APPL-IED AND ENVIRONMENTAL MICROBIOLOGY. June 1989, p. 1617-16230099-2240/89/061617-07$02.0O/OCopyright © 1989, American Society for Microbiology

Freon 11 Extraction of Volatile Metabolites Formed by CertainLactic Acid Bacteria

RALPH P. TRACEY'-,* AND TREVOR J. BRITZ-Viticultural an d Oenological Researc(h Inistitute, Stellenibos(ch, antid Depar-tmlenut of' Mi(-robiology, University of 'tlie

OIrantige Free State, 9300 Bloel,nfotmtein,2 Soidtli Afiri(ca

Received 7 November 1988/Accepted 3 March 1989

The volatile metabolites formed by 18 lactic acid bacteria, representing three genera, were extracted froma complex medium by using a Freon 11 extraction method. The Freon extracts were then analyzed by capillarygas chromatography, and certain extracts were analyzed by gas chromatography-mass spectrometry. A totalof 35 major peaks, of which 20 were positively identified, were used to differentiate between the various strains.On the basis of the results obtained, it was possible to differentiate between the members of the genera

Lactobacillus, Pediococcus, and Leuconostoc, as well as between various species within the genus Leuconostoc.Of the 10 Leuconostoc oenos strains included in this study, 9 yielded similar results, but it was still possible todifferentiate between the various strains. L. oenos B66 differed from the other L. oenos strains. Use of the Freon11 extraction technique to determine volatile metabolites formed by lactic acid bacteria was shown to be highlyreproducible and of great value. Furthermore, certain compounds not previously known to be formed by lacticacid bacteria were found.

The use of end product analysis by gas-liquid chromatog-raphy for the characterization and identification of bacteriais well documented. In the eighth edition of Bergev 's M,iailitilof Determinatiie Bactaeriology (3), as well as in Bergevy'sManiual ofSystematic Bacteriology (7, 13), a large number ofgeneric descriptions include metabolic end products as cri-teria for the inclusion of an organism in a particular genus.Most gas-liquid chromatography methods suffer from the

disadvantage that a pretreatment of the sample is oftennecessary and that this is sometimes followed by a derivati-zation procedure (14). Even though Thornhill and Cogan (14)regard pretreatment and derivatization as a disadvantage, itmust be borne in mind that pretreatment or derivatization ofsamples can contribute greatly to the analyses and, in so

doing, can increase the value of the analyses and subsequentgrouping or classification of species or strains. Direct injec-tion of samples to measure the fermentation products formedby bacteria has only a limited application, because in mostcases only a few compounds are determined. Because onlythe main compounds of fermentation are determined, organ-isms may be grouped together when, in reality, their rela-tionship to each other is not very high. It is thereforenecessary that additional tests be carried out to confirm therelationships.

Recently a reproducible technique, involving the use of a

Freon 11 extraction method, was published to determine theamounts and identities of terpenes in grape juice and wine bycapillary gas chromatography (10). The use of Freon 11 forthe extraction of volatiles has also gained wide acceptance inthe field of wine flavor research. It has been used todetermine the amounts and identities of volatile yeast metab-olites in fermenting grape musts, for which the gas-chro-matographic analysis of Freon 11 extracts revealed over 72esters and other metabolites (1). Furthermore, Freon 11 hasvarious other advantages: it is relatively cheap, easily ob-tainable, noninflammable, and nontoxic (11). Two disadvan-

* Corresponding author.t Present address: Department of Microbiology. University of the

Orange Free State, P.O. Box 339, 9300 Bloemfontein. South Africa.

tages, however, are that Freon 11 does not extract ethanol,which is an important metabolite formed by some bacteria,and that because Freon has a boiling point of 23.6°C, all thework must be carried out below this temperature in air-

conditioned rooms.Since ethanol is not one of the major metabolites formed

during glucose catabolism by lactic acid bacteria, it ispossible that the Freon 11 metabolite extraction techniquewill be of value in the differentiation of species or even

strains of the lactic acid bacteria. The purpose of this studywas to determine whether this metabolite extraction tech-nique could be used in the differentiation of lactic acidbacteria and, more specifically, strains of LeuconostocOeUIOS which are of importance to the wine industry.

MATERIALS AND METHODS

Bacterial strains. The strains used in this study include thetype strains Lactobacillus casei NCIB 8010, Pediococcusdalumltiosius DSM 20331, Leuconostoc crelnoris DSM 20346,L. Dextraniculm DSM 20484, L. lactis DSM 20202, L.mesenteroicdes DSM 20343, L. palraimeseniteroides DSM20288, and L. oenios DSM 20252, as well as reference strainsLactobacillius pl(uiitai-rin NCIB 6376, L. oenos DSM 20255,and L. oeCios DMS 20257. L. oenios B66, 37D, 19, and 36were selected as representative centrotype strains of fourclusters obtained during a numerical taxonomic study onmalolactic bacteria from the main wine-producing areas inthe world (15). Three L. oenios strains (Lc5x, 30, and 7B)were selected on the basis of having the lowest similarity tothe centrotype strain (15) of each of the three particularclusters. The strains were preserved by lyophilization onfilter paper disks (6).

Culture conditions. For each strain, 50 ml of enrichedtomato juice broth (15) was inoculated with a filter paper diskand incubated at 25TC for 48 h. A 10-ml sample of this culturewas then used to inoculate 300 ml of the same medium.Excellent growth was found after incubation at 25°C for 4days, whereafter the cells were removed by centrifugationfor 20 min at 18,000 x g. The supernatant was recovered and

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1618 TRACEY AND BRITZ

immediately cooled to 0°C for extraction with Freon 11 andsubsequent gas-chromatographic analysis.

Extraction procedure and concentration of the extract. Theprocedure and extraction apparatus as described by Marais(10) were used. The internal standard, 2-ethyl-1-hexanol (80p.g/liter) was added to 250 ml of the cooled sample in ameasuring flask and carefully mixed. A 20-ml portion ofFreon 11 and the cooled sample containing the internalstandard was carefully poured into the extraction apparatusdescribed previously (10). This was then installed with itsbottom in 50 mm of ice to prevent the formation of anemulsion. A collecting funnel and condenser, through whichethanol (at -5°C) was circulated, were fitted to the extrac-tion unit. A 25-ml pear-shaped flask containing 20 ml ofFreon was fitted to the extraction unit, and the fitted flaskwas then immersed in a water bath at 35°C. The extractionwas performed for 20 h at a controlled temperature of 20°C.To concentrate the extract, a Vigreux fractioning column(270 by 20 mm) and an air condenser (550 by 13 mm) werefixed onto the flask containing the extract to facilitate reflux.The flask was placed in a water bath at 35°C, with the roomtemperature at about 20°C, and the extract was concentratedto 2 ml. The flask was then placed in solid CO, to freeze outany traces of water from the extract. The concentratedextract was then transferred to a 3-ml pear-shaped flask witha Pasteur pipette. A small condenser (220 by 8 mm), fittedwith a spiral made from Teflon, was fitted to the flask, andconcentration was continued under partial reflux to approx-imately 0.1 ml. The extracts were then stored at -12°C priorto analysis.Gas chromatography conditions. The gas-chromatographic

analyses were carried out on a Hewlett Packard 5880A gaschromatograph equipped with a hydrogen flame ionizationdetector and dual integrators. A Carbowax 20M fused-silicacapillary column (50 m by 0.31 mm [inner diameter]) wasused under the following operating conditions: injectiontemperature, 200°C; detector temperature, 250°C; columntemperature, 60°C for 10 min, then increased by 1°C/min to190°C and held at 190°C for 30 min; flow rate of carrier gas(helium), 1.5 ml/min; split flow rate, 120 ml/min; split ratio,90:1; septum purge, 6 ml/min; hydrogen flow rate, 30 ml/min;air flow rate, 300 ml/min; injection volume, 1 [Ll.

Identification of the major peaks. A Finnigan gas chro-matograph-mass spectrometer (model 4500) equipped with aCarbowax 20M fused-silica column (50 m by 0.31 mm [innerdiameter]) and gas-chromatographic operating conditions, asdescribed above, were used to identify the major peaksformed by the L. oeilos strains on the basis of retention timeand mass spectra. The identities of the various chemicalcompounds were confirmed by comparing their mass spectrawith those of authentic standards analyzed under similarconditions. The following operating conditions were used:interface temperature, 190°C; manifold temperature, 100°C;column flow rate, 1.4 ml/min; split ratio, 48:1, electronmultiplier voltage, 1,600 V; scanning rate, 35 to 450 amu/ehswith a 0.1-s delay between scans.

Determination of the relative concentrations. The relativeconcentrations of the major compounds were calculated byusing the internal standard calibration method: relative con-centration (b) = [area (b) x concentration (al)]/area (a), wherea is the internal standard and b is an unknown compound.

Reproducibility. The reproducibility of the technique wasdetermined by analyzing the Freon 11 extracts of fourseparately grown cultures of each of Lacztobacillits caseiNCIB 8010 and L. oCelos DMS 20252. The mean, standarddeviation, and coefficient of variation for each separate peak

was calculated from the relative concentrations of eachpeak.

RESULTS AND DISCUSSION

Reproducibility. The data for the four separately growncultures of Latowbacillits casei NCIB 8010 and L. oeniosDSM 20252 are given in Table 1. The coefficients of variationwere very low, and it was therefore possible that all thepeaks could be used as an aid in the differentiation of thestrains. The high level of reproducibility also indicates thatthis technique is reproducible when determining volatilemetabolites formed by lactic acid bacteria.

Basal medium. The composition of the Freon extract of thebasal medium (enriched tomato juice broth) is given in Table2. The relative amounts of volatile products were found to besimilar for each batch of basal medium used. A chromato-gram showing the volatiles present in the basal medium ispresented in Fig. IA. With the use of the Freon 11 extractionmethod, 12 compounds were found in the basal medium.This complex medium was specifically used because it waspreviously found to contain all the required amino acids andother growth factors (15).

Metabolites produced by the various lactic acid bacteria.The relative concentrations of the major peaks of all thestrains are given in Table 2. A total of 35 major volatilescould be differentiated, of which 20 were positively identi-fied. By using the Freon 11 extraction technique, it ispossible to identify several previously unknown compoundsformed by lactic acid bacteria. No previous literature on theformation of these compounds by lactic acid bacteria couldbe found. These compounds include 3-methyl-2-butanol,ethyl-2-hydroxypropionate, benzaldehyde, 3-(methylthio)-1-propanol, (x,c-dimethylbenzenemethanol, and benzenemeth-anol. Of these compounds, two were present in the basalmedium but at much lower concentrations than produced bythe organisms used in the study. It is possible that byapplying this technique to other bacteria, even more com-pounds formed by bacteria will be discovered. These uniquemetabolites could play a vital role in differentiating betweenbacteria and thus possibly contribute to an even betterclassification of bacteria. A fact that must be taken intoconsideration is that a rich complex basal medium was usedin the study, and this will greatly influence the type andconcentration of metabolites produced.

Differentiation between the various lactic and bacteria.After visual comparison of the chromatograms of the variousstrains on the basis of the presence or absence and/or therelative concentrations of the compounds, we used thefollowing compounds to differentiate between the variousstrains (peak numbers also given): isobutanol (peak 1),isoamyl alcohol (peak 2), acetoin (peak 3), 3-methyl-2-butanol (peak 4), ethyl-2-hydroxypropionate (peak 5), aceticacid (peak 6), butanoic acid (peak 9), c,cx-dimethylbenzen-emethanol (peak 13), hexanoic acid (peak 14), heptanoic acid(peak 17), octanoic acid (peak 19), nonanoic acid (peak 21),decanoic acid (peak 22), dodecanoic acid (peak 25), tetrade-canoic acid (peak 31), and the unidentified compounds inpeaks 8, 10, 18, 23, 24, 27, 28, 33, and 34.The various strains of lactic acid bacteria (Table 2: Fig.

1B) could easily be differentiated. Lactobacilulis casei (Fig.iB) could be separated from the other strains on the basisthat it formed the largest amount of ethyl-2-hydroxypropi-onate (peak 5) of all the strains studied (Table 2) and alsoproduced large amounts of acetic acid (peak 6), (x-dimeth-ylbenzenemethanol (peak 13), benzenemethanol (peak 15),

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FREON 11 EXTRACTION OF VOLATILE METABOLITES

TABLE 1. Relative concentrations, means, standard deviations, and coefficients of variation for the four Freon 11 extractsof each of Lactobacillus casei NICB 8010 and L. oenos DSM 20252

Lactobacilluds casei L. oeniosCompound Peak Retention

no. time (min) Mean concn ± SD Coefficient of Mean concn SD Coefficient ofMeancoen ± SD variation" K) MaconSD variation e(

Isobutanol 1 7.26 152.80 ± 3.08 2.01 133.58 ± 1.10 0.83Isoamyl alcohol 2 10.20 252.83 ± 5.92 2.34 248.05 ± 2.90 1.17Acetoin 3 14.81 7.10 ± 0.20 2.823-Methyl-2-butanol 4 18.31 25.53 ± 0.43 1.67 24.18 ± 0.22 0.92Ethyl-2-hydroxypropionate 5 20.23 1,122.05 ± 24.81 2.21 482.12 ± 8.69 1.80Acetic acid 6 29.95 599.53 ± 12.87 2.15 601.13 ± 14.76 2.45Benzaldehyde 7 43.52 88.50 ± 1.09 1.23 86.00 ± 0.78 0.91Unidentified 8 48.53 118.23 ± 1.57 1.33Butanoic acid 9 50.07 203.23 ± 1.73 0.85 217.93 ± 2.59 1.19Unidentified 10 53.92 95.83 ± 0.35 0.37Pentanoic acid 11 54.92 121.85 ± 0.72 0.59 126.40 ± 1.97 1.563-(Methylthio)-1-propanol 12 59.41 233.83 ± 1.56 0.67 192.90 ± 4.62 2.39(x,o-Dimethylbenzenemethanol 13 64.59 86.35 ± 0.76 0.88 37.35 ± 1.12 3.01Hexanoic acid 14 74.16 41.45 ± 0.52 1.25 37.55 ± 1.46 3.88Benzenemethanol 15 76.15 233.23 ± 0.81 0.35 211.38 ± 6.24 2.952-Phenylethanol 16 79.63 375.25 ± 0.97 0.26 342.50 ± 9.19 2.68Heptanoic acid 17 85.32 52.40 ± 0.14 0.27 54.78 ± 1.74 3.17Unidentified 18 92.02 24.30 ± 0.22 0.89 42.13 ± 0.28 0.65Octanoic acid 19 96.12 120.70 ± 0.65 0.54 101.88 ± 2.28 2.23Unidentified 20 99.22 44.75 ± 0.34 0.76 32.13 ± 1.08 3.37Nonanoic acid 21 106.40 20.88 ± 0.26 1.26 10.63 ± 0.25 2.35Decanoic acid 22 116.21 119.00 ± 1.60 1.34 46.03 ± 0.59 1.27Unidentified 24 127.60 46.90 ± 0.42 0.90Dodecanoic acid 25 134.71 233.95 ± 0.82 0.35 58.50 ± 2.10 3.59Unidentified 27 138.37 30.18 ± 0.39 1.28Tetradecanoic acid 31 154.85 229.70 ± 5.71 2.49 98.15 ± 3.73 3.80Unidentified 34 160.40 143.90 ± 2.90 2.04 65.98 ± 0.25 0.38

" Coefficient of variation = (standard deviation/mean) x 100.

nonanoic acid (peak 21), decanoic acid (peak 22), andtetradecanoic acid (peak 31).The formation of the highest concentrations of hexanoic

acid (peak 14) and dodecanoic acid (peak 25) by the Lacto-bacillus planttariin strain and the formation of high concen-trations of acetoin (peak 3), octanoic acid (peak 19), anddecanoic acid (peak 22) and a small amount of acetic acid(peak 6), as well as the absence of two unidentified com-pounds (peaks 8 and 10) and nonanoic acid (peak 21), couldbe used to differentiate this strain from the other strainsincluded in this study (Table 2).The P. dainnltlosl(s strain was differentiated from the others

on the basis that it formed the largest amount of isoamylalcohol (peak 2) of all the strains studied; it also formed highconcentrations of ethyl-2-hydroxypropionate (peak 5), and itformed two unidentified compounds (peaks 8 and 10). Theabove-mentioned differences for these organisms can also becomplemented with the use of morphology and cell wallcomposition as previously described (15).When the various Leliconostoc species were compared

with each other, it was found that although they generallyyielded similar profiles, they could still be separated (Table2). L. crZemo)ris was separated mainly on the basis of the highconcentrations of 2-phenylethanol (peak 16) and dodecanoicacid (peak 25) formed and the presence of high concentra-tions of the unidentified peaks 26 and 33. This strain was theonly organism that produced the unidentified compound inpeak 33.The L. devtroaniiciimi strain could be differentiated on the

basis of the amounts of butanoic acid (peak 9), nonanoic acid(peak 21), decanoic acid (peak 22), and an unidentifiedcompound (peak 34) formed.

The high concentration of 3-(methylthio)-1-propanol (peak12) and the low concentrations of (x,o.-dimethylbenzeneme-thanol (peak 13) and heptanoic acid (peak 17) formed wereused to separate L. lactis from the other strains. Accordingto Cogan et al. (4), L. lactis forms acetoin (a fact that wasconfirmed in this study). even though only at low concentra-tions (Table 2).

L. iiesenter-oides was differentiated from the other strainson the basis of the formation of the largest amount of acetoin(peak 3), as well as the relatively high concentrations of3-methyl-2-butanol (peak 4), the presence of the unidentifiedcompounds in peaks 18 and 24, and the absence of aceticacid (peak 5) and the unidentified compound in peak 10(Table 2).The formation of the largest amounts of o,a-dimethylben-

zenemethanol (peak 13), heptanoic acid (peak 17), and theunidentified compounds in peaks 18, 20. 27, and 28 made itpossible to separate L. pt1E(lra1ieseniter-oides from all the otherstrains. This was the only strain to form the unidentifiedcompound 28, no ethyl-2-hydroxypropionate (peak 5), noacetic acid (peak 6), and no unidentified compounds 8 and10. and it characteristically produced only low concentra-tions of butanoic acid (peak 9), hexanoic acid (peak 14).octanoic acid (peak 19), and tetradecanoic acid (peak 31).Garvie (5) found that it was difficult to separate L. paiw-t/ite-seniteioides from the non-dextran-forming strains of L. mie-seniter-oides, whereas this study (Table 2) showed that thesespecies can easily be distinguished. The formation of aceticacid (peak 6) can also be used to separate them from variousother Leuconostoc species, since the L. ,nesetitenroides andL. p(irIilnesetlter-oiles cultures used in this study did notform any acetic acid (peak 6).

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1620 TRACEY AND BRITZ

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VOL. 55, 1989 FREON 11 EXTRACTION OF VOLATILE METABOLITES 1621

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1622 TRACEY AND BRITZ

TABLE 3. Peaks used to differentiate between the various L. oenos strains

Relative concns" of peak no.':Strain Cluster"

2 3 5 6 8 it) 23 24 26 27 29 3t) 35

DSM 20252 4+ - 6+ 7+ - - - - - - - - -DSM 20255 4+ + 7+ 6+ 3+ 2+ - + - + - - -DSM 20257 3+ + 2+ 5+ + - - + - + - - -B66 A (centrotype) 5+ + 6+ 5+ 8+ 8+ - + 3+ + - - 7+Lc5x A 6+ - 8+ 7+ - + - 2+ - - - - +37D B (centrotype) 3+ + 5+ 8+ 2+ 2+ - + - + - - -

30 B 4+ + 4+ 7+ 3+ 2+ - + - - 8+ - -19 C (centrotype) 4+ 5+ - + - - 3+ - - + - 1+ -36 D (centrotype) 3+ + 7+ 5+ 3+ 3+ - + - + - 2+ -

7B D 3+ - 8+ 6+ - - - - - - - 2+ -

Strains not designated as centrotype straiins are the strains selected as having the lowest similarity to the centrotype strain." Relative concentrations are expressed as follows: -, t); +, 1 to 5(); 2+, 51 to 1i)(); 3+, 11)1 to 20)0; 4+, 2111 to 3t)(); 5+, 3t)1 to 40)1); 6+, 4t1 to 50t); 7+, 5()1

to 750); 8 +, > 75).Peak identities arl-e given in Table 2.

Garvie (5) reduced the species in the genus Leiwconostocfrom six to four by including L. de.vtranicum and L. (remnorisas subspecies of L. /nesente-oidIes. The results from thisstudy (Table 2) generally confirm that L. cremoris and L.dextrani(cum are closely related. However, L. Inesenteroictesdoes not appear to be closely related to them. The L.mesenteroides strain appeared to be more closely related tothe L. paralnesenteroides strain than to the L. dexrwanicumand L. cremnoris strains. More representative strains fromthese four species (or subspecies, as proposed by Garvie [5])will have to be studied to confirm the above observations.

In a previous study (15), it was shown that the differenti-ation between the leuconostocs and pediococci can beproblematic and that cell wall analysis and morphology areimportant criteria in differentiating the leuconostocs fromother genera. Morphologically, Lactohacillus can easily bedistinguished from the coccoid genera Leiconostoc andPediococcus. Separation of the three genera Lacto bacilluts,Pediococcus, and Leitconostoc by using volatile metaboliteswas comparatively easy, especially when comparing thedifferences in the amounts of isoamyl alcohol (peak 2),acetoin (peak 3), ethyl-2-hydroxypropionate (peak 5), aceticacid (peak 6), and hexanoic acid (peak 14) formed. Thepresence or absence of several other metabolites is also ofvalue. However, the results obtained in this study, with theexception of results for the L. oe)Cos species, were based ononly a single representative of each species and must there-fore be carefully interpreted before any general conclusionsor recommendations can be made. One positive conclusionthat can be made is that the Freon 11 technique can be ofgreat value in differentiating between lactic acid bacteria.

Differentiation of the L. oenos strains. Overall, the majorityof L. oenios strains yielded similar metabolic profiles, but therelative amounts of each compound formed differed mark-edly. Of the 35 important compounds detected in this study,only 13 yielded major differences that could be used todifferentiate between the L. oeuos strains (Tables 2 and 3).The use of these 13 compounds in the separation of the L.oeCUos strains used in this study is shown in Table 3. Theseinclude acetoin (peak 3), ethyl-2-hydroxypropionate (peak5), and the unidentified compounds in peaks 8, 10, 24, and 27(Table 3). Although the L. oeiios strains compared favorablywith the L. oenos reference strains regarding the presence orabsence of compounds 1 to 22, marked differences werefound for the remaining compounds.

Strain B66 (Table 2) was separated from the other strainssince this organism formed the highest concentrations of the

unidentified compounds in peaks 8, 10, 34, and 35 of all thestrains studied. Several other differences are also evident.Strain B66 is the centrotype strain of cluster A (15), and thevolatile metabolites formed by this strain is a further confir-mation that it differs from the other strains included in thisstudy. In contrast to this, strain LcSx did not form or formedmuch less of the unidentified compounds in peaks 8, 10, 34,and 35 (Table 3).

L. oenos 37D and 30 were selected as representatives ofthe largest phenotypic cluster (cluster B) from a previoustaxonomic study (15). The volatile-metabolite profiles ofthese strains differed to a certain degree, and they could beseparated on the basis of the presence or absence and/orconcentrations of compounds 5, 6, 9, 27, 29, 31, and 34(Tables 2 and 3).

Strain 19 could be separated, mainly because it was theonly L. oenios strain to form large concentrations of acetoin(peak 3) and was the only strain to form the unidentifiedcompound in peak 23. Further profile characteristics of thisorganism were the absence of ethyl-2-hydroxypropionate(peak 5) and the unidentified compounds in peaks 8 and 10(Table 3). This strain had a different overall volatile-metab-olite profile from those of the other L. oenios strains. It wasthe centrotype strain of cluster C and was phenotypicallyfairly closely related to cluster B (15).The two representative strains from cluster D (36 and 7B)

were separated from the other strains on the basis of theformation of less isobutanol (peak 1) and isoamyl alcohol(peak 2). They could also be separated from each other byusing acetoin (peak 3) and the unidentified compounds inpeaks 8, 10, 24, and 27 (Table 3). The overall volatile-metabolite profiles of these two strains compared favorablywith those of the other L. oenios strains.Once again, it must be stressed that the data obtained and

used in the differentiation of the different L. oenos strains arebased on single representatives of the phenotypic clusters(15) and that any conclusions made must be seen in this light.Further work is also required on these and other strains thatare to be used to induce malolactic fermentation. It has beenshown that L. oenos strains are the most reliable organismswhich can be used for inducing malolactic fermentation (2, 8,9). Therefore, to find the L. oenos strain that is best suitedfor inducing malolactic fermentation and that will have theleast negative effect on the wine, the strains will have to betested individually in wine, and these wines must subse-quently then undergo a sensory evaluation and analysis bythe Freon extraction method.

APPL. ENVIRON. MICROBIOL.

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FREON 11 EXTRACTION OF VOLATILE METABOLITES

Utility and value of the Freon I I extraction technique.Although the Freon extraction technique is more time-consuming and elaborate than previously published gas-chromatographic techniques for the study of volatile metab-olites produced by bacteria, we believe that the amount ofwork and time taken for the extraction are duly rewarded inthe results obtained, since a wider range of metabolites aredetermined than by conventional direct-injection chromnatog-raphy. Furthermore, it is possible to differentiate betweenspecies of a particular genus and between strains of aparticular species.When organisms are used industrially, as with the lactic

acid bacteria, this technique could help to determine thecontribution of the organisms to the flavor of the variousproducts. In 1965, Pilone and Kunkee (12) showed thatflavor differences in wines in which malolactic fermentationhad been induced by bacteria were small. The use ofdifferent and newer strains thus warrants a new look at thepossible contribution of the microorganisms to the flavor ofwine. It is also important to try and correlate the sensoryevaluation of wines which have undergone malolactic fer-mentation with chemical analyses by the technique de-scribed in this paper.

ACKNOWLEDGMENTS

The technical assistance of Helena Kritzinger and Isabeau Bronnis acknowledged. Johan Marais is thanked for his critical review ofthe paper.

LITERATURE CITED1. Akhtar, M., R. E. Subden, J. D. Cunningham, C. Fvfe, and A.

Meiering. 1985. Production of volatile yeast metabolites infermenting grape musts. Can. Inst. Food Sci. Technol. J.18:280-283.

2. Beelman, R. B., A. Gavin, and R. M. Keen. 1977. A new strainof Leuiconostoc oeno.s for induced malo-lactic fermentation inEastern wines. Am. J. Enol. Vitic. 28:159-165.

3. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergevs*smanual of determinative bacteriology. The Williims & WilkinsCo.. Baltimore.

4. Cogan, T. M., M. O'Dowd, and D. Mellerick. 1981. Effects ofpH and sugar on acetoin production froni citraite by Leuconos-to( Iulalis. AppI. Environ. Microbiol. 41:1-8.

5. Garvie, E. 1. 1986. Genus Lcuuouo.soc. p. 1071-1075. InP. H. A. Sneath. N. S. Mair. M. E. Sharpe. and J. G. Holt (ed.).Bereevs manual of systematic bacteriology. vol. 2. TheWilliams & Wilkins Co.. Baltimore.

6. Joubert, W. A., and T. J. Britz. 1987. A simple and inexpensivemethod for the long-term preservation of microbial cultures. J.Microbiol. Methods 7:73-76.

7. Krieg, N. R., & J. G. Holt (ed.). 1984. Bergey's manuail ofsystematic bacteriology. vol. 1. The Williams & Wilkins Co..Baltimore.

8. Kunkee, R. E. 1967. Malo-lactic fermentattion. Adv. AppI.Microbiol. 9:235-279.

9. Lafon-Lafourcade, S., E. Carre, and P. Ribereau-Gavon. 1983.Occurrence of lactic acid bacteria during the different stages ofvinification and conservation of wines. Appl. Environ. Micro-biol. 46:874-880.

10. Marais, J. 1986. A reproducible capillary gias chromnatogr-aphictechnique for the determination of specific terpenes in grapejuice and wine. S. Afr. J. Enol. Vitic. 7:21-25.

11. Marais, J., and A. C. Houtman. 1979. Quantitative gas chro-niatographic determination of specific ester-s 'and higher alcoholsin wine using Freon extraction. Am. J. Enol. Vitic. 30:250-252.

12. Pilone, G. J., and R. E. Kunkee. 1965. Sensory characterizationof wines fermented with several malo-laictic strains of bacteria.Am. J. Enol. Vitic. 16:224-230.

13. Sneath, P. H. A., N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.).1986. Bergey's manual of systematic bacteriology. vol. 2. TheWilliams & Wilkins Co.. Baltimore.

14. Thornhill, P. j., and T. M. Cogan. 1984. Use of gas-liquidchromatography to determine the end products of growth oflactic acid bacteria. AppI. Environ. Microbiol. 47:125(-1254.

15. Tracey, R. P.. and r. J. Britz. 1987. A numerical taxononlicstudy of Leoiubonswc oenos strains from wine. J. Appl. Balcte-riol. 63:523-532.

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