Effect of chitosan on the growth of human colonic bacteria.

Growth of 6 bacterial strains representing dominant members of the human colonic microflora was measured in the presence of 0.025, 0.05 and 0.5 % chitosan (from shrimp shells, with a 97 % final degree of deacetylation). The effect of chitosan was variable and dependent on bacterial species. The most susceptible to chitosan were bacteria belonging to genera Bacteroides and Clostridium (91-97% growth inhibition). On the other hand, Roseburia sp., Eubacterium sp. and Faecalibacterium sp. were more resistant (63-83 % inhibition of growth). Chitosan can thus be considered as one of the means for influencing the bacterial population in the human colon.

Classical and molecular approaches as a powerful tool for the characterization of rumen polycentric fungi.

Ribosomal ITS1 and ITS2 fragments from 8 isolates of polycentric rumen anaerobic fungi were PCR-amplified and sequenced; the sequences obtained were aligned with published data and phylogenetic analyses were performed. Analysis of the ITS1 fragment clearly differentiated between the two polycentric genera Orpinomyces and Anaeromyces and this classification is supported by morphological observation. A multi-order phylogram based on ITS2 sequences proved that anaerobic rumen fungi are separated from aerobic chytrids, which form a well-supported monophylum with the highest possible bootstrap proportion values of 100%. Sequence analysis of ITS regions is a powerful tool for classification of anaerobic fungi but morphological description of strains is still necessary because some genera of rumen fungi display a high genetic heterogeneity.

Chitinolytic enzymes from Clostridium aminovalericum: activity screening and purification.

A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.

Identification and characterization of Clostridium paraputrificum, a chitinolytic bacterium of human digestive tract.

A strictly anaerobic, mesophilic and chitinolytic bacterial strain was isolated from human feces. Based on morphological and physiological properties and 16S rRNA sequence analysis the strain was identified as Clostridium paraputrificum. The strain utilized chitin and N-acetyl-D-glucosamine, grew on glucose and hydrolyzed starch. Cultivation of the strain with colloidal chitin as the growth substrate resulted in the production of gas (hydrogen and carbon dioxide) and formation of acetate and lactate (21.6 and 18.9 mmol/L, respectively) and only small quantities of propionate and butyrate (1.7 and 2.6 mmol/L, respectively). In the course of a 10-d cultivation with chitin, the endochitinase activity was detected after 1 d and gradually increased, reaching maximum after 3 d (251 nkat/L N-acetyl-D-glucosamine). The beta-N-acetylglucosaminidase activity appeared just at the beginning of the cultivation, increased to day 2 and then remained nearly constant. More than 90% of chitin added was degraded within 2 d of cultivation. On the zymogram of the extracellular chitinolytic complex were visible at least 6 isoenzymes with molar mass 43.5-65.0 kDa. The temperature optimum of endochitinase and beta-N-acetylglucosaminidase activities was 50 degrees C; the optimum activity of both enzymes was found at pH 4-6.

Chitinolytic bacteria of the mammal digestive tract.

Chitinolytic bacteria were isolated from the digestive tract of different mammals and characterized. All isolates were facultatively anaerobic, long Gram-positive, straight rods resembling Clostridium sp. Only one isolate consisted of Gram-positive ovoid cells. All cultures grew on glucose, N-acetylglucosamine, glucosamine, galactose, starch, hemicellulose and xylan. Fermentation products were mainly formate, acetate, butyrate and lactate. The isolates were identified as Clostridium sartagoforme (2 species), C. aminovalericum, C. bifermentans and Enterococcus durans (1 isolate of each species). Exocellular fractions of all strains exhibited higher activities of all enzymes than cellular ones. Inductive effects of hemicelluloses, pectin and laminarine on chitinases were demonstrated. High exocellular endochitinase activity was found in cultures grown on chitin. N-Acetylglucosaminidase activity was low with the exception of exocellular fractions of two strains of C. sartagoforme.

Chitinolytic enzymes produced by ovine rumen bacteria.

Two strains of clostridia, isolated from the rumen fluid of sheep as potential antagonists toward anaerobic fungi showed a complete array of chitinolytic enzymes. Enzyme tests in cultures demonstrated endochitinase, exochitinase, N-acetyl-glucosaminidase, chitosanase and chitin deacetylase activities mainly in the extracellular fractions. In all samples, the highest was the activity of exochitinase (600-1100 nmol mL-1 h-1); the activity of endochitinase (280-500 nmol mL-1 h-1) was also significant. Chitinases were stimulated in the presence of reducing compounds and no dependence on cations was observed. In both strains different isoforms of chitinases of molar mass 36-96 kDa were detected. The chitinases from our isolates lyzed cell walls of anaerobic fungi in vitro and inhibited the activity of fungal beta-1,4-endoglucanases. Of the two bacteria examined, one was more effective in both antifungal effects.

Cellulolytic enzymes of rumen anaerobic fungi Orpinomyces joyonii and Caecomyces communis.

The rumen anaerobic fungi Orpinomyces joyonii A4 and Caecomyces communis JB1 were grown on microcrystalline cellulose (MC) and alfalfa hay. The cellular distribution of cellulases produced by these organisms was monitored. Fungal cultures were separated into extracellular, intracellular and cell wall fractions and assayed for endoglucanase (EG) and beta-glucosidase activity. In both fungal isolates, EG activity was the highest in the extracellular fraction regardless of the substrate used. The beta-glucosidase activity produced by O. joyonii was mainly found in the cell wall fraction. On the contrary, the same enzyme activity in C. communis predominated in the extracellular fraction. The polycentric isolate A4 more efficiently utilized both substrates, produced more short chain fatty acids (up to 31 mmol/l) and showed higher total levels of EG (2744 nmol glucose/h/ml) than the monocentric strain JB1. On the other hand, beta-glucosidase (9033 nmol glucose/h/ml) activity was the highest in cultures of C. communis grown on cellulose. In cultures of O. joyonii grown on MC, the production of yellow affinity substance (YAS) with similar properties compared with yellow substance from Clostridium thermocellum was observed. This compound increased the adsorption of fungal cellulases to MC the temperature and pH range tested.

The effect of yellow affinity substance on cellulases of Ruminococcus flavefaciens.

Cellulolytic cultures of Ruminococcus flavefaciens produced a yellow affinity substance (YAS) with a strong affinity to microcrystalline cellulose (MC). YAS was bound to MC in the range of pH from 5 to 8 and at temperatures from 10 degrees C to 60 degrees C. The positive effect of YAS on adsorption of ruminococcal cellulases was demonstrated by comparing the adsorption behaviour of endoglucanases and cellobiohydrolases onto MC and YAS-treated MC. HPLC chromatography proved the presence of two yellow compounds with affinity to cellulose as well as to ruminococcal cellulases. Both YAS compounds were sensitive to oxidation. The observed YAS properties showed a close relation to YS of Clostridium thermocellum.

The isolation and characterization of a rumen chitinolytic bacterium.

Chitinolytic bacteria were detected in faeces and digesta of wild and domesticated herbivores. The presence of chitinolytic bacteria in two cows was verified following enrichment culture of rumen fluid on colloidal chitin. In three other cows, direct counts on chitin agar showed that the numbers of these bacteria in the rumen fluid ranged from 5 x 10(4) to 2 x 10(8) ml-1. Most of these bacteria were Clostridium-like spore producers. The most typical strain, Clostridium sp. ChK5, was characterized further. This bacterium degraded colloidal chitin and produced mainly acetate, butyrate and lactate. Endochitinase and chitobiase were produced when chitin was the growth substrate. Endochitinase was also detected in cultures grown on N-acetylglucosamine and glucose. Optimal conditions for endochitinase activity were 37 degrees C and pH 4.5-6.1. The Michaelis constant (Km) for this enzyme was 19.3 mg ml-1. Strain ChK5 shows strong phenotypic similarity to Clostridium tertium.

The effect of rumen chitinolytic bacteria on cellulolytic anaerobic fungi.

The polycentric anaerobic fungus Orpinomyces joyonii A4 was cultivated on microcrystalline cellulose alone and in association with the rumen chitinolytic bacterium Clostridium sp. strain ChK5, which shows strong phenotypic similarity to Clostridium tertium. The presence of strain ChK5 significantly depressed the solubilization of microcrystalline cellulose, the production of short-chain fatty acids (SCFA) and the release of endoglucanase by the fungus. Co-culture of the monocentric anaerobic fungus Neocallimastix frontalis strain RE1, Neocallimastix sp. strain G-1 and Caecomyces sp. strain SC2 with strain ChK5 also resulted in depressed fungal cellulolysis. Cell-free supernatant fluids from strain ChK5 inhibited the release of reducing sugars from carboxymethylcellulose by cell-free supernatant fluids from O. joyonii strain A4. Strain 007 of the cellulolytic anaerobe Ruminococcus flavefaciens was also shown to produce small amounts of soluble products upon incubation with colloidal chitin. Mixtures of culture supernates from this bacterium and from O. joyonii strain A4 showed cellulase activity that was less than that of the component cultures. It is suggested that the ability of some rumen bacteria to hydrolyse or transform chitin may be an important factor in the interactions between bacteria and fungi in the rumen.

Interaction of the rumen fungus Orpinomyces joyonii with Megasphaera elsdenii and Eubacterium limosum.

The degradation and fermentation of microcrystalline cellulose were studied in monoculture of the polycentric anaerobic fungus Orpinomyces joyonii and in co-cultures with the rumen bacteria Megasphaera elsdenii and Eubacterium limosum. More than 25% of cellulose hydrolysis products (glucose and cellodextrins) were released by the fungus into the medium after 8 d of cultivation. These products were metabolized by bacteria in mixed cultures. In co-culture with the fungus M. elsdenii and E. limosum increased the extent of microcrystalline cellulose degradation by 10.12% and 7.96%, respectively. Biomass yield in co-cultures was increased by 89.9% and 59.4% for M. elsdenii and E. limosum. Y cellulose for fungus alone was 52.29 g dry matter mol-1 glucose. These values were 64.93 and 55.92 g mol-1 glucose unit in co-culture with M. elsdenii and E. limosum, respectively.

Pectinolytic enzymes of anaerobic fungi.

Pectinolytic enzymes of four rumen fungi have been described. Three fungal species were monocentric Neocallimastix spp. H15, JL3 and OC2, and one isolate was a polycentric strain of Orpinomyces joyonii, A4. They differed in degree of pectin degradation and utilization. Only the strain Neocallimastix sp. H15 and partially Orpinomyces joyonii A4 were able to utilize pectin to a higher extent. The most important pectinolytic activity in all these isolates represented pectin lyase (EC 4.2.2.10) and polygalacturonase (EC 3.2.1.15). Their specific activities were in the range of 100-900 and 10-450 micrograms galacturonic acid h-1 mg protein-1 for pectin lyase and polygalacturonase, respectively. Polygalacturonase, located mainly in the endocellular fraction, was inhibited by calcium ions and had the main pH optimum at pH 6.0. All strains produced pectate lyase (EC 4.2.2.2). None of the strains tested produced pectinesterase (EC 3.1.1.11).