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Journal of Culture Collections, Volume 1, 1995, pp 28-33

THE POLYAMINE CELL COMPOSITION AS A CHEMOTAXONOMIC MARKER IN LACTIC ACID BACTERIA IDENTIFICATION

¹National Bank for Industrial Microorganisms and Cell Cultures, 1113 Sofia, P.O.Box 239, Bulgaria;
²The Sofia University, Biological Faculty, 8 "Dragan Tsankov" st., 1421 Sofia, Bulgaria

Analysis of the cell polyamines - cadaverine, putrescine, spermine and spermidine of 11 newly isolated strains of lactic acid bacteria from different regions of West Bulgaria was performed. The obtained data were compared with the physiological and biochemical characteristics of these strains, so that the polyamine analysis could be used as a taxonomic criterion. The results showed that spermidine was the main polyamine in all tested cultures. Spermine and putrescine were found in small quantities in two strains, whereas cadaverine - in none of the studied strains. Spermidine values were high and varied in the different strains and showed good correlation with their physiological and biochemical characteristics. On the basis of the obtained results a conclusion was made that the polyamine analysis can serve as a chemotaxonomic criterion only on the strictly standardised cultivation conditions and in the presence of data-base for qualitative and quantitative composition of the corresponding type cultures.

Investigations on microbial chemistry have shown that cell component analysis can be used in bacterial classification and identification [2]. Recently, the polyamine cell composition is used as a chemotaxonomic marker in addition to determination of cell wall composition, protein patterns, fatty acids and enzyme characterisation [6, 7, 8, 9, 15, 16, 11, 10].

Polyamines as putrescine, spermidine, spermine and cadaverine are polycationic compounds, which are widely distributed in nature. Several lines of evidence show that these polyamines are essential for growth and the regulation of macromolecular synthesis in almost all organisms [1]. Putrescine and spermidine are the main polyamines present in most prokaryotic organisms: eubacteria, archaebacteria and cyanobacteria [16]. Considerable amounts of spermidine have been found in thermophilic bacilli [15], thermophilic actinomycetes [8], acetobacteria [11] and agrobacteria [17]. Not considerable quantities of it were found in lactic acid bacteria [7]. Cadaverine is also synthesised but not by all microorganisms. Even if spermine is detected in certain eubacteria, it does not necessarily mean that they synthesise spermine, because polyamines can be taken up from culture media by growing bacteria. Trace amounts of spermine can be detected even in some eubacteria that lack spermine synthetic activity such as Escherichia coli [9] and staphylococci [13].

Recently the so-called “unusual” polyamines have been found in various microorganisms. Norspermidine is synthesised by halophilic strains of Vibrionaceae family [18] while symhomospermidine - by photosynthetic bacteria [5, 6], nitrogen-fixing bacteria [6, 14] and sulphur-oxidising bacteria [6]. Strains from Thermus [10] and Clostridium [11] genera possess norspermidine and thermospermidine.

The present work focuses on the application of polyamine analysis for a more complete characterisation of newly isolated strains of lactic acid bacteria.

Microorganisms and media. Eleven newly isolated strains of lactic acid bacteria from different starters named at the present stage of investigation as N-9-11, R-26-3, R-11-5, P-1, R-11, F-29-4, R-29, R-9-1, R-14-10, F-35-3 and F-1-9 had been used for analysis of the cell polyamines.

The strains were cultivated on MRS medium containing (g/l): bactopeptone - 10, meat extract - 10, yeast extract - 5, glucose - 20, diamonium citrate - 2g, MgSO₄.7H₂O - 0.2, K₂HPO₄- 2, CH₃COONa - 5, MnSO₄.7H₂O - 0.05, Tween-80 - 2.1ml (pH of the media was adjusted to 5.2). Three-stage cultivation was performed: 1) The freeze-dried strains were suspended in 3 ml skim milk and cultivated for 24 h; 2) 0.1 ml of this first culture was inoculated in 3 ml MRS liquid medium for 24 h; 3) 6 ml of the second culture were inoculated in 250 ml MRS medium for 48 h. The cultivation temperatures of each strain were shown in Table 1. The biomass was separated by centrifugation at 8 000 rpm, 4°C on Beckman JA-21 centrifuge and triply washed with physiological solution under the same conditions.

Physiological and biochemical tests. Colonial morphology was examined on isolated colonies grown on MRS agar for 2 - 4 days. Cellular morphology was examined in Gram-stained smears. The fermentation of different sugars was studied in liquid MRS medium without meat extract and glucose and with 0.05% (w/v) chlorphenol red. The sterilised by filtration sugar and alcohol solutions were added in the medium to a final concentration of 1%. The test cultures cultivation was performed at optimal for the growth of the different strains temperatures. The presence of metachromatic grains in the cells was detected microscopically after staining with methylene blue, and milk coagulation - after inoculation of the strains in sterile skim milk.

The growth of the cultures in the presence of 2% NaCl was established at temperatures 15 and 45°C.

The tests for NH₃ production from arginine, gelatinase and catalase activities had been performed according to the methods described in [3].

Bergey's manual [2] was used for identification of the strains and Lactobacillus bulgaricus NBIMCC 1382 was used as a referent strain.

Polyamine analysis. 1 g fresh biomass was washed with distilled water and homogenised with equal volume 10% 3-chloracetic acid. The polyamine extraction had been performed for 2 h at 4°C, followed by centrifugation at 15 000 rpm for 20 min at 4°C. The supernatant was lyophilized and after solubilisation of the dry matter in 0.6 ml 0.1N HCl was used for analysis. Polyamines were determined quantitatively by use of Amino acid analyser type AAA-Prague and ninhydrin reaction.

The investigation of the polyamine pattern cannot be applied in isolation without classical taxonomic information. All of the isolated strains are Gram positive rods with rounded ends, 0.5-0.8 by about 2-9 mm, occurring singly and in short chains. Growth on solid medium showed that they form R and S colonies. No catalase and gelatinase activity was observed. According to Bergey’s manual some physiological and biochemical characteristics were determined. The results are presented in Table 2. Nine of the studied strains showed phenotypic characteristics close to that of L. delbrueckii subsp. bulgaricus - they ferment only some sugars, possess metachromatic grains, do not form NH₃ from arginine, grow at temperature 45°C in medium with 2% NaCl, but growth at 15°C was not observed. Strain F-1-9 showed high phenotypic similarities with L. delbrueckii subsp. bulgaricus, with exception of its ability metabolise maltose. This places the strain close to L. delbrueckii subsp. lactis and L. casei subsp. casei. The obtained results for strain P-14-10 (positive characteristics for esculin, maltose, sucrose and NH₃ from arginine) distinguish it from the other strains and include it in the group of L. delbrueckii subsp. lactis. Further investigations are necessary for correct classification of these strains into corresponding species.

For a more complete characterisation of newly isolated strains of lactic acid bacteria and to evaluate the possibility to use as taxonomic criterion, polyamine analysis was carried out. The obtained results showed that spermidine is the main polyamine in all tested cultures (Table 1). Spermine appeared only in R-14-10 and F-35-3, while putrescine - in R-14-10. Spermidine values in N-19-11, F-26-3, R-11-5, P-1, F-29-4, R-29, F-35-3 and R-11did not vary significantly: 0.013 - 0.027 mol/g fresh biomass. Chromatograms of some of the strains are presented on Fig. 1 (b, c and d).

Guirard and Snell [4] showed that spermine was present in various lactic acid bacteria after analysing polyamines by a bioassay procedure. The trace amount of spermine has also been detected in these organisms in the study of Hamana et al. [7]. These authors suggested that this seems to be due to the uptake or contamination from the medium containing peptone, yeast extract or tryptose. Our results did not confirm also the spermine biosynthesis by lactic acid bacteria. Small quantities were found only in R-14-10 and F-35-3.

Putrescine was not present in the cells of the studied strains with the exception of R-14-10. Cadaverine was not found in anyone of them (Table 1). Hamana et al. [6] had found considerable amount of this polyamine only in L. acidophilus which probably was due to induction of the cadaverine biosynthesis by the specific growth conditions.

The results of the polyamine analysis of 11 lactic acid bacteria strains showed that species differentiation could be done using quantitative determination of spermidine. Its values were high and variable for the strains belonging to the different species. Comparing our results for the spermidine with those of Hamana et al. [6], we found out that N-19-11, F-26-3, R-11-5, P-11, F-29-4, R-29, P-9-1, F-35-3 belonged to the group of L. delbrueckii subsp. bulgaricus. Strains F-1-9 and R-14-10 showed differences not only in their physiological and biochemical characteristics but also in their polyamine content (0.086 mol/g fresh biomass for R-14-10 and 0.216 mol/g for F-1-9). The insufficient information in literature about the polyamine composition of the corresponding type strains did not allow us to classify R-14-10 and F-1-9 into a certain group.

Summarising the data of the present investigation and those in the literature it can be concluded that the polyamine analysis gives reliable proofs for the physiological status of certain species of the Lactobacillus genera. Strict standardisation of the cultivation condition and presence of database for the quantitative and qualitative polyamine composition of the corresponding type cultures should be done in order to use it as a chemotaxonomic marker.

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