I. Koppová

The antimicrobial action of low-molar-mass chitosan, chitosan derivatives and chitooligosaccharides on bifidobacteria

The crude fractions of chitooligosaccharides (COS) and low-molar-mass chitosans (LMWC) were prepared by enzyme hydrolysis of chitosan (CS). Specific growth rate of B. adolescentis, B. bifidum, B. breve, B. catenulatum, B. infantis and B. longum ssp. longum was determined in the presence of 0.025 and 0.5 % COS (<5 kDa), LMWC (5–10 kDa), and 0.025, 0.1 and 0.5 % of CS, chitosan succinate and chitosan glutamate in vitro. Minimum inhibitory concentrations (MIC; assayed by colony counting on TPY agar plates) of COS-LMWC and CS ranged from 0.025 % to 0.75 % of CS-LMWC. The growth of all bifidobacterial strains in the presence of chitosan, its derivatives and LMWC decreased at a concentration of 0.025 %; the bacterial growth was completely inhibited at a concentration of 0.5 %. COS did not show any inhibitory effect, an increased growth rate was even observed in the case of B. bifidum, B. catenulatum and B. infantis.

Postnatal development of bacterial population in the gastrointestinal tract of calves

Bacterial 16S rDNA from fecal samples of two calves were amplified by PCR and analyzed by denaturing gradient gel electrophoresis; selected bands were sequenced.Escherichia coli andBifidobacterium animalis were the initial colonizers, followed by species closely related to the generaBacteroides, Clostridium andFaecalibacterium. Change of diet was connected with shifts of bacterial population and with the occurrence of many bacterial species that have not been cultured up to now. The diet change corresponded with an alteration in a volatile-fatty-acid concentration in fecal samples.

PCR-DGGE-based study of fecal microbial stability during the long-term chitosan supplementation of humans.

A feeding study was performed to monitor the effect of chitosan intake on the fecal microbiota of ten healthy human subjects. Diversity of microflora was monitored during 8 weeks including 4 weeks of chitosan supplementations. Using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons and quantitative PCR method we revealed possible changes originating in the overall bacterial composition and also in the subpopulation of Bifidobacterium group. DGGE profiles displayed high complexity and individuality for each subject. Considerable variations in the composition of band patterns were observed among different persons. A raised level of fecal Bacteroides in response to chitosan intake was found in all samples. Bifidobacterium levels following chitosan intake increased or remain unchanged. Non-significant increase was, surprisingly, found in the numbers of butyrate-producing bacteria.

Effect of fatty acids on growth of conjugated-linoleic-acids-producing bacteria in rumen

Microorganisms with high activity of linoleic acid Δ12-cis,Δ11-trans-isomerase were isolated from the digestive tract of ruminants and characterized. The isolate with the highest isomerase activity was identified asPseudobutyrivibrio ruminis. The susceptibility of this strain to 3 fatty acids added to the grow medium was determined. A significant inhibition of bacterial growth (during a 3-d period) by linoleic acid (0.1 %) and oleic acid (5 ppm) was observed; no inhibition was found in the presence of stearic acid.

Chitinolytic activities of Clostridium sp. JM2 isolated from stool of human administered per orally by chitosan

The novel chitinolytic bacterium Clostridium beijerinckii strain JM2 was isolated from the stool of healthy volunteers supplied daily per orally with 3 g of chitosan. The bacterium grown on colloidal chitin produced a complete array of chitinolytic enzymes. Significant activities of endochitinase, exochitinase and chitosanase were excreted into the medium (301, 282 and 268 nkat/μg protein, respectively). The high cellular activity of N-acetyl-β-glucosaminidase (NAGase) and chitosanase were detected (732.4 and 154 nkat/μg protein, respectively). NAGase activity represented the main activity associated with the cellular fraction. The activities of both enzymes tested increased from 20 to 50 °C; the optimum reaction temperature estimated being 50 °C. Endochitinase as well as NAGase showed an activity in the pH interval of 4.0–8.0; the optimum pH values were 6.5 and 6.0, respectively. The extracellular endochitinase complex consisted of six isoenzymes with molar mass of 32–76 kDa; in the cellular fraction five bands with molar mass of 45–86 kDa were detected. Exochitinase activity was demonstrated in the form of three bands (with molar mass of 30–57 kDa), NAGase activity displayed one band of 45 kDa.

Excretome of the chitinolytic bacterium Clostridium paraputrificum J4

A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-d-glucosaminide, N,N´-diacetyl-β-d-chitobiose, or N,N´,N˝-triacetyl-β-d-chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.

Analysis of fatty acid composition of anaerobic rumen fungi

The fatty acid (FA) composition of fresh mycelia of anaerobic rumen fungi was determined. The fatty acids methyl esters (FAME) of six strains belonging to four genera (Neocallimastix, Caecomyces, Orpinomyces, Anaeromyces) and one unknown strain were analyzed by gas chromatography. All studied fungi possess the same FAs but differences were found in their relative concentrations. The FA profile of anaerobic fungi comprises carbon chains of length ranging from 12 to 24; the most common fatty acids were stearic (C18:0), arachidic (C20:0), heneicosanoic (C21:0), behenic (C22:0), tricosanoic (C23:0) and lignoceric (C24:0) with relative amount representing >4% of total FA. Significant differences were determined for heptadecanoic, oleic, behenic and tricosanoic acids. Rumen anaerobic fungi can contain very long chain fatty acids; we found unsaturated fatty acids including cis-11-eicosenoic (C20:1), cis-11,14-eicosadienoic (C20:2), erucic (C22:1n9), cis-13,16-docosadienoic (C22:2) and nervonic (C24:1) acids in very small amounts but their presence seems to be unique for anaerobic fungi.

Extracellular complex of chitinolytic enzymes of Clostridium paraputrificum strain J4 separated by membrane ultrafiltration

Membrane diafiltration was used for separation of the extracellular complex of chitinolytic enzymes of C. paraputrificum J4 free from contaminants with molar mass higher than 100 kDa and lower than 30 kDa. The enzyme complex containing β-N-acetylglucosaminidase (NAGase) and six endochitinases was concentrated on a membrane with cut-off 30 kDa. In this retentate, the NAGase/endochitinase specific activity was 13.5/6.5-times higher than in the initial culture filtrate. The proportion (in %) of endochitinases: 23 (90 kDa), 42 (86 kDa), 8 (72 kDa), 16 (68 kDa) and 8 (60 kDa) was calculated from their peak areas (determined by densitometry) in images of zymograms. NAGase (38 kDa) was less active and stable at pH lower than 4 and higher than 8 but it was more temperature-stable than endochitinases, especially at 40–60 °C. In contrast to endochitinases, the pH optimum of NAGase activity was shifted by ca. 0.7 pH units to the alkaline region. Extracellular NAGase together with six endochitinases secreted by C. paraputrificum J4 were separated by membrane diafiltration and characterized by molar mass, stability and activity in dependence on pH and temperature. The knowledge of composition of chitinolytic enzymes, their pH and temperature stability is useful for optimization of the separation process.