from methyl anthranilate (inclusive) and thereafter were trapped into wine base D; and (e) all components eluting except Freon solvent were trapped into wine 6ase E to serve as control. The results of the sensory evaluation of the solutions obtained in this experiment are presented in Table IV. Sensory evaluation of samples was done by a group of faculty, staff and graduate students of the Chemistry Department, 3lcMaster University, who had been trained to recognize the Labnlsca flavor.

Results and Discussion The results of the GC experiments presented in Table I show that in all cases where the voIatile flavor extract was of the Labnwca origin, the introduction of the flavor extract into the wine base through the GC frac tion collectorproducednon homogeneous solutions. Note that when the volatile flavor extract was of the Vinifera origin, homogeneous solutions were obtained regardless of the origin of the wine bases. The results shown in Table I1 were obtained by introducing the flavor extracts directly into the wine bases thus elim inating the GC step for sample introduction used for obtaining the results presented in Table 1. Since both methods of sample introduction info the wine bases produced non homogeneous solutions, it was concluded that the results obtained in Tables I and I1 were not caused 'by the method of sample introduction, but may have been caused by processes occurring prior to the sample. introduction step, i. e., the isolation concentration procedures. Any changes caused by the isolation concentration procedures wouid affect either the flavor extract orr the wine base or both. In an attempt to identify the source zpd cause of the change in the Labmsca varieties, it was observed that the flavor extract of Pichon Lalange and White Riesling wines, 'both made from Vingma grapes, dissolved readily into their o m wine bases and all the' wine bases of Labmsca origin to regenerate the characteristic Cabernet Sauvignon or Riesling aroma. This obser vation led us to conclude that it is the flavor eitract of ,the 4bbrusca rather than the wine basg that may have 'hnilergobe change during the sample prepaiation stages. Ext, ensive review of the'liteqture dnd our own "* l" A l" L "_.. 1 * .I L 3  . .  conducted under carefully controlled inert atmospheric conditions (22), it became possible to produce homo geneous solutions and therefore reconstitute all the Vitis lafrrusca wines as shown in Table 111. Any further sample extraction and concentration procedures relat ing to the LQbncsca varieties were therefore performed under the modified operating conditions. Having developed a method that retains the original nature of the volatile flayor extract to permit the reconstitution of Lahcsea \vines, it was observed. from the results of the experiments. conducted to assess the aroma contribution of the eluting components to the wine that, besides'the control sample E, only fractions B and C trapped into the wine bases produced solutions that typsed the Lubncsca character; fraction D (methyl anthranilate) produced a solution that could hardly be described as typically Lubncsca. It is noteworthy to observe that these results and conclusions were the same for all thetabntscu varietiesinvestigated includ ing, E l v h andDelaware, which even showedno detectableconcentrations of methyl anthranilate. Since ,

fraction €3 also encompasses fraction C, which by itself imparts .the Labrusca flavor to the wine, it may be concluded that fraction C must be the most organo leptically significant portion of the profile of the vola tile flavor composition of Vitis labnlsca varieties on the Superpak 20 M column. This conclusion is rein forced by the results of the preliminary experiments to evaluate regions of organoleptic significance in the chromatographicprofile (Figure 1). The results in Figure 1 deiine even more precisely the region of Organoleptic interest in the chromatographic profile by confining it to the labelled section which is comprised of compounds with similar retention times to octanoic acid. Any experiments designed to detect and identify the compound( s) responsible for imparting the typical Labrusca flavor to the native North American vari eties should therefore be confined to the section of the chromatogram labelled 'kegion of organoleptic inter est" (Figure 1).

Conclusions The results of these experiments confii the suspi cions of previous investigators that the presence or absence of methyl anthranilate alone cannot explain: the typicalflavorassociated with the native North . '  L Investigation of Volatile Flavor Composition in Wines tant odorous compound in these varieties. Unlike Vitis vinifera varieties, isolation and concentration proce dures for the examination of the volatile flavor compo sition of Vitis labnrsca grape musts and wines must be conducted under. carefully controlled inert atmos pheric conditions t o prevent any possiblechemical/ structural modifications to the nature of the volatile flavor extract. The experimental evidence presented in this paper demonstrates that compounds other than methyl anthranilate are responsible for imparting the typical Labmca flavor. Such compound( s) are those with similar retention times to that of octanoic acid on the Superpak 20 M column. This conclusion should be of considerable significance to vinters and viticultuy alists in the eastern United States and Canada in help ing them to review not only their vinification and viti cultural practices but also their perception of the role  played by methyl anthranilate in the total aroma picture of the Labntsca varieties. "

... ". . ::

ps; References L ,l I .

1. F. B. Power and V. K. Chestnut, J. h e r . Chem. Soe., L3.1741 (1921). 2. 3. 4.

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9s R. D. Scott, Ind. Eng. Chem., IS, 732( 1923). J. W. Sale and J. B. Wilson, Agric. Res., 33, 301 (1926). W. B. Robinson and N. J. Shaulis, Proc. Amer. Hortic. Soc., 64, 214 (1953). W. 3. Robinson and N. J. Shautis, J. Amer. Food Manut, f9( 2), 36 (1949). W. B. Robinson and N. J. Shaulis, ibid., 09( 2), 54 0949). W. B. Robinson and N. J. Shaulis, ibid., 29( 2), 62 (1949). R. W. Holley, B. Stoyla and A. D. Holley, J. Food Res., PO, 326 (1953). W. J. Clore, A. M. Neubert, G. H. Carter, D. W. Ingalsbe and V. B. Dnunmond, Wash. Agric. Exp. Stat. Bull., 48 (1961. J. J. Casimir and J. C. Moyer, Proc. N. Y. WineInd. Tech. Adv. Corn, N. Y. Agric Exp. Stat.. Geneva, (1965). D. J. Stem, A. Lee, W. H. Mcfadden and K. L. Stevens, J. Agric. Food Chem., 1s. 1100 (1967). 1. E. Friedman, New York State Hortic. Soe. F'roc., 121,132 (1976). T. Fuieki, Can. J. Plant Sei., 52,863 (19n). R. R. Neison and T. E. Acree, Amer. J. Enol. Viticult,. 29( 2), 83 (1978). W. L. Stanley, J. E. Brekke, .and R. Teranishi, U. S. Patent 13113031, Dee. 3 1963 to US. Government. A. C. Rice in "Chemistry of Winemaking" Ed. A. D. Webb, Adv. in Chemistry Series #137, p. 88( 1974): A. H. Jackson, PbD. Thesis ZncliMaster University, Canada. 19i7. J. S. Caldwell. J. Agric. Res., SO. 1133( 1925). 11. A. Amerine, E. B. R w s s I e r and F. Filipello, Hilgadia, 28( 18), 500 (1959). A. D. Webb and R. ' E. Kepner, Amer. J. Enol. Vitidt,, 7, 8 (1956). R. R. Xeison. T. E. Acree and R. B i. Butts, J. Agric Food Chem.. 26, 1188 (1978). J. 0. Boison. Ph. D. Thesis McMister University, Canada, 1984.

 A Cornparison of the Effect of Toxic and Non Toxic Metals on the Aromatic System of Salicylic Acid Wiodzimierz Lewandowski Contribution from Department of Physico Chemical Analyses, Agricultuml University .of Warsaw, 03 528 Warsaw, Rakoutiecka 26/ 30? Poland Received: November 10, 1986 Accepted (inrevised form): May 29, 1987 R6sumC Les spectres d'abso? ption IR et Raman des salicy lates de mercure( II), cadmium et aluminium ont dtd 6tudi6s et interpritks. On acompark l%= fluence de mereure( 1) et "( II), pLomb( II), cadmium, mgne sium, zinc, aluminium, fer( III), lanthane( II0, neodyme( III), disprosium( III), ytterbiumllll), .sodium et potassium sur le noyau aromatiQue de l'aeide salicilique. Pour les investigations on a applipud de la spectroscopie d'absorption Llectronique (UV, VIS) et la spectroscopie d'oscillation (celle $absorption a l'infrarouge et celle de Raman). On a constat6 que tes , . . .. . .  , ;. .  . . . .  contraire de aluminium, lunthadIII), n& iynte( III), disprosium( III), ytterbium( III), fer( III), zinc et magwsiz~ ml perturbent le systi? meammatique de l'acide salicilique.

Abstract ' Assignments areproposed for the IR absorptionand ' Raman spectra of mercury( II), cadmium and aluminium salicylates. Th effect of mercury( I) and 4 1 1 , l e d l l ), cadmiuwi as weld as of magnesium, zinc. aluminium, iron( III), lanthanum( III), ., f . .. ..

&. r 1188 J. A&. Food Chem., Vd. 26, No. 5,. 1978 Jeon, I. J., Reineccius, G. A, Thomas, E L., J . Agric. Food Chem, 24.433 (1976). Kinsella, J. E., Chem. fnd., 36 (1969). . Kirk, J. R., Hedrick, T. I., She, C. M., J. Dairy Sci. 51,492 (1968); Langler, J. E., Day, E. A., J. Dairy Sci. 47,1291 (1964). ; Ldlard, D. A., Day, E. A., J. Dairy Sci. 44, 623 (1961). Muck, G. A,, Tobias, J., U'hitney, R. M., J. Dairy Sci. 46, 774: (1963).

Whitney, R. M. J . Dairy Sci. 46, 671 (1963).

6,232 (1964).

(1963). Nawar, U'. W., Lombard, S . H., Dall, H. E. T., Ganguly, A. S.,

Parks, 0. W., Keeney, M., Katz, I., Schwartz, D. P., J. Lipid Res.

Parks, 0. W., Keeney, M., Schwartz, D. P., J. Dairy Sci. 46,295 Parks, 0. W., Patton, S., J. Dairy Sci. 44, 1 (1961). Parks, 0. W., Schwartz, D. P., Keeney, M., Nature (London) 202, 185 (1964). patel, T. D., Calbert, H. E., Morgan, D. G., Strong, E'. M., J. DLrY Sci. 45,601 (1962). **"* rrw *

Patton, S., J. Dairy Sci. 36, 1053 (1952). Patton, S., Josephson, V., Food Res. 22,316 (1957). scan la^, R A, U y , R C., Libbey. L. M., %, E A, J. Dairy schwartz, D. P., Parks, 0. W., Y o n d e , R A, J. Am. Oil Cbm. , i Stark, W., Forss. D. A, J. Dairy Res. 33,31 (1966). .1 Thomas. E. L., Burton, H., Ford, J. E., Perkin, A. C., J. Bairn ; Si. 51, 1001 (1968).

Soc. 43,128 (1966). 1 Res. 42,285 (1975). 1 Toothill, J., Thompson, S. Y., Edwards Webb, J.. J. Dairy R ~~. 1 4 37,29 (1970). 7 . withers, M. K., J. Chromatogr. 66,249 (1972). Zadow, J. G., BimRistle, R., J. Dairy Res. 40,169 (1973).

Isolation and Identification of Volatiles from Catawba Wine Richard R. Nelson, Terry E. Acree,* and Robert M. Butts .

The volatile composition of three Catawba wines prepared from grapes grown in the vineyards of the New York State Agricultural Experiment Station during the 1976 vintage were analyzed by instrumental and sensory means. The three wines differed according to the enological technique employed for their production. Volatiles were isolated by solvent extraction, separated and quantified by gas chroma tography, arid identified by combined gas chromatography mass spectrometry. Although some variation in volatile composition due to processing technique was observed, sensory analyses comparing the win? with corresponding model solutions indicate that the major identifiable components are of little im portance in determining the aroma of Catawba wine as influenced by processing technique. 4 Catawba vines have been cultivated in the northeastern United States for over 150 years. Currently, in New York, over 10 000tons are produced annually and over 90% of that is used for wine production (New York State Crop Reporting Service, 1976). Catawba grapes can be used in the production of either white or ros6 wines depending upon enological technique, and much of the white wine produced is used in sparkling wine cuv6es. The literature dealing with the volatile composition of wines and winegrapesisextensive. Kahn (1969) and Webb and Muller (1972) have tabulated hundreds of compounds that have been identified in wines and other 'alcoholic beverages. Many native American grape varieties including Concord and Catawba have characteristic aroma components that appear to be unique to some varieties with iabrusca parentage. Although many workers (Holley et al., 1955; Neudoerffer et al., 1965; Stevens et J., 1965; and Stern et al., 1967) have studied the volatile composition of the Concord variety, no such investigations have been con ducted with Catawba. Methyl anthranilate, a compound long thought to be of major importance in the aroma of labrum varieties (Sale and Wilson, 19261, now appears to be far less important than previously thought (Nelson et d., 1977a). Friedman (1976) believes that methyl anthranilate is of little im

Department of Food Science and Technologyv New Yoyk state A& ultural Experiment Station, Geneva, New Yo& 14456.  _ "l .".1 ." .."". n*, fi portance in the aroma of Concord grapes, even though ita concentration in that variety & relatively high. h e h e et aL (1959) noted that the distinctive Catawba aroma was apparently not due to methyl antbrdte and that other more important compounds must be present. This report examines the volatile composition of Ca tawbawines prepared by three different enological techniques. In addition, it attempts to assess the effect of processing technique on the occurrence of these volatils in Catawba wine. MATERIALS AND METHODS Wine Preparation. Catawba grape6 were harvested at 16.6" Brix in October of 1976. The fruit was divided into three 20 kg lots for fermentation. From one lot a white Catawba wine was prepaied by immediately pressing the 7 crushed grapes while rose wines were prepared from the other two. One of the rose wines was prepared. by fer. meriting the juice in contact with the ekins for 5 days [d (FS)] whil'e the other was thermally vin% ed [rose (m71. "hemal Mlcation comista of hating the crushed grapes in a steam kettle to 60 O C for 16 min, followed by im mediate pressing. Fermentations were conducted at 20 *C and other standard enological procedures as described b' Nelson et al. (1977b) were followed in each case. Volatile Isolation. The Catawba wine volatile6 were isolated using organic solvent extraction with Freon 113 (1~ 1P tricbloro 1,2,2uor~~~ e, "Precision Cleaning Agent, Du Pont"). 33qd volumes of wine and fieon (2700 &I were stirred fdr 1 h. The Freon phase was then draws bff, dried over anhydrous magne+ m sulfate, and Con * retention concentration, ppm time, rose rosd compound min white (TV) (FS) " ethyl acetate p ) jsobutyl acetate (c) ethyl butyrate (d) isoamyl acetate (e) isoamyl alcohol (f) ethyl hexanoate (g) hexyl acetate (h) ethyl lactate ii j n hexanol (j) cjs 3 hexene 1 01 (k) ethyl actanoate (1) ~inaloiil (m) butyric acid (n) ybutyrolactone ' (0) ethyl Gecanoate (p) diethyl succinate (9) 2 phenylethyl acetate (r) hexanoic acid (6) 2 phenylethyl alcohol (t) octanoic acid (u) methyl anthranilate 3.25 6.22 6.74 9.14 11.37 12.84 14.07 16.18 16.43 17.41 19.61 23.18 24.13 25.02 26.07 27.39 31.70 32.76 33.17 38.87 46.41 0.15 0.16 0.02 0.03 0.04 0.03 i 0.17 0.12 0.09 2.57 3.19 1.68 6.27 3.19 1.68 0.57 0.49 0.47 0.04 0.13 0.20 0.17 0.24 0.45 0.34 0.12 0.76 0.06 0.03 0.13 1.16 0.74 0.55 0.02 0.05 0.10 0.04 'I" ' 0.12 ndb Tr 0.06 0.53 0.33 0.26 0.30 0.56 0.95 1.09 3.79 2.90 3.23 1.35 5.51 5.00 7.45 7.50 9.20 7.35 1.80 ndb 0.07 Tf 'a only trace quantity detected. Not detected.

centrated in a'rotary evaporator with water bathat 20 "C. The final extract concentration was 13500 fold (0.2 mL), md each extract had a characteristic Catawba like aroma while the aqueous phase was nearly odorless. Instrumental Analysis. The Catawba extracts were analyzed by combined gas chromatography mass spec trometry using 3 pL injections. The system consisted of a Sarian Series 1400 gas chromatograph with a 4 m X 2 mm i. d. glass column packed with 10% SP lo00 on 100 120 mesh Chromosorb W. A temperature program from 60 to 200 "C at 4 "C/ min was employed. The gas chromatograph was interfaced through a LleweUyn type methyl silicone membrane separator to a Bendix Model 12 Time of Flight mass spectrometer equipped with CVC Mark IV solid state electronics and with a computerized data collection system. Spectra were taken at 70 eV and identification was done by comparison of experimental spectra with published spectra and with those of authentic standards. Comparison of component retention times with those of the authentic standards was considered to be confirmatory. Quantification of volatile components was done using a Hewlett Packard 5830 A gas chromatograph Chromatographic conditions were the same as above exckpt that a stainless steel column of the same dimensiow was used. Quantitative estimation was done by comparing ;the peak area of 2 phenylethyl alcohol, a major component, with that of an internd standard (n decanol) added to the Freon extract at a level of 2 ppm relative to the original wine. The concentrations of the other components were then calculated directly using relative peak area ratios. Sensory Evaluation. The odors of the individpal components in the three extracts were characterized by the authors using a sniffing device (Acree'etm a]., 1976) attached to the effluent port of a Packard Model 800 gas chromatograph. Chromatographic conditions were the same as in the GC MS system. In order tb examine the role, if any, of the identified compounds in determining Catawba odor, model wine solutions were prepared using 12% v/ v ethanol 1% w/ w tartaric acid, pu; ified Concord grape anthodyanin gigmeht, and the identified volatiles in distilled water. The v& tiles Were added to the three model solutions at the level de termined for each compound iq the corresponding au t i ..

I 30 40 50 RETENTION nME (MIN)

Figure 1. Chromatogram of the 13500 fold Freon extract of Catawba wine fermented in contact with the skins.

tbentic wine. The concentration of each compound added to model solutions we listed in Table I. All compounds identified were added to the corresponding model solution, except when only a trace was detected none was added. To eliminate panel bias due to color differences, pigment was added to the wins and to the model solutions at levels such that each sample had a t ypical ros& color. An ex perienced 1Zmember panel completed three sets of randomized triangle. difference tests. Only odor was Considered by the panel 90 the samples were not tasted. The Catawba wines were first tested against each other, and then the model solutions were examined for aroma , differe? ces. Finally each winewas tested against its corresponding model solution. Samples were presented at room temperature in standard wine glasses in individual tasting booths. Sample size was 50 mL. RESULTS A N D DISCUSSION In the white Catawba wine, 36 compounds were present in concentrations sufficient for quantitative estimation. In the rose wine fermented in contact with the skins and in the thermally vinified ros6 wine 42 and 50 compounds were detected, respectively. Of these, 19 compounds found

in the white wine were identified and 21 compounds in the other two extracts were identified. Methyl anthranilate and y butyrolactpne were not detected in the white Ca tawba wine. The compounds that were identified were generally those present in the largest quantities. The ide'ntified'compounds had a total concentration that was ne+ y constant at 30.8 ppm in all three wines and rep res, ented' from 95.7 to 97.5% of the total extractable volatiles; F i 1 shows a chromatogram of the 13 5oO fold extract of the wine fermented in contact with the skins. The letters "a" through "v" correspond to the identified cornpourids listed in Table I with their concentrations in the differmt wines. The concentrations of the maj0rit. y of $he'compunds appear to be little affected by enologid. technique. However, the acetate esters, particularly isohmy1 acetate tpd 2 phenylethyl acetate. are distinctly more +bundant in 'the thermally vinified wine. Decanoic acid (v) was identified in each of the wine extiacts but its concentrktion could not be reliably estimated due to excessivk chromatographic tailing. I!@ ii$ J~ skrongly oro ow compounds were detected by gas chrpdaxikaphip ,effluent sniffing. Some, with extremely lo? && Fent thresholds, were present in concentrations, ,, nble E. Results of Randomized Triangle Teb Comparing the Aroma of Catawba Winea and of Model Solutions correct comparison response% significance wines vs. wines white vs. rosh (TVIb 10 0.001 white vs. r o d (E'S)= 9 0.01 res; (TV) vs. rosd (FS) 9 0.01 white vs. r& (TV) 9 0.01 white vs. ros6 (FS) 7 NSd ro& (TV) vs. rose (FS) 9 0.01 white 11 0.001 rosi (TV) 1 2 0.001 ros6 (FS) 10 0.001 Maximum nurpber of correct responses is 12. Ther mally vinified rose wine. Rosd .wine fermented in con tact with the skins. Not significant.

too low to give any detector response. No single compound was thought to have a distinctly Catawba like aroma.. The results of the triangle difference tests are shown in Table 11. The panel found that the aroma of each Ca tawba wine was significantly different from that of the other twowines (99% level'of confidenceor better). Clearly, if processing technique had no significant effect on Catawba wine aroma, further sensory investigation would be unproductive but this is apparently not the case. The panel was then asked to distinguish among the three model solutions. The aroma of the solution imitating the thermally vinified wine was significantly different from that of the other two solutions (99% level of confidence). .

Apparently some compositional difference in the identified compounds is organoleptically significant and peculiar to the thermally vinified wine. Although proof is not yet available, the mostobviouscompounds to which this difference can be attributed are isoamyl acetate and 2 phenyktiiyl acetate because of their high concentration in that sample. The solutions corresponding to the wine fermented in contact with the skins and the white wine were indistinguishable by the panel. This indicates that the identified compounds, even though they represent over 95% of the total extractable volatiles, do not account for the significant aroma differences that occur in Catawba wine due b processing technique. In the final triangle test each wine was judged against its corresponding, model, solution. A highly significant difference (99.9%) was found in each case. The three model solutions were, in fact, very poor imitations of the authentic wine. The high concentratiofi of acetate esters in the thermally vinified wine may contribute to its dis. tinctive aroma but it certainly does not define that aroma. models vs. models wines vs. models . ."" ., " q *

It appears that the identified components, although they do contribute aroma, contribute little or nothii to Ca. tawba varietal character as influenced by enologicd technique. The nature of wine and wine grape aroma is not we0 understood. Brander (1974) has suggested that e n m y the same volatile Components are present in all wine varieties and that the aroma differences 0ng. Virietia are due to these components beiig present in differing ratios. On the other hand, Stern (1975) stresses the h portance to wine aroma of compounds present in trace quantities. The concentrations of these compoun& is often too low to give any detector response whataoever cment methodology, but they may be of great 0rganoIeptii importance if they have sufficiently low thresholds. The major volatile components detected and identified in the three Catawba wines do contribute aroma to those wines. However, the model solutions containing these compounds at their appropriate concentrations are easily disthguished from the authentic wine. It can be condud& that the unidentified trace components, although they comprise less than 5% of the total Freon extractable volatiles, are of critical importance to the aroma of Ca tawba wine as influenced by processing technique. LlTERATURE CITED Acree, T. E, Butts, R. M., Nelson, R. R., Lee, C. Y., Am!. Chem. 48( 12), 1821 (1976). Amerine, M. A,, Roessler, E. B., Filipello, F.,. Hifgardia 28( 1&), i 500 501 (1959). Brander, C. F., Am. J . Enol. Vitic. W( l), 13 18 (1974). I Friedman, I. E., N. Y. Hortic. SOC. Roc. 121, 132 136( 1976). 1 Holley. R. W., Stoyla, B., Holley, A. D., Food Res. 20.326330 I (1955). " "_

Kahn, J. H., J. Assoc. Off. Anal. Chem 52( 6), 1166 1123 (1% 9). Nelson, R. R., A~ ree, T E., Lee, C. Y., Butts, R. M., J. Food Sci.

Nelson, R R, A m , T. E., Robinson, W. B., Pool, R M., Bertino,

Neudoerffer, T. F., Sbdler, S., Zubeckis, E., Smith, M. D., J .

New York Crop Reporting Service, Survey of Wineries and Grape . Sale, J. W., Wilson, J. B., J. Agric. Res. 33( 4), 301 310 (19261. Stern, D. J., Guadagni, D., Stevens, K. L, Am. J.% nol. Vitic. 26( 4).

Stern, D. J., Lee, A., McFadden, R . H., Stevens, K. L., J. Agric.

Stevens, K. L, McFadden, W. H., Teranishi, R., J. Food Sci. 30,

Webb, A. D., Muller, C., Ado. Appl. Micro& ioZ. 15,75 146 (1972). 42,57 59 (1977a).

J. J.,. N. Y. Food Life Sci. Bull. No. 66 (July 1977b).

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208 213 (1975).

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1006 1007 (19651.

Received for review October 25,1977. Accepted May 4,1978. Presented at the 174th. National Meetingof the American ChemicalSociety, Chicago, Ill., Aug1977. Approved by the Director of the New York State Agricultural Experiment Station as Journal Paper No. 3107. .. f  .~

R. R. NELSON. 1. E. ACREE, C Y. LEE and R. M. BUTTS Dept. of Foodscience & Technology New York Sfate Agricultural Experiment Station, Geneva, NY 14456 METHYL; ANTHRANILATE AS AN AROMA CONSTITUENT OF AMERICAN WINE i INTRODUCTION lf[ f` WINE INDUSTRY of the Eastern United States and Can s based on the native American grapes and on hybrids and rlr,; ions thereof. Certain native grapes have a characteristic r!,,, nF flavor which is disagreeable t o many wine consumers. 1 rwrilc over 100 yr of intensive grape breeding, this "foxy" ,.& c?? s, t, r still prohibits production of European style wines from \\ h. 7 The origin of the term "foxy" and its association with the grapes I: . ._  ~

 Amric n varieties is unclear, but the most reasonable theory ,, b K d on the similarity of the wild musky odor of the grapes r: th the characteristic animal like odor of a fox or of a fox's dtr. The gapes, which were originally called Fox Grapes were fuflhCr subdivided into the Xorthern Fox Grapes (Viris iabrus r;) and the Southern Fox Grapes (Vilis rotundifolio) (Bailey, i PO8 ). Methyl anthranilate has been suggested as an important rr. mpofient of the characteristic Concord (the most important ra:: vc' American variety) aroma for over 50 yr (Power, 1921). has since been implicated as a major contributor to "foxi

e a *' in American wines (Sale and Wilson, 1926; Winkler, f Q 7 2 ). Amerine et al. (1959), using Catawba as an example, rqgcsted that "foxy" varieties need not contain methyl mthranilate. This paper is intended to evaluate the importance of methyl 3n: hranilate as an odor constituent of wines produced from *dely differing grape varieties.

MATERIALS & METHODS *Hca i( different varieties of wine were obtained from the experimental *2 z k :I ~o n of the New York State Agricultural Experiment Station for * w. this experiment. AU wines were prepaied according to the stan d *. ` rmt dure of Pool et al. (1976). The wines represent four snecies ( '1 T ~c wineswere extracted with Freon 11 3, concentrated, andana ' 4 k r r methyl anthranilate by the gas chromatographic method of ~c !s .~t r e1 at. (1976) Sensory evaluation Two separate sensory analyses were performed. First, the threshold of methylanthraniiate in wine (V. viuiferu cv. WhiteRiesling) was estimated by comparing the odor of wines with varying concentrations of added methyl anthranilate. Rankings were done by the taste panel to determine the point at which methyl anthranilate could no longerbe detected. Second, an evaluation of relative foxiness in selected varieties wasperformed by the same panel. The wines were selected to vary in nature andintensity of odor and to containmethylanthranilate at various levels. This permits a direct comparison of foxiness and methyl anthranilate concentration. The sevenpanelists employed inthiswork are experienced in the critical sensory evaluation of wines.

RESULTS & DISCUSSION THE THRESHOLD of methyl anthranilate in wine was esti mated by sensory evaluation. The panel was given clean, young White Riesling wine samples with methyl anthranilate added at various levels. The ranking was conducted in two groups of four samples each (0.00, 0.01, 0.03, 0.1 and 0.1,0.3, 1.0, 3.0 ppm). The results, shown in Table 1 , indicate that the panel could successfullf distinguish higher levels of methyl anthranil ate. They were, howeker, unable to correctly rank the less ' concentrated set. The threshold can be estimated from these results, Each set of four wines contained a sample with 0.1 ppm methyl anthranilate. The panelists could correctly rank this sample within the set of higher concentrations while they could not do so within the less Concentrated set. This indicates that 0.1 ppm could not actually be detected but was ranked . correctly by the process of elimination only when samples of higher concentrptipn were available for reference. The sample containing 0.3 pp, m was consistently ranked correctly so it can be assumed that this concentration of methyl anthranilate is at or near the threshold level in White Riesling wine. With this information, a mdre meaningful estimation of the importance of methyl anthranilate in wine aroma can be made.

Table 1 The threshold of methyl anthranilate in white riesling wine Methyl anthranilate (ppm) Correct rank AVQ ran@

0.00 1 3.1 0.01 2 2.3 0.03 3 1.9 0.1 0 `4 2.7.  0.10 4 4.3 0.30 5 5.3 1 .oo 6 5.9 3 00 7 6.6 ,a Average rank of seven experienced panelists Volume 42 (1977) JOURNAL OF FOOD SCIENCE 57 . ... ~~ ~ ~~~~ ~

f "JCIlJRNAL OF FOOD SCIENCE Volume 42 (? Y// J The majority of the 45 varieties analyzed were found to be ; free of methyl anthranilate. Table 2 fists these varieties, their parentages, and the vintage of each sample. Only six varieties 3. Additional samples of Niagara, Concord and Delaware were (? ' analyzed and considerable variation in the level of methyl anthranilate due .to vintage and enological technique was found. In 1949, Robinson et al. reported that methyl anthra nilate was formed in grapes during the final stages of ripening.

sample was harvested at 16.0° B and showed 3.1 ppm methyl t i 9 % were found to contain methyl anthranilate as shown in Table L %,/

i This certainly seems to b e t h e case for Niagara. The 1970 i !4 1 '

Table 2 Wine varieties without methyl anthranilate Variety Vintage  French hybrid9 Aurore (5.788 X 5.29) Baco 22A (Folle blanche X Noah) Chancellor (S. 5163 X S. 880) Chelois (S. 5163 X s. 5593) OeChaunacb 6.51 63 x S. 793) Fochb (Mgt. 101 14 X Gold Riesling) b n d o t 451 1 (L244 X S. V. 12 375) Ravat 51 (5.6095 X Pinot blanc) S e i b e l 10868 6.5163 X 5.5593)

Geneva hybrids Canada Muscat Cayuga White GW 1 (Catawba. X Seneca)

i/.;..., ?, GW 2 (Seyval X Schuylerl I GW 4 (SeyvaI X Sene= ') L T GW 5 (Pinot Blanc X Ontario)

~

GW 6 (S e y ~l X Seneca)

GW 7 fSeywI X Schuyler) GW 8 (Pinot Blanc X Aurore) GW 10 (SeyvaI X Chardonnay) GR 1 (Buffalo X Baco Noir) GR 3 (Buffalo X Baoo Noir) GR 6 (Buffalo .x Baco Noir) GR 7 (Buffalo, X Baco Noir) N. Y. Muscat Vitis aestivalis Wild Summer Vir;$ labrusca (and crosses) Diamond 1970 1972 1969 1970 1970 1973 1970 1973 1973 1969 1974 1970 ~

1967 1967 1971 1967 1968 anthranilate while the 1975 sample matured to only 12 50 and showed 0.6 ppm. , Of the six varieties containing methyl anthranilate, Catau ba, Delaware and Elvira may have'insufficient methyl anthr nilate to influence the aroma of these wines since the COncel trations determined are near the threshold level. These wine however, are decidedly American in character. A wide range of native American wines are called "fox) but methyl anthranilate appears t o be important in only a fe of them. In order to examine the correlation of "foxiness with methyl anthranilate concentration, a series of wines w; selected for further taste panel work. The: wines were selected to have a wide. range of apparer foxiness and to subject the panelists to other varietal aromi that may or may not be considered foxy by the individu judges. The six varieties selected were. Canada Muscat, Carlo Concord, Moored, Niagara and White Riesling. A seventh sari ple was prepared by adding 0.7 ppm methyl anthranilare t the same White Riesling wine. In the experiment panelists we1 asked to smell the wines and rank them according to decrea ing foxiness. The results are shown in Table 4. There appeal to be no correlation between methyl anthranilate concentr; tion and the relative foxiness of these wines. The fact that the White Riesling sample with 0.7 p p ~ added methyl anthranilate was ranked higher in foxiness tha the pure Riesling may indicate that the ester makes some cor tribution to the foxy character. However, other varieties wit less methyl anthranilate were consistently judged higher in thl character. In fact, three of the four wines judged highest i Table 3 Wine varieties with methyl anthranilate Methyl anthranilate V a r i e t y V i n t a g e Parentage bpm)

1972 Niagara 1970 V. labrusca 1972 Concord. 1970 V. labrusca 1 967 Concord 1975 V. Iabrusca 1972 Concord 1972 V. labrusca 1972 Niagara 1975 V. labrusca . 1973 lves 1972 . V. labrusca 1969 Catawba 1972 V. Iabrusca X ? 3.102 1.752 1 .110 0.699 0.61 7 0.381 0.1 78 Delewaren 1975 , V. labrusca X ? 0.102 Elvira . 1972 V. labrusca X V. riparia trace Oelaware 1973 V. iebrusca X ? 0. M) O "

a Hot pressedsamples . 1970 1966 Dutchess 1967 Eumelan 1967 lsabella .  1966 Moored 1975 Table 4 Evaluation of "'foxiness" in selected varietal wines Vincent . 1970 Methyl Vitis rotundifolia nnthranilate Carlos 1974 Variety foxiness' . . , fppml Fry 1974 Hunt 1974 Carlos 5.1 ' 0.0 I Concord 49 1.7  Canada Muscat 4 4 Pinot Blanc 1971 0.0 Moored Pinot Blanc 4.1 1972 0.0 A: 1 ..\

Sylvaner Niagara 1973 3.7 0.6 Rieslingb 3.3 White Riesling 1972 Riesling 2.4 0.0 0.7 Vitis vinifera :r  i ", ._

a Seedling abbreviations; S = Seibel; Mgt. = Millardet et de Grasset; a Rank averages, seventasfers on a seven point scale bHot pressed samples L = Lendot; S. V. = Seyve Villard, . . . . Methyl anthranilate added (0.7 ppm) using standard solution in EtOH  ..

I,, ..

.. . , .  .. ataw * inthra , oncen. win? . 4 rhb &Some "foxy" wine varieties frm of methyl snttiranilstr " Variety Parantap d WiW Omda Muscat V. vinifera X V. labrusce Carlos V. rotundifofia Diamond labrusca X ? outchess V. labrusca X 7 FrY V. rotundifolia M, wouri Riesling V. labrusca X V. riparia Eumetan V. aestivalis tabella V. labrusca Moored V. labrusca (hybrid) N. y. Muscat V. vinifera X V. labrusca vtncent V. labrusca (hybrid) R d foxiness were entirely free of methyl anthranilate including Carlos which was rated the most foxy of all wines tested. The case of Carlos (a bronze variety of the muscadine grape, I? rc, rundifoliu according to Ferree, 1975) is not unique. Table 5 lists several more varieties, both red and white, of various par entages that are generally considered t o produce foxy wines and art' free of methyl anthranilate a t levels determined in this work.

CONCLUSIONS METHYL ANTHRANILATE occurs with relative infrequency in wine varieties. It is limited to some varieties of V. hbrusca and a few crosses thereof. In addition, the concenlration methyl anthranilate present in many cases appears lo be bel the threshdld level which furlher minimizes its importance win e. aroma. If present in large quantities, as in some samples of Concc and Niagara wines, methyl anthranilate prohahly contribu to the characteristic "grapey" or "fruity" aroma of thc wines. However, any correlation of methyl anthranilate wi foxiness seems to be absent. It cannot he said that mett anthranilate does not contribute t o foxiness but it is clear t h it is not the primary source of the odor. Unlike the occuren of methyl anthranilate, foxiness is not limited to varieties K . lahrusca but is found in a wide range of native Americ. wines of dissimilar parentage. Further analytical and sensory work yifl be required determine the cause of the native American wine aron known as foxiness.

REFERENCES Amerine, h1. A.. Roesrler, E. B. and Filipello. F. 1959. Modern sensol methods of evaluating wine. Hilgardia 28: 477. Bailey, L. H. 1898. "The Evolution of our Native Fruits." The Ma millan Co.. New York. Ferree. M. E; 1975, Muscadine grape culture. Bull. 739. Univ. of G,

Nelson. R. R.. Aeree, T. E.. Lee, C. Y. and Butts, R. M. 1976. Gas liqui CoL of A&, Athens. Ga chomatographic determination of methyl anthranilate in win1 JAOAC. In pres Pool. R. M.. Robinson. W. B.. Einset. J., Kimball. K. H.. Watson J. P. an Bertino, J. J. 1976. Vineyard and cellar notes 1958 197a. Specis Power, F. B. 1921. The Detection of Methyl Anthranilate in Fmi Report No. 22.. N. Y. St. Ag. Exp. Sa.. Geneva. N. Y.

Juices J. Amer. Chem. SOC. 43: 377. Robinson. W. B.. Shaulis. N. J. and Pederson, C. S. 1949. Ripenin Studies of Grapes Grown in 1948 for Juice Manufacture. .Fruit Prod J. and A m Food Mfg. 29( 2): 36. 'Sa'e. J. WI and Wilson. J. B. 1926. Distribution of voIatile flavors L Winkler. A. J. 1972. ''General Viticulture." University of Caliiorni; grapes and grape juices J. Agr. R e s 33: 301.

hfs received 6/ 2/ 76; revised 7/ 9/ 76; accepted 1/ 16/ 16. bess. Berkeley. Calif. iNHERITANCE OF METHYL ANTHRANILATE AND TOTAL A. G. Reynolds, T. Fuleki, and W. D. Evans Respectivelyformergraduatestudent. Department of HorticulturalScience. University of Guelph. Guelph, Ontario. Canada N1G 2W1; Research Scientist, Horticultural Products Laborato ry. Horticultural Research Institute of Ontario, Vineland Station, Ontario LOR 2EO; and Associate Professor, Department of Horticultural Science, University of Guelph. Mr. Reynolds is currently pursuing further graduate study in the Department of Pomology and Viticulture, New York State Agricultural Experiment Station, Cornell University, Geneva, New York 14456. Taken in part from the MSc Thesis of the senisr author.

Thesenior authorwishes to thank Dr. R. E. Subden, Department of Botany and Genetics, University of Guelph, forhisencouragementandguidancethroughoutthecourse of this work. Presented at the Fifth Annual Meeting of the Eastern Section, American Society of Enologists, August 8. 1980. Erie, Pennsylvania.

Financial Assistance from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged.

Manuscript 25 May 1981 ,

Revised manuscript received 2 November 1981.

Accepted for publication 7 November 1981.

ABSTRACT Methyl anthranilate and total volatile esters concen trations were determined for two families of grape ' seedlings which resulted from crosses made at Vineland, Ontario in 1972. Chi square analysis of the segregation patterns suggested that three dominant complementary genes were involved in the inheritance of methyl anth

,., c ~

ranilate and two for total volatile esters. High heterosis r\. j and broadsense heritability values for both characters In Ontario, ,grapes are grown on approximately 10000 ha, about half of which are still planted to the traditional Vitis labruscana cultivars. Improvement of the Ontario grape and wine industry to allow it a greater competitiveness domestically has involved both the planting of French hybrid and V. uinifera cultivars, and the introduction of new wine grape cultivars from grape breeding programs such as that of the Horticultural ' Research Institute of Ontario a t Vineland Station, On tario. This latter breeding program has, as one of its, objectives, the elimination of the undesirable "foxy" or labrusca flavor character from its select, ions; however, due to the climatic constraints of the area, the objectives of winter hardiness, disease resistance, and vigor often ' take precedence over this objective, such that the utili

' zation of labrusca flavored but hardy cultivars as par en'ts is often necessary. Because of this problem, this study was initiated, to elucidate a mode of inheritance for the labrusca flavor charact. er, and to make recom mendations for its avoidance in wine grape breeding programs. dyid'

~

. .. An early reference to "foxiness" in grapes was made ;: by Hedrick et al. (9), who described the aroma of certain /,; ' V labruscana cultivars as being reminiscent of a fox's den or burrow. Early attempts to characterize this flavor Liq;/ j chemically' were reported by Power and Chesnut (Is), ,II I l l suggested dominance. Statistical differences between the families and examination of the ancestries of the parental cultivars allowed the postulation of genotypic formulae for the parents. No correlation wasfound between methyi anthranilate and volatile esters, or among either of these characters and soluble solids, winter hardiness, or vigor.

. " . ?' .; ,

who found a relationship between labrusca flavor char acter, V. labrusca ancestry, and the ester methyl anth ranilate (MA). Sale and Wilson (18) reported similar results, but indicated that other volatile esters such as ethyl acetate also made an important contribution to the flavor. Ho'lley et al. (11) detected eight components in the essence of Concord juice, of .. which MA, methyl acetate, and ethyl acetate were predominant. They concluded, however, that many other important aroma tic substances were probably overlooked due to the iimitation of their methods. The development of gas chromatography has allowed much more sophisticated and detailed investigations int. 0 the volatile composition of fruit crops. Neudoerffer et al. (13) identified 32 compounds in Concord essence using gas chromat. ography: Methyl anthranilate was not detected because it did not elute from the column. Similar, but less extensive was the list of Stevens et al. (201, who, $sing gas liquid chromatography mass spec trometry, sugg, ested methyl 3 buten 2 01 to be of great significance, along with ethyl acetate. Sixty components of Concord essence were detected by gaschromato graphy mass spectrometry by Stern et a/: (191, of which a series of crotonate esters and an alkylthioester were viewed as being important, as well as ethyl acetat. e, MA, and several other esters. For the facilitation of' selection of non" foxy" culti

14 ',; I Am. J. Enol. Vitic., Vot. 33, No. 1, 1982 in the wine grape breeding program in Ontario, ;r7..>, Li (6, r) has devised an index for t. he labrusca flavor acter based on the concentration of MA and total ,,, latile esters (T\ 'E) in the fruit. This score, known as \'ineland Grape Flavor Index (VGFI), has intro dltced a high degree of speed and objectivity into the celec'ti() n process. The derivat. ion of this index was based i,,, the high correlation between labrusca flavor charac ter M A and TVE concentration. The inheritance of specific flavor characters in Craps. most notably muscat flavor, have been investi

Pa ted by a number of European researchers .i n . V. l .i ,l i f p m . Wagner (231, using organoleptic evaluation, ,tlggel; ted t h a t a t least five dominant complementary plies were involved in muscat flavor inheritance. Tsek nlislrenk<> and Filinova (22), considered the character to be monogenic and dominant. M'agner et ai. (24) p0st. u laled a, mechanism for muscat flavor inheritance based o n the heritability of the five major terpenes normally Ihought to comprise the flavor. It was found that four of the five were inherited identically via a two gene system, ahereas the fifth. linaloijl, possessed a more complex inheritanre. Later studies (25,26) part. ially refuted t. his hypothesis, by indicating a correlation between terpenes previously thought to be unrelated, and a lack ofcorrela titm between terpene concentration and intensity of muscat flavor as perceived organoleptically.

. .. Genetic investigations into the labrusca flavor char

" ': have not been previously carried out. The objec

" of this research were to elucidate a mode of mdritance for the labrusca flavor charact. er based on 31.4 and TV E content, and to make recommen'dations on its minimization in wine grape breeding programs. MATERIALS AND METHODS Ayailable genet. ic material: All mat. eria1 for our investigations was grown and harvested at. the Grape Research Station, Vineland, Ontario. Cultural methods were carried out according to Ontario recommendations (141, and those of Bradt( 1). Two families of seedlings, namely V. 7218 and V. 7219 were chosen for investigation, tn, th ofwhich were the result of the hybridization of one high and one low VGFl parent carried out a t Vineland in 1972. Such a parentage maximized t. he probability of ohraining a significant number of non labrusca and !at) rusca flavored seedlings in the progenies. Family V. 7218 was a cross between V. 5407i (de Chapnac X Concord, Yineland 1954; MA = 0.14 ppm, TVE = 21 ppm) and Bertille Seyve 5563 (S. 6905 X B. S. :U 4% M A = nil, TVE = 3 ppm). A total of 229 seedlings wab available in the autumn of 1978. Family V. 7219 was a cross between V. 54077 and V. 50061 (Alden X Lo manto, Vineland 1950; hlA = nil, TVE = I pprn). In the autumn of 1978, 226 seedlings were available in this family. Fifty seedlings were chosen randomly from each fa2 ,. '.. for our investigations. of vigor and hardiness: In May, 1978, ne^ in both families were rated on a scale of 0 to 4 r( k vigor and wint. er hardiness. For vigor, shoot length and number constituted the criterion for the rating, Uihere ''4'' represented the most vigorous. I n assessing METHYL ANTHRANILATE  15 winter hardiness, the amount of killed buds was consid ered such that a rating of "0" indicated the hardiest vine. H a r v e s t i n g a n d s t o r a g e : M a n y s t u d i e s (3,4,15,17,18) have indicat. ed t. bal MA concentration increases with advancing fruit mat. urit. y, and decreases somewhat as the fruit becomes overripe. It was thus imperative to harvest each seedling at it. s optimum maturit. g. To accomplish. this, preharvest checks were initiated on September 1, 1978, and were continued twice weekly until all fruit was harvested. The fruit was considered ripe if the soluble solids content attained 20" Brix or greater, or was t. he same for three consecutive sampling dates, as measured by a hand refractometer. Harvesting took place between September 15 and Oct o. ber 18, 1978. Once the fruit of each genotype was harvested, it was placed in a plastic bag and transported to the Biochem istry Laboratory, Department. of Horticultural Science, University of Guelph. Each sample was then washed, . destemmed, and replaced int. o its bag. Subsequent st. or age t. ook place at 31OC. Sample preparation: Each sample was ground in a hand mill, and 50 g of the homogenate was steam distilled (SGA Scientific, Bloomfield, N. J. No. JD 2115) until 100 mL of distillate was collected. This procedure was duplicated for each genotype. A 50 mL beaker of the homogenate was also retained for subsequent soluble solids determination using an Abbe type refractornet. er. Methyl anthranilate determination: The determi nation of MA concentration was done according to the .

fluorometric procedure of Casimir et d l . (2).. Fluoromet ric determination was carried out on a Turner Model 110 fluorometer (G . K. Turner ASSOC., Palo Alto, Calif.) equipped with an attachment for accepting glass cu vettes. A narrow pass (360 nm) and a sharp cut (415 nm) filter served as primary and secondary filters re spectively. Sample fluorometric readings were related to hlA concentration through the use of st. andard curves. using a series of aqueous MA solutions that ranged in concentrat. ion from 0.05 to 10 ppm. Total volatile esters' determination: The det. ermi nation of TVE concentration was carried out by Hill's method (10) as described by Thompson (21). Spectro photometric determination was done using a Coleman Junior I1 Model 6/ 20 spectrophotomet. er (Coleman In struments, Maywood, Ill.) set. at a wavelength of 540 nm. Absorbance was related to TVE concentrat. ion by the use of standard curves, using a series of aqueous ethyl acetate solut. ions that ranged in concent, ration from 0 to 125 ppm.

RESULTS AND DISCUSSION Variability within families: The variability en countered within the families was . great, but specific patterns were nonetheless existent. Hist. ograms (Figs. 1 and 2) relating seedling frequency and MA or TVE concentration illustrate this variability, suggesting an absence of simple Mendelian inheritance in either sys tem. The lack of a normal distribution in t. he data also suggested that. a typical polygenic system was also not Am .I Fnol Vitir 16  METHYL ANTHRANILATE ..

f 0 V. 7219 SEEDLING CLASS PPm M A x 100 Fig. 1. Distribution of seedlings in families V. 7218 and V. 7219 according to their methyl anthranilate concentration.

40.

> V 2 Y V. 7218 V. 7219 SEEDLING CLASS ppm TVE Fig. 2. Distribution of seedlings in families V. 7218 and V. 7219 according to their volatile esters concentration.

present. Most noticeable vias a distinct. bimodal distribution which could be observed for both characters in each family, whereby the majority of seedlings lag to the extreme left as one group, and the others were scattered to the right. in a less homogeneous group. This distribu tion conveniently placed the seedlings into one of two categories, that of labrusca" or "non labrusca", in terms of MA and TVE. The line of demarcation bet. ween the two categories for M A appeared to be close to 0.10 ppm (Fig. 11, a value suggested by Nelson et al. (13) as being the compound7s organoleptic threshold in wine. For TVE, the point of delineation bet. ween the two classes was found to be about 12 ppm (Fig. 2), a level suggested by Fuleki (5) as being that. where labrusca flavor be comes noticeable organoleptically. Number of genes involved After placement of the seedlings into either the %on labrusca'' or "labrusca"

,/ ' class i n terms of MA and TVE. the data were subjected to Chi square goodness of fit tests. Table 1 indicates, faF MA, the ratios were 5: 3 and i :l for families V. 7218 and Y. 7219 respectively. These ratios corresponded with a hypothesis o f t hre. e dominant complementary genes for t. he inheritance of this compound. The large variation io MA concentration in the "labrusca" class could be at. trib uted to modifier genes, an explanation offered by Wag. ner (231, for the control of muscat flavor. The year to. year variation in the concentration of MA (and TVE well) inmost V. labruscana cultivars could also be explained by modifier genes.

Table 1. Chi square goodness of fit analysis of methyl anthranila; concentration in families V. 7218 and V. 72193 FamilyNon labruscab Labruscac Ratio P

ObservedExpectedObservedExpected V. 7218 32 31.25 18 18.75 5 3 .80. 9< V. 7219 44 43.75 6 6.25 7: l .90. 95 a Observedand expected valuesarebased on a sample of 50 b Methyl anthranilate concentration = 0 to 0.1 ppm. c Methyl anthranilate concentration = 0.1 ppm or higher.  seedlmgs.

In the case of TVE, 5: 3 ratios were obtained for both families (Table 2). These corresponded in simplest t. erms to a hypothesis of two dominant complementq genes for the control of this group of compounds. As previously indicated for MA, modifier genes were likely responsible for the. large variation in TVE concent. ration  in the "labrusca" class.

Table 2. Chi square goodness of ftt analysis for total volatile ester; concentration in familiesV. 7218andV. 7219a . `

Famity Non IabruscabLabruscac Ratio P ObservedExpectedObservedExpected V. 7218 31 31.25 19 18.75 513 .90. 95 V. 7219 32 31.25 18 18.75 5: 3 .80. 90 a Observed and expected valuesarebased on a sample of 50 b Total volatile esters concentration'= 0 to 12 ppm. c Total volatile esters concenfration = 12 pprn or higher.

It must be stressed that these dat. a possess some degree of weakness due to the small sample sizes. Other rat, ios teg. 3: l or 9 7 ) are possible, although not as statistically acceptable. Insofar as gene nomenclature is concerned, three genes, henceforth designated as M, A, and F, are sug gest. ed for the synt. hesis of hlA in grapes. Seedlings in the "labruscar class, which. were characterized by rela tively high hfA concentrations, could logically result from all three loci being in the dominant condition. The "non labrusca" class contained seedlings either with no MA or with concentrations less than 0.10 ppm. Those cont. aining no MA whatsoever would have recessiveness at all three loci, whereas those containing minute amounts of the compound could have at least one dominant locus. In the case of TVE, two genes, designated as V and E. are suggest. ed for volatile ester synthesis in grapes. A seedling possessing both genes in the dominant condi tion would be one characteristic of the "labrusca" class: seedlings.

Am. 4. Enol. Vitic., Vol. 33, No. I , 1982 Heterosis =  x 100 F1 F, + P, ,, here F, is the averagcvalue of a particular character in the F, generation and P, is the midparent value for that. The high values displayed for both hk4 and T\ 'E i i the two families suggested that dominant genes were most likely present (Table 3).

&e 3. Transgressive segregation (heterosis) of methyl anthranilate a, s total volatile esters over the parental cultivars In families V. 7218 and V. 7219 " f a d y Heterosis (0. b)

Methyl Total volatile anthranilate v 7218 491.4 65.0 \' 7219 55.7 65.5 .

i (i : High broadsense heritability (h') has also been impli : cated as a criterion for dominance. It is expressed as the percentage of the total variance of a progeny that can be 1. attributed to genotype, and can be summarized in the 1. following formula:

z 0, '

3 ff, ' + 4 2 ..

4 hz = x 100 I where o i is variance due to genot. ype and u: is variance i due to environment. The quantity u: was broken down I  into ua and c;,? which represent variance found between duplicate samples wit. hin a genotype and variance due to error respectively. An estimate of the u,", values for MA i and TYE were obtained from an analysis of variance of i 51.4 and TVE concentrations in several 1, '. labruscana cultivars over a nine year period. The u3 values were t ohtained from a one way model LI analysis of variance of '. each family.

1 .. The h2 values for both characters viere relatively high * In both families (Table 4), with the exception of that. for 2 ,It.\ infarn'ily V. 7219. Such information uas evidence i .'that dominant. genes were present. t I t 8  .

Ta% 4. Variance components and broadsense heritability of methyl anthranilate and total volatile esters in families V. 7218 and V. 721ga . L ,>_

.z I Character 4 I , 3rnnlly METHYL ANTHRANILATE  17 Parental genotypic formulae: The compilation of information previously reported here and the consider ation of the ancestrx of the parental cultivars allowed the postulation of genotypic formulae for the parents. Since the seed parent V. 5407i was common for the two families. any statistical differences between the families were attributable t. o the pollen parents B S 5563 and Y. 50061. Basic stat. istics such as mean and variance indicated large differences between the families in terms of hfA and TVE (data not shown). Further statistical analysis examining coefficients of variation and equalit_ v of variances confirmed these differences, and strongly suggested t. hat the pollen parents were not the same genotypically, at., least in terms of MA (Tables 5 and 6).

Table 5. Coefficients of variation formethylanthranilateand total volatile esters concentration in families V. 7218 and V. 7219. Family Character Methyl anthranilateTotalvolatileesters . V .7 2 1 8 5 7 3 .4 8 V. 7219 3336.62 27.93 5622.44 Table 6. Equality of variancesformethylanthranilateand total volatile esters in families V. 7218 and V. 7219. Character Methyl anthranilate 24.87" Total volatile esters 2.0342'

* p = 0.05 ** p = 0.01 .  With this in mind, genotypic formulae. were 'esta6 lished for the parental cultivars in terms of MA and TYE. A collation of the information from the Chi square t. ests (Tables 1 and 21, the statistical analysis (Tables 5 and 61, and the ancest. ry of the parental cultivars (16) .

provided the basis for this. With consideration to MA in family V. 7218, it is suggested that V. 54077 (MA = 0.14 ppm) is of the genotype MmAaFf, while B. S. 5563 (MA = nil) is MM. 4aff or any similar genotype cont. aining a homozygous dominant, a heterozygous, and a recessive . .

locus. For family V. 7219, V. 54077 is as previously noted, while V. 50061 (MA = nil) is rnrnaaff. If one examines the ancestries of these cultivars, these genotypic formulae can be qualified. The cultivar V 54O'ii is a cross between Concord, probably a pure t'. labrusca cultivar (and likely a homozygote in terms of .

MA and TVE), and de Chaunac, whose background is V. labrusca free for OUT generations, (and is likely ti, be a homozygous recessive). An F, progeny which is heterozy gous at all three loci is thus probable. For B. S. 5563, the parents are both French hybrids, but the pollen parent, B. S. 34 45, has the V. lhbruscana cuhivar Othello as a 1 ~~ ~. ~~~ ~~ ~ ~ .~ . . ~ ~~ ~ ~ " ~~

~ ~