Piotr Jarocki

A new insight into the physiological role of bile salt hydrolase among intestinal bacteria from the genus Bifidobacterium.

This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.

Genetic Diversity of Bile Salt Hydrolases Among Human Intestinal Bifidobacteria

This study analyzes the application of degenerative primers for the screening of bile salt hydrolase-encoding genes (bsh) in various intestinal bifidobacteria. In the first stage, the design and evaluation of the universal PCR primers for amplifying the partial coding sequence of bile salt hydrolase in bifidobacteria were performed. The amplified bsh gene fragments were sequenced and the obtained sequences were compared to the bsh genes present in GenBank. The determined results showed the utility of the designed PCR primers for the amplification of partial gene encoding bile salt hydrolase in different intestinal bifidobacteria. Moreover, sequence analysis revealed that bile salt hydrolase-encoding genes may be used as valuable molecular markers for phylogenetic studies and identification of even closely related members of the genus Bifidobacterium.

Comparison of various molecular methods for rapid differentiation of intestinal bifidobacteria at the species, subspecies and strain level.

BackgroundMembers of the genus Bifidobacterium are anaerobic Gram-positive Actinobacteria, which are natural inhabitants of human and animal gastrointestinal tract. Certain bifidobacteria are frequently used as food additives and probiotic pharmaceuticals, because of their various health-promoting properties.Due to the enormous demand on probiotic bacteria, manufacture of high-quality products containing living microorganisms requires rapid and accurate identification of specific bacteria. Additionally, isolation of new industrial bacteria from various environments may lead to multiple isolations of the same strain, therefore, it is important to apply rapid, low-cost and effective procedures differentiating bifidobacteria at the intra-species level. The identification of new isolates using microbiological and biochemical methods is difficult, but the accurate characterization of isolated strains may be achieved using a polyphasic approach that includes classical phenotypic methods and molecular procedures. However, some of these procedures are time-consuming and cumbersome, particularly when a large group of new isolates is typed, while some other approaches may have too low discriminatory power to distinguish closely related isolates obtained from similar sources.ResultsThis work presents the evaluation of the discriminatory power of four molecular methods (ARDRA, RAPD-PCR, rep-PCR and SDS-PAGE fingerprinting) that are extensively used for fast differentiation of bifidobacteria up to the strain level. Our experiments included 17 reference strains and showed that in comparison to ARDRA, genotypic fingerprinting procedures (RAPD and rep-PCR) seemed to be less reproducible, however, they allowed to differentiate the tested microorganisms even at the intra-species level. In general, RAPD and rep-PCR have similar discriminatory power, though, in some instances more than one oligonucleotide needs to be used in random amplified polymorphic DNA analysis. Moreover, the results also demonstrated a high discriminatory power of SDS-PAGE fingerprinting of whole-cell proteins. On the other hand, the protein profiles obtained were rather complex, and therefore, difficult to analyze.ConclusionsAmong the tested procedures, rep-PCR proved to be the most effective and reliable method allowing rapid differentiation of Bifidobacterium strains. Additionally, the use of the BOXA1R primer in the differentiation of 21 Bifidobacterium strains, newly isolated from infant feces, demonstrated slightly better discriminatory power in comparison to PCR reactions with the (GTG)5 oligonucleotide. Thus, BOX-PCR turned out to be the most appropriate and convenient molecular technique in differentiating Bifidobacterium strains at all taxonomic levels.

Enterobacter sp. LU1 as a novel succinic acid producer - co-utilization of glycerol and lactose.

Succinic acid is an important C4‐building chemical platform for many applications. A novel succinic acid‐producing bacterial strain was isolated from goat rumen. Phylogenetic analysis based on the 16S rRNA sequence and physiological analysis indicated that the strain belongs to the genus Enterobacter. This is the first report of a wild bacterial strain from the genus Enterobacter that is capable of efficient succinic acid production. Co‐fermentation of glycerol and lactose significantly improved glycerol utilization under anaerobic conditions, debottlenecking the utilization pathway of this valuable biodiesel waste product. Succinic acid production reached 35 g l−1 when Enterobacter sp. LU1 was cultured in medium containing 50 g l−1 of glycerol and 25 g l−1 of lactose as carbon sources.

Media optimization for economic succinic acid production by Enterobacter sp. LU1.

Enterobacter sp. LU1 could efficiently convert glycerol to succinic acid under anaerobic conditions after the addition of lactose. In this study, media constituents affecting both Enterobacter sp. LU1 biomass and succinic acid production were investigated employing response surface methodology (RSM) with central composite design. Statistical methods led to the development of an efficient and inexpensive microbiological media based on crude glycerol, whey permeate as carbon sources and urea as a nitrogen source. The optimized production of bacterial biomass in aerobic conditions was predicted and the interactive effects between crude glycerol, urea and magnesium sulfate were investigated. As a result, a model for predicting the concentration of bacterial biocatalyst biomass was developed with crude glycerol as a sole carbon source. In addition, it was observed that the interactive effect between crude glycerol and urea was statistically significant. Response surface methodology was also employed to develop the model for predicting the concentration of succinic acid produced. Validity of the model was confirmed during verification experiments wherein actual results differed from predicted values by 0.77%. The applied statistical methods proved the feasibility for anaerobic succinic acid production on crude glycerol without expensive yeast extract addition. In conclusion, the RSM method can provide valuable information for succinic acid scale-up fermentation using Enterobacter sp. LU1.