Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag.

AIMS: The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB. METHODS AND RESULTS: Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2-10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and alpha-chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N-termini were blocked hampering straightforward Edman degradation. CONCLUSIONS: Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998).

Assessment of methanolysis for the determination of composite sugars of gelling carrageenans and agarose by HPLC.

The characterization of the main composite sugars of commercial gelling red algae galactans (agarose, iota and kappa carrageenans) by methanolysis and separation of the methyl glycosides produced by high performance liquid chromatography is described. The methanolysis (methanolic hydrochloric acid strength, temperature, and reaction time) was optimized in order to release monosaccharides in near quantitative yield. The results were compared to those obtained by (1) gas chromatography of the alditol acetates of the neutral sugars released by acid hydrolysis and (2) specific colorimetric determination of the acid-labile 3,6-anhydrogalactosyl residue. Conditions such as methanolic 0.125 M HCl for 1 h at 85 degrees C were sufficient to release all of the galactosidic and 3,6-anhydrogalactosidic bonds for iota carrageenan without apparent degradation of the anhydrogalactosyl unit. However, with the same conditions, the yields of 3,6-anhydrogalactosyl residues were 80 and 70% for kappa carrageenan and agarose, respectively. These yields were not improved by stronger conditions. At the opposite extreme, under very mild methanolysis conditions such as methanolic 0.01 M HCl at 100 degrees C for 1 h, agars and gelling carrageenans were well differentiated by the respective determination of agarobiose- and carrabiose-dimethyl acetal which are well-separated on octadecyl reversed phase HPLC columns with water as eluent.