JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 68, NO. 4, 282-285. 1989

Monitoring of Alcoholic Fermentations of Fruit Juices Via a Gas Membrane Sensor

MARIE-NOi~LLE PONS, DENIS P I C H O N , AND M A R T I A L A U T H I E R

Laboratoire des Sciences du Gdnie Chimique, CNRS-ENSIC-INPL, BP 541, F-54001 Nancy, Cedex, France

Received 20 February 1989/Accepted l0 August 1989

Volatiles (ethanol, acetaldehyde, ethyl acetate, fusei alcohols) during alcoholic fermentations of various natural (grape, apple, and pineapple juices) and synthetic (glucose, fructose, sucrose) media have been monitored via a gas membrane sensor connected to a gas chromatograph. For all media similar behavior was observed concerning acetaldehyde production and disappearance and ethanol, ethyl acetate, and n-propyl, isobutyl, and isoamyl alcohol production. The concentration of acetaldehyde reaches its maximum when the production rates of ethanol and detected fusel alcohols as well as the carbon dioxide production are maximal. The observed levels of fusel alcohols depend on the composition of the broth.

During recent years the interest in factors influencing the quali ty of alcoholic beverages has risen, first to insure cons- tant organolept ic propert ies for existing fermented drinks and second to provide guidelines for the creation of new ones. One has to be fully aware that the definition and the quantif icat ion of the quali ty of a beverage are a very diffi- cult task, because of the high variabi l i ty in time and space of the natura l substrates and the slow matura t ion process of some beverages like wines, as repor ted by Barre (Barre, P., Proc. 8th Int. Biotechnol. Symp. , Vol. 2, p. 899-909, 1988). Some hints have been given concerning the possible impor tan t effects of fusel alcohols on the strength and a roma of beverages. However kinetics of format ion of these volatile components are difficult to obtain because most of them are present as traces and direct sensors are still scarcely available. As an example, in the scheme pro- posed by Webb and Ingraham (1) acetaldehyde is a key intermediary but its detect ion by convent ional means (i.e. liquid sampling of fermentat ion broth and off-line analysis) is made difficult by its high volati l i ty (boiling point 20°C).

We propose here to investigate the kinetics of format ion of volatile metaboli tes during the alcoholic fermentat ion of various natural substrates (fruit juices) by an on-line mem- brane sensor connected to a gas chomatograph . This detec- t ion is based on the gas phase diffusion of solute molecules through a microporous hydrophobic membrane , made of polytetraf luorethylene (PTFE) or polypropylene, as pro- posed by several workers (2, 3). One side of the membrane is in contact with the liquid phase, when the other is swept by a carrier gas which t ransports the molecules towards a suitable detector, here a gas chromatograph equipped with a flame ionizat ion detector (FID). This technique is suitable to detect a large range of volatiles once a correct choice of the chromatographic method has been made as shown by Pons et al. (4) and has already been applied to alcoholic fermentat ion of beet molasses by Pons and Engasser (5). As we are not specialists in oenology or fermented drinks product ion , we will not a t tempt here to discuss the quali ty of the beverage obtained. Our purpose is to demonst ra te the use of such a membrane device to ob- tain useful data on this kind of fermentat ion.

MATERIALS A N D METHODS

Fermentat ion: the experiments were run in a 2-I jacketed stainless steel reactor at constant temperature (Fig. 1). Mild agitat ion (magnetic stirrer) was maintained throughout the fermentat ion. A condenser was placed at the gas outlet to prevent the str ipping of volatiles. The in- oculum was prepared by suspending a given amount of commercial dry Saccharomyces cerevisiae (Fermipan TM

from Soci6t6 des Produi ts du Ma/s, Clamart , France) in 50 ml of fruit juice and 50ml of water during 15 min at 30°C. One and six-tenths liters of commercial fruit juice (made of fruit juice concentrate except for the white grape juice) was placed in the fermentor. The synthetic media used for comparison contained (per liter): (NH~)iSO4, 10 g; KHzPO4, 6g ; MgSO4-7HzO, 3g; NaC1, 0.1 g; CaCli, 0.1 g; yeast extract, 5 g; and 100 g of sugar (glucose, fructose, or sucrose).

The s tandard fermentat ion condit ions were: tem- perature (T) 30°C, initial biomass concentrat ion (X0) 6.5

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282

VOL. 68, 1989 MONITORING OF ALCOHOLIC FERMENTATIONS 283

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FIG. 2. Conductimetry-based carbon dioxide detection system.

g/l. The glucose and fructose contents of the juices were measured by enzymatic assays (Boehringer-Mannheim kit ref. 139106). Due to the presence of pulp in the pine- apple juice, it was filtrated on a 0 . 2 p m membrane before the test.

CO2 detection: in absence of any sophist icated CO2- meter and due to the small volume of the reaction vessel a crude gas detection system was built to moni tor the produc- tion of carbon dioxide: it was based upon the fluctuations induced by the gas bubbles on the response of a conduc- timetric probe (Tacussel CM01) connected to a conduc- t imeter (Tacussel CD61) and a recorder (Fig. 2). The ampl i tude of the fluctuations can be linearly related to the gas flow rate as shown in Fig. 3.

Moni tor ing of volatiles: a 121 FL Delsi Instruments gas chromatograph equipped with a FID and an automat ic gas sampling valve (sample v o l u m e = 0 . 3 ml) was used. The 4 m × 1/8" stainless steel column was packed with 1 0 ~ Car- bowax 20M. The gas chromatograph carrier gas was nitrogen. The temperature settings were: oven: 80°C, injec- tor: 135 o C, detector: 150 ° C. The electrometer signal treat- ment was done by an ICR-IB Shimadzu integrator (peak area calculations). The following volatiles were detected: acetaldehyde, ethyl acetate, ethanol , and n-propyl , isobutyl, and isoamyl alcohols. The flat membrane of the gas membrane sensor was made out of microporous hydrophobic PTFE (pore equivalent diameter 0 .2pro , porosi ty 70%, thickness 140 pm, surface 1 cm 2) and the car-

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20 410 60 8'0 180 120 Gas fl0w raLe (cm3/mn)

Relat ion between the conduct imetr ic signal and the

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F1G. 4. Typical chromatogram obtained in alcoholic fermenta- tion with the gas membrane. Sensor: 1, acetaldehyde; 2, ethyl acetate; 3, ethanol; 4, propanol; 5, isobutanol; 6, isoamyl alcohol.

rier gas was nitrogen (flow rate 50 ml /min) . Peaks were well separated except for n-propyl alcohol as shown on the chromatogram of Fig. 4. The cal ibrat ion curves were linear for all tested components within the range of in-

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FIG. 5. A lcohol ic fermentat ion on glucose. Symbols: e, acetaldehyde; ( , ethyl acetate; II, n-propyl alcohol; rT, ethanol; ~, isobutyl alcohol; A, isoamyl alcohol.

284 PONS ET AL. J. FERMENT. BIOENG.,

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FIG. 6. Alcoholic fermentation on sucrose with carbon dioxide monitoring.

terest and checked up to 150 g/l for ethanol. The lower detection limits were: for acetaldehyde 0.2 mg/l, for ethyl acetate 0.8 mg/l, for ethanol and n-propyl , isobutyl , and isoamyl alcohols 10 mg/l. The sampling time is 30 min.

RESULTS AND DISCUSSION

Synthetic media Figure 5 presents the kinetics of a 100g/ / initial glucose concentrat ion fermentat ion under s tandard condit ions. Aceta ldehyde is first produced along with the other metaboli tes . Its later d isappearance could be due to reconsumpt ion by the yeast but its stripping by carbon dioxide off-gas cannot be over looked, due to the high volati l i ty of acetaldehyde. The product ion of acet- a ldehyde is closely related to the product ion of carbon dioxide and to the presence of sugar as indicated on Fig. 6. A similar behavior has been found by Groboi l lo t (6) for alcoholic fermentat ion on beet molasses and this is in agreement with the metabol ism proposed by Webb and Ingraham (1). The maximal value of ethanol product ion rate is obta ined when the acetaldehyde concentrat ion is maximal (Fig. 7), and this corresponds too to the maximal product ion rates of both isobutyl and isoamyl alcohols.

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FIG. 7. Production rates of volatiles during alcoholic fermenta- tion on glucose.

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T i m e (h ) FIG. 8. Alcoholic fermentation of red grape juice. Symbols: o,

acetaldehyde; o , ethyl acetate; 5 ethanol; :1, isobutyl alcohol; &, isoamyl alcohol.

However the product ion pat tern of ethyl acetate is slightly different and its maximal product ion rate is obta ined a little later. This could indicate that ethyl acetate is not produced from acetaldehyde, in disagreement with the scheme proposed by Webb and Ingraham (1).

Table 1 summarizes the results obtained on synthetic media. The maximal value reached by acetaldehyde is similar for the three substrates. Isoamyl alcohol is the most abundant fusel alcohol (yield 0.86 mg/g initial sugar at 30°C) and higher temperature tends to favor its produc- tion as well as the one of isobutyl alcohol (yield 0.33 mg/g initial sugar at 30°C).

Natural substrates Three different red grape juice brands, of average sugar content 165g/1 (80g fruc- tose/l+85 g glucose//) have been tested. A typical run is presented in Fig. 8. The rate of product ion of ethanol is slightly lower than on synthetic substrates while the pro- ductions of isobutyl and isoamyl alcohols are higher:

VOL. 68, 1989 MONITORING OF ALCOHOLIC FERMENTATIONS 285

TABLE 1. Global results of alcoholic fermentation on synthetic media

Isoamyl Total fusel % ~!6 Run Sugar Ethanol Fermentation Max. Ethyl Propanol lsobutanol alcohol alcohols Isobut. Isoamyl no. time Temp. Inoculation acetaldehyde acetate

g/I h °C g/l dry yeast mg/I mg/l mg/I mg/I mg/I mg/l

1 Glucose 52 16 30 6.5 64.3 11.2 44.9 48 140 188 26 74 2 Glucose 69.7 19 21 6.5 43.4 22 62.5 36 92 128 28 72 3 Fructose 50 14.5 30 6.5 64.3 10 50.9 51 133 184 28 72 4 Fructose 43.9 15 21 6.5 40 14 41.7 22 48.7 71 31 69 5 Sucrose 50 15 30 6.5 60 10 50 50 130 180 28 72

TABLE 2. Global results of alcoholic fermentation on pineapple juices

. . . . Isoamyl Total fusel °4 ~ Run Fermentation Max. Ethyl Propanol lsooutanol alcohol alcohols Isobut. Isoamyl no. time Temperature Inoculation Ethanol acetaldehyde acetate

h °C g/I dry yeast g/l mg/l mg/l mg/I mg/I mg/I mg/I

I 10.5 30 6.5 74 28.6 10 30 150 279 429 35 65 2 10.5 30 6.5 59 43 9.6 0 136 251 387 35 65 3 22 21 6.5 55.6 51.3 17 0 94 281 375 25 75 4 13.5 27 6.5 42.8 35.7 9.6 0 108 230 338 32 68 5 12 30 6.5 54.6 52.6 9 0 119 256 375 32 68 6 10 30 9.7 54.8 35.6 11 0 147 256 403 36 64 7 16 30 3.2 54.7 62.5 10 0 114 294 408 28 72

1 . 7 2 m g isoamyl a l c o h o l / g init ial sugar and 0 . 6 7 m g isobuty l a l c o h o l / g ini t ial sugar . The synthesis o f fusel a lcohols is usual ly closely related to the presence o f amino - acids in the m e d i u m as descr ibed by var ious au thors (7, 8). Grape ju ice is r icher in those e lements than the synthet ic med ia p rev ious ly used and the fusel a lcohol p r o d u c t i o n is enhanced in ag reemen t wi th Yosh izawa (7) on grape juice. T h e r e f o r e the overa l l yield o f fusel a lcohols is 2.4 m g / g in- itial sugar and is close to the value indica ted by G u y m o n et al. (9). The percen tage o f i sobutyl a lcohol is s imilar to the one observed on synthet ic media and is coheren t wi th the l i terature . No p r o p a n o l is detected. The e thano l yield varies f r o m one b rand to ano the r while the max ima l value o f ace ta ldehyde does not depend upon the origin o f the ju ice .

Similar kinetics are ob ta ined by fe rment ing a white grape ju ice con ta in ing 80 g f r u c t o s e / / a n d 95 g g lucose / / . The p r o d u c t i o n o f volat i les is similar to the one ob ta ined with red grape ju ice except that here p r o p a n o l is detected.

Three brands o f apple ju ice o f average sugar con ten t 80 g/I (20 g f r u c t o s e / l + 6 0 g g lucose / / ) have been tested. The kinetics are s imilar to the ones observed for grape juice. The e thano l yield is close to the theore t ica l value (0 .51g e t h a n o l / g initial sugar). M o r e isobutyl (yield 1.75 m g / g initial sugar) and i soamyl (yield 3.75 m g / g in- itial sugar) a lcohols are p r o d u c e d than on red grape juice. The percen tage o f i sobutyl a lcohol is abou t the same in these three brands o f apple juice.

T w o different b rands o f p ineapple ju ice with an ap- pa ren t sugar con ten t o f 50 g/ l have been tested and the results are presented in Tab le 2. The e thano l yield could seem higher than the theore t ica l one but off-l ine enzymat ic analysis used here does not accoun t for the sucrose con ten t o f p ineapple ju ice . The final e thano l concen t r a t i on is wi thin the range indica ted by Koua ta et al. (10). The in- crease o f t empe ra tu r e or o f the inocu la t ion size shor tens the f e rmen ta t i on t ime while it does not m o d i f y the final me tabo l i t e concen t r a t ions significatively. The fusel a lcohol r epa r t i t ion is s imilar to the one observed on the prev ious substrates .

A l t h o u g h these f e rmen ta t i on tests with high inocu la t ion

size are not fully representa t ive o f a lcohol ic beverage pro- duc t ion , the gas m e m b r a n e sensor, connec ted to a gas c h r o m a t o g r a p h , is a system of reasonable cost , avoid ing l iquid sampl ing and m e d i u m losses, and is a useful tool for a more adap ted inves t iga t ion o f f e rmen ta t ions involv ing volat i le c o m p o n e n t s , especial ly those related to the qual i ty o f the beverages . It can help physiologis ts to clar ify the metabo l i c pa thways o f the yeast (aceta ldehyde, ethyl acetate) , process engineers to insure compos i t i on guidel ines in the final dr inks, and labora tor ies o f p roduc t deve lopmen t to create new drinks.

REFERENCES

1. Webb, A.D. and lngraham J.L.: Fusel off. Adv. Appl. Microbiol., 5, 317-353 (1963).

2. Phillips, D. H. and Johnson, M.J.: Measurement of dissolved oxygen in fermentations. J. Biochem. Microbiol. Technol. Eng., 3, 261-275 (1961).

3. Yaman~, T., Matsuda, M., and Sada, E.: Application of porous teflon tubing method to automatic fed-batch culture of microorganisms. 1. Mass transfer through porous teflon tubing. Biotechnol. Bioeng., 23, 2483-2507 (1981).

4. Pons, M. N. and Ducouret, P., Engasser, J. M.: Mass transfer characteristics of hydrophobic tubing membrane sensors. Biotechnol. Lett., 8, 407-410 (1986).

5. Pons, M. N. and Engasser, J. M.: Monitoring of alcoholic fed- batch culture by gas chromatography via a gas-permeable mem- brane. Analyt. Chim. Acta, 213, 231-236 (1988).

6. Groboillot, A., Pons, M. N., and Engasser, J. M.: Monitoring of volatiles in alcoholic fermentations on molasses via a gas mem- brane sensor. Appl. Microbiol. Biotechnol. (1989) in press.

7. Yoshizawa, H.: On various factors affecting formation of isobutanol and isoamyl alcohol during alcoholic fermentation. Agr. Biol. Chem., 30, 634-641 (1966).

8. Borzani, W., Vario, M. L. R., Koshimizu, L. H., De Melo, C., and Perego, L. Jr: Kinetics of amyl alcohol production during ethanol fermentation of blackstrap molasses. Biomass, 1, 115- 126 (1981).

9. Guymon, J. F., Ingraham, J. L., and Crowell, E. A.: Influence of aeration upon the formation of higher alcohols by yeasts. Am. J. Enol. Viticult., 12, 60-66 (1961).

10. Kouakou, A., Agbo, N'zi G., and Yaboua, A.: Ethanol produc- tion from pineapple juice in C6te d'lvoire with preselected yeast strains. J. Ferment. Technol., 65, 475-481 (1987).