1 Winemaking as a Biotechnological Sequence
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F. D R A W E R T Institut für Lebensmitteltechnologie und Analytische Chemie der Technischen Universitat Munchen, D-8050 Freising-Weihenstephan, München, Germany
The chemistry of winemaking is characterized by intensive metabolism of the ingredients, beginning with the grape and ending with wine. We therefore view the chemistry of winemaking as a biotechnological sequence. This sequence originates with the grape, proceeds to the grape destemming, crushing, and pressing technology and, finally, is decisively influenced by the fermentation. The ingredients of the grapes, their dependence on variety, climate, vineyard management (fertilization), and biochemical processes during the crushing and pressing operations, are important to the biosynthesis of aroma substances.
he chemistry of winemaking is still i n the formative stages, and most of the chemical problems are analytical i n nature. W i n e contains hundreds of components (1, 2, 3) of widespread concentrations (nggrams/1. ). It is not clear what sensorial contribution each of these com ponents makes to the taste and aroma of a particular wine. Neither is it clear how grape variety influences aroma and taste, even though the influence is obviously great. The varietal influences are pronounced i n wine made from such grapes as Concord, Muscat, Burgundy, and Riesling, but analysis indicates little difference i n such gross measures as alcohol content, extract composition, sugar content, acidity, and ash content This implies that the sensorial characteristics of wine are derived from specific components. W i t h modern analytical techniques, such as gasliquid chromatography and mass spectrometry, it is possible to identify the specific components. In the past few years several groups (1, 2, 3) have made inventories of several hundred aroma components i n various wines. However, experience and critical evaluation of the inventories suggest that the inventories are still insufficient to characterize wine A
1 In Chemistry of Winemaking; Webb, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
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CHEMISTRY O F W I N E M A K I N G
adequately or to describe winemaking chemistry. W e believe that wine production and winemaking chemistry are best understood b y consid ering the specific components to be end products of a biotechnological sequence ( 2 ) , beginning with the vine and ending with bottled wine.
QRAPtt
TECHNOLOGY
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RIPENESS STATE CONDITION
YEAST FERMENTATION
YOUNG AGING PROCESSES WINE RIPENING, TECHN01ÛGY
Figure 1. Results and
GRAPE MUST
WINE
The biological-technological sequence of wine
Discussion
The Biotechnological Sequence. Figure 1 shows the biotechnological sequence for winemaking, beginning with the vine and including the effects of climate, soil, and grape variety. During the sequence, grape components are modified by ripening and by the physical condition of the berries. The technology involved i n crushing may also greatly influ ence the components of the grape must. This influence is of particular importance (4, 5,6) i n the subsequent yeast fermentation caused b y the extraordinary reduction potential and the ability of the yeasts to syn thesize various aroma components depending on the composition of the Table I.
Amino Acids, mg/liter
Fertilizer Variety
Ν
Κ
Ρ
Nitrate, N0 2
Aris
6
Riesling
0,5 2,0 2,0 2,0 4,0 2,0 6,0 2,0
6
His 20 15 36 16 23 48
29,0
960 1598 2651 2972
0,5 2,0 2,0 2,0 2,0 2,0 4,0 2,0 2,0
1,5 2,0 4,4
800 1064 2445
2
3,5
Total Amino Acids
2,0 2,0 2,0 2,0
• Ser + G l u - N H
Dependence of the Content
+
Lys
+ + + + + + 13
Arg
NH
S
Asp
480 801 853 828
29 39 82 87
39 22 57 70
174 314 810
10 20 53
49 36 63
+ Asp-NH . 2
In Chemistry of Winemaking; Webb, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
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1.
DRAWERT
3
Biotechnological Sequence
substrate. Finally w e have the aging processes. These depend, for ex ample, on the reduction potential and/or the oxygen content and are very influential on the final character of the wine. The components of the grape vary according to variety during the first stages of the sequence. This is shown b y analytical data on Sugar acids, amino acids, proteins, phenolic compounds, coloring matter, and aroma compounds i n the wine. W e should, however, be extremely careful in interpreting specific differences between samples of the same types of wines because the differences are only quantitative. The differences may change i n magnitude depending on the climate, soil, and particularly on the kind of fertilization. Little is known, for example, about the influence of nitrogen fertilizer on the nitrogen content of grapes or on the nitrogen content of free amino acids. Table I shows remarkably increasing amounts of free amino acids occurring i n the grapes if the amount of fertilizer applied (ammonium nitrate) is increased. The grapes come from Aris (a new hybrid) and Riesling vines ( 7 ) . The vines were grown i n culture vessels according to Mitscherlich's method. The applied doses of potassium sulfate and calcium hydrogen phosphate were kept constant throughout the experi ments. During the yeast fermentation, the amino acids enter the yeast metabolism with a definable biochemical valence with respect to the fermentation side products which are aroma substances. W e recently succeeded i n differentiating vine varieties (Figure 2) by using the distri bution patterns of grape proteins which we obtained from disc electro phoresis and isoelectrical focusing i n polyacrylamid gels (8, 9 ) . If w e dye the enzymes distributed i n the gels together with the proteins according to substrate specificity, we obtain the so-called zymo grams for phenolase, peroxidase, esterase, and malatdehydrogenase ( 9 ) . The resulting zymograms exhibit heterogenity of the isoenzymes, a probof Free Amino Acids on Fertilization Amino Acids, mg/liter
Thr
Ser
Glu
Pro
Gly
Ala
Val
Met
lieu
Leu
Phe
26 38 44 88
128 204 470 495
222 203 216 268
+ + 480
+ + 7,5
63 107 252 318
19 27 27 46
15 21
+
12 25 31 31
20 35 50 45
22 62 66 67
30 49 123
65 78 305
132
123 136 179
+
86 136 454
22 40 45
21 30 24
26 31 23
33 42 32
31 48 49
a
—
370
590
4,5
7,5 22,5
—
New wine variety.
In Chemistry of Winemaking; Webb, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
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CHEMISTRY O F W I N E M A K I N G
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lem i n its own right. This is noticed, for example, when purified poly phenol oxidase (isolated from grapes )is allowed to react on catecholcontaining base systems. W h e n measuring the oxygen consumption with a Warburg apparatus, we found oxygen consumption to vary with the addition of certain amino acids (JO) (Table I I ) . Therefore, the following reactions are possible:
Grapes release numerous hydrocarbons i n the adjacent gas volume. W e have found alkanes, alkenes, and aromatic hydrocarbons (11). Accord ingly, we extracted the skin waxes of the grapes with n-pentane and investigated the extracts b y using a combination G L C - m a s s spectrometer. The total amounts of the wax extracts contained about 0.5% paraffins, 89% of which were saturated hydrocarbons ranging from C i to C i . 3
Table II.
3
Consumption of Oxygen after A d d i n g Amino Acids to the System Phenolase—Catechol (1,2-Dihydroxybenzene) Phenolase + Catechol + Amino Acid
—
glycine valine methionine threonine arginine-HCL histidine-HCL gluthathione aspartic acid glutamic acid
μΖ 0^/60 min 140 270 230 230 220 200 170 110 90 70
About 1 1 % were unsaturated hydrocarbons ranging from C i to Q29. The main components were the odd-numbered, saturated hydrocarbons with carbon numbers 23, 25, and 27. T h e even-numbered hydrocarbons are found i n lesser amounts and have a maximum at Q24 (12) (Table I I I ) . 4
In Chemistry of Winemaking; Webb, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
1.
DRAWERT
Biotechnological Sequence
5
Considering the biotechnical sequence, it is possible to explain the presence of hydrocarbons i n wine. T o characterize a further step i n the biotechnical sequence which bears a vital influence on the aroma events, an example from the many biochemical and chemical processes which take place i n connection with the crushing and pressing of the? grape berries is made. During crushing and at the moment of destruction DE
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START
FRONT
1
1 1 1 1 1 1
l l l l
1 +
VARIETIES
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FARBER
il III : :
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il h
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RULÀNDER
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Figure 2. Protein pherograms (disc electrophoresis) of different grape varieties (harvest 1971). The separation distance was redrawn to a 10 cm scale. Scale: Rf values, Arrows: Main bands common to all varieties.
In Chemistry of Winemaking; Webb, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
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CHEMISTRY OF W I N E M A K I N G
of the cell structure, enzymatic-hydrolytic and enzymatic-oxidative processes occur with amazingly high speed (13, 14, 15). Depending on p H and temperature, the first ones cause a more or less rapid cleavage of natural esters; the effects of the latter are especially severe and are causing new synthesis of aroma substances. T o determine these enzymatic-oxidative processes quantitatively on a laboratory scale, we homogenized, for example, grape berries or vine leaves without inhibiting the enzymes in a phosphate buffer. W i t h i n 10 min, increased amounts of hexanal were apparent ( as indicated i n Table I V , column b ) ; they increased from 90 to 410 /xg/100 grams, and trans2-hexen-l-al increased 130 to 13600 /xg/100 grams. W e proved i n numerous, thorough experiments that these enzymatic-oxidative processes occur if oxygen (in air) had access to the substrate and if the enzymes had not been inhibited previously. Hexanal and hexenals are essential components of the grass taste and odor; this is why grape juices made Table III.
Hydrocarbons i n the Waxlayer of Grapes (Riesling) 3.924 mg/10 kg n-Alkanes: 3481 /xg = ca. 88.8% n-Alkenes: 443 /xg = ca. 11.2%
Number of Carbon Atoms 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 * Traces.
n-Alkenes
n-Alkanes μρ/10 kg tr" tr tr 4 6 8 12 15 15 21 29 195 90 725 112 1090 72 550 30 312 15 180 tr
%
— — — 0.1
0.15 0.2 0.3 0.4 0.4 0.6 0.7 5.0 2.3 18.5 2.8 27.8 1.8 14.0 0.8 8.0 0.4 4.6
—
\ig/10 kg tr
—6 —7 —2 —3
5 9 9 80 49 115 52 51 30 15 10 tr tr tr
%
— — 0.2 — 0.2 — 0.05 — 0.05 0.1 0.2 0.2 2.0 1.2 3.0 1.3 1.3 0.8 0.4 0.2
In Chemistry of Winemaking; Webb, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
— — —
1.
DRAWERT
Table I V .
7
Biotechnological Sequence
Formation of Q-aldehydes and C -alcohols (itg/100 grams) in Grapes and i n Vine Leaves e
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Grape, Inhibition of Enzymes"
Hexanal m-3-Hexene-l -al 2rans-3-Hexene-l -al £rans-2-Hexene-l-al c 1-Hexanol as-3-Hexene-l -ol C=C< REDUCTASE
XH OH 2
HEXANOL
Figure 4.
r
1 -HEXANOL
u
CHjOH
Enzymatic cleavage of linolenic acid
spectrometer ( V a r i a n - M A T , Bremen, W . Ger., model C H 7 ) . The separa tion column was 2.5 m stainless steel, 1/16-inch diameter, filled with 10 w t % F F A P on Chromosorb (80/100 mesh). Carrier gas flow rate was 5 m l N / m i n .Temperature was 100°-270°C (2°/min). W e have reported i n detail the isolation and separation of C -aldehydes and -alcohols elsewhere (13). 2
e
Literature Cited 1. Webb, A . D., Advan. Appl. Microbiol. (1972) 15, 75. 2. Drawert, F., Rapp, Α., Vitis (1966) 5, 351-376. 3. Drawert, F., Rapp, Α., Chromatographia (1968) 1, 446-457.
In Chemistry of Winemaking; Webb, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
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CHEMISTRY OF WINEMAKING
4. Drawert, F., Rapp, Α., Vitis (1964) 4, 262-268. 5. Drawert, F., Rapp, Α., Ulrich, W., Vitis (1965) 5, 20-23. 6. Drawert, F., Tressl, R., "Abstracts of Papers," 164th National Meeting, ACS, Aug.-Sept. 1972, A G F D 066. 7. Drawert, F., Alleweldt, G., unpublished data. 8. Drawert, F., Görg, Α., Chromatographia (1972) 5, 268-274. 9. Drawert, F., Görg, Α., Ζ. Lebensm. Unters. Forsch., in press. 10. Gebbing, H., "Uber pflanzliche Polyphenoloxidasen," Doctoral Dissertation, University of Karlsruhe, 1968. 11. Drawert, F., "Moderne physikalisch-chemische Methoden in der Erforschung der Aromastoffe des Weines," in "Chemie und landwirtschaftliche Produktion," Wien, 1971. 12. Speck, M . , "Enzymatisch-oxidative Bildung von Aldehyden und Aldehydcarbonsäuren beim Zerstören pflanzlicher Zellverbande und über die Oberflächenwachse von Bananen und Trauben," Doctoral Dissertation, Technische Universität München, 1971. 13. Drawert, F., Tressl, R., Heimann, W . , Emberger, R., Speck, M . , Chem. Mikrobiol. Technol. Lebens. (1973) 2, 10-22. 14. Drawert, F., Heimann, W . , Emberger, R., Tressl, R., Naturwissenschaften (1965) 52, 304-305. 15. Drawert, F., Heimann, W . , Emberger, R., Tressl, R., Ann. (1966) 694, 200-208. 16. Drawert, F., Rapp, Α., Ullemeyer, W., Vitis (1967) 6, 177-197. 17. Drawert, F., Vitis (1963) 4, 49-56. 18. Drawert, F., Heimann, W., Rolle, Κ., Z. Lebensm. Unters. Forsch. (1970) 144(4), 237-245. 19. Watson, I. T., Bieman, K., Anal. Chem. (1964) 36, 1135. RECEIVED September 20, 1973.
In Chemistry of Winemaking; Webb, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
