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An improved medium for distinguishing between homofermentative and heterofermentative lactic acid bacteria
International Journal of Food Microbiology, 18 (1993) 37-42 © 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1605/93/$06.00
37
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 microorganisms that include the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, 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-phosphate 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-reproducible 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 heterofermentative 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 distinguishing 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 bacteria
and MLO for homo- and heterofermentative lactic acid
Component ( g / l )
M5
HHD
MLO
Tryptone Yeast extract Casaminoacids Bactosoytone
10
10
10
5
1
5
3 1.5
Glucose
10
Fructose
2.5
2.5
5
Nit 2 PO4 Tween80
2.5 1 ml
2.5 1 g
1 ml
L-Cysteine H C 1
0.5
MgSO 4 •7H 20
0.2
-
MnSO 4-1H20
0.05
-
0.05
(NH4) 2 citrate
0.Ill
-
3.5
-
100 ml
Calcium pantothenate Tomato juice Bromocresol green " Agar pH
-
0.5
20 ml
20 ml
20
20
6.5
7.0
:~ Stock solution, 0.1 g of bromocresol green in 30 ml of 0.01 N N a O H .
0.2
2(/ 4.8
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 Lactobacillus) 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) described, 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 cellobiosus 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, Carnobacteriurn 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
Species
and M5 media
Strain
Carnohacterium C. dil,ergens C. piscicola Enterococcus E. ,faecalis E. mundtii Lactohacillus L. acidophilus L. alimentarius L. hrel,is L. case, ssp. case1 1,. ccllobiosus L. cuwatus L. fermentum L. fructil,orans L. hilgardii L. plantarum L. plantarum L. sake I.. ~Yridescens Lactococcus L. lactis ssp. cremoris I.. lactis ssp. lactis L. raffinolacti5 Leuconostoc L. mesenteroide.c- ssp. dextranicum I.. mesrnteroides ssp. mesenteroidrs 1.. oenos L. oenos L. ocnos I.. oenos L. oenOS L. ornos L. oenos L. oenos L. paramesenteroides Pediococcus P. acidilactici P. damnosus P. purc~ulus P. pentosaceus Streptococcus S. mitis S. mutans S. salif,arius ssp. sahr~arius S. sanguis * Aberrant result. “ Blue colonies. ” White colonies. ’ Assay not carried ” No growth.
on HHD
out.
Medium HHD
M5
CECT 4016 CECT 4020
B ~’ B*
B w ”
CECT I87 CECT 972
B _c
B B
CECT 289 CECT 570 CECT 216 CECT 475 CECT 562 CECT 904 CECT 285 UR 3839 UR 3817 CECT 220 UR 3809 CECT 906 CECT 283
W* w* W W” W B B” Nd W B B
B B W B W B W W W B B B B
CECT 697 CECT 916 CECT 988
_
B B B
(‘ECT 912 CECT 215 U R 3840 UR 3841 UR 3843 UR 3866 UR 3872 UR 3873 UR 3874 ML34 (CECT IJR 3816
_
B
218)
N N N N N N N N W
W W W W W W W W W W W B B B B
CECT CECT CECT CECT
9X 793 813 923
B
CECT CECT CECT CECT
804 479 805 480
B _ _
_
B B B B
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 homofermentarive. 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 production under aerobic conditions for two reasons: first, more fructose can be converted 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 heterofermentative strains behaved as homofermentatives both under aerobic and anaerobic conditions. This is also suggested by the fact that the heterofermentative Lactobacillus 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
Tecnologia
was
supported
(BIO89-0430)
by the
Comisi6n
lnterministerial
and by a grant from the Generalitat
de
Valenciana
Ciencia
y
to M.Z.
References Claus, D., Lack, P. and Neu, P. (1983) D.S.M. Catalogue of strains. Deutsche Sammlung yon Mikroorganismen. Condon, S. (1987) Responses of lactic acid bacteria to oxygen. FEMS Microbiol. Rev. 46, 269-280. De Bruyn, I.N., Louw, A.I., Visser, L. and Holzapfel, W.H. (19871 Lactobacillus dicergens is a homofermentative organism. Syst. Appl. Microbiol. 9, 173 175. De Bruyn, I.N., Holzapfel, W.H., Visser, L. and Louw, A.I. (1988) Glucose metabolism by Lactobacillus dit,ergens. J. Gen. Microbiol. 134, 2103- 21 (19. de Man, J.C., Rogosa, M. and Sharpc, M.E. ( 19601 A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130 135. Garvie, E.I. (1978) Streptococcus raffinolactis (Orla-Jensen and ttansen): a group N streptococcus found in raw milk. Int. J. Syst. Bacteriol. 28, 19(I-193. Gibson. T. and Abd-EI-Malek, Y. (1945) The formation of C O , by lactic acid bacteria and Bacillus licheniformis and a cultural method of detecting the process. J. Dairy, Res. 14, 35 44. ftolzapfel, W.H. and Gerber, E.S. (19831 Lactobacillus dil'ergens sp. nov., a new heterofermentative Lactobacillus species producing i.( + )-lactate. Syst. Appl. Microbiol. 4, 522--534. Kandler, O. (19831 Carbohydrate metabolism in lactic acid bacteria. Antonie van Leeuwenhoek. 4t). 209-224. McDonald, L.C., MeFeeters, R.F., Daeschel, M.A. and Fleming, H.P. (1987) A differential medium for the enumeration of homofermentativc and heterofermentative lactic acid bacteria. Appl. Environ. Microbiol. 53. 1382-1384. Sheath, P.|t.A., Mair, N.S., Sharpe, M.E. and Holt, J.G. (1986) Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams & Wilkins, Baltimore, MD. Terzaghi. B. and Sandinc, W.E. (1975) Improved medium for lactic streptococci and their baclcriophages. Appl. Microbiol. 29, 807 813. van Beelen, P., van der Hoeven. J.S., dc Jong, M.H. and Itoogendoorn, H. (1986) The effect of oxygen on the growth and acid production of Streptococcus mutans and Streptococcus sangtds. FEMS Microbiol. Ecol. 38, 25 3(/.
37
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 microorganisms that include the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, 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-phosphate 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-reproducible 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 heterofermentative 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 distinguishing 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 bacteria
and MLO for homo- and heterofermentative lactic acid
Component ( g / l )
M5
HHD
MLO
Tryptone Yeast extract Casaminoacids Bactosoytone
10
10
10
5
1
5
3 1.5
Glucose
10
Fructose
2.5
2.5
5
Nit 2 PO4 Tween80
2.5 1 ml
2.5 1 g
1 ml
L-Cysteine H C 1
0.5
MgSO 4 •7H 20
0.2
-
MnSO 4-1H20
0.05
-
0.05
(NH4) 2 citrate
0.Ill
-
3.5
-
100 ml
Calcium pantothenate Tomato juice Bromocresol green " Agar pH
-
0.5
20 ml
20 ml
20
20
6.5
7.0
:~ Stock solution, 0.1 g of bromocresol green in 30 ml of 0.01 N N a O H .
0.2
2(/ 4.8
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 Lactobacillus) 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) described, 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 cellobiosus 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, Carnobacteriurn 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
Species
and M5 media
Strain
Carnohacterium C. dil,ergens C. piscicola Enterococcus E. ,faecalis E. mundtii Lactohacillus L. acidophilus L. alimentarius L. hrel,is L. case, ssp. case1 1,. ccllobiosus L. cuwatus L. fermentum L. fructil,orans L. hilgardii L. plantarum L. plantarum L. sake I.. ~Yridescens Lactococcus L. lactis ssp. cremoris I.. lactis ssp. lactis L. raffinolacti5 Leuconostoc L. mesenteroide.c- ssp. dextranicum I.. mesrnteroides ssp. mesenteroidrs 1.. oenos L. oenos L. ocnos I.. oenos L. oenOS L. ornos L. oenos L. oenos L. paramesenteroides Pediococcus P. acidilactici P. damnosus P. purc~ulus P. pentosaceus Streptococcus S. mitis S. mutans S. salif,arius ssp. sahr~arius S. sanguis * Aberrant result. “ Blue colonies. ” White colonies. ’ Assay not carried ” No growth.
on HHD
out.
Medium HHD
M5
CECT 4016 CECT 4020
B ~’ B*
B w ”
CECT I87 CECT 972
B _c
B B
CECT 289 CECT 570 CECT 216 CECT 475 CECT 562 CECT 904 CECT 285 UR 3839 UR 3817 CECT 220 UR 3809 CECT 906 CECT 283
W* w* W W” W B B” Nd W B B
B B W B W B W W W B B B B
CECT 697 CECT 916 CECT 988
_
B B B
(‘ECT 912 CECT 215 U R 3840 UR 3841 UR 3843 UR 3866 UR 3872 UR 3873 UR 3874 ML34 (CECT IJR 3816
_
B
218)
N N N N N N N N W
W W W W W W W W W W W B B B B
CECT CECT CECT CECT
9X 793 813 923
B
CECT CECT CECT CECT
804 479 805 480
B _ _
_
B B B B
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 homofermentarive. 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 production under aerobic conditions for two reasons: first, more fructose can be converted 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 heterofermentative strains behaved as homofermentatives both under aerobic and anaerobic conditions. This is also suggested by the fact that the heterofermentative Lactobacillus 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
Tecnologia
was
supported
(BIO89-0430)
by the
Comisi6n
lnterministerial
and by a grant from the Generalitat
de
Valenciana
Ciencia
y
to M.Z.
References Claus, D., Lack, P. and Neu, P. (1983) D.S.M. Catalogue of strains. Deutsche Sammlung yon Mikroorganismen. Condon, S. (1987) Responses of lactic acid bacteria to oxygen. FEMS Microbiol. Rev. 46, 269-280. De Bruyn, I.N., Louw, A.I., Visser, L. and Holzapfel, W.H. (19871 Lactobacillus dicergens is a homofermentative organism. Syst. Appl. Microbiol. 9, 173 175. De Bruyn, I.N., Holzapfel, W.H., Visser, L. and Louw, A.I. (1988) Glucose metabolism by Lactobacillus dit,ergens. J. Gen. Microbiol. 134, 2103- 21 (19. de Man, J.C., Rogosa, M. and Sharpc, M.E. ( 19601 A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130 135. Garvie, E.I. (1978) Streptococcus raffinolactis (Orla-Jensen and ttansen): a group N streptococcus found in raw milk. Int. J. Syst. Bacteriol. 28, 19(I-193. Gibson. T. and Abd-EI-Malek, Y. (1945) The formation of C O , by lactic acid bacteria and Bacillus licheniformis and a cultural method of detecting the process. J. Dairy, Res. 14, 35 44. ftolzapfel, W.H. and Gerber, E.S. (19831 Lactobacillus dil'ergens sp. nov., a new heterofermentative Lactobacillus species producing i.( + )-lactate. Syst. Appl. Microbiol. 4, 522--534. Kandler, O. (19831 Carbohydrate metabolism in lactic acid bacteria. Antonie van Leeuwenhoek. 4t). 209-224. McDonald, L.C., MeFeeters, R.F., Daeschel, M.A. and Fleming, H.P. (1987) A differential medium for the enumeration of homofermentativc and heterofermentative lactic acid bacteria. Appl. Environ. Microbiol. 53. 1382-1384. Sheath, P.|t.A., Mair, N.S., Sharpe, M.E. and Holt, J.G. (1986) Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams & Wilkins, Baltimore, MD. Terzaghi. B. and Sandinc, W.E. (1975) Improved medium for lactic streptococci and their baclcriophages. Appl. Microbiol. 29, 807 813. van Beelen, P., van der Hoeven. J.S., dc Jong, M.H. and Itoogendoorn, H. (1986) The effect of oxygen on the growth and acid production of Streptococcus mutans and Streptococcus sangtds. FEMS Microbiol. Ecol. 38, 25 3(/.