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Comparison of methods for differentiation between homofermentative and heterofermentative lactic acid bacteria
Zentralbl. Mikrobiol. 145 (1990), 363-366 VEB Gustav Fischer Verlag lena
[Institut fur Futterproduktion Paulinenaue der Akademie der Landwirtschaftswissenschaften der DDR]
Comparison of Methods for Differentiation between Homofermentative and Heterofermentative Lactic Acid Bacteria Vergleich von Methoden zur Differenzierung zwischen homofermentativen und heterofermentativen Milchsaurebakterien THOMAS MULLER Key words: Silage fermentation, lactic acid bacteria, gas production, taxonomy, standard methods
Summary Five methodsfor determiningthe type of fermentation in lactic acid bacteria were tested in a comparativestudy. Gas detectingmethods withDurhamtubes dependingon the mediumused providedthe most rapidand reliableresults.
Zusammenfassung Funf Methodenzur Bestimmung des Fermentationstyps von Milchsaurebakterien wurden in einer vergleichenden Untersuchung getestet. Methoden des Gasnachweises mit Durharn-Rohrchen lieferten in Abhangigkeit vom verwendeten Medium die schnellsten und zuverlassigsten Resultate.
Lactic acid bacteria (LAB) are divided into two physiological group s : the homofermentative LAB , which produce primarily lactic acid, and the heterofermentative, which produce lactic acid, acetic acid, ethanol , mannitol and CO 2 from hexoses (KAN DLER 1983). Different iation between homo- and heterofermentative activity is one of the first steps in systematic classification of LAB (KANDLER und WEISS 1986). The procedure s of the differentiation are usually based on the production of CO 2 by heterofermentative LAB . There have been described many method s for the detection of gas production differin g in their gas detect ing arrangements or in the media used or in both. For example , ROGOSAet al. (1953) detected gas produ ction by the appearance of cracks in a solid agar medium , HAYWARD (1957) and WOOLFORD and SAWCZYC (1984) used conventional Durham tubes in different liquid media , that were sealed with a layer of agar. SPERBER and SWAN (1976 ) described the hot-loop test: A heated inoculating loop is plunged into a liquid culture . There are developing gas bubbles if the fermentation type is heterofermentative. Recently McDONALD et al. (1987) developed a medium, that enables a differentiation between the two physiological types by their difference in acid production . Bromcre solgreen is used as pH indicator during fermentation of fructose . In comparative studies we found that these method s in many cases differ in reliability and sensitivity . It was the aim of our study to pick out a simple , but sensitive, rapid and reliable method for determining the type of fermentation in LAB .
Material and Methods Procedures compared A) Gas detecting method with Durham tubes in a medium as described by HAYWARD (1957). B) Gas detecting method with Durham tubes in a medium as described by WOOLFORDand SAWCZYC (1984).
18 21 45 104 105 107 149 152 23 92 98 19 85 86 87 93 94 106 123 124 125
Lb. brevis Lb. brevis Lb. brevis Lb. brevis Lb. confusus Lb. conf usus Lb. f ermentum Lb. f ermentum L. mesenteroides L. mesenteroides L. mesenteroides L. paramesent. L. param esent. L. paramesent. L. param esent. L. param esent. L. paramesent. L. param esent. Lb. plantarum Lb. plantarum Lb. plantarum
-
+ + + + + + + + + + + + + + -
-
1st
A
= positive (gas producti on) + +w = weak positive = ' negative (no gas production) = blue colour of medium b = green colour of medium g b-g = blue-green colour of medium Lb. = Lactobacillus L. = Leuconostoc
No . of strain
species
-
-
+ + + + + + + + + + + + + + + + +
2nd
-
-
+ + + + + + + + + + + + + + + + + +
3rd day
-
-
-
-
+
-
+ + + + + + +
1st
B
-
-
+ + + + + + + + + + + + + + +
2nd
-
-
+
-
+ + + + + + + + + + + + + + +
3rd day
-
-
+ + + + + + + + + + + + + +
-
-
1st
C
+ + + + + + + + + + + + + + + + + + -
2nd
+ + -
+
+ + + + + + + + + + + + + + +
3rd day
-
-
-
-
-
-
-
-
-
1st
D
-
-
-
+ +w
-
+w + + + +w + +w +w +w +w
-
-
-
-
+w
-
-
+ +
-
-
+
-
+ +w
3rd day
2nd
Table I. Comparison of 5 procedure s for the determin ation of homo- or heterofermentative activity by 2 1 isolates of lactic acid bacteria.
b b b b b b b b b b b b b b b b b b b-g b-g b-g
1st
E
b b b b b b b g b b b-g b-g g b b b b-g b-g g g g
2nd b b b b b b b-g g b b b-g b-g g b b-g b b-g b-g g g g
3rd day
:>0
'"
,... ,...
-l
:.: ::: e,
~
loU
Comparison of Methods
365
C) GasdetectingmethodwithDurhamtubesin APTbroth(EVANSandNIVEN 1951)plus 1.0% glucose and0.5 % sodium acetate (ADCA medium) according to SPERBER and SWAN (1976). Following inoculation each tube in A, Band C was sealed with a 10mm deep layer of agar (20 g agar litre- I in water). Homo- and heterofermentative activitywas assessedby the absenceor presenceof gas in the Durhamtubes. D) Hot-loop test in ADCA medium as described by SPERBER and SWAN (1976) for the determination of CO2 production. In heterofermentative cultures gas bubbles rose after plunging of the red hot loop, while in homofermentative cultures they did not so. E) Inoculation into differential broth (HHD)according to McDoNALD et al. (1987). HHDbroth inoculated with homofermentative LAB was green, while HHD broth containing heterofermentative LAB remained blue. In all procedures uninoculated controls were also prepared. All test tubes were incubated at 30°C and were controlled daily up to 3 days.
Strains used and inoculation procedure 18 heterofermentative and 3 homofermentative LAB-strains used in the comparative study were isolatedeither fromgrass or grass silage. Theyweresystematically identifiedby meansof characteristicsdescribedby GARVIE (1986) or KANDLER and WEISS (1986). A drop of each MRS culture was implanted into the test tubes containing 5-ml quantities of the test specific media.
Results and Discussion The results of the comparative study are presented in Table 1. The determination of the homofermentative type of fermentation was possible by all procedure s tested (strains no . 123-125). In the case ofheterofermentative fermentation the gas detecting methods with Durham tubes rapidly and reliably enabled the determination . There were no differences in suitability of media used in procedures A and C for all species tested . But there were some problems in determining the physiological type by method B. It seems that the medium described by WOOLFORD and SAWCZYC (1984) is not suitable for the genus Leuconostoc. In two cases results were false negative . Many false negative or uncertain results were also provided by the hot-loop test (method D). It seems that CO2-bubbles were shown surely only in cultures with very strong gas production. The differentiation between homo- and heterofermentative activity by means of colour change from blue to green in the HHD broth (method E) sometimes is difficult because in some cases the colour was bluish-green . In other cases the quantity of acid produced was enough to change the colour from blue to green by heterofermentative species, too. Thus, the method provided false positive results . According to the results presented in this paper the use of the gas detecting method with Durham tubes is recommended for determination of homo- or heterofermentative activity by LAB . Suitable media are that described by HAYWARD (1957) or APT with added glucose and sodium acetate .
References EVANS, J. B., NIVEN , C. F.: Nutrition of the heterofermentative Lactobacilli that cause greening of cured meat products. J. Bact. 62 (1951), 599-603. GARVIE, E. I.: GenusLeuconostoc VAN TIEGHEM 1878, 198AL emend mut. char. HUCKER and PEDERSON 1930,66AL• In: Bergey's manual of systematicbacteriology, vol. 2 (Eds.: SNEATH , P. H. A., MAIR, N. S., SHARPE, M. E., HOLT, J. G.) Williams and Wilkins Co., Baltimore 1986, 1071-1075 . HAYWARD, A. C.: Detection of gas production from glucose by heterofermentative lactic acid bacteria. J. gen. Microbiol. 16 (1957), 9-15 . KANDLER, 0 .: Carbohydrate metabolism of lacticacid bacteria. Antonie van Leeuwenhoek 49 (1983), 2099- 2224. - WEISS, N.: Genus Lactobacillus BEIJERINCK 1901, 22AL • In: Bergey's manual of systematicbacteriology, vol. 2 (Eds.: SNEATH, P. H. A., MAIR, N. S. , SHARPE, M. E., HOLT, J. G.) Williams and WilkinsCo., Baltimore 1986, 1209-1234 . McDONALD, L. c., Mc FEETERS, R. F., DAESCHEL, M. A., FLEMING, H. P.: A differential medium for the enumeration of homofermentative and heterofermentative lactic acid bacteria. Appl. Environ. Microbiol. 53 (1987), 1382-1384.
366
TH. MOLLER
ROGOSA, M., WISEMAN, R. F., MITCHELL, J. A., DISRAELY, M. N., BEAUMAN , A. J.: Species differentiation of oral lactobacilli from man including descriptions of Lactobacillus salivarius nov. spec. and Lactobacillus cellobiosus nov. spec. J. Bact. 65 (1953), 681. SPERBER, W. H., SWAN , J.: Hot-looptest for the determination of carbondioxideproduction from glucoseby lactic acid bacteria. Appl. Environ. Microbiol. 31 (1976), 990-991. WOOLFORD, M. K., SAWCZYC, M. K.: An investigation into the effect of cultures of lactic acid bacteria on fermentation in silage. I. Strain selection. Grass and Forage Science 39 (1984), 139-148. Author's address: Dr. rer. nat. THOMAS MOLLER, Institut fur Futterproduktion der Akademie der Landwirtschafts. wissenschaften der DDR, Paulinenaue, DDR - 1551.
Zentralbl. Mikrobiol. 145 (1990), 366 VEB Gustav Fischer Verlag Jena
Buchbesprechung SCRAGG, A. H.: Biotechnologyfor Engineers - BiologicalSystems in Technological Processes. Ellis Horwood Limited, Publishers Chichester, Wiley & Sons 1988, 390 Seiten, Preis: 45,OO£. ISBN 74580226. In 19 Kapiteln gibt das Buch eine relativ umfassende Einfiihrung in die Biotechnologie. Nach einem kurzen historischen AbriB wirdauf Cytologieund Molekularbiologie der Pro- undEukaryoten, auf Stoffwechselprozesse und ihre Regulation, Wachstumsphysiologie, Enzymkinetik und -technologie, Sterilisationsmethoden, Bioreaktoren und Aufarbeitungsprozesse eingegangen. Als Beispielefur die groBtechnische Anwendung biotechnologischer Prozesse werden die Citronensaureproduktion, die Antibiotikaherstellung, die biologische Abwasserbehandlung und die Brauereibehandelt. Damitist das Buch sehr breitangelegt. Der Schwerpunkt liegt in einer Vermittlung biologischen Grundwissens an Nichtbiologen, die technischen Aspekte sind nur knapp beriicksichtigt. Biochemie und Stoffwechselphysiologie sind ausfiihrlich dargestellt, unzureichend im Umfang sind jedoch Photosynthese und Chemosynthese sowie der Sekundarstoffwechsel behandelt. Es fehlt auch eine Ubersicht iiber biotechnologisch relevante Mikroorganismengruppen und deren Eigenschaften. Die zahlreichen Abbildungen und Tabellen sind - bis auf unzulassige Vereinfachungen in der Cytologie iibersichtlich und informativ. Insgesamtordnet sich das Buch in eine Reiheweiterer, in den letztenJahren mit ahnlichem Profil erschienener Biicherein. Fiirden Verfahrenstechniker, dessen ArbeitimZusammenhang mitder Biotechnologie steht, ist das Buch eingeeigneterEinstieg. G. STRAUBE, Merseburg
[Institut fur Futterproduktion Paulinenaue der Akademie der Landwirtschaftswissenschaften der DDR]
Comparison of Methods for Differentiation between Homofermentative and Heterofermentative Lactic Acid Bacteria Vergleich von Methoden zur Differenzierung zwischen homofermentativen und heterofermentativen Milchsaurebakterien THOMAS MULLER Key words: Silage fermentation, lactic acid bacteria, gas production, taxonomy, standard methods
Summary Five methodsfor determiningthe type of fermentation in lactic acid bacteria were tested in a comparativestudy. Gas detectingmethods withDurhamtubes dependingon the mediumused providedthe most rapidand reliableresults.
Zusammenfassung Funf Methodenzur Bestimmung des Fermentationstyps von Milchsaurebakterien wurden in einer vergleichenden Untersuchung getestet. Methoden des Gasnachweises mit Durharn-Rohrchen lieferten in Abhangigkeit vom verwendeten Medium die schnellsten und zuverlassigsten Resultate.
Lactic acid bacteria (LAB) are divided into two physiological group s : the homofermentative LAB , which produce primarily lactic acid, and the heterofermentative, which produce lactic acid, acetic acid, ethanol , mannitol and CO 2 from hexoses (KAN DLER 1983). Different iation between homo- and heterofermentative activity is one of the first steps in systematic classification of LAB (KANDLER und WEISS 1986). The procedure s of the differentiation are usually based on the production of CO 2 by heterofermentative LAB . There have been described many method s for the detection of gas production differin g in their gas detect ing arrangements or in the media used or in both. For example , ROGOSAet al. (1953) detected gas produ ction by the appearance of cracks in a solid agar medium , HAYWARD (1957) and WOOLFORD and SAWCZYC (1984) used conventional Durham tubes in different liquid media , that were sealed with a layer of agar. SPERBER and SWAN (1976 ) described the hot-loop test: A heated inoculating loop is plunged into a liquid culture . There are developing gas bubbles if the fermentation type is heterofermentative. Recently McDONALD et al. (1987) developed a medium, that enables a differentiation between the two physiological types by their difference in acid production . Bromcre solgreen is used as pH indicator during fermentation of fructose . In comparative studies we found that these method s in many cases differ in reliability and sensitivity . It was the aim of our study to pick out a simple , but sensitive, rapid and reliable method for determining the type of fermentation in LAB .
Material and Methods Procedures compared A) Gas detecting method with Durham tubes in a medium as described by HAYWARD (1957). B) Gas detecting method with Durham tubes in a medium as described by WOOLFORDand SAWCZYC (1984).
18 21 45 104 105 107 149 152 23 92 98 19 85 86 87 93 94 106 123 124 125
Lb. brevis Lb. brevis Lb. brevis Lb. brevis Lb. confusus Lb. conf usus Lb. f ermentum Lb. f ermentum L. mesenteroides L. mesenteroides L. mesenteroides L. paramesent. L. param esent. L. paramesent. L. param esent. L. param esent. L. paramesent. L. param esent. Lb. plantarum Lb. plantarum Lb. plantarum
-
+ + + + + + + + + + + + + + -
-
1st
A
= positive (gas producti on) + +w = weak positive = ' negative (no gas production) = blue colour of medium b = green colour of medium g b-g = blue-green colour of medium Lb. = Lactobacillus L. = Leuconostoc
No . of strain
species
-
-
+ + + + + + + + + + + + + + + + +
2nd
-
-
+ + + + + + + + + + + + + + + + + +
3rd day
-
-
-
-
+
-
+ + + + + + +
1st
B
-
-
+ + + + + + + + + + + + + + +
2nd
-
-
+
-
+ + + + + + + + + + + + + + +
3rd day
-
-
+ + + + + + + + + + + + + +
-
-
1st
C
+ + + + + + + + + + + + + + + + + + -
2nd
+ + -
+
+ + + + + + + + + + + + + + +
3rd day
-
-
-
-
-
-
-
-
-
1st
D
-
-
-
+ +w
-
+w + + + +w + +w +w +w +w
-
-
-
-
+w
-
-
+ +
-
-
+
-
+ +w
3rd day
2nd
Table I. Comparison of 5 procedure s for the determin ation of homo- or heterofermentative activity by 2 1 isolates of lactic acid bacteria.
b b b b b b b b b b b b b b b b b b b-g b-g b-g
1st
E
b b b b b b b g b b b-g b-g g b b b b-g b-g g g g
2nd b b b b b b b-g g b b b-g b-g g b b-g b b-g b-g g g g
3rd day
:>0
'"
,... ,...
-l
:.: ::: e,
~
loU
Comparison of Methods
365
C) GasdetectingmethodwithDurhamtubesin APTbroth(EVANSandNIVEN 1951)plus 1.0% glucose and0.5 % sodium acetate (ADCA medium) according to SPERBER and SWAN (1976). Following inoculation each tube in A, Band C was sealed with a 10mm deep layer of agar (20 g agar litre- I in water). Homo- and heterofermentative activitywas assessedby the absenceor presenceof gas in the Durhamtubes. D) Hot-loop test in ADCA medium as described by SPERBER and SWAN (1976) for the determination of CO2 production. In heterofermentative cultures gas bubbles rose after plunging of the red hot loop, while in homofermentative cultures they did not so. E) Inoculation into differential broth (HHD)according to McDoNALD et al. (1987). HHDbroth inoculated with homofermentative LAB was green, while HHD broth containing heterofermentative LAB remained blue. In all procedures uninoculated controls were also prepared. All test tubes were incubated at 30°C and were controlled daily up to 3 days.
Strains used and inoculation procedure 18 heterofermentative and 3 homofermentative LAB-strains used in the comparative study were isolatedeither fromgrass or grass silage. Theyweresystematically identifiedby meansof characteristicsdescribedby GARVIE (1986) or KANDLER and WEISS (1986). A drop of each MRS culture was implanted into the test tubes containing 5-ml quantities of the test specific media.
Results and Discussion The results of the comparative study are presented in Table 1. The determination of the homofermentative type of fermentation was possible by all procedure s tested (strains no . 123-125). In the case ofheterofermentative fermentation the gas detecting methods with Durham tubes rapidly and reliably enabled the determination . There were no differences in suitability of media used in procedures A and C for all species tested . But there were some problems in determining the physiological type by method B. It seems that the medium described by WOOLFORD and SAWCZYC (1984) is not suitable for the genus Leuconostoc. In two cases results were false negative . Many false negative or uncertain results were also provided by the hot-loop test (method D). It seems that CO2-bubbles were shown surely only in cultures with very strong gas production. The differentiation between homo- and heterofermentative activity by means of colour change from blue to green in the HHD broth (method E) sometimes is difficult because in some cases the colour was bluish-green . In other cases the quantity of acid produced was enough to change the colour from blue to green by heterofermentative species, too. Thus, the method provided false positive results . According to the results presented in this paper the use of the gas detecting method with Durham tubes is recommended for determination of homo- or heterofermentative activity by LAB . Suitable media are that described by HAYWARD (1957) or APT with added glucose and sodium acetate .
References EVANS, J. B., NIVEN , C. F.: Nutrition of the heterofermentative Lactobacilli that cause greening of cured meat products. J. Bact. 62 (1951), 599-603. GARVIE, E. I.: GenusLeuconostoc VAN TIEGHEM 1878, 198AL emend mut. char. HUCKER and PEDERSON 1930,66AL• In: Bergey's manual of systematicbacteriology, vol. 2 (Eds.: SNEATH , P. H. A., MAIR, N. S., SHARPE, M. E., HOLT, J. G.) Williams and Wilkins Co., Baltimore 1986, 1071-1075 . HAYWARD, A. C.: Detection of gas production from glucose by heterofermentative lactic acid bacteria. J. gen. Microbiol. 16 (1957), 9-15 . KANDLER, 0 .: Carbohydrate metabolism of lacticacid bacteria. Antonie van Leeuwenhoek 49 (1983), 2099- 2224. - WEISS, N.: Genus Lactobacillus BEIJERINCK 1901, 22AL • In: Bergey's manual of systematicbacteriology, vol. 2 (Eds.: SNEATH, P. H. A., MAIR, N. S. , SHARPE, M. E., HOLT, J. G.) Williams and WilkinsCo., Baltimore 1986, 1209-1234 . McDONALD, L. c., Mc FEETERS, R. F., DAESCHEL, M. A., FLEMING, H. P.: A differential medium for the enumeration of homofermentative and heterofermentative lactic acid bacteria. Appl. Environ. Microbiol. 53 (1987), 1382-1384.
366
TH. MOLLER
ROGOSA, M., WISEMAN, R. F., MITCHELL, J. A., DISRAELY, M. N., BEAUMAN , A. J.: Species differentiation of oral lactobacilli from man including descriptions of Lactobacillus salivarius nov. spec. and Lactobacillus cellobiosus nov. spec. J. Bact. 65 (1953), 681. SPERBER, W. H., SWAN , J.: Hot-looptest for the determination of carbondioxideproduction from glucoseby lactic acid bacteria. Appl. Environ. Microbiol. 31 (1976), 990-991. WOOLFORD, M. K., SAWCZYC, M. K.: An investigation into the effect of cultures of lactic acid bacteria on fermentation in silage. I. Strain selection. Grass and Forage Science 39 (1984), 139-148. Author's address: Dr. rer. nat. THOMAS MOLLER, Institut fur Futterproduktion der Akademie der Landwirtschafts. wissenschaften der DDR, Paulinenaue, DDR - 1551.
Zentralbl. Mikrobiol. 145 (1990), 366 VEB Gustav Fischer Verlag Jena
Buchbesprechung SCRAGG, A. H.: Biotechnologyfor Engineers - BiologicalSystems in Technological Processes. Ellis Horwood Limited, Publishers Chichester, Wiley & Sons 1988, 390 Seiten, Preis: 45,OO£. ISBN 74580226. In 19 Kapiteln gibt das Buch eine relativ umfassende Einfiihrung in die Biotechnologie. Nach einem kurzen historischen AbriB wirdauf Cytologieund Molekularbiologie der Pro- undEukaryoten, auf Stoffwechselprozesse und ihre Regulation, Wachstumsphysiologie, Enzymkinetik und -technologie, Sterilisationsmethoden, Bioreaktoren und Aufarbeitungsprozesse eingegangen. Als Beispielefur die groBtechnische Anwendung biotechnologischer Prozesse werden die Citronensaureproduktion, die Antibiotikaherstellung, die biologische Abwasserbehandlung und die Brauereibehandelt. Damitist das Buch sehr breitangelegt. Der Schwerpunkt liegt in einer Vermittlung biologischen Grundwissens an Nichtbiologen, die technischen Aspekte sind nur knapp beriicksichtigt. Biochemie und Stoffwechselphysiologie sind ausfiihrlich dargestellt, unzureichend im Umfang sind jedoch Photosynthese und Chemosynthese sowie der Sekundarstoffwechsel behandelt. Es fehlt auch eine Ubersicht iiber biotechnologisch relevante Mikroorganismengruppen und deren Eigenschaften. Die zahlreichen Abbildungen und Tabellen sind - bis auf unzulassige Vereinfachungen in der Cytologie iibersichtlich und informativ. Insgesamtordnet sich das Buch in eine Reiheweiterer, in den letztenJahren mit ahnlichem Profil erschienener Biicherein. Fiirden Verfahrenstechniker, dessen ArbeitimZusammenhang mitder Biotechnologie steht, ist das Buch eingeeigneterEinstieg. G. STRAUBE, Merseburg