Europaisches Patentamt (19) European Patent Office Office europeeneen des brevets EP 0 821 055 A2
(12) EUROPEAN PATENT APPLICATION
(43) Date of publication: (51) |nt CI C12G 3/06, C12C 5/02, 28.01.1998 Bulletin 1998/05 A23L 1/236 A23L 1/226 aa^ol. (21) Application number: 97304614.7
(22) Date of filing: 27.06.1997
(84) Designated Contracting States: • Kossiakoff, Nicholas AT BE CH DE DK ES Fl FR GB GR IE IT LI LU MC 78240 Chambourcy (FR) NL PT SE • Hawn, Regina D. Matawan, New Jersey 07747 (US) (30) Priority: 25.07.1996 US 22597 P • Scharpf, Lewis G., Jr. 06.03.1997 US 813884 Fair Haven, New Jersey 07704 (US)
(71) Applicant: INTERNATIONAL FLAVORS & (74) Representative: Brown, John David et al FRAGRANCES INC. FORRESTER & BOEHMERT New York New York 1 001 9 (US) Franz-Joseph-Strasse 38 80801 Miinchen (DE) (72) Inventors: • Bolen, Paul L. Middletown, New Jersey 07701 (US)
(54) Use of monellin in enhancing the flavour of beverages and foodstuffs
(57) Described is the use of monellin and single- ing or enhancing the organoleptic properties of food- chain analogues of monellin taken alone or further to- stuffs and beverages, particularly alcohol-containing gether with sclareolide and/or succinic acid in augment- beverages; and improving the properties of flavorants such as isobutyl thiazole.
Printed by Jouve, 75001 PARIS (FR) EP 0 821 055 A2
Description
Our invention is directed to fermentation-derived beverages and distilled spirits which are consumable or ingestible and which comprise: 5 (i) from 1 .5% up to 70% by weight of an alcohol or ester selected from the group consisting of ethyl alcohol, isoamyl alcohol, n-hexanol, /.-menthol and cis-3-hexenyl acetate;
(ii) from 0.5 parts per million (ppm) up to 15 ppm of monellin or an analogue thereof; and 10 (iii) from about 0.1 parts per billion (ppb) up to about 2 by weight of a fermentation-derived beverage or distilled spirit flavorant.
Our invention is also directed to a process for augmenting or enhancing the aroma and/or taste of a fermentation- 's derived beverage or distilled spirit comprising the step of adding to a fermentation-derived beverage or distilled spirit from 0.5 ppm up to 1 5 ppm of monellin or an analogue thereof. Our invention is also directed to monellin (or an analogue thereof) containing flavorants containing from about 0.5 up to about 1 5 ppm of monellin or an analogue thereof and from about 0.01 up to about 200 ppm of at least one flavorant selected from the group consisting of: 20 isobutyl thiazole; trimethyl pyrazine; 2- methoxy-3-isobutyl pyrazine; menthol; 25 isopulegol; trans-2-hexenol; 6-methyl-5-hepten-2-one; methyl acetophenone; vanilla; 30 vanillin; 3- methyl-2-buten-1-ol; a mixture of 3-methyl-2-buten-1-ol and ethyl acetate; a mixture of 3-methyl-2-buten-1-ol, ethyl acetate and CITRAL ; natural banana essence; 35 ethyl decanoate; natural raspberry flavor essence in admixture with a-terpineol; P-ionone; bis(methylfuryl)disulfide; farnesene; 40 germacrene; ethyl pyruvate; 1 ,3,5-undecatriene; and indole.
45 Our invention is also directed to monellin (or an analogue thereof) in admixture with peanut butter, preferably low- fat peanut butter. As will be seen in the examples provided, infra, the monellin or analogue thereof has an extremely favorable flavor and mouthfeel impact upon the fermentation-derived beverage or distilled spirit. Examples of fermentation-derived beverages and distilled spirits are beer (both low alcohol beer, medium alcohol beer and high alcohol beer), wine, so liqueurs; and hard liquors including vodka, rum, brandy, bourbon, Scotch whiskies and the like. The term "monellin or an analogue thereof" can mean natural monellin protein such as that disclosed by Somoza, et al, in J. Mol. Biol. (1993), 234, pages 390-404, title "Two Crystal Structures of a Potently Sweet protein/Natural Monellin at 2-75 A Resolution and Single-Chain Monellin at 1-7 A Resolution" defined according to Figures 1, 2 and 4B described, supra, or analogues thereof as set forth in the following list: 55
2 EP 0 821 055 A2
TABLE I
ANALOGUES OF NATIVE MONELLIN
Monellin analogue Native monellin of Figures 1, 2 and 4B GluB49Asn,AsnB50Glu AspA22Asn, GluA2 5Gln, AspA2 6Asn, GluB4 9Asn, AsnB50Glu GluB4 9Asn, AsnB50Glu CysB41Ser AspA2 2Asn GluA2 5Gln AspA2 6Asn AspB7Asn AspB7Abu (Abu - 1-g-aminobutyr ic acid) AspB7Glu AspB7Gly AspB7D-Asp lleB6Gly lleB6Ala lleB6D-Ala lleB8Gly lleB8Ala lleB8Phe lleB8D-Ala TyrAl 3Gly TyrA13Phe GlyB9Ala GlyB9D-Ala LysA4Ahx (Ahx - 1-a-amino hexanoic acid) LysAl 7Ahx LysA2 8Ahx LysA33Ahx LysB17Ahx LysB25Ahx LysB3 6Ahx GlnBl 3Ahx LysB44Ahx GlnBl 3Ahx, LysB55Ahx EP 0 821 055 A2
In addition, the "monellin or an analogue thereof" of our invention can be a single-chain monellin or mutant thereof as set forth in Figure 3A or Figure 3B(ii) and prepared according to Published Japanese Application No. JP05/070494 (abstracted at Chemical Abstracts, Volume 119:48069g, thusly: 11 9:48069G Single-stranded monellinsfor improved thermostability, lijima, Hiroshi; Sone, Hidetaka (Kirin Brewery) 5 Jpn. Kokai Tokkyo Koho JP 05 70,494 [93 70,494] (CI. C07K7/10), 23 Mar 1993, JP Appl. 90/196,983, 25 Jul 1990; 29 pp. The single-stranded (ss) monellins (I) retain the sweetness of the wild type two-stranded I and have enhanced thermostability, esp. in lower pH, and protease resistance. The ss-l are prepd. by linking the C-terminus of B chain of I with the N-terminal Phe of A chain with a linker amino acid Xaa (Xaa = Gly, Ser, Glu, or Thr). Prepn. of a ss-l using Gly as the linker amino acid by expression of the synthetic gene in Escherichia coii and Saccharomyces cerevisiae o was shown. The ss-l retained its sweetness after incubation for 20 mins at 100°, but the wild type lost its sweetness at 50°. Moreover, the ss-l but not the wild type counterpart was resistant to proteinase degrdn. with e.g. Achromobacter proteinase I. Replacing the Val12 or Val37 of the ss-l with Ala did not alter the thermostability and protease resistance, or according to the procedure or U.S. Letters Patent No. 5,478,923 issued on December 26, 1995, the specification for which is incorporated by reference herein. Examples of mutants of single-chain monellin are as follows:
TABLE II
SINGLE -CHAIN MONELLIN MUTANTS 20
Single-chain monellin of Figure 3A Single-chain monellin of Figure 3B(ii) Lys43Glu Arg70Glu Lysl7Cys, Phe34Cys (disulfide) Lys44Cys, Glu57Cys (disulfide) Lys44Gln, Glu48Gln, Glu50Gln, Arg57Gln, Glu52Gln, Lys54Gln Gln59Cys, Asp72Cys, Cys41Ser Phe34Asp GlylMet, Glu2Met Glu59Cys Cys41Ser Met42Val Trp3Thr, He5Glu,Met42Asn Deletion of Pro94 Deletion of Pro92 to Pro94 Deletion of Pro90 to Pro94
As stated above, the amount of monellin or analogue thereof may be used in the beverage at levels of from 0.05 ppm up to 15 ppm depending upon the nature of the beverage and the type of flavorant used. Indeed, good results are obtained as will be shown in the Examples, infra, at levels of 0.1 ppm with chocolate flavor using natural monellin or recombinant monellin. When used with cis-3-hexenyl acetate, levels of monellin or analogues thereof of from 3 and 5 ppm are useful. When used with liqueurs such as blueberry liqueur or Liqueur Perle de Brillet, levels of 10 ppm are useful. In addition to the monellin or analogue thereof and flavor added to the alcoholic beverage composition, other materials have been found by us to be useful in augmenting or enhancing the aroma and taste of the fermentation- derived beverage or distilled spirit. These other materials are as follows:
4 EP 0 821 055 A2
(i) thaumatin, including Thaumatin I, Thaumatin II, Thaumatin B and mixtures thereof such as TALIN® (trademark of Tate and Lyle Limited of the United Kingdom) ;
(ii) succinic acid;
(iii) sclareolide having the structure:
35 The amount of succinic acid that can be used can vary from 0.05 ppm up to 2%. The amount of sclareolide that can be used may vary from 0.05 ppm up to 2%. The amount of thaumatin that can be used may vary from 0.1 ppb up to about 15 ppm by weight of the fermentation-derived beverage or distilled spirit. When thaumatin is used with the monellin or analogue thereof, it is preferable to use a ratio of thaumatin:monellin or analogue thereof of about 1:1. However, for the purposes of our invention, a ratio of from about 0.05 parts monellin: 40 1 part thaumatin up to 1 part thaumatin:0.05 parts monellin is within the scope of our invention. The following examples are given to illustrate embodiments of the invention as it is preferred to practice it. It will be understood that these examples are illustrative and the invention is not to be considered as restricted thereto except as indicated in the appended claims.
45 EXAMPLE I
EXAMPLE l(A)
CHOCOLATE FLAVOR FORMULATION 50 The following formulation is prepared:
"AAA COMPOSITION" Ingredients Parts by Weight Sclareolide 50 Caryophyllene beta 50
5 EP 0 821 055 A2
(continued) "AAA COMPOSITION" Ingredients Parts by Weight 5-Methyl-2-phenyl-2-hexanal 130 Isovaleric acid 25 Phenyl acetaldehyde 80 Phenyl ethyl acetate 10 Geranyl acetate 30 Linalool 150 Furaneol15% 240 2-Ethyl-3-methyl pyrazine 0.4 2-Ethyl-5-methyl pyrazine 0.1 2,3,5-Trimethyl pyrazine 0.4 Propylene glycol 234.1 Total:1,000
EXAMPLE I (B) 25 COMPARISONS
Water bfatural Monellin (1 ppm) Reccntoinant Monellin 30 of Figures 1, 2 and 4B of Figure 4A (1 ppm) AAA 1 ppm: Typical AAA 1 ppm: Typical AAA AAA 1 ppm: Typical AAA (chocolate) chocolate note with a AAA chocolate note, note. larger mouth feel . but here the There is also a metallic/acid 35 metallic/acid aftertaste is aftertaste in the reduced, back. The monellin Ranking (on a scale seems to boost this of 1-10 with 10 of aftertaste being the 40 type . Ranking (on a scale best) :9/10 of 1-10 vith 10 being the best) :7/10
45 The water has now been replaced with filtered water in order to reduce the metallic/acid aftertaste and the result are as follows:
50
55
6 EP 0 821 055 A2
Water Monellin (1 ppm) Re 20 The chocolate flavor of Example IA is now used at a level of 0.1 ppm in 70% aqueous ethyl alcohol: Water bfetural Monellin (1 ppm) Beccrnbinant Monellin of 1, 2 and 4B of Figure 3B(ii) 25 Figures (1 ppm) AAA 0.1 ppm (10 AAA 0.1 ppm (10 AAA 1 ppm (10 drops drops of alcohol drops of ethanol of ethanol solution) : Typical solution) : Typical solution) : Typical 3o AAA note. AAA, no difference AAA note, but in the flavor but alcohol is enhancement of ethyl stronger . alcohol traces. Ranking (on a scale Ranking (on a scale of 1-10 vith 10 35 of 1-10 vith 10 being the best) :8/10 being the best) :7/10 EXAMPLE II 40 EXAMPLE 11(A) The following mint flavor formulation is prepared: Ingredients Parts by Weight Sclareolide 50 Caryophyllene beta 50 Methanone 50 Limonene 25 Thymol 20 Eucalyptol 10 Terpineol 4 4 Myrcene 4 7 EP 0 821 055 A2 (continued) Ingredients Parts by Weight para Cymene 2 gamma Terpinene 2 Peppermint oil 783 Total: 1,000 10 EXAMPLE ll(B) COMPARISONS 15 Water Natural Moral J in (1 ppm) Reocmbinant Monellin of Figures 1, 2 and 4B of Figure 4A (1 ppm) Mint flavor of Mint flavor of Mint flavor of Example 11(A) (0.1 Example 11(A) at 0.1 Example 11(A) (1 ppm) : Typical mint ppm:Mint impact is ppm: Typical mint flavor impact. depressed with ethyl impact with a sweet alcohol impression alcohol note. The covering the mint product is rounder, note. Ranking: 6/10 Ranking: 8/10 EXAMPLE III 30 Additional comparisons were carried out as follows: 35 Water Natural Monellin (10 ppm) Raoombinant Monellin of of Figures 1, 2 and 4B Figure 3B(ii) (10 ppm) AAA flavor of AAA flavor of AAA flavor of Example 1(A) (0.5 Example 1(A) (0.5 Example 1(A) (0.5 ppm) : Typical AAA ppm) : Typical AAA note ppm) : Typical AAA 40 (chocolate) note. with a depression of (chocolate) note; the floral chocolate better flavor impact impact. The sweet with alcohol impact is impression. The instantaneous with no sweet nuance is 45 delay. i nstantaneous , but Ranking: 8/10 with a small licorice aftertaste . Ranking: 9/10 50 8 EP 0 821 055 A2 Water Natural Monellin (10 ppm) Reocrnbinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Isoamyl alcohol Isoamyl alcohol Isoamyl alcohol (1 ppm): Typical (1 ppm) : Typical , but (1 ppm) : Typical , but isoamyl alcohol we have a stronger much stronger impression, isoamyl alcohol isoamyl alcohol fruity, winey. impact together with impact. Isoamyl a sweet aftertaste . alcohol is around Ranking: 8/10 your mouth with a coating impression. Ranking: 9/10 Water Natural Monellin (10 ppm) Reocrnbinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Hexanol (1 ppm) : Hexanol (1 ppm) : Hexanol (1 ppm) : Typical hexanol Typical stronger, Typical, but much note with a winey hexanol note stronger with a green, skinny, with a sweet vegetable , full, fruity, winey aftertaste . strong, green note note. Ranking: 8/10 with a sweet impact. Ranking: 9/10 Water Natural Monellin (10 ppm) Recombinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Hexanol (5 ppm) : Hexanol (5 ppm) : Hexanol (5 ppm) : Typical hexanol Typical stronger with Typical, but much note with' a waxy, a dry impression of stronger with a fatty, green rum. nice, sweet impact, beans note; the Ranking:7/10 Ranking: 9/10 profile is flat. Water Natural Monellin (10 ppm) Recombinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Cis-3-hexenyl- Cis-3-hexenyl -acetate Cis-3-hexenyl-acetate acetate (5 ppm): (5 ppm) : Typical (5 ppm): Typical, but Typical green, stronger with a more much stronger with a fruity, beany tea green, living note natural, full, note. with a sweet impact. strong, green note. Ranking: 9/10 The dosage is too strong. Ranking: 8/10 9 IP 0 821 055 A2 Water Natural Monellin (10 ppm) Reccnbinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Cis-3-hexenyl- Cis-3-hexenyl-acetate Cis-3-hexenyl- acetate (3 ppm): (3 ppm) : Typical acetate (3 ppm): Typical green, stronger with a more Typical, but no more fruity, straw- fresh green, living chemical impact, berry note. note. Rank±ng:7/10 Ranking: 9/10 Water Natural Monellin (10 ppm) Recombinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) L-Menthol (5 I-Menthol (5 ppm) : L-Menthol (5 ppm) : ppm) : Typical Typical stronger with Typical stronger. green, fruity, a more fresh green, Ranking: 9/10 strawberry note. living note. Ranking: 8/10 Water Natural Monellin (10 ppm) Recombinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Citral (0.5 ppm): Citral (0.5 ppm): Citral (0.5 ppm): Typical citral, Depression of citral, Depression of the lemon note. lemon impact. typical citral note. Ranking: 4/10 Ranking: 4/10 Water Natural Monellin (10 ppm) Recombinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Citral (5 ppm) : Citral (5 ppm) :Very Citral (5 ppm) :Very Typical citral, strong depression of strong depression of lemon note. the citral, lemon the typical citral impact. Ranking: 4/10 note. Ranking: 4/10 Water Natural Monellin (10 ppm) Recombinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Gamma decalactone Gamma decalactone (5 Gamma decalactone (5 (5 ppm) -.Typical ppm):Butter lactone ppm):£?utter lactone butter lactone. with a pungent taste with a fruity and with a linoleic impression, taste. Ranking: 5/10 Ranking: 7/10 10 EP 0 821 055 A2 Water Natural Monellin (10 ppm) Recombinant Monellin of of Figures 1, 2 and 43 Figure 4A (10 ppm) Oxyphenylon (15 Oxyphenylon (15 ppm) : Oxyphenylon (15 ppm) -.Sweet, warm, Boosting the rasp- ppm) -.Direct impact of fruity rasp- berry flavor, but oxyphenylon notes, berry, powdery with a strong, soapy, but the soapy note is taste. powder note. very strong?? [Oxyphenylon has Ranking: 4/10 Ranking: 3/10 the structure : Water Natural Monellin (10 ppm) Recombinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Acetaldehyde (5 Acetaldehyde (5 ppm) : Acetaldehyde (5 ppm) : Aceta 1 dehyde No working depressing ppm) : No working impact. the acetaldehyde depressing the impact. Ranking: 2/10 acetaldehyde impact. Ranking: 3/10 Blueberry liquor Blueberry liquor plus Blueberry liquor plus (be Kuyper) Natural Monellin (10 ppm) Recombinant Monellin of of Figures 1, 2 and 4B Figure 4A (10 ppm) Standard product More fruity, more The fruity impact is with blueberry volatility; the more ripe type, but flavor plus alcohol impact is the alcohol impact is extract. stronger. very high. Ranking:7/10 Ranking: 9/10 11 IP 0 821 055 A2 Blueberry Blueberry liquor Blueberry liquor Blueberry liquor liquor plus "Thaumatin" plus Ifetural plus Recombinant (De Kuypar) (TALIN*) (0.5 ppn) Monellin (10 ppm) of Monellin of Figure Figures 1, 2 and 4B 4A (10 ppm) Standard More natural Fruity with Fruity, but very product. fruit flavor; strong alcohol strong alcohol no action on impact. impact. the alcohol Ranking: 7/10 Ranking: 8/10 1 evel . Ranking: 6/10 Liqueur Liqueur Perle de Liqueur Perle de Liqueur Perl e de Perle de Brillet plus Brillet plus Natural Brillet plus Brillet "Thaumatin'' Monellin (10 ppm) of Recxarbinant 24° Ale (TALIN®) (0.5 ppm) Figures 1, 2 and 4B Monellin of Figure 4A (10 ppm) Apple More natural More body; amyl Fruity, but very Pear type fruit flavor; alcohol and strong alcohol of no action on hexanol is higher impact may be 20- flavor. the alcohol with strong ethyl 25% stronger. level. alcohol impact, Ranking:8/10 Ranking: 6/10 20% more. Ranking: 7/10 Neutral liquor Neutral liquor plus Neutral liquor plus Natural Monellin (5 ppm) Recombinant Monellin of of Figures 1, 2 and 4B Figure 4A (5 ppm) Standard neutral More fruity, more The alcohol impact product. volatility; the is very high. alcohol impact is Ranking: 9/10 stronger . Ranking: 7/10 12 EP 0 821 055 A2 Liquor 12° Liquor 12° brix liquor 12° brix 20% Liquor 12° brix 20% brix 20% 20% alcohol with alcohol with Lemon alcohol with lemon alcohol Lemon flavor flavor (40 ppm) plus flavor (40 ppm) plus with Lemon (40 ppm) plus "Thaumatin" (TAT_EN®) "Thaumatin" (TALIN®) flavor tetural Monellin (1 ppn) (1 ppm) plus (40 ppm) (5 ppm) of R_ccnbinant Figures 1, 2 and Monellin of Figure 4B 4A (1 ppm) Lemon Stronger ethyl The citrus impact Nice citrus impact. alcohol is better, impact with a impact, stronger, more stronger alcohol depression of natural, no impression. the lemon impact on Ranking: 8/10 impact. alcohol. Ranking: 5/10 Ranking: 6/10 The lemon flavor of the foregoing Example III has the following formulation: Lemon Flavor Ingredients Parts by Weight Lemon essential oil 13.0 Citral 2.0 Cis-3-hexenyl acetate 1.0 Citric acid 2.0 The "apple pear type of flavor" of the foregoing Example III contains the following ingredients and parts by we Pear Flavor Ingredients Parts by Weight Amyl acetate 1,340.0 Amyl valerate 130.0 Benzyl acetate (10% solution in 95% food grade ethyl alcohol) 25.0 Benzyl buty rate 1.0 Bergamot essential oil 1 .0 n-Butyl acetate 1.0 Ethyl acetate 80.0 Ethyl butyrate 13.0 Ethyl decanoate 2.0 Ethyl heptylate 2.0 Ethyl hexylate 1.0 Ethyl octanoate 2.0 Geranyl propionate 100.0 Hexyl acetate 2.0 13 EP 0 821 055 A2 (continued) Pear Flavor Ingredients Parts by Weight a-lonone (1% solution in 95% food grade ethyl alcohol) 1 .0 Isoamyl acetate 1.0 Isoamyl valerate 1.0 CITRAL™ 3.0 Methyl acetate 1.0 Methyl heptenone 1.0 Orris resinoid (1% solution in 95% food grade ethyl alcohol) 5.0 Propyl acetate 1.0 Citronellol (1% solution in 95% food grade ethyl alcohol) 1 .0 y-Undecalactone 1.0 Vanillin 5.0 Propylene glycol 279.0 Total 2,000.0 The blueberry flavor used in the foregoing Example III has the following formulation: Blueberry Flavor Ingredients Parts by Weight Amyl acetate 500.0 Amyl butyrate 15.0 Anethol 0.5 Benzyl salicylate 10.0 Acetyl methyl carbinol 8.0 Dimethyl anthranilate 1 .0 Ethyl acetate 60.0 Ethyl butyrate 74.0 Ethyl methylphenylglycidate 20.0 Ethyl succinate 1 .0 Ethyl valerate 1 .0 Geraniol 13.0 Hexanal 1 .0 Cis-3-hexen-1-ol 1.0 2-Hexenyl acetate 1 .0 2-Hexenyl butyrate 1 .0 Hexyl butyrate 1 .0 4-(p-Hydroxyphenyl-2-butanone) 16.0 a-lonone 210.0 14 EP 0 821 055 A2 (continued) Blueberry Flavor Ingredients Parts by Weight Isobutyl cinnamate 1 .0 Jasmine, absolute (10% solution in 95% food grade ethanol) 7.0 Lemon essential oil 13.0 Maltol 3.0 Methyl butyrate 1 .0 Methyl capronate 1 .0 Methyl disulfide 1.0 methyl p-naphthyl ketone 1 .0 50-50 Weight-weight mixture of orris resinoid and a-irone) 60.5 Rose, absolute 10.0 Terpenyl acetate 1 .0 y-Undecalactone 10.0 Ethyl vanillin 86.0 Propylene glycol 870.0 Total 2,000.0 EXAMPLE IV FLAVORANTS AND PEANUT BUTTER CONTAINING, IN ADDITION, NATURAL MONELLIN The following table sets forth various flavorants used at various levels in aqueous solutions of ethanol, their eval- uations and the ranking of same on a scale of 1 -5, with 5 being the best of the overall sensory perception value thereof when using 5 ppm of natural monellin of Figures 1 , 2 and 4B. 15 iP 0 821 055 A2 Quality Ranking cn a Scale of 1- 5, with 5 Baing the Bast of Molecule CX^rall Sensory Molecule Ccncentration Evaluation Percepticn "Value Natural 2 ppm (using a The monellin decreases 2 dimethyl 1% solution) volatility, but causes a sulfide larger flavor impact. Also a 'Yon-sugar" sweetness impact is created. Ethyl acetate 5 ppm (using a No impact. 0 1% solution) Ethyl acetate 40 ppm (using a The monellin increases the 1 10% solution) volatility together with the sweet perception. Acetaldehyde 5 ppm (using a A higher, sweet impact; 1 1% solution) with no change in volatility. Isobutyl 0.1 ppm (using The green vegetable tomato 5 thiazole a 0.1% nuance impact is greatly (TCMATOZOLE®1 ) solution) increased. The natural tomato nuance is also improved. Natural 2,5- 1 ppm (using a The pyrazine impact is 2 dimethyl 1% solution) increased. The naturalness pyrazine of the product is increased. Trimethyl 1 ppm (using a Pyrazine impact 3 pyrazine 1% solution) strengthened. (TCKPAZINEf 001) 2-Methoxy-3- 0.01 ppm (using Pyrazine note is enhanced 3 isobutyl a 0.1% with a more natural impact. pyrazine solution) The taste can be described (GAlBAZINEf) as "extremely powerful, earthy, herbaceous, vegetable, green bell pepper flavor" having use levels in the finished consumer product of from 0.0001-0.05 ppm. 16 EP 0 821 055 A2 :ontinued/ Qjality Ranking 5, vd.th'i'5 Being the Best of Molecule CX^raH Sensory Molecule Conoantration Evaluation Percepticn Value Menthol 20 ppm (using a Freshness, effervescent 4 10% solution) effect and fresh impact is improved. Trans-2- 1 ppm (using a A green, fruity, stronger 1 hexenal 1% solution) arcma and taste with waxy nuances . Trans-2- 4 ppm (using a A green arcma and taste 1 hexenal 1% solution) with soapy nuance; but not "natural" . Natural 2 ppm (using a A minty, cooling arcma with 3 isopulegol 1% solution) a larger perception of cooling on the tongue. Natural 5 ppm (using a The monellin causes a 3 isopulegol 1% solution) larger impact having a rounder flavor with a sweet, sugar background. Trans-2- 2 ppm (using a The monellin causes a real 4 hexenol 1% solution) increase of intensity, a fuller greater impact on the side of the tongue. 6-Methyl-5- 1 ppm (using a The monellin increases the 4 hepten-2-one 1% solution) power of the methyl heptenone giving it a more natural impact. Methyl 2 ppm (using a The monellin causes the 5 acetophenone 1% solution) product to be more natural and causes it to be a strong "living fruit" aroma and taste. Vanilla 200 ppm The monellin causes the 5 extract material to have a richer vanilla arcma, more creamy with a fuller impact. Vanillin 5 ppm (using a A stronger impact with a 4 1% solution) sweet, sugar increase. "Prenol" (3- 0.1 ppm (using The monellin causes the 4 methyl-2- a 0.1% prenol to have a greater buten-l-ol) solution) volatility and a "third" dimension is given to the green, fruity nuance with a more natural impact. "Prenol" (3- 1 ppm (using a The monellin gives a slight 2 methyl-2- 1% solution) increase to the green, buten-l-ol) fruity aroma; but the best impact is at 0.1 ppm rather than at 1 ppm of prenol. 17 r U B_l Ubb A_ ontinuea/ on a Scale of 1- . 5, with 5 Eteing the East of Molecule Cu-rall Sensory Molecule Concentration Evaluation Perception Value Mixture of 2 mixture (2 ppm An enhanced, natural fruity 4 ppm Prenol and and 10 ppm of note. 10 ppm ethyl each compound) acetate Mixture of 1 mixture (1 ppm The monellin gives to this b and ppm prenol; 10 prenol; 10 ppm mixture a greater lift ppm ethyl ethyl acetate; a real, fresh lemon juice acetate; and 5 and 5 ppm nuance. ppm CITRAL CITRAL ) 4 Peanut butter 0.1 ppm A natural, rich, fresh (reduced fat) monellin in roast peanut nuance with peanut butter oumnished, bitter, salty- peanut skin nuance. 3 Peanut butter 1 ppm monellin A natural, fresh roast (reduced fat) peanut nuance with diminished, acid, salty, bitter impact. Natural banana 10 ppm (using a The flavor impact is 3 essence 10% solution) stronger with a more (produced via natural impression, oervaporation) 1 Peach essence 10 ppm (using a The ethyl decanoate note is (produced via 10% solution) boosted causing the impact pervaporation) to be a "fatty" impact. Ethyl 5 ppm (using a The fatty, waxy impact is 4 decanoate 1% solution) increased. Raspberry 10 ppm (using a The impression of fhionone b essence 10% solution) is vastly reduced; causing (produced via a real improvement with a pervaporation) natural, fresh, warm, (also fruity note, containing 10% a-terpineol) monellin the 5 P-Ionone 3 Pf311 3 ^ causes 1% solution) ionone impact to be stronger and the chemical note is eliminated causing a real ripe, fresh, fruity, raspberry note to be created; cutting the floral impact and causing a natural fruit nuance to develop. Pistrrethylfuryl) 0.04 ppm (using The monellin causes the 5 disulfide a 0.001% product to have a fuller, solution) more juicy flavor with a natural roasted meat impact. 1 8 EP 0 821 055 A2 continued/ Cuality I_ffiking m a Srnl onf 1- 5 5, with 5 Being the Bast of Molecule Overall Ssnsory Molecule Concentration Evaluation Peroapticn Value r^rnesere 4 ppm (using a The monellin causes the 5 10 1% solution) flavor impact to be natural. Germacrere 4 ppm (using a The natural nuance is 5 1% solution) vastly increased. Ethyl pyruvate 5 ppm (using a The natural impact is 5 15 1% solution) vastly increased. 1,3,5- 5 ppb (using a The galbanum impact is 5 tir±3C3txicre 0.001% vastly increased, solution) Natural irrble 0.5 ppm (using The chemical taste is 5 20 a 0.1% eliminated and a natural solution) honeysuckle, LIVING FLCWER arcma and taste nuance is created using the monellin. 25 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the amino acid sequence of the A- and B-chains of the natural monellin protein. Figure 2 is a representation of the three-dimensional conformation of the natural, native monellin protein. 30 Figure 3A shows the amino acid sequence of a singlechain monellin protein analogue prepared according to the process of U.S. Letters Patent No. 5,487,983, and the nucleotide sequence of a synthetic gene useful in synthesizing the singel-chain monellin protein. Figure 3B(i) shows the amino acid sequence (leaving room for substitution at position 35 using the symbol "Xaa") of Japanese Published Application No. JP05/070494 (abstracted at Chemical Abstracts, Volume 11 9:48069g, entitled 35 "Singlestranded monellins for improved thermostability", the contents of which abstract are incorporated herein by reference. The symbol "Xaa" represents either of glycine, serine, glutamic acid or threonine. Figure 3B(ii) shows the amino acid sequence of a complete single-chain monellin protein of Published Japanese Application No. JP05/070494 abstracted at Chemical Abstracts, Volume 119:48069g. Figure 4A sets forth the a-carbon trace of the backbone of a single-chain monellin produced according to the 40 process of U.S. Letters Patent No. 5,487,983 issued on January 30, 1996 with dots at the a-carbon positions of residues that have been probed by mutagenesis. Figure 4B sets forth the a-carbon trace of the backbones of natural monellin with dots at the a-carbon positions of residues that have been probed by mutagenesis. Figure 5 is a liquid chromatogram profile for TALIN® (trademark of Tate and Lyle Limited of the United Kingdom), 45 a mixture of Thaumatin I, Thaumatin II and Thaumatin B (conditions: S-Sepharose column operating at 7 ml per minute; gradient: 0-25mM NaCI (2 x 750 ml); fraction size: 45 ml). Figure 6 is the high pressure liquid chromatography profile for the same TALIN® as set forth concerning the brief description of Figure 5. so DETAILED DESCRIPTION OF THE DRAWINGS As stated at column 9, line 1 2 of U.S. Letters Patent No. 5,487,983, the protein of the amino acid sequence shown in Figure 3A is encoded by a DNA sequence as they are shown. As shown in Figure 3A, nucleotides 1-141 encode residues 1 -45 of the native B-chain preceded by a MET encoding ATG start code on, nucleotides 1 42-1 65 encode the 55 linking "C" portion of eight amino acids, and nucleotides 166-285 encode residues 6-45 of the native A protein. The protein of the monellin amino acid sequence shown in Figure 3B(ii) is also encoded by a DNA sequence as thereshown. As shown in Figure 3B(ii), the nucleotides shown thereon encode residues 1-95 of the single-chain pre- ceded by a MET encoding ATG start code on. The amino acid sequence of Figure 3B(ii) is a replication of the amino 19 EP 0 821 055 A2 acid sequence of Figure 3B(i) whereat at position 51, "Xaa" is replaced by a glycine ("Gly") moiety. As stated, supra, the "Xaa" protein at position 51 can be either a glycine, a serine, a glutamic acid or a threonine moiety and is produced according to the procedure of Japanese Published Application No. JP05/070494 abstracted at Chemical Abstracts, Volume 119:48069g, which abstract is set forth as follows: 5 119:48069G Single-stranded monellins for improved thermostability, lijima, Hiroshi; Sone, Hidetaka (Kirin Brewery) Jpn. Kokai Tokkyo Koho JP 05 70,494 [93 70,494] (CI. C07K7/10), 23 Mar 1993, JP Appl. 90/196,983, 25 Jul 1990; 29 pp. The single-stranded (ss) monellins (I) retain the sweetness of the wild type two-stranded I and have enhanced thermostability, esp. in lower pH, and protease resistance. The ss-l are prepd. by linking the C-terminus of B chain of I with the N-terminal Phe of A chain with a linker amino acid Xaa (Xaa = Gly, Ser, Glu, or Thr). Prepn. of a ss-l using 10 Gly as the linker amino acid by expression of the synthetic gene in Escherichia coii and Saccharomyces cerevisiae was shown. The ss-l retained its sweetness after incubation for 20 mins at 100°, but the wild type lost its sweetness at 50°. Moreover, the ss-l but not the wild type counterpart was resistant to proteinase degrdn. with e.g. Achromobacter proteinase I. Replacing the Val12 or Val37 of the ss-l with Ala did not alter the thermostability and protease resistance. Referring to Figure 4A, the a-carbon trace of the backbone of single-chain monellin, the numbered dots at the 20 EP 0 821 055 A2 Asp137J [^Lys46, Asp113, where "Lys" stands for "lysine" moiety; and "Asp" stands for an "Aspartic acid" moiety. The peaks indicated by reference numerals 2312 and 2314 are for "Thaumatin B" and "Thaumatin II" as described in United States Letters Patent No. 4,771 ,000, the specification for which is incorporated by reference herein. The features disclosed in the foregoing description, in the followiny claims and/or in the accompanyiny drawings may both separately and in any combination thereof, be material for realising the invention in diverse forms thereof. 21 EP 0 821 055 A2 SEQUENCE LISTING ( 1 ) GEN EKAL 1 N FORMAT 1 ON : (i) APPLICANT: (A) NAME: International Flavors & Fragrances Inc. (B) STREET: 521 West 57th Street (C) CITY: New York (E) COUNTRY: USA (F) POSTAL CODE (ZIP) : 10019 (G) TELEPHONE: 001 (212) 7G5 5500 (H) TELEFAX: 001 (212) 708 7132 (ii) TITLE OF INVENTION: Use of Monellin in Enhancing the Organoleptic Properties of Beverages and Foodstuffs and Improving the 15 Properties of other Flavorants (ill) NUMBER OF SEQUENCES : 7 (IV) COMPUTER READABLE FORM : (A) MEDIUM TYPE: Floppy disk 20 (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC - DOS /MS - DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) l_J INFORMATION FOR SEQ ID NO : 1: 25 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 30 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 1: 35 Phe Arg Glu lie Lys Gly Tyr Glu Tyr Gin Leu Tyr Val Tyr Ala Ser 15 10 15 Asp Lys Leu Phe Arg Ala Asp lie Ser Glu Asp Tyr Lys Thr Arg Gly 20 25 30 40 Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 35 40 45 (2) INFORMATION FOR SEQ ID NO: 2: 45 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 01/ (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2: Gly Glu Trp Glu lie He Asp He Gly Pro Phe Thr Gin Asn Leu Gly 55 1 5 10 15 !2 EP 0 821 055 A2 Lys Phe Ala Val Asp Glu Glu Asn Lys He Gly Gin Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val He Arg Pro Cys Met. Lys Lys Thr He Tyr Glu 35 40 45 Asn Glu 50 (2) INFORMATION FOR SEQ ID NO : 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 285 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear l IX) r hi/V-L UK_ : (A) NAME /KEY : CDS (B) LOCATION: 1 ..285 XI J £>_yu_iNC_ Ubbl-KIFI 1UJM : __Q ID WO : J: Al_ __A __a n.7i_ bM Ail ATC GAT ATT GGA CCA TTC ACT CAA AAC TTG 48 Met Gly Gly Trp Glu He He Asp He Gly Pro Phe Thr Gin Asn Leu 25 1 5 10 15 GGT AAG TTC GCT GTT GAC GAA GAA AAG AAG ATT GGT GAA TAT GGT AGA 9 6 Gly Lys Phe Ala Val Asp Glu Glu Lys Lys He Gly Gin Tyr Gly Arg 20 25 30 30 TTG ACT TTC AAC AAG GTT ATT AGA CCA TGT ATG AAG AAG ACT ATT TAC 14 4 Leu Thr Phe Asn Lys Val He Arg Pro Cys Met Lys Lys Thr He Tyr 35 40 45 GAA AAC GAA AGA GAA ATT AAG GGG TAC GAA TAC CAA TTG TAT GTT TAC 192 ' 35 Glu Asn Glu Arg Glu He Lys Gly Tyr Glu Tyr Gin Leu Tyr Val Tyr 50 55 60 GCT TCT GAC AAG CTT TTC AGA GCT GAC ATT TCT GAA GAC TAC AAG ACC 24 0 Ala Ser Asp Lys Leu Phe Arg Ala Asp He Ser Glu Asp Tyr Lys Thr 65 70 75 80 40 CGC GGT AGA AAG TTG TTG AGA TTC AAC GGT CCA GTT CCA CCA CCA 28 5 Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 « (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B) TYPE: amino acid (D) 50 TOPOLOGY: linear (11) MOLECULE TYPE : protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 4et Gly Gly Trp Glu He He Asp He Gly Pro Phe Thr Gin Asn Leu 1 5 10 15 3 EP 0 821 055 A2 Gly Lys Phe Ala Val Asp Glu Glu Lys Lys He Gly Gin Tyr Giy Arg 20 25 30 Leu Thr Phe Asn Lys Val He Arg Pro Cys Met Lys Lys Thr He Tyr 35 40 45 Glu Asn Glu Arg Glu He Lys Gly Tyr Glu Tyr Gin Leu Tyr Val Tyr 50 55 60 Ala Ser Asp Lys Leu Phe Arg Ala Asp He Ser Glu Asp Tyr Lys Thr 65 70 75 80 Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 35 (2) INFORMATION FOR SEQ ID NO : 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 96 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Gly Glu Trp Glu He He Asp He Gly Pro Phe Thr Gin Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys He Gly Gin Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val He Arg Pro Cys Met Lys Lys Thr He Tyr Glu 35 40 45 Asn Glu Xaa Phe Arg Glu He Lys Gly Tyr Glu Tyr Gin Leu Tyr Val 50 55 60 Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp He Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 288 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 4 .. 288 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 24 EP 0 821 055 A2 ATG GGC GAG TGG GAA ATC ATC GAT ATC GGT CCA TTC ACT CAA AAC TTG 4 8 Met Gly Glu Trp Glu He He Asp He Gly Pro Phe Thr Gin Asn Leu 15 10 15 GGT AAA TTC GCT GTT GAT GAA GAA AAC AAG ATT GGC CAA TAC GGT AGA 9 6 Gly Lys Phe Ala Val Asp Glu Glu Asn Lys He Gly Gin Tyr Gly Arg 20 25 30 TTG ACC TTT AAC AAG GTT ATC AGA CCA TGC ATG AAG AAG ACT ATT TAC 144 Leu Thr Phe Asn Lys Val He Arg Pro Cys Met Lys Lys Thr He Tyr 35 40 45 GAA AAC GAA GGT TTT AGA GAA ATT AAG GGT TAC GAA TAC CAA TTG TAC 192 Glu Asn Glu Gly Phe Arg Glu He Lys Gly Tyr Glu Tyr Gin Leu Tyr 50 55 60 GTA TAC GCT TCT GAC AAG TTG TTC CGT GCT GAC ATT TCC GAA GAC TAC 24 0 Val Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp He Ser Glu Asp Tyr 65 70 75 AAG ACA CGT GGT CGT AAG TTG TTG AGA TTC AAC GGT CCA GTC CCA CCA 2 88 Lys Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro 80 85 90 95 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7: Gly Glu Trp Glu He He Asp He Gly Pro Phe Thr Gin Asn Leu Gly 15 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys He Gly Gin Tyr Gly Arg Leu 2 0 2 5 3 0 Thr Phe Asn Lys Val He Arg Pro Cys Met Lys Lys Thr He Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu He Lys Gly Tyr Glu Tyr Gin Leu Tyr Val 50 55 60 Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp He Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro 85 90 95 Claims 1. A consumable, ingestible fermentation-derived beverage or distilled spirit, alcoholic beverage composition com prising: (i) from 1 .5% up to 70% by weight of an alcohol or ester selected from the group consisting of ethyl alcohol isoamyl alcohol, n-hexanol, /.-menthol and cis-3-hexenyl acetate; 25 EP 0 821 055 A2 (ii) from 0.05 parts per million (ppm) up to 15 ppm of monellin or an analogue thereof; and (iii) from about 0. 1 ppb up to about 2% by weight of a fermentation-derived beverage or distilled spirit flavorant. 5 2. A process for augmenting or enhancing the aroma and taste of a fermentation-derived beverage or distilled spirit comprising the step of adding to a fermentation-derived beverage or distilled spirit from 0.05 ppm up to 1 5 ppm of monellin or an analogue thereof. 3. The process of Claim 2 wherein the fermentation-derived beverage or distilled spirit is a fruit liqueur. 10 4. The process of Claim 3 wherein the fruit liqueur is pear liqueur. 5. The composition of Claim 1 , also containing from 0.05 ppm up to 2% by weight of succinic acid. is 6. The composition of Claim 1 , also containing from 0.05 ppm up to 2% by weight of sclareolide. 7. The composition of Claim 1 , also containing from 0.1 ppb up to 15 ppm of thaumatin. 8. A flavor formulation containing from about 0.5 ppm up to about 15 ppm of monellin or an analogue thereof; and 20 from about 0.01 ppm up to about 200 ppm of at least one flavorant selected from the group consisting of: isobutyl thiazole; trimethyl pyrazine; 2- methoxy-3-isobutyl pyrazine; 25 menthol; isopulegol; trans-2-hexenol; 6-methyl-5-hepten-2-one; methyl acetophenone; 30 vanilla; vanillin; 3- methyl-2-buten-1-ol; a mixture of 3-methyl-2-buten-1-ol and ethyl acetate; a mixture of 3-methyl-2-buten-1-ol, ethyl acetate and CITRAL ; 35 natural banana essence; ethyl decanoate; a mixture of natural raspberry essence and 10% a-terpineol; P-ionone; bis(methylfuryl)disulfide; 40 farnesene; germacrene; ethyl pyruvate; 1 ,2,5-undecatriene; and indole. 45 9. A process for augmenting, enhancing or imparting a flavor in or to a foodstuff comprising the step of adding to said foodstuff the composition defined according to Claim 8. 10. The composition of Claim 8 wherein the material added to the monellin is isobutyl thiazole. 50 11 . A fermentation-derived beverage or distilled spirit containing from 0.05 ppm up to 1 5 ppm of monellin or an analogue thereof. 55 26 EP 0 821 055 A2 AMINO ACID SEQUENCE OF NATURAL MONELLIN A-Chain 1 *PHE ARGGLUILE LYS GLY TYR GLU TYR GLN LEU TYR VAL TYR ALA SER ASP LYS LEU PHE ARG ALA ASP ILE SER G LU ASP TYR LYS TH R ARG GLY ARG LYS LEU LEU ARG PHE ASN GLY PROVAL PROPROPRO 45 e- [- B-Chain 1 GLY GLU TRP GLU ILE ILE ASP ILE GLYPRO PHE THR GLN ASN LEU GLY LYS PHE ALA VAL ASP GLU GLU ASN LYS ILE GLY GLN TYR GLY ARG LEU THR PHE ASN LYS VAL ILE ARGPRO CYS METLYS LYS THR ILE TYR GLU ASN GLU 50 * PHE presents only in 10 % of natural monellin. 27 < w O < < _ So $z b b 81 oj= S3 <^ oo ( r\ «o>> o>5^ £<5 KiPjH f^o^ Vj^ oo Ph << NPn. Moo Pa, <£. < 2 F 3 < »> Ii 5= vyo oo o-i << o< oo_i-h <-i Fa. Fo 5^ "s < = <_l Ss g- g3 oo < _i <_i o< F-j o _ <3To < >» 3> FF OCT <-l FF <£-J o^ 3=2 "S F« o? oo oo / 28 % 31 =: =r cn*<^ < -« 3 -« w «< > > go TJ TJ O <3 5T °E J J E O (/> X co> > — i ^- TO 03 wiw ST -3 ^ -i D) 3 > > -o r- ro < Q cri r- r~ > < > cn = CJlv< *< 0) (0 TO O TO — TO — TO II C C c c r -o « = > o > E C7) c o ca -g > > r tj > = -t -i «< O -i (a TO / . I CO CO W O 3 \J4 tj > Q O £>r o I ^ TO > H S = ^ TJ UJ 3 TO ^ ° s» — cn ro — *< — 3- —— • «< c «< £ O ^ W 3 -« — c 3 -« ^ 3 -o > r = co o > "J (A) TO TO O — (/) CO TJ —I —I H > -»r cn »< «< < cn to ' tj cor < o £ O o w - c c < 29 EP 0 821 055 A2 °°r-J> < O OO r- -H OO S3> <=> ^ w-H ci> ^"O o — 1 O 5> i> <= O — 1 ~ O ;t» o> -h -n 3> 1> -1 3> l 3> — *■ O O n m < > wi> =*" o **=■*> - o 3> O J> O 01 o o -a ~a —i o o -< O =r — I to » 2 <=> t= ~3> — I -a — i o o o o —i m r > O O =r — l o — o o ~ CD o — 1 cn 3> O —| cn O OT O 10 >■ 3> J>o — i —i S3> =:r> — -ao o <» 3> i» > 3=* to —i «> n 0 "< o =»ot30 -*o — O —| °> O O ~>J = 1> OO r~> OO T7 — 4 < S^0 H E"3> ^ 3> ^ O =r -J — • — 1 j> °° O O ■< O OO O O *»■ -H J» —1 —1 OO 1>0 r--H ^J^coctjo 5>J> SO o>5> ra H 0 ~ O v>j> 10 T O —1—1 — i-h — 1 — 1 3> 5> ,— 5> -"O ~«0 -«0 to^^co 30 EP 0 821 055 A2 FIG.4-A 31 EP 0 821 055 A2 FIG.4-B 32 EP 0 821 055 A2 I << ^ ^ o>« oo o< hO Ph oo