Vol. 51, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1986, p. 539-5450099-2240/86/030539-07$02.00/0Copyright C 1986, American Society for Microbiology

Growth and Metabolism of Lactic Acid Bacteria during and afterMalolactic Fermentation of Wines at Different pH

C. R. DAVIS, D. J. WIBOWO, T. H. LEE,t AND G. H. FLEET*

School ofFood Science and Technology, The University of New South Wales, Kensington, New South Wales,Australia 2033

Received 16 September 1985/Accepted 9 December 1985

Commercially produced red wines were adjusted to pH 3.0, 3.2, 3.5, 3.7, or 4.0 and examined during andafter malolactic fermentation for growth of lactic acid bacteria and changes in the concentrations ofcarbohydrates, organic acids, amino acids, and acetaldehyde. With one exception, Leuconostoc oenos

conducted the malolactic fermentation in all wines and was the only species to occur in wines at pH below 3.5.Malolactic fermentation by L. oenos was accompanied by degradation of malic, citric, and fumaric acids andproduction of lactic and acetic acids. The concentrations of arginine, histidine, and acetaldehyde also decreasedat this stage, but the behavior of hexose and pentose sugars was complicated by other factors. Pediococcusparvulus conducted the malolactic fermentation in one wine containing 72 mg of total sulfur dioxide per liter.Fumaric and citric acids were not degraded during this malolactic fermentation, but hexose sugars were

metabolized. P. parvulus and species of Lactobacillus grew after malolactic fermentation in wines with pHadjusted above 3.5. This growth was accompanied by the utilization of wine sugars and production of lactic andacetic acids.

Lactic acid bacteria make a significant contribution to thequality of wines by conducting malolactic fermentation andby causing spoilage at later stages of vinification (3, 17, 18,20). Wine composition and, consequently, wine quality arealtered by these bacteria, because some wine componentsare utilized as substrates for growth and metabolic endproducts are excreted into the wine.Many qualitative studies (for a review, see references 3,

17, and 18) and more recent quantitative data (9, 11, 19) haveshown that Leuconostoc oenos is the species most fre-quently occurring in wine and is mainly responsible for themalolactic fermentation. Species of Pediococcus andLactobacillus have been isolated to a lesser extent and aremore likely to occur at stages after malolactic fermentationin wines of higher pH (3.5 to 4.0). A major weakness of theecological studies so far reported has been the infrequencyand irregularity of analysis of wines during the criticalperiods when lactic acid bacteria grow. Such a problem hasa-risen because wineries from which wine samples are ob-tained are generally located at considerable distances fromresearch laboratories (9). Consequently, a complete ecolog-ical description of the growth of lactic acid bacteria in aparticular wine at close time intervals during vinification isstill lacking; this ecology would be particularly complex inthe higher-pH wines, in which the successive growth anddeath of several species may occur (9, 11).The chemical changes in the composition of wine that are

caused by lactic acid bacteria are not well defined, except forthe conversion of malic acid to lactic acid during malolacticfermentation by L. oenos and to a lesser degree by species ofPediococcuts and Lactobacillus (3, 17, 18, 20, 27). Isolatedstudies suggest that wine carbohydrates (10, 24, 28) andamino acids (3, 23, 34) may be utilized by these bacteriaduring the malolactic fermentation and that this metabolismas well as that of organic acids (3, 17, 20, 27, 28) can lead to

* Corresponding author.t Present address: The Australian Wine Research Institute, Glen

Osmond, South Australia, Australia 5064.

changes in the concentration of constituents which affect thesensory quality of wines. The metabolism of wine compo-nents by lactic acid bacteria after malolactic fermentation islargely unstudied. Quantitative studies that correlate thekinetics of bacterial growth in wines with changes in theconcentration of specific wine constituents have not beenconducted. Consequently, the biochemical mechanisms bywhich lactic acid bacteria grow in wines are still poorlyunderstood.

In this paper we report the enumeration, isolation, andidentification of lactic acid bacteria from several wines atclose time intervals during and after malolactic fermentation.The growth of isolated species is correlated with theirutilization of wine sugars, organic acids, and amino acids.The effect of wine pH on both the species isolated and theirutilization of wine components is also reported.

MATERIALS AND METHODS

Wine samples. Shiraz wine was taken from wineries A andB (50 liters from each) immediately after completion ofalcoholic fermentation (about 7 days after the crushing of thegrapes) and was transported to the University of New SouthWales laboratory for storage and analysis. The two wineriesare located in the Hunter Valley district ofNew South Wales,Australia. At the laboratory, the wines were racked off thelees and dispensed as 4-liter volumes in sterile glass flasks thatwere fitted with air locks. Duplicate volumes of each winewere adjusted to the desired pH by the addition of eithersterile 50% tartaric acid or 5 M potassium hydroxide. The pHof one set of duplicates remained unadjusted. Wines werestored at 20°C and were sampled regularly for analysis oflactic acid bacteria, organic acids, monosaccharides, pH, and(at some sampling times) acetaldehyde and amino acids.Samples (30 ml) for analysis were taken from the center of theflask after thorough mixing of the contents. The flasks wereflushed with CO2 at each sampling. Microbiological and pHanalyses were conducted immediately after sampling. The

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sample was then filtered through a membrane (pore size, 0.45,um) and frozen until required for chemical analysis.Wines from several other wineries were obtained and

stored as described above, but without pH adjustment.Enumeration and identification of lactic acid bacteria. Lac-

tic acid bacteria were enumerated by spread-inoculating 0.1ml of the wine sample (diluted if necessary) over the surfaceof plates of MRS agar (Oxoid Ltd., London, England) andtomato juice agar (9, 25). Plates were incubated in anatmosphere of 5% CO2 for 7 days at 30°C. Plating mediacontained 100,ug of cycloheximide per ml to suppress thegrowth of yeasts. Colonies that grew on the surface of theplates and which were gram positive and catalase negative(9, 25) were counted and identified as lactic acid bacteria.Several representatives of each colony type were subcul-tured and identified to genus and species level by the testsdescribed previously for L. oenos (13), for Pediococcusparvulus (2, 14), and for isolates of Lactobacillus (29, 30).Carbohydrate fermentation tests were conducted in API 50CH galleries (La Balmes les Gottes-38390, MontalieuVercien, France).Chemical analyses. Hexose and pentose sugars were sep-

arated and determined by gas-liquid chromatography (GLC)of their per-o-acetylaldonitrile derivatives. Glucitol andmyo-inositol were also determined by GLC but as acetatederivatives. Procedures for the derivatization of the sugarshave been previously described (35). The derivatized sugarswere separated by injection into a 2100 Aerograph chromato-graph (Varian Associates, Palo Alto, Calif.) equipped with aglass column (1 m by 3 mm inner diameter) packed with 10%DEGS on Chromosorb W/AW 80/100; nitrogen was thecarrier gas at a flow rate of 60 ml/min. Injector and detectortemperatures were 190 and 250°C, respectively, and the oventemperature was programmed from 140 to 190°C at 1°C/min.Before derivatization, wine samples were treated to removesubstances that interfered with the resolution of the sugarsby GLC. This involved passage of 1.0 ml of the wine samplefirst through a small column (1.0 ml) containing mixed-anion(Bio-Rad AG-1 x 8, acetate form, 100/200 mesh; Bio-RadLaboratories, Richmond, Calif.) and cation (Dowex-1 x 8,H+ form, 100/200 mesh) resins and then through a C18Sep-Pak (Waters Associates, Inc., Milford, Mass.). Thecolumns were eluted with distilled water, and the eluatecontaining the sugars was concentrated by freeze-drying.The dried residue was used for derivatization. Studies withspiked wine samples indicated that the pretreatment andderivatization procedures gave greater than 85% recovery ofeach sugar. The identification of wine carbohydrates wasconfirmed by combined GLC-mass spectrometry on aFinnigan 4021 instrument at the Australian Wine ResearchInstitute.GLC performed by the method described above did not

satisfactorily separate either glucose from mannitol or man-nose from 2-deoxyglucose. Consequently, the concentra-tions of glucose and mannose, as well as those of fructoseand glycerol, were determined enzymatically by using kitsfrom Boehringer GmbH, Mannheim.

Organic acids were measured by high-pressure liquidchromatography (HPLC) with a chromatograph (WatersAssociates) equipped with an ion exclusion, cation exchangecolumn (Bio-Rad HPX-87H) and a detector operating at 210nm. The column was eluted with 0.1% phosphoric acid at55°C at a flow rate of 0.5 ml/min. Before HPLC, winesamples were filtered through a membrane (pore size, 0.45,um) and passed through a C18 Sep-Pak. This procedureyielded >95% recovery of the organic acids found in wine.

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FIG. 1. Effect of growth of lactic acid bacteria on pH andconcentration of organic acids and carbohydrates during and afterthe malolactic fermentation of Shiraz wine (pH 3.2) from winery A.(a) L. oenos (0). (b) Malic acid (0), lactic acid (0), citric acid (E),acetic acid (U), fumaric acid (A), and pH (A). (c) Glucose (0),fructose (0), Ul (LI), myo-inositol (A), and glycerol (0). (d)Rhamnose (0), ribose (OI), arabinose (-), xylose (A), and mannose(A). The concentration of galactose (80 mg/liter) and that of glucitol(80 mg/liter) remained constant throughout storage and are notshown.

The concentration of malic acid was also determined by anenzymatic method with a kit from Boehringer.The free amino acids were separated by HPLC and

estimated by post-column derivatization with ortho-phthalaldialdehyde (16). The technology and equipment usedfor these analyses were from Waters Associates. The chro-matograph was equipped with an amino acid analysis column(Waters Associates) that was gradient eluted with sodiumeluent A and sodium eluent B buffers (Waters Associates).Acetaldehyde was measured enzymatically by using a kit

from Boehringer.

RESULTSShiraz wine from winery A. The alcohol and total sulfur

dioxide (SO2) concentrations of the Shiraz wine from wineryA were 14.2% and 11.2 mg/liter, respectively. Samples wereadjusted to pH 3.0, 3.2, 3.7, or 4.0, and one sample remainedunadjusted at pH 3.5. The growth of bacteria in the wines atpH 3.2 and 3.7 is shown in Fig. 1 and 2 along with somechanges in wine composition.

L. oenos was present at initial levels of 10 to 100 cells perml, and after a lag period of 1 to 2 days, grew to a maximumpopulation of 107 to 108 cells per ml in wines at pH 3.2, 3.5,

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FIG. 2. Effect of growth of lactic acid bacteria on pH andconcentration of organic acids and carbohydrates during and afterthe malolactic fermentation of Shiraz wine (pH 3.7) from winery A.(a) L. oenos (0), P. parvulus (A), and Lactobacillus cellobiosus (O).Symbols for panels b, c, and d are the same as for Fig. 1.

3.7, and 4.0. The growth rate increased as the wine pH wasincreased. The maximum population was achieved at 44days after crushing in wine at pH 3.2 and at 17 days aftercrushing in wine at pH 4.0. No growth occurred in wine atpH 3.0. The malolactic fermentation occurred in conjunctionwith the growth of L. oenos and was indicated both by thecomplete degradation of malic acid and by a parallel increasein concentration of lactic acid to about 1.8 g/liter. The pH ofthe wines increased by approximately 0.3 U during thisperiod. No species other than L. oenos was detected in thewines during the malolactic fermentation.

Other chemical changes that occurred during malolacticfermentation in wines at pH 3.2 and 3.7 are shown in Fig. 1and 2, respectively. The trends were the same for the winesat pH 3.5 and 4.0, although the time scales were different.Citric and fumaric acids were completely degraded, and theconcentration of acetic acid increased by about 0.1 g/liter.Glucose concentration doubled from 90 to approximately160 mg/liter by the end of the fermentation, and fructoseconcentration also increased, although to a lesser extent.Both arabinose and mannose concentrations decreased dur-ing malolactic fermentation. However, the concentration ofgalactose, ribose, xylose, rhamnose, glycerol, myo-inositol,glucitol (data not shown), and an unidentified monosac-charide (Ul) remained unchanged. Mass spectral data indi-cated that Ul is probably a hexose glycoside, but further

identification studies are required. Increases in the concen-trations of glucose and fructose and decreases in the con-centrations of mannose and arabinose were also observed inthe wine at pH 3.0, in which there was no bacterial growthand no malolactic fermentation.The microbiological and chemical changes that occurred

in the wines after malolactic fermentation depended uponpH. Data for the wines at pH 3.2 and 3.7 are presented inFig. 1 and 2, respectively. L. oenos survived at a level ofapproximately 106 cells per ml in the wine at pH 3.2 and wasthe only species isolated from this wine. The concentrationsof glucose and fructose continued to increase, and those ofmannose and arabinose continued to decrease. The concen-trations of ribose, xylose, and, to a lesser extent, rhamnoseincreased slightly.

L. oenos remained at a level of 106 cells per ml in the wineat pH 3.5 during the postmalolactic period. However, anunidentified species of Lactobacillus was also isolated fromthis wine at 60 days after crushing, and it subsequently grewto a population of 105 cells per ml to coexist with L. oenos.The growth of this species coincided with small but signifi-cant decreases in the concentrations of glucose, fructose,myo-inositol, and ribose, indicating that they were beingutilized as growth substrates. The concentrations ofarabinose and mannose continued to decrease in the samefashion as did the wine at pH 3.2 and did not specificallycorrelate with the growth of the Lactobacillus sp.Both P. parvulus and Lactobacillus cellobiosus were

isolated from the wine at pH 3.7 during the postmalolacticfermentation period. These species grew to populations of106 to 107 cells per ml, and when this occurred, L. oenos wasno longer isolated from the wine (Fig. 2). The growth of P.parvulus and Lactobacillus cellobiosus was accompanied bysubstantial changes in the chemical composition of the wine.Lactic and acetic acid concentrations increased from 1.8 and0.4 g/liter, respectively, at the end of malolactic fermentationto 3 and 1.65 g/liter, respectively, and wine pH decreasedfrom 3.95 to 3.72. The concentrations of glucose, fructose,myo-inositol, glycerol, ribose, xylose, and Ul decreasedmarkedly (Fig. 2). The concentration of arabinose alsodecreased, although not in coincidence with bacterialgrowth. Mannose, after virtually complete utilization, laterincreased to a level between 25 and 30 mg/liter.

P. parvulus and an unidentified species of Lactobacillusgrew in the wine at pH 4.0 during the period of postmalo-lactic fermentation and achieved populations of 107 cells perml by 30 and 45 days, respectively, after grape crushing.However, both L. oenos and the Lactobacillus sp. disap-peared by 71 days, leaving P. parvulus as the survivingorganism. The chemical changes that occurred in this winewere similar to those of the wine at pH 3.7 except thatgalactose was also utilized.

Acetaldehyde, present at 15 mg/liter at the end of alcoholicfermentation, was partially degraded in all wines duringmalolactic fermentation. The proportion degraded increasedfrom 13.6 to 29% as the wine pH increased from 3.2 to 4.0.

Table 1 shows the concentration of some amino acids inthe wine at pH 3.7 just before malolactic fermentation, at theend of this reaction (62 days) and at a later stage (100 days)of storage. With the exception of histidine and arginine,which were completely utilized during malolactic fermenta-tion, the concentrations of all individual amino acids in-creased slightly during this fermentation and storage. Similardata were found for the wine at pH 3.2, except that theincreases in concentrations were slightly smaller.

Shiraz wine from winery B. Shiraz wine from winery B

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contained 11.9% alcohol and 72 mg of total SO2 per liter.Samples were adjusted to pH 3.0, 3.2, 3.7, and 4.0, and onesample remained unadjusted at pH 3.5. L. oenos, P.parvulus, and Lactobacillus buchneri were isolated from thewine at a level of 10 to 100 cells per ml at the end of alcoholicfermentation. Thereafter, no lactic acid bacteria were de-tected in the wines at pH 3.0, 3.2, 3.5, or 3.7 at any stageduring storage for 150 days. P. parvulus commenced growthin the wine at pH 4.0 after a lag phase of approximately 40days (Fig. 3). It achieved a maximum population of almost107 cells per ml and survived in the wine until the end ofsampling. Malolactic fermentation occurred concomitantlywith the growth of P. parvulus. Citric and fumaric acids werenot degraded during this period, and acetic acid was notproduced; the concentrations of acetic and lactic acids,however, increased substantially during storage aftermalolactic fermentation (Fig. 3). The concentrations of glu-cose and fructose in the wine increased before malolacticfermentation, but then decreased along with those of galac-tose and Ul during the fermentation. The concentrations ofarabinose, xylose, and glucitol remained constant during andafter malolactic fermentation, but those of ribose and myo-inositol decreased slightly at the time that acetic acid wasproduced. Mannose, which was not detected in the winebefore malolactic fermentation, subsequently increased toabout 40 mg/liter.Other wines. L. oenos was the only species isolated from

Shiraz and Cabernet Sauvignon wines obtained from sevenother wineries throughout Australia. This species conductedthe malolactic fermentation during laboratory storage ofthese wines at their unadjusted pH (3.5 to 3.6). In all wines,the malolactic fermentation was accompanied by the degra-dation of citric and fumaric acids and production of aceticacid (Fig. 1). Changes in the concentration of sugars were

TABLE 1. Effect of malolactic fermentation and subsequentconservation on the concentration of some amino acids of a Shiraz

wine at pH 3.7Conc (mmol/liter)

Amino acid Before(10 (62After AfterAminoacid BeMFore1 completion storagedayS

(1 of MLF10dy)das" (62 dayS)b 10dy)

Aspartic acid 0.015 0.055 0.045Threonine + asparaginec 0.085 0.125 0.17Serine + glutamined 0.085 0.175 0.09Glutamic acid 0.08 0.16 0.18Glycine 0.085 0.16 0.205Alanine 0.15 0.235 0.21Valine 0.005 0.06 0.105Methionine NDe 0.01 0.01Isoleucine 0.005 0.035 0.055Leucine 0.015 0.115 0.135Tyrosine 0.03 0.056 0.06Phenylalanine ND 0.045 0.055a-Aminobutyric acid 0.085 0.145 0.195Lysine 0.01 0.095 0.075Histidine 0.035 ND NDArginine 0.02 ND ND

a MLF, Malolactic fermentation.b Days after crushing (Fig. 2).' Acids not resolved on chromatogram; concentration expressed as milli-

moles of threonine per liter.d Acids not resolved on chromatogram; concentration expressed as milli-

moles of serine per liter.e ND, Not detected.

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concentration of organic acids and carbohydrates during and afterthe malolactic fermentation of Shiraz wine (pH 4.0) from winery B.(a) L. oenos (0), P. parvulus (Li), and Lactobacillus buchneri (A).(b) Malic acid (0), citric acid (El), lactic acid (0), acetic acid (U),fumaric acid (A), and pH (A). (c) Glucose (0), fructose (0),galactose (U), Ul (El), and myo-inositol (A). (d) Xylose (0), ribose(o),arabinose (p),and mannose (A).

measured for a Shiraz wine from the McLaren Vale district,South Australia. The concentration of ribose, arabinose,myo-inositol, and particularly of Ul decreased duringmalolactic fermentation; the concentration of the other sug-ars, including glucose and fructose, remained constant. L.oenos (106 cells per ml), P. parvulus (105 cells per ml), andLactobacillus brevis (103 cells per ml) were isolated from oneShiraz wine at pH 3.85 on completion of the malolacticfermentation.

DISCUSSIONThe growth cycle of lactic acid bacteria in wines is

complex and consists of distinct phases that occur duringalcoholic fermentation, malolactic fermentation, and conser-vation after malolactic fermentation (11, 19, 28). In thisstudy we give a detailed, quantitative description of theecological and chemical behavior of lactic acid bacteriaduring and after malolactic fermentation. Overall, our datastrengthen and extend the conclusions of previous studies (3,17, 18, 20).The effect of pH on the growth rate of lactic acid bacteria

in wines is well demonstrated in the literature (4, 5, 22) andby the data in Fig. 1 and 2. Generally, the rate of bacterialgrowth and malolactic fermentation increased as wine pHwas increased from 3.0 to 4.0. In addition, pH had a

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profound, selective effect upon the species that grow inwines. Usually, L. oenos was the only species isolated fromwines with a pH below 3.5. In such wines, L. oenos grewexponentially to conduct the malolactic fermentation, andthereafter remained in a viable but nonproliferating state forlong periods (Fig. 1). The long-term survival of L. oenosunder practical winery conditions, however, is determinedby the addition of SO2 (19). As noted by ourselves and others(28), Pediococcus and Lactobacillus spp. rarely grow inwines with a pH below 3.5, but do occur in wines of pH 3.5and above, particularly after the malolactic fermentation hasbeen completed by L. oenos (Fig. 2). The growth of thesebacteria at this stage seems antagonistic to the survival of L.oenos (Fig. 2); we have noted this behavior previously (9).The molecular basis of this antagonistic interaction and itsenological significance warrant further study.The concentration of SO2 may also selectively influence

the species of lactic acid bacteria that grow in wines. This iswell illustrated by the Shiraz wine from winery B, which hada total SO2 concentration of 72 mg/liter and did not supportthe growth of any lactic acid bacteria until the pH wasadjusted to 4.0. Despite the initial presence of L. oenos,Lactobacillus buchneri, and P. parvulus in this wine, P.parvulus was the only species able to grow, and it conductedthe malolactic fermentation (Fig. 3). According to otherstudies (18, 33), a total SO2 concentration of >50 mg/litergenerally restricts the growth of lactic acid bacteria in wines,especially at the lower pH values, when a greater proportionof the SO2 is in the undissociated, antimicrobial form. Thefrequent occurrence of Pediococcus spp. in Australian wines(9, 25) may be indicative of wines with a high concentrationof SO2 and a high pH (e.g., 3.5 to 4.0).The populations of 106 to 107 cells per ml formed by lactic

acid bacteria either during or after malolactic fermentationwere quantitatively significant and produced measurablechanges in the concentrations of some wine components.Although published evidence is not strong, it is generally

considered that wine sugars are utilized as carbon substratesfor the growth of lactic acid bacteria (18, 24, 28). Our dataprovide experimental evidence to support this conclusion,but also show that the behavior of sugars in some wines isquite complex.The concentrations of glucose, fructose, and the uniden-

tified monosaccharide Ul decreased in direct proportion tothe growth of P. parvulus during the malolactic fermentationof the Shiraz wine at pH 4 from winery B (Fig. 3), and it maybe concluded that these carbohydrates were metabolized bythis species. Similar conclusions can be made for the utili-zation of glucose, fructose, Ul, myo-inositol, glycerol,ribose, and xylose during the growth of pediococci andlactobacilli after malolactic fermentation of the Shiraz wineat pH 3.7 from winery A (Fig. 2). Metabolism of the hexoseand pentose sugars (18, 28) probably accounted for the largeincreases in the concentration of acetic and lactic acids alsoobserved at this stage. The metabolism of glycerol by lacticacid bacteria was related to production of the bitter sub-stance acrolein (10, 18). The utilization of myo-inositol bywine lactic acid bacteria is not well documented in theliterature, but was reported earlier by Peynaud and Domercq(26). Further studies are required to understand the metab-olism of this substance as well as that of Ul. Myo-inositol,Ul, ribose, and arabinose, but not glucose or fructose, werealso metabolized by L. oenos during malolactic fermentationof the Shiraz wine from the McLaren Vale district.Data for the Shiraz wines (winery A, pH 3.2 or 3.7) (Fig.

1 and 2) suggest that the growth of L. oenos during

malolactic fermentation was not accompanied by the specificutilization of any hexose or pentose sugars. Strains of L.oenos isolated from these wines fermented glucose andfructose as determined by the API tests, but, contrary toexpectation, the concentrations of these two sugars in-creased significantly during malolactic fermentation. Theseincreases were not directly related to growth, as mightappear from Fig. 1 and 2, because they occurred in otherwines (e.g., the same Shiraz wine at pH 3.0 and the Shirazwine described in Fig. 3), in which there was no growth of L.oenos. Other researchers have also noted increases in theconcentrations of some wine sugars during malolactic fer-mentation (8, 10), but the mechanism of this behavior has notyet been satisfactorily explained. Residual enzymatic activ-ities from grapes and yeasts could be involved. The hydro-lysis of sucrose would produce both glucose and fructose,but sucrose was not found in our wine samples even beforemalolactic fermentation (data not shown). Trehalose is an-other important wine disaccharide (32) whose hydrolysiswould yield glucose. We did not specifically analyze thissugar, but some preliminary measurements indicated that itsconcentration decreased during malolactic fermentation.The hydrolysis of phenolic glycosides of wine (18) could alsolead to an increase in the concentration of monosaccharidesugars. However, the utilization of glucose and fructose byL. oenos still remains a possibility, since they may begenerated at a rate faster than they are utilized.

Decreases in the concentration of mannose and arabinoseduring malolactic fermentation (Fig. 1 and 2) were notspecifically related to the growth of L. oenos, as they alsooccurred under conditions at which no bacterial growth wasevident (e.g., in the same wine at pH 3.0 and in the Shirazwine described in Fig. 3). Moreover, the strains of L. oenosisolated from the wines at this stage were not able to fermentarabinose, as measured by the API tests. We are not able toprovide a satisfactory explanation for these decreases; fur-ther studies are needed. Increases in the concentration ofmannose at later stages of vinification (Fig. 2 and 3) are alsodifficult to interpret, but could possibly arise from thehydrolysis of mannan polysaccharide of the yeast cell wallduring autolysis (1).As expected from what is now a well-established fact (3,

17), malic acid was degraded by L. oenos and P. parvulusduring malolactic fermentation, with the concomitant pro-duction of lactic acid. The degradation of citric and fumaricacids and production of acetic acid were important second-ary reactions during the malolactic fermentation by L. oenosbut not during the malolactic fermentation by P. parvulus.Other researchers (6, 7, 10, 28, 31, 36) have also observedthese secondary reactions; there is probably direct metabo-lism of citric acid to acetic acid. The metabolic pathway ofthis reaction has been studied for dairy lactic acid bacteria(15). The metabolism of citric acid in wine is also related toan increase in the concentration of diacetyl and acetoin (12,28, 37).

Significant changes in the concentration of wine aminoacids have been reported to occur during malolactic fermen-tation. Some amino acids decrease in concentration,whereas others increase, but no consistent trends haveemerged for any individual amino acid, except for a decreasein arginine, which is probably converted to ornithine (3, 23,28, 34). We were able to confirm the utilization of arginine byL. oenos during malolactic fermentation, and in addition, wenoted the utilization of histidine. It is possible that histidinewas decarboxylated to histamine, which is also known tooccur in wines (18). An increase in the concentration of all

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other amino acids could be explained by the degradation ofwine proteins by proteases produced by lactic acid bacteria,but the production of such enzymes by these bacteria has notyet been studied. Dairy lactic acid bacteria are well knownfor their protease production (21). Increases in the concen-tration of amino acids might also occur through yeastautolysis (1). Amino acid metabolism by wine lactic acidbacteria has not been studied. By analogy to dairy fermen-tation (21), such metabolism is likely to affect wine quality.The HPLC technology that now permits rapid measurementof free amino acids will facilitate further study of thebehavior of amino acids during vinification and shouldenable a better understanding of their contribution to winequality.The degradation of acetaldehyde by L. oenos is signifi-

cant, since SO2, which is strongly bound to this component,is freed to become active against further bacterial growth(33).The data described in this paper, together with those

reported by others (18, 20, 33), have provided furtherevidence of the important influence of pH and S02 concen-tration upon the conduct of the malolactic fermentation and,consequently, upon the quality of red wines. They alsoindicate the potential of bacteria to grow in wines afterconclusion of the malolactic fermentation and the need forsound winemaking practices to prevent spoilage at thisstage. Furthermore, our findings suggest that the metabolismof wine components, especially the carbohydrates, duringand after malolactic fermentation is more complex thanpreviously thought and requires more detailed investigation.

ACKNOWLEDGMENTSWe thank the Australian Wine Research Institute for financial

support and the Australian Government for CommonwealthPostgraduate Awards to C.R.D. and D.W.

LITERATURE CITED1. Arnold, W. N. 1981. Autolysis, p. 135-137. In W. N. Arnold

(ed.), Yeast cell envelopes. Biochemistry, biophysics, and ultra-structure. CRC Press, Inc., Boca Raton, Fla.

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