International Journal of Food Microbiology, 18 (1993) 37-42 37

© 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1605/93/$06.00

FOOD 00562

An improved medium for distinguishing between

homofermentative and heterofermentative lactic
acid bacteria
M. Zfifiiga, I. Pardo and S. Ferrer
Departament de Microbiologia, Facultat de CiOnciesBiolbgiques, Universitat de Valencia, Burjassot,
Valencia, Spain
(Received 30 April 1992; accepted 3 September 1992)

An improved solid medium for differentiating between homofermentative and heterofermentative lactic
acid bacteria is proposed. It was developed to support the growth of wine strains unable to grow in
other media. However, it can be employed as a general medium for the lactic acid bacteria that utilize
fructose.
Key words: Lactic acid bacteria, homofermentative, heterofermentative; Differential medium

Introduction
Lactic acid bacteria (LAB) are an extensive group of Gram-positive microorgan-
isms that include the genera Carnobacterium, Enterococcus, Lactobacillus, Lacto-
coccus, Leuconostoc, Pediococcus and Streptococcus. All are obligate fermentative
bacteria with complex nutritional requirements (Kandler, 1983; S n e a t h e t al.,
1986). They are found in a wide variety of fermented beverages and foods, the
intestinal tract of animals, plant materials, etc. (Sneath et al., 1986). Lactic acid
bacteria play an important role in winemaking: they transform L-malic acid
naturally present in wines into L( + )-lactic acid and CO2, or, in other words,
perform malolactic fermentation.
LAB can be divided into two physiological groups, depending on their hexose
fermentation pathway: homofermentative LAB and heterofermentative LAB. The
homofermentative LAB degrade hexoses via glycolysis, producing lactic acid as the
major end-product, whereas the heterofermentative LAB use the pentose-phos-
phate pathway and yield lactic acid, CO2, acetic acid a n d / o r ethanol. LAB can
also produce other end-products, depending on the species and growth conditions
(Condon, 1987; Kandler, 1983; Sneath et al., 1986).
The methods usually employed to distinguish between homofermentative and
heterofermentative LAB are based on the production of CO 2 by heterofermenta-

Correspondence address: Sergi Ferrer, Departament de Microbiologia,Universitat de Valencia, E-46100

Burjassot-Val6ncia, Spain. Tel. + 34 (6) 3864390; Fax + 34 (6) 3864372.
38

tive LAB (Gibson and Abd-E1-Malek, 1945). We have routinely used this system to
identify LAB strains isolated from Spanish musts and wines. However, non-repro-
ducible results were obtained with some of these strains (unpublished results).
McDonald et al. (1987) designed a differential medium named H H D that made
it possible to distinguish between homofermentative and heterofermentative LAB
both in solid or liquid media. This method is based on the greater acid production
from a fixed amount of fructose by homofermentative LAB. As the method
involves a pH indicator, differences in pH can be observed by means of detectable
color changes. Homofermentative LAB produce blue colonies, whereas heterofer-
mentative LAB remain white. McDonald et al. (1987) tested H H D medium with
several genera of LAB and obtained clear differentiation.
We evaluated H H D medium with LAB isolated from musts and wines and
found that several strains were unable to grow or did so very poorly. We therefore
evaluated an alternative medium (M5) able to support the growth of wine strains.
Further studies showed that M5 could be used as a general medium for distin-
guishing homofermentative from heterofermentative LAB that utilize fructose.

Materials and Methods

The components of M5 medium are given in Table I. It is based on the MLO

medium (Claus et al., 1983), with the following modifications: omission of glucose,

TABLE l
Composition of differential media M 5 , H H D and MLO for homo- and heterofermentative lactic acid
bacteria

Component ( g / l ) M5 HHD MLO

Tryptone 10 10 10
Yeast extract 5 1 5
Casaminoacids 3
Bactosoytone 1.5 -
Glucose 10
Fructose 2.5 2.5 5
Nit 2 PO4 2.5 2.5 -
Tween80 1 ml 1 g 1 ml
L-Cysteine H C 1 0.5 0.5
MgSO 4 •7H 20 0.2 - 0.2
MnSO 4-1H20 0.05 - 0.05
(NH4) 2 citrate - - 3.5
Calcium pantothenate 0.Ill
Tomato juice - - 100 ml
Bromocresol green " 20 ml 20 ml
Agar 20 20 2(/
pH 6.5 7.0 4.8

:~ Stock solution, 0.1 g of bromocresol green in 30 ml of 0.01 N N a O H .

 39

tomato juice and diammonium citrate, and the addition of potassium phosphate,
calcium pantothenate, and also of fructose as the sole carbon source, and
bromocresol green as a p H indicator. The p H was adjusted to 6.5. Strains used in
this study are listed in Table II, and were obtained from the Colecci6n Espafiola
de Cultivos Tipo (CECT) or directly isolated from wines. Streptococcus and
Enterococcus strains were routinely grown in Brain H e a r t Infusion (Oxoid) at 37°C,
Lactococcus strains in M17 broth (Terzaghi and Sandine, 1975), with glucose
added instead of lactose, at 32°C. Carnobacterium strains (synonymous Lactobacil-
lus) were grown in Y G P B (Garvie, 1978) at 28°C, and Lactobacillus, Pediococcus,
and Leuconostoc strains except Leuconostoc oenos in MRS broth (de Man et al.,
1960) at 28°C. L. oenos strains were grown in M L O medium (Claus et al., 1983) at
28°C. All the media were sterilized by autoclaving for 30 min at 115°C.
Inocula for the assays with H H D or M5 were prepared as follows: cells were
grown until the early stationary phase in the appropriate medium, harvested by
centrifugation, and washed twice with sterile distilled water. The pellet was
resuspended in distilled water and appropriate dilutions were spread onto plates of
H H D or M5 media. Unless otherwise indicated plates of H H D were incubated
under aerobic conditions (ambient atmosphere), as McDonald et al. (1987) de-
scribed, and plates of M5 in anaerobic jars (Oxoid) under an atmosphere of 100%
CO 2 at 25°C to improve the use of fructose as an electron acceptor.
The differentiation between homofermentative and heterofermentative LAB in
mixed populations was investigated, and variations in viable counts in M5 and
standard media for isolation of LAB (de Man et al., 1960) were also tested.
Lactobacillus plantarum C E C T 220 (homofermentative) and Lactobacillus cellobio-
sus C E C T 562 (heterofermentative) were used because they produce colonies with
different morphologies. Cell suspensions of both strains were spread on M5 plates
at ratios of 1:1, l : 2 a n d 1:10.

Results and Discussion

The results of the assays of the strains of LAB tested in H H D and M5 are
shown in Table II. As in H H D medium, homofermentative LAB colonies were
blue in M5, while heterofermentative colonies remained white. None of the L.
oenos strains nor Lactobacillus fructiuorans U R 3839 could grow in H H D even
after 20 days of incubation. Minor modifications in H H D composition or culture
conditions (variations in temperature or presence of CO 2) did not allow the growth
of these strains. Furthermore, Carnobacterium diL,ergens C E C T 4016, Carnobac-
teriurn piscicola C E C T 4020, Lactobacillus acidophilus C E C T 289, Lactobacillus
alimentarius C E C T 570, Lactobacillus casei ssp. casei C E C T 475 and Lactobacillus
fermentum C E C T 285 showed an inappropriate response in H H D . Most of the
strains tested showed a better growth in M5 medium than in H H D . L. acidophilus
C E C T 289, Streptococcus mitis C E C T 804, Streptococcus mutans C E C T 479 and
Streptococcus saliearius ssp. salit~arius C E C T 805 could not grow on M5 plates at
25°C, but they did at 37°C. For the rest of LAB strains the incubation temperature
40

TABLE II

Differential growth of lactic acid bacteria on HHD and M5 media

Species Strain Medium

HHD M5
Carnohacterium
C. dil,ergens CECT 4016 B ~’ B
C. piscicola CECT 4020 B* w ”
Enterococcus
E. ,faecalis CECT I87 B B
E. mundtii CECT 972 _c B
Lactohacillus
L. acidophilus CECT 289 W* B
L. alimentarius CECT 570 w* B
L. hrel,is CECT 216 W W
L. case, ssp. case1 CECT 475 W” B
1,. ccllobiosus CECT 562 W W
L. cuwatus CECT 904 B B
L. fermentum CECT 285 B” W
L. fructil,orans UR 3839 Nd W
L. hilgardii UR 3817 W W
L. plantarum CECT 220 B B
L. plantarum UR 3809 B B
L. sake CECT 906 B
I.. ~Yridescens CECT 283 B
Lactococcus
L. lactis ssp. cremoris CECT 697 _ B
I.. lactis ssp. lactis CECT 916 B B
L. raffinolacti5 CECT 988 B
Leuconostoc
L. mesenteroide.c- ssp. dextranicum (‘ECT 912 _ W
I.. mesrnteroides ssp. mesenteroidrs CECT 215 W
1.. oenos U R 3840 N W
L. oenos UR 3841 N W
L. ocnos UR 3843 N W
I.. oenos UR 3866 N W
L. oenOS UR 3872 N W
L. ornos UR 3873 N W
L. oenos UR 3874 N W
L. oenos ML34 (CECT 218) N W
L. paramesenteroides IJR 3816 W W
Pediococcus
P. acidilactici CECT 9X B
P. damnosus CECT 793 B
P. purc~ulus CECT 813 B
P. pentosaceus CECT 923 B B
Streptococcus
S. mitis CECT 804 _ B
S. mutans CECT 479 B B
S. salif,arius ssp. sahr~arius CECT 805 _ B
S. sanguis CECT 480 _ B

* Aberrant result.
“ Blue colonies.
” White colonies.
’ Assay not carried out.
” No growth.
 41

did not modify the response in M5, and the time required to observe the test result
was strain-dependent. Most of the strains grew in 7 days or less, except the L.
oenos and Lactobacillus fructivorans U K 3839 strains, which needed more than 10
days.
In the development of the M5 medium, different amounts of nutrients were
tested, and their concentration optimized: e.g., too much yeast extract (10 g / l ) or
calcium pantothenate (0.1 g / l ) could yield anomalous results. Factors affecting the
final p H of the M5 medium were also analysed, because an increase in the acid
production can result in heterofermentative LAB being assessed as homofermenta-
rive. We optimized the phosphate concentration, the initial p H value, the type and
amount of sugar, and the influence of oxygen. As LAB can use oxygen as an
electron acceptor, this can lead to a change of the end-products (Condon, 1987;
van Beelen et al., 1986). When M5 was used, incubation under aerobic conditions
led to inappropriate response in some cases due to a higher acid production by the
heterofermentative strains. Heterofermentative LAB can increase the acid produc-
tion under aerobic conditions for two reasons: first, more fructose can be con-
verted into acids because it is not reduced to mannitol; second, more acetic acid
can be produced (Condon, 1987). Reduction of fructose to mannitol is essential to
obtain the appropriate results from all strains. If the reduction of fructose to
mannitol is not required for the reliability of the method, the replacement of this
sugar by glucose will not modify the results in either aerobic or anaerobic
conditions. When fructose was replaced by glucose in the M5 medium, we
observed that for some strains the growth was very poor, and many heterofermen-
tative strains behaved as homofermentatives both under aerobic and anaerobic
conditions. This is also suggested by the fact that the heterofermentative Lacto-
bacillus viridescens behaves as homofermentative even on M5 plates incubated
under CO 2 atmosphere (Table II), because it does not produce mannitol from
fructose (Holzapfel and Gerber, 1983). Although Carnobacterium diuergens (syn.
Lactobacillus dicergens) was initially described as an atypical heterofermenter
(Holzapfel and Gerber, 1983), further studies reclassified it as a homofermentative
organism (De Bruyn et al., 1987; 1988). So the results provided by this organism
both in H H D and M5 media were correct.
The possibility of using M5 medium for direct isolation and differentiation of
LAB from natural environments was also tested by inoculating in the same plates
both L. plantarum and L. brevis at different ratios. Alter incubation it was
observed that colonies of both strains yielded the appropriate results, and the
relative ratios were maintained. Similar results had been reported by McDonald et
al. (1987) with H H D for combinations of several strains of LAB.
Therefore, M5 can be employed as a general medium for differentiating
between homofermentative and heterofermentative lactic acid bacteria that utilize
fructose. Best results are achieved when plates are incubated under an atmosphere
of 100% CO 2 at 25°C.
42

Acknowledgements

This research was supported by the Comisi6n lnterministerial de Ciencia y

Tecnologia (BIO89-0430) and by a grant from the Generalitat Valenciana to M.Z.

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