Z Lebensm Unters Forsch (1982) 175:253-257 Zeitschrift far

Lebensmittel- Untersuchung

und-Forschung @~I. F. Bergmann Verlag 1982

Oxygen Isotope Studies on Some New Zealand Grape Juices

John Dunbar

Chemistry Department, University of Waikato, Hamilton, New Zealand*

Untersuchungen yon Sauerstoffisotopen in Neuseel~indischen Traubens~iften

Zusammenfassung. Bet der Messung der Wasser-lSO/ i60_Verh/iltniss e einer Anzahl yon Traubens/iften wur- den folgende Ergebnisse ermittelt: 1. Innerhalb yon 24 Std vedindert sich das ~ 80/160-Verhfdtnis im Trau- bensaft bet einer bestimmten Pflanze nur wenig. 2. Die Aufnahme yon Regenwasser in reife Trauben bewirkt eine Verdiinnung der i so-Anreicherung des Saftes. 3. D as 180/160_Verhfiltnis yon mehreren durch R ota- tionsverdampfung hergestellten Traubensaftkonzen- traten wurde gemessen und die Ergebnisse waren denen yon Kontrollweinen/ihnlich. 4. Bet der Verdunstung yon Wasser durch die Schale gelesener Trauben wird der iibrige Saft mit l so angereichert. 5. Die Vergfirung yon Most zu Wein findert das 180/a60-Verh/iltnis des Wassers nicht wesentlich.

Summary. The ~ 80/160 ratios of the water of a number of grape juices were measured and the following ob- servations were made: (1) The 180/160 ratio of the juice of a given grape does not change significantly dur- ing the course 0f24 h. (2) Rain will only affect i80/160 ratios when the fruit is ripe. Uptake of this water in ripe grapes has a diluting effect on the t so enrichment of the juice. (3) The 180/160 ratios of several juice con- centrates that had been prepared by rotary evaporation were found to be very similar to those of the control wines. (4) As harvested grapes lose water by evapora- tion through the skin, the juice remaining becomes more enriched in 180. (5) Fermenting grape juice into wine does not appreciably change the 180/16 ratio of the water.

* Present address: Lehrstuhl f/ix Allgemeine Chemie und Bioche- mie, Technische Universit/it Miinchen, D-8050 Freising-Weihen- stephan, Federal Republic of Germany

Introduction

For many years it has been desirable to detect added tap water in wine so that the customer can be better protected from fraudulent wine-making practices. To this end the results of a study of the oxygen isotope en- richment in grape juice will be presented and the factors that affect this enrichment discussed.

It is known that water in fruits can become isotopi- cally enriched in deuterium and ~ 80 due to the process of evapotranspiration [1-4]. Little work in compar- ison however has been done to determine which physi- cal parameters affect this enrichment, and to what de- gree. Bricout [5] studied 24 grape juices and found a geographical effect between grapes grown in different lands. The waters of those grown in countries with hot climates, e.g. Algeria, Morocco and Turkey, were found to be more enriched in deuterium and ~80 than those grown in cooler climates, e. g. France. The waters of different varieties of grapes grown in the same area were found to have similar isotopic compositions.

An experiment was also performed by Bricout to determine if there was any correlation between the 6D value of the water of the leaves and that of the fruit of a grape vine throughout the course of a day. Large variations were measured in the leaf water isotope ratio, however very little change was observed in the fruit. It was also noted that immature grapes were more enriched in 180 than were mature grapes.

The water of grapes infected with Botry t is cinerea, a disease that causes the grapes to lose water, was found to be richer in t so than that in unaffected gra- pes. This also applied to grapes that were left on the vine throughout the change from warm to cold weather. These grapes were found to be 1%o richer in 180 at the end of this time period.

It is therefore the aim of this work to study further the physical parameters that affect the enrichment of 1sO in the water of grapes.

0044-3026/82/0175/0253/$1.00

254 J. Dunbar: Oxygen Isotope Studies on Some New Zealand Grape Juices

Method

Oxygen Isotope Determinations The lso/160 ratios were determined by directly equilibrating CO 2 with the grape juice using the method as described by Epstein and Mayeda [6]. A 10-ml portion of filtered grape juice was equilibrated with 20 ml of CO2 at 25 °C for 48 h, after which the CO2 was re- moved, freed from water by fractional distillation and then admitted into an isotope-ratio mass spectrometer (Micromass 602C).

The results of the analyses are reported in the ~-notation where:

blso= [ lsO/16~O0 sample 1] x 1000%o. [ isOy~Os~ow J

i80/16Os~ow is the lso/~60 ratio of the international standard SMOW (Standard Mean Ocean Water). The absolute 1 so/1 ~O ratio of this standard as reported by Gonliantiui is (2005.20 + 0.45) x 10- 6 [7].

Analyses could be replicated to within ± 0.05%o.

Results

a) Changes in the i 80 / i 60 Ratio of Grape Water Throughout a 24-h-Period

At 4 hourly intervals bunches of ripe grapes were pick- ed from a local grape vine, the juice was extracted and filtered, and the 180/160 ratio was determined. The day chosen was warm, sunny and s011, with a maximum temperature of 26 °C, and a minimum of 16 °C.

The results of these analyses are shown in Table 1. From these results it can be seen that there is a vari-

ation of 0.5%0 between the maximum and minimum 61so values. Zundel et al. [8] when measuring the 180 content of leaves and branches, found that the repro- ducibility during a daily enrichment cycle was hardly better than 1%o. I f this magnitude of scatter is to be ex- pected, then the values in Table 1 must be considered as indistinguishable, i. e. there was no change in the 18 O content of the grapes throughout the course of the day.

This is not the same situation as that which applies in the case of leaves [8-10], however it is reasonable considering the volumes of water involved. A grape berry contains much more water in relation to its sur- face area, and is also more impervious, hence daily changes in the 1so content of the water of the berry would be more buffered.

b) Effect of Grape Maturity on the 180/~60 Ratio of the Grape Juice Water

Grapes from a Cabernet Sauvignon vine were picked at various intervals throughout a six-week period from immaturity through to ripeness and harvesting. The 180/160 ratios of the juices were then measured with the results given in Table 2.

The values show that there is very little change in the i 8 0 / i 6 0 ratio of the grape juice water as the grapes approach maturity and over the last two weeks, within the limits of experimental error, there was no change at all.

Table 1. ~80/~60 ratios of grapes picked at 4 hourly intervals over a 24 h period

Time 618OsMow %0

6.00 am + 2.7 8.30 am + 2.4 1.30 am + 2.4 6.00 pm + 2.6 9.45 pm + 2.2 1.00 am + 2.5

Table 2. Changes in the 180/160 ratio of grape juice water during ripening of the grapes

Days before harvest 6 lSOsMow %0

36 +0.6 26 + 1.5 23 40.9 14 +1.5 7 41.5 0 +1.6

Table 3. The effect of rain on the i sO/16 0 ratio of grape juice water

6 iSOsraow %0

Immature grapes: Before rain + 1.3 After rain + 1.5

Mature grapes: Before rain + 1.2 After rain + 0.5

Rain water 0.0 (approx)

c) The Effect of Rain on the 180/i60 Ratio of Grape Juice Water

It is known that when rain falls on a grape vine bearing ripe fruit, rapid uptake of water into the grapes can oc- cur. This effect can sometimes be so great that the gra- pes are split due to the extra water content. It would therefore be expected that the isotopic composition of the grape juice would change if water uptake to this lev- el occurred.

In this experiment both mature and immature gra- pes were studied with the following conditions apply- ing at the time of sampling. Immature Grapes. An 8-ram shower of rain fell on gra- pes that were at a point of maturity such that they were to be harvested in two weeks i.e. they were still small and had not started to soften. No rain had fallen for the previous 6 days and the grapes were sampled 12 h after the rain. Mature Grapes. The grapes were sampled at the time of harvest. On this particular day 17 mm of rain fell in the morning, which by the afternoon had caused the grapes to split. The grapes were sampled after the rain. The re- sults are shown in Table 3.

J. Dunbar: Oxygen Isotope Studies on Some New Zealand Grape Juices 255

In this case, within the limits of biological error, no change in the isotopic ratio of the immature grapes had occurred. However this does not apply in the case of mature grapes in which a dilution of the enriched grape water had occurred. By using the 180/160 ratio of the grape water before the rain and the ratio for rain water the dilution factor can be calculated. In this case a change in the 180/160 ratio of the grape juice of 0.7%0 corresponds to a 60% dilution by the rain.

d) 180/160 Ratios of Grape Juice Concentrates

It is quite common practice in the New Zealand wine industry to use grape juice concentrate as:

a) a source of added sugar before fermentation b) a source of residual sweetness and "fruity char-

acter" added after fermentation, i.e. back blending. For case (b) the additions are usually up to 5%,

whereas for case (a) they can be anywhere between 0 and 100%.

Rotary evaporation is one of the techniques used to produce the concentrate, which contains approximate- ly 60% of sugar. It is difficult to remove more water than this, because by this stage the concentrate has the consistency of a heavy syrup. During the rotary evapo- ration process it is possible that isotopic fraetionation of the water in the concentrate will occur, due to one or both of the following effects:

a) Liquid-vapour fractionations at 60 °C (Rotary evaporator operating temperature)

b) Fugacity fractionation effects, i. e. fraetionation between water and water vapour due to dissolved solutes.

The wines and concentrates shown in Table 4 were studied to determine the magnitude of these combined effects.

From these measurements the following points can be noted.

a) Two concentrates from 1978, as well as one of their respective wines (which was made from normal unconcentrated juice), were measured. (Unfortunately no Baco 22A wine sample was available). The Siebel concentrate 6180 value was 0.2%0 lighter than the cor- responding wine, indicating that very little isotope frac- tionation had occurred during its production.

b) The three Pinot Chardonnay wines listed were made from juices that had been treated in the following manner:

i) Sample 1 had a natural sugar content of 16.6%. No additional sugar was added.

ii) Sample 2 was the same juice, but the sugar level was increased to 19% with cane sugar, i.e. 13 % of the sugar was of cane origin, 87% of grape origin.

iii) Sample 3 was also the same juice but in this case the sugar level was raised to 19% with Baco 22A con- centrate.

Table 4. The effect of concentrates on the ' 80 / ' 60 ratios of wines

Variety Year 6 ' 8OsMow %0

Baco 22A concentrate 1978 - 0.7 Siebel 5437 concentrate 1978 0.0 Siebe15437 wine 1978 + 0.2 Pinot Chardonnay wine 1 1979 - 2.4

(no added concentrate) Pinot Chardonnay wine 2 1979 -2.3

(with added cane sugar) Pinot Chardonnay wine 3 1979 - 2,4

(with added Baco 22A concentrate)

In all three cases the 180/160 ratios of the resultant wines were the same within the limits of experimental error, indicating that the source of the additional sugar used in the production of the wine was unimportant.

It is known however that oxygen exchange in cer- tain sugars can occur. Using glucose, Titani and Goto [11, 12] showed that one of the oxygen atoms can ex- change via the free aldehyde form. This was also con- firmed by Bently and Bhate [13] and Rittenberg and Graft [14]. It is therefore likely that exchange had oc- cured between the oxygen atoms of the glucose/fruc- tose in the added concentrate and the oxygen in the grape juice water. In the case of concentrate sugars the exchangeable oxygen would have the same 180/160 ratio as that of the concentrate water, i.e. when ex- change occurs with a "foreign" grape juice with water of a similar lso/160 ratio there would be no effect on the 1so/160 ratio of the resultant mixture. This of course assumes that there is no fractionation effect in- volved in the exchange. It is unlikely that oxygen ex- change between water and sucrose occurs, because in sucrose the oxygen atom that is involved in the free aldehyde form is now bonded. Therefore addition of sucrose to a grape juice should have no effect on the 180/160 ratio of the water present.

In summary, concentrates can have an effect on the 180/160 ratio of the water to which they are added via two mechanisms. These are:

1) Exchange between the oxygen atoms of the sugar and grape water.

2) Modification of the 180/160 ratio of the wine because of the addition of the concentrate water, which may have a different 180/160 ratio.

In the wines and concentrates studied these effects have been found to be small.

e) Evaporation of Water from Picked Grapes

This investigation was carried out to determine what effect loss of water by evaporation from picked grapes would have on the 180/160 ratio of the grape water re- maining.

256 J. Dunbar: Oxygen Isotope Studies on Some New Zealand Grape Juices

~o

*4

*3 small shrivelled berries

*2 .--e

÷1 ,

=

0

-11

-2 2 4 6 8 10 12 14

number of days

Fig. 1. The effect o f evaporation on the 3180 content of picked grapes

Table 5. 180/160 ratios of the water of grape juices and, after fer- mentation, of the corresponding wines

Variety Year Juice Wine zJ (wine- 518OsMow 618OsMow juice) %0 %0 %0

Riesling Sylvaner 1978 + 0.4 + 0.7 + 0.3 (Type 1)

Riesling Sylvaner 1978 + 1.7 + 0.1 - 1.6 (Type 2)

Pinotage 1978 0.0 + 1.2 + 1.2 Pinot Meunier 1978 + 0.9 - 0.8 - 1.7 Cabernet 1978 + 1.6 0.0 - 1.6

Sauvignon Pinot Noir 1978 +0.1 +0.9 +0.8 Pinard 1977 - 1.3 - 1.9 - 0 . 6 Siebel 5437 1978 + 0.9 + 0.2 - 0.7 Siebel 7053 1978 + 1.0 + 1.3 + 0.3 CD 18/92 1978 +0.7 +2.0 + 1.3 Pinot Chardonnay 1977 0.0 +0.6 +0.6 Golden Chasselas 1976 +0.1 +1.4 +1.3 Baco 22A 1977 +0.1 - 1 . 6 - 1 . 7

Bunches of Cabernet Sauvignon grapes were picked then stored in a cool dry place for up to 12 days. At various time intervals approximately 20 berries were crushed and the 180/160 ratio was determined.

The results of these measurements are shown in Fig. 1.

It was expected that as these grapes lost water the 180/i 60 ratio of the juice would become enriched, and this was in fact found. After 12 days the enrichment of the water remaining in the berries (which by this stage had lost most of their water) was 4%o. This enrichment could be due to two possible factors:

1) Normal unrestricted evaporation as would be expected from an open body of water, e. g. a beaker of water left to evaporate on a laboratory bench.

2) Evaporation through a biological membrane. A grape skin can be considered a semi-permeable mem- brane because it will allow the easy through passage of some compounds but not others. In the case of water, this process is likely to be associated with some form of fractionation against the heavier form resulting in en- richment of the remaining water.

Figure 1 shows that this enrichment increases recti linearly up to seven days, after which it becomes con- stant. However due to the lack of data points in the lat- ter region of the graph this will be discussed no further.

Up to seven days the isotope enrichment under the particular conditions prevailing at the time, was 0.4%o per day. However for a different set of conditions (e. g. temperature, humidity, air flow) this could easily change. In the context of wine making there could clearly be an effect on the 180/160 ratio of the wine if the picked grapes were stored for one to two days prior to crushing. This would however be unlikely because spoilage would be a problem after this length of time.

M e a n = -0.2%0 or= 1.2%0

f) The Effect of Fermentation on the 180/160 Ratio of Grape Juice Water

It was also necessary to determine if changes in the 180/160 ratio of the water occur when grape juice is fermented into wine. For this experiment 13 varieties of grape juice were fermented and bottled, the 180/160 ratios being determined before and after fermentation. The results are shown in Table 5 where it can be seen that in most cases there are differences in isotope ratio between a juice and its resultant wine.

For seven of the samples the 180/160 ratio of the wine was greater than that of the juice, however in six cases it was smaller. The juice 6180 value has been sub- tracted from the 6180 value of the corresponding wine and the result shown in the (A%o) column. If these values are then averaged a slightly negative (-0.2%o) result is obtained. However with such a large standard deviation (1.2%o) no significant conclusions can be reached.

During fermentation large quantities of COz are re- leased. If this is allowed to equilibrate or even partially equilibrate with the water before being released, some' of the 180 from the grape water could be lost due to the C O 2 / H 2 0 fractionation effect [6]. An approximation of this loss can be made if it is assumed that total equili- bration of the CO 2 and water in a closed system occurs. For the purposes of this calculation the following con- ditions have been used: Sugar content of the grape juice=20% 6180 value of the CO2 released from the sugar (assum- ing this is released without fractionation)= +25%o

J. Dunbar: Oxygen Isotope Studies on Some New Zealand Grape Juices 257

6~80 value of the water in the grape juice before fer- mentation = 0%0 Temperature 1 = -t-25 °C, c~= 1.0412 Temperature 2 = + 5 °C, e = 1.0455 (c~ = the C02/H20 fractionation factor).

It then follows that, per litre of juice, the CO2 released and the water present will contain 71 gm and 711 gm of oxygen respectively. Therefore at tempera- ture 1 (25 °C) the 180 depletion of the water will be 1.5%o and at temperature 2 (5 °C) it will be 2.0%0.

The following points however should be consid- ered:

1) The system is not closed, therefore as C02 (in equilibrium with the water) is released the 6180 value of the water will fall, causing the amount of 180 re- moved by the CO2 also to decrease. The total depletion of the water will consequently not be as great.

2) It is unlikely that full equilibration of the CO2 is going to occur, this being especially true during the log- arithmic growth phase of the yeast.

In order to produce certain characteristics in a wine it is sometimes necessary to carry out the fermentation slowly and at a low temperature. In this case full equili- bration of the CO2 and water could occur, and con- sidering that the fractionation factor, ~, is higher at this temperature, 180 depletion of the water approaching that of the maximum could occur.

Conclusions

The results can be summarised as follows: a) The 180/160 ratio of the juice from the grapes

of a given vine does not change significantly during the course of 24 h.

b) The 180/160 ratio of the juice from the grapes of a given vine does not change significantly as the gra- pes ripen, and up to 14 days before harvest there is no detectable change.

c) Rain will only affect grape juice 180/160 ratios when the fruit is ripe. Uptake of this water in ripe gra- pes has a diluting effect on the 180 enrichment of the juice.

d) The 180/160 ratios of several grape juice con- centrates that had been prepared by rotary evaporation were found to be very similar to those of control wines.

e) As picked grapes lose water by evaporation through the skin, the juice remaining becomes more en- riched in 180.

f) Fermenting grape juice into wine does not ap- preciably change the water 180/160 ratio.

These results show that many of the factors that were initially considered to have an effect on the oxy- gen enrichment of the water of grapes in fact change this enrichment only marginally. As an exception, rain may play an important role in the final 6180 value of a wine, especially if it comes at the time of harvest. If however a particularly low 180/160 ratio has been measured and there is suspicion that tap water has been intentionally added, the use of meteorological data should help decide if this was in fact the case.

This preliminary study therefore indicates that it should be possible to ultimately develop a method for the detection of added water in wine.

References

1. Bricout J (1973) Ann Fals Exp Chim 66:195 2. Bricout J, Merlivat L, Fontes JC (1973) Ind Agr Aliment 90:19 3. Bricout J, Fontes JC, Merlivat L (1972) C R Acad Sc (Paris) D

274:1803 4. Bricout J, Mouaze Y (1971) Fruits 26:777 5. Bricout J (1978) Rev Cytol Biol V6g&-Bot 1:133 6. Epstein S, Mayeda T (1953) Geochim Cosmochim Acta 4:213 7. Gonfiantini R (1978) Nature 171:534 8. Zundel G, Miekeley N, Grisi B, F6rstel H (1978) Radiat Environ

Biophys 15:203 9. Farris F, Strain BR (1978) Radiat Environ Biophys 15:167

10. Dongmann G, Niirnberg H, F6rstel H, Wagener K (1974) Radiat Environ Biophys 11:141

11. Titani T, Goto K (1940) Proc Imp Acad (Tokyo) 16:398 12. Titani T, Goto K (1941) Japan Academy Nikon Gakashiin (To-

kyo Proc) 15:298 (1941) Bently R, Bhate DS (1960) J Biol Chem 235:1225 Rittenberg D, Graft C (1958) J Am Chem Soe 80:3370

13. 14.

Received March 8, 1982