Journal of Applied Bacteriology 1988,64, 505-513 2713/01/88

Identification and physiological characteristics of heterofermentative strains of Lactobacillus from South African red wines

L.M.T. DICKS & H.J.J. V A N VUUREN* Department of Microbiology and Institute for Biotechnology, University of Stellenbosch, Stellenbosch 7600, South Africa

Received 4 January 1988 and accepted 4 March 1988

DICKS, L . M . T . & V A N VUUREN, H.J.J. 1988. Identification and physiological characteristics of heterofermentative strains of Lactobacillus from South African red wines. Journal of Applied Bacteriology 64, 505-513.

Nineteen atypical heterofermentative strains of Lactobacillus spp. isolated from South African red wines were identified with the API 50 CHL system and by com- puterized comparison of their total soluble cell protein patterns. Eleven strains were identified as Lactobacillus hilgardii and eight strains as L. breuis. Three strains of L. hilgardii and three strains of L. breuis produced small amounts of H,S in peptone water supplemented with 0.01% L-cysteine. All strains produced mannitol from fructose. Proteolytic activity was detected in all except two strains of L. hilgardii. Strains capable of digesting gelatine, hydrolized casein, but not uice versa. The excessive production of sulphur-containing compounds, mannitol and acetaldehyde by lactic acid bacteria could have serious quality implications for the wine industry. Three strains of L. hilgardii and all strains of L. breuis decarboxylated L-malic acid to L-lactic acid.

During the primary fermentation of wine, grape must is fermented to ethanol, CO, and flavour compounds by Saccharomyces cerevisiae (Kunkee & Amerine 1970; Kunkee & Goswell 1977). In a secondary fermentation, malic acid is decarboxylated to lactic acid by certain Lacto- bacillus, Leuconostoc and Pediococcus spp. (Kunkee 1967; Davis et al. 1985; Wibowo et al. 1985). The conversion of malate to lactate ensures microbiological stability, deacidification and affects the organoleptic properties of wine (Kunkee 1967; Davis et al. 1985). Malolactic fer- mentations occur naturally or can be induced by introduction of starter cultures. Strains of Leuconostoc oenos are usually employed for inoculation (Davis et al. 1985; Wibowo et al. 1985).

Corresponding author: Professor H.J.J. van Vuuren, Department of Microbiology and Immu- nology, College of Medicine, 858 Madison Avenue, Memphis, Tennessee 38163, USA.

Heterofermentative Lactobacillus spp. thus far isolated from wines, include L. trichodes and L. jkctivorans (Amerine & Kunkee 1968; Peynaud & Domercq 1970), now considered to belong to the species L. fructivorans (Kandler & Weiss 1986); L. desidiosus (Vaughn et al. 1949; Peynaud & Domercq 1970), reclassified as L. hilgardii (Vescovo et al. 1979; Kandler & Weiss 1986); L. fermentum (Vaughn 1955); L. brevis (Vaughn 1955; du Plessis & van Zyl 1963; Pilone et al. 1966; Chalfan et al. 1977; Sharpe 1981); L. buchneri (Vaughn 1955; du Plessis & van Zy1 1963; Pilone et al. 1966; Sharpe 1981); and L. hilgardii (Vaughn 1955; du Plessis & van Zyl 1963; Sharpe 1981). Lactobacillus brevis, L. buchneri and L. hilgardii are the only hetero- fermentative malolactic Lactobacillus spp. thus far reported in South African wines (du Plessis & van Zyl 1963; du Plessis 1964). However, various unidentified Lactobacillus spp. and atypical strains of L. brevis and L. buchneri have been isolated from South African red wines (Van der Westhuizen 1980).

506 L. M . T. Dicks & H . J . J . van Vuuren

Tnble 1. Fermentation of carbohydrates by heterofermentative lactobacilli. Results were recorded with the API 50 CHL system

Reaction in tests

- -~ ~

+ - (+) - - - - - - - - - - fermenturn ATCC 23272 - - - + + - - - - + + - - - - - - - - - - -

- - - + + - _ - - + + - - - - - - - - - - - reureri DSM 20016 + + + (+) - - - - - - - - - fermenrurn ATCC 11976 - - - + + + - - -

ferntenrum ATCC 14932 - - - ( + ) + + - - - + + ( + ) ( + ) - - - - - N T - - - - (+) collrnordes ATCC 27612 - - - + + + - - - + + + - - - - - - - -

burhneri ATCC 12935 - - - + + - N T - - ( + ) + ( + ) - - - - - - - - (+) - + + (+ ) - - - - - - - - - - hilqordii ATCC 82W - - - -

+ (+) - - - - - - - - - - b r r m ATCC I1577 - - - + + + - - - - - - - - + + - - - (+) + (+ ) - - - - - - - - - - Ba I

+ (+ ) - - - - - - - - - - + + - - - (+) Ba3 - - _ - + + - - - (+) (+ ) (+) - - - - - - - - - - Bb3

Bc2 - - - + + + - - - ( + ) + ( + ) - - - - N T - - N T - - + (+) - - - - - - - - - - Gal + (+) - - - - - - - - - - Fa5

- - - - + + - - - ( + ) + ( + ) - - - - - - - - - - Fbl - - - - + + - - - ( + ) ( + ) (+ ) - - - - - - - - - - Ba4

Lacr fermenturn ATCC 23271 - - - - + + - - - ( + I + (+) - - - - - - - - - - buchnerr ATCC 11579 - - - + + + - - - (+)

huchnrri ATCC 12936 - - - + ( + ) + - - - ( + ) + ( + ) - - - - - - - - - - confusus ATCC 27646 - - - + + + - - + (+ ) + ( + ) - - - - NT (+) - - (+) (+)

+ + + - - + ( + ) ( + ) I + ) - - - - - - - - (+) (+) breiis ATCC 8287 _ - - + + ( + ) - - - ( + ) + + - - - - - (+) - - (+) (+) breiu ATCC 367 + + + - - + (+) ( + ) (+ ) - - - - - (+) - - (+) (+) IbS

- (+) + + - - - + ( + ) ( + ) - - - - - - - - (+) (+) Ib3 + + + - - - + + + - - - - - (+) - - (+) (+) Gel

Eb2 - - - ( + ) + + - - - ( + ) (+) (+ ) - - - - - - - - (+) (+) + + + - - - ( + ) (+ ) (+ ) - - - - - (+) - - (+) (+) Fc5

Fc2 - - - ( + ) + + - - - (+) (+) ( + ) - - - - - - - - (+) (+) t + + - - + ( + I + ( + ) - - - - - - - - I+) (+) Ec3

Lacr breiisATCC 13648 - - - ( + ) + + - - - ( + ) + (+) - - - - - (+) - - (+) (+) + + + - - - (+) (+) (+ ) - - - - - (+) - - (+) (+) + + + - - - ( + ) + ( + ) - - - - - - - - - (+)

- (+) uzrldescens ATCC 12706 - - - - - - - - - - + + (+) - - - - - - - ~ N C I Z W T ( I ~ S ATCC 8288 - - - - - - - - - - + + - - - - - - - - - -

+ + - - - - ( + ) (+ ) + (+) - - - - - NT - - (+) kandleri DSM 20593 - - - buchnerr ATCC 11307 - - - + + - - - - ( + I + ( + ) - - - - - - + - - - cellobiosus ATCC 11739 NT - - + + - - - - + + + + - - - - - - - - -

- (+) fermenturn ATCC 8289 - - - + + + - - - + + + + - - - - - - - cellobiusus ATCC 1 1 7 4 0 - - - + + + - - - + + + + + + - ( + I + + - + +

+ + + - - - + + + + - (+) - - + + (+) (+) + Gc4 Bbl ( + ) - - + + + - - - + + + + - (+) (+) - + + - + (+) Id3 - - - ( + I + + - - - + + + + - - - - + + - + (+) 1c I ( + I - - + + + - - - + + + + - + + - + + - + + Locr huchneri ATCC 9460 - - - + + + - - - + + + + - ( + ) - - + + - + +

+ ( + ) - - - - Lncr breiis ATCC 8037 - - -

+ + - - -

- - - -

+ + - - - (+) + + - - - ( + )

- - - - - - - _

+ + + - - - - - - - - -

- - - - - - - - - - -

- - -

- - -

brews ATCC 4006 - - - brew ATCC 14869 - - -

- - -

+ + + - - - + + + + - - - - (+) - - - (+) brecis ATCC 14434 - - -

+ + + - - - ( + ) + ( + ) - - - - - (+) - - ( + I (+) buchneri ATCC 4005 - - -

fennenrurn ATCC 9338 - - - + + + - - - + + + + - ( + ) - - + - - ( + ) + collinoldec ATCC 27610 - - - + + + - - - + + + - ( + ) ( + I - - + - - - ( + I

+, positive reaction: a wlour change of the pH indicator lrom purple to yellow. The hydrolysis ofaesculin produas a precipitation of ferrous sulphide which i s black; (+), Weak positive reaction; - . negative reaction; NT. not tested

Lactobacilli in wine 507

Table 1. (continued)

- ( + I - - ( + I - - ( + I - - ( + I - - ( + I -

- ( + I - - (+) -

- + -

- + - - + - - + - - + - - + - - + - - + - - ( + I -

- ( + I - - (+) -

- + -

- + - - + - - + - - ( + I - - + - - + - - ( + I - - + - - + - - + - - ( + I - - + - - + - - + - - ( + I - - ( + I - - ( + I -

- + ( + I - + ( + I

- t -

+ + + + + + + + ( + I - + t + + + + + +

( + I + (+) - + ( + I

( + I + ( + I + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

(+) (+) -

+ + + + + + + + + + + + +

+ (+) + + + + + + + + + + + + + + ( + I + + + +

L. M . T . Dicks & H . J . J . van Vuuren In the present study, the unidentified and

atypical heterofermentative strains of Lacto- bacillus isolated by Van der Westhuizen (1 980), were characterized by numerical analysis of API 50 CHL results and by comparison of their respective electrophoretic total soluble cell protein patterns. The latter technique enables grouping of strains with high genetic similarity (Kersters & de Ley 1980). Furthermore, pro- duction of H,S, mannitol, proteolytic activity, and the ability to convert malic acid to lactic acid by the wine lactobacilli were investigated.

Use of the API 50 CHL biochemical kits for rapid identification of heterofermentative wine lactobacilli was evaluated.

Materials and Methods

S T R A I N S

Nineteen heterofermentative Lactobacillus iso- lates from wine and 30 Lactobacillus reference strains were included (Table 1).

P H E N O T Y P I C PROPERTIES O F ISOLATES D E T E R M I N E D BY A P I 50 C H L KITS

The bacteria were grown in MRS broth (E. Merck, Darmstadt, Federal Republic of Germany) at 30°C until active growth was observed. Cultures were centrifuged at 3000 g for 10 min and washed once with sterile saline. The cell density was adjusted to two on the McFarland scale (ca 6 x lo8 cells/ml) with sterile saline. Each compartment of the API 50 CHL kit (API system, S.A., Montalieu Vercieu, France) was inoculated with the bacterial sus- pension with a sterile Pasteur pipette according to the manufacturer’s instructions. Com- partments were filled with sterile liquid parafin after inoculation. Strips were incubated at 30°C and results recorded at 48 h. The phenotypic tests are listed in Table 1 .

C O M P U T E R ASSISTED N U M E R I C A L A N A L Y S I S OF R E S U L T S R E C O R D E D W I T H API 50 C H L BIOCHEMICAL KITS

Results were recorded as positive or negative. The data were analysed by computer and the resemblance of each strain with every other strain was calculated using the simple matching coeficient (La) of Sokal & Michener (1958).

Strains were clustered by the unweighted average pair-group method. Redundant charac- ters were not used for calculating coefficients of association.

P R O D U C T I O N OF H2S

Production of H,S was tested in MRS broth (E. Merck, Darmstadt, Federal Republic of Germany) and peptone water supplemented with 0.01Y0 (w/v) L-cysteine. The lead citrate paper strip method (Harrigan & McCance 1976) was used. Results were recorded after 48 h.

P R O D U C T I O N O F M A N N I T O L

Production of mannitol from fructose was tested by the methods described by Chalfan et al. (1975). Results were recorded after 88 and 1 1 1 h. Solutions (2% w/v) of mannitol (Griffin and George, Birmingham, England), dulcitol (Baltimore Biological Laboratory, Baltimore) and sorbitol (BDH) were used as standards. Uninoculated HC1-treated broth, uninoculated broth without HC1 and concentrated HCI were included as controls.

H Y D R O L Y S I S OF G E L A T I N E A N D C A S E I N

Hydrolysis of 0.4% and 0.6% gelatine and 0.4% and 0.6% casein, incorporated into MRS agar, (Merck) were recorded after 48 or 72 h. The methods of Clarke & Cowan (1952) were used.

C O N V E R S I O N OF L - M A L I C A C I D TO

L - L A C T I C A C I D

Malolactic fermentation was recorded in MRS broth (de Man et al. 1960) containing 0.5% (w/v) glucose and 0.4% (w/v) L-malic acid (Sigma). Results were recorded daily for 1 week and again after 2 weeks. The paper chromato- graphy method of Rankine (1969) was used.

G R O U P I N G A N D I D E N T I F I C A T I O N OF S T R A I N S BY C O M P A R I S O N OF THEIR T O T A L S O L U B L E P R O T E I N P A T T E R N S O B T A I N E D BY P O L Y A C R Y L A M I D E GEL ELECTROPHORESIS

Culturing of strains, preparation of cell-free extracts. Dolvacrvlamide gel electroDhoresis,

Lactobacilli in wine 509 densitometry, normalization of densitometric tracings, photography and normalization of photographs were carried out according to the methods described by Dicks & van Vuuren (1987). Each protein extract was investigated in at least three independent electrophoretic runs. The normalized densitometric tracings were converted as described by Kersters & de Ley (1975) into a sequency of 120 numbers, rep- resenting the optical densities (expressed in millimeters height) of each position on a scan. The Pearson product-moment correlation coef- ficient (I), between any pair of densitometric

Percentage similarity 60 70 80 90 100

i

ins! bnws 4TCC 8007

Iarm.ntum ATCC 23272

r#ut.ri DSM 20016 f.rm."lUm 4TCC , 1976

f.rmemlum 4TCC I4932 coi1rnord.r &TCC 27612

bUChnw, ATCC 12935 1 h,lgordir 4TCC 8290

brews 4TCC 11577

801

803 Bb3 852 GO 1

FnS

Fbl b 4 Loct farmmum 4TCC 23211

buchnwi ATCC I I579 buchwn 4TCC 12936

eonfvlvi ATCC 27646 brews 4TCC 8287 brows ATCC 367

Ib5

Ib3 GI I

Eb2

Fc5

Fc2

Et3 LOCI brwi t 4TCC 13648

bmw. 4TCC 4006

Fig. 1. Simplified dendrogram of S,, similarity coefi- cient ( x lOO), grouped by the unweighted average pair-group method, showing phenotypic similarities among 19 heterofermentative Lactobacillus wine strains and L. brevis, L. fermenturn, L. reuteri, L. collin- oides, L. buchneri, L. hilgardii, L. confusus, L. virides- cens, L. fructitrorans, L. kandleri and L. cellobiosus. Results were obtained with the API 50 CHL system.

, 0 50 060 0 70 0 8 0 0 9 0 I 0 0 y n g

Lobbr,",r 4TCC 8007 9 u s ; 0,

3 2 2 s ~ ~ , ~ ~ ~ c ~ 7 0 6 brmmtum ATCC 11976 ]. ~

c e l l o b ~ ~ ~ ~ ATCC 11739 fwmnrum ATCC 23271 fwmnwm ATCC 14932

fwmntum 4TCC 8289 f,,mn,"m 4TCC 9338

koondlw, OSH 205'13

1- odlmmbk ATCC 27610 wlnoid. l ATCC 27612 rrurrmnr m c 8288

Fig. 2. Simplified dendrogram showing genomic rela- tedness among 19 heterofermentative Lactobacillus wine strains, and reference strains of L. brevis, L. fer- menturn, L. reuteri, L buchneri, L. celtobiosus, L. hil- gardii, L. confusus, L. viridescens, L. kandleri, L. collinoides and L. fructivorans based on computerized numerical analysis of total soluble cell protein pat- terns. Grouping was by the unweighted average pair- group method.

tracings of protein patterns was calculated for 147 gels (three gels/strain).

Results and Discussion

Results recorded with API 50 CHL biochemical kits are listed in Table 1. Nineteen strains from wine were grouped into three phenons at > 88% S by computerized numerical analysis of API 50 CHL results (Fig. 1). Computerized numerical analysis of protein electropherograms grouped the same wine strains into three clus- ters at r = 0.79 (Fig. 2). Protein profiles of respresentative strains from each cluster and 12

510 L. M . T . Dicks & H . J . J . van Vuuren strains which did not group into any cluster are shown in Fig. 3. A summary of results obtained by API 50 CHL and polyacrylamide gel electro- phoresis and the final identity of the wine strains are presented in Table 2.

Wine strains Bal, Ba3, Bb3, Bc2, Gal, Fa5, Fbl and Ba4 were phenotypically related to the type strain of L. hilgardii (ATCC 8290), L. brecis ATCC 11 577, L. fermenturn ATCC 23271 and L. buchneri ATCC 11579 and ATCC 12936 (phenon 11, Fig. I ) . Wine strains Gc4 and Bbl grouped with L. cellobiosus ATCC 11740 and L. buchneri ATCC 9460 (phenon VI, Fig. 1).

However, protein patterns of wine strains Bal, Ba3, Bb3, Bc2, Gal , Fa5, Fbl, Ba4, Gc4 and Bbl compared well with each other and with that of the type strain of L. hilgardii (ATCC 8290) and L. brevis ATCC 11577 (cluster 11, Fig. 2). Lactobacillus brevis ATCC 11577 has been reclassified as a strain of L. hilgardii (Dicks & van Vuuren 1987). Wine strains Bal, Ba3, Bb3, Bc2, Gal , Fa5, Fbl, Ba4, Gc4 and Bbl are therefore regarded as strains of L. hilgardii (Table 2).

Wine strains Ib5, Ib3, Gcl, Eb2, Fc5, Fc2 and Ec3 were phenotypically related to L. confusus

Fig. 3. Normalized protein patterns of 18 representative strains of Loctobacillus from clusters I-V and 12 strains which did not group into any cluster (Fig. 2).

Lactobacilli in wine 51 1 Table 2. Identification of strains isolated from wine according to API 50 CHL results and poly-

acrylamide gel electrophoresis

Grouping of strains isolated from wine according to: Final identity of strains isolated from

API 50 CHL tests PAGE* wine based on Isolate (see Fig. 1) (see Figs 2 and 3) comparison of API 50 number Phenon Cluster CHL and PAGE results

Fa5 Fbl Ba4

I1

Ib5

Eb2 Fc5

Ec3

Gc4 B b l i

111

VI

Ia3 VI

Icl VI

I1

111

IV

I1

t

I11

L. hilgardii

L. brevis

L. brevis

L. hilgardii

L. hilgardii (atypical)

L. brevis

* Polyacrylamide gel electrophoresis. t Not clustered by numerical analysis into a group or cluster

ATCC 27646 and L. brevis strains ATCC 8287, ATCC 367, ATCC 13648, ATCC 4006 and ATCC 14869 (type strain) (phenon 111, Fig. 1). However, L. confusus ATCC 27646 is geno- mically related to L. brevis (Dicks & van Vuuren 1987). Numerical analysis of polyacryla- mide gel electropherograms grouped wine strains Ib5, Ib3, Gcl, Eb2 and Fc5 with L. brevis ATCC 8287 and ATCC 367 at r = 0.82 in cluster I11 and wine strains Fc2 and Ec3 with L. brevis strains ATCC 13648, ATCC 4006 and the type strain (ATCC 14869) at r = 0.80 in cluster IV (Figs 2 and 3). Wine strains Ib5, Ib3, Gcl Eb2, Fc5, Fc2 and Ec3 belong to L. brevis (Table 2).

Wine strain Ia3 was phenotypically closely related to L . cellobiosus ATCC 11740 and L. buchneri ATCC 9460 (phenon VI, Fig. 1) However, protein patterns of wine strain Ia3 showed some degree of similarity to protein pat- terns of the type strain of L. hilgardii (ATCC 8290) and the L. hilgardii wine strains in cluster

I1 (Figs 2 and 3). Wine strain la3 is therefore considered to be an atypical strain of L. hilgard- ii (Table 2).

Wine strain Icl was phenotypically related to L. cellobiosus ATCC 11740 and L. buchneri ATCC 9460 (phenon VI, Fig. 1). Lactobacillus cellobiosus ATCC 11740 was originally mis- classified and its identify remains uncertain (Dicks & van Vuuren 1987). However, the overall protein pattern of strain Icl corresponded well with that of L. brevis ATCC 8287 and ATCC 367 (cluster 111, Fig. 2). Strain Icl is thus regarded as L. brevis.

A good overall agreement was found between results obtained by APT 50 CHL and polyacry- lamide gel electrophoretic methods. Strains placed in the same phenotypic group generally displayed similar, and in some cases, almost identical protein patterns. We found the API 50 CHL system a reliable and rapid method for the identification of Lactobaciilus syp. in wine.

The occurrence of excessive sulphur-

512 L. M . T . Dicks & H . J . J . van Vuuren containing compounds in wine is undesirable (Kunkee & Amerine 1970). Sulphur-containing compounds in alcoholic beverages can be pro- duced by yeasts (Beech & Davenport 1970; Kunkee & Amerine 1970; Rainbow 1970) and bacteria (Harrison & Graham 1970; van Vuuren et al. 1980). Lactobacillus hilgardii Ba3, Bb3 and Ba4 and L. brecis Ib3, Eb2 and Fc2 produced small amounts of sulphur-containing compounds in peptone water supplemented with 0.01% (w/v) L-cysteine. The production of volatile sulphur-containing compounds by Lac- tobacillus spp. in wine has not yet been reported and should therefore be investigated.

All strains of L. hilgardii and L. breuis isolated from wine, produced mannitol from fructose. This phenomenon is characteristic for hetero- fermentative lactic acid bacteria (Fornachon 1964; Kunkee 1967; Sharpe 1981). The pro- duction of ethanol and glycerol decreases as fructose is reduced to mannitol (Eltz & Vande- mark 1960). Furthermore, the excess formation of mannitol may result in mannite spoilage of wine (Amerine & Kunkee 1968). Mannite spoil- age is accompanied by formation of excess acetic acid (Dittrich 1977).

Lactic acid bacteria are weakly proteolytic compared with many other groups of bacteria (Sharpe 1962). Minute amounts of soluble nitro- gen from casein is produced by some strains of certain Lactobacillus spp. (Kandler & Weiss 1986). However, intracellular and extracellular proteinases and several peptidases have been found in lactobacilli (Law & Kolstad 1983). The presence of proteolytic activity in Group I (subgenus Thermobacterium) and Group 111 (subgenus Betabacterium) (Kandler & Weiss 1986) was reported by El Soda and co-workers (Law & Kolstad 1983). Dellaglio et al. (1974) reported high proteolytic activity for certain strains of L. helveticus. Proteolytic activity in strains of I.. hilgardii and L. breuis isolated from South African red wines and its effect on wine quality have not previously been reported. The production of acetaldehyde from threonine by lactobacilli couid well affect the quality of wine and should be investigated.

Malolactic fermentation assures a microbio- logically stable wine in bottle (Rankine 1977; Davis er al. 1985). Furthermore, it may contrib- ute favourably to the flavour and aroma of a wine by giving it more mature complexity (Ingraham & Cooke 1960; Rankine 1977), espe-

cially red wines (Lafon-Lafourcade et al. 1983). Malolactic fermentation may be detrimental to wines with high pH, such as those found in South Africa, California and Australia, resulting in a flat, insipid wine and growth of spoilage bacteria (Davis et al. 1985). The fermentation of certain carbohydrates by lactic acid bacteria may result in increased levels of volatile acids, diacetyl, or other flavour compounds (Kunkee et al. 1964). It is therefore important to identify lactic acid bacteria in wine and to study their metabolism. The effect of Lactobacillus spp. on the organoleptic quality of wine should be investigated.

We thank Stellenbosch Farmer’s Winery and the Distillers Corporation for financial support.

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