Folia Microbiol. 48 (3), 339–345 (2003)
http://www.biomed.cas.cz/mbu/folia/
Is Pseudobutyrivibrio xylanivorans Strain Mz5T Suitable as a Probiotic? An in Vitro Study T. ČEPELJNIKa, M. ZORECa, R. KOSTANJŠEKb, F.V. NEKREPa, R. MARINŠEK-LOGARa* aZootechnical Department, Biotechnical Faculty, University of Ljubljana, 1230 Domžale, Slovenia fax +386 1 7241 005 e-mail
[email protected] bDepartment of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia Received 30 September 2002
ABSTRACT. Rumen bacterium Pseudobutyrivibrio xylanivorans strain Mz5T possessed a potent xylanolytic enzyme system consisting of at least 7 different xylan hydrolases with molar mass 27–145 kDa. Three of them were successfully isolated in active native form. This strain produced butyrate and lactate on different saccharides. cis-9,trans-11-Conjugated linoleic acid was also detected in the culture medium. Bacteriocin-like inhibitory substances of Mz5T were active against some strains of rumen bacteria and against selected Salmonella and E. coli isolates from poultry meat. The strain Mz5T retained viability and xylanolytic activity also under not fully anaerobic conditions; its cells attached to the Caco-2 cells so that its successful association with gut epithelial cells may be expected. These in vitro results confirmed several probiotic traits of the isolate Mz5T and justified further in vivo experiments to test its ability to improve animal health and performance.
During the last decades the use of growth promoters was the usual practice in extensive animal production. Low doses of antibiotics kill a great proportion of pathogens but still allow the growth of beneficial microflora. The net effect is a reduced loss of feed intake and prevention of bacterial toxin production. Nevertheless, in the last years the use of growth promoters has been discouraged and much effort has been directed toward the search for new alternatives (e.g., Morovský et al. 2001). Enhanced digestion can be achieved by other feed additives that efficiently contribute to the forage degradation (e.g., hydrolytic enzymes) or influence the growth of pathogens (e.g., organic acids, bacteriocins, probiotics). Furthermore, the quality of the final product (meat, milk, eggs) can also be improved. Many animals depend on symbiotic microflora that colonizes the gastrointestinal tract (GIT) soon after birth and later establish stable association with the animal host. Gastrointestinal microflora is believed to contribute to healthy gut function in a variety of ways, including protection against pathogens and production of nutrients for the colonic mucosa. Short-chain fatty acids (SCFA), butyrate in particular, formed by microbial fermentation have important role in the metabolism and normal development of colonic epithelial cells. Butyrate is also implicated in protection against colon cancer and ulcerative colitis (Engelhart et al. 1998) and, together with glutamine, represents the major energy substrate for colonocytes in pigs, rats and humans. Through butyrate production the intestinal flora can even control the expression of target genes involved in the metabolism of epithelial colonic cells (Cherbuy et al. 2001). Butyrate production by diverse microflora is strongly influenced by feed composition (Topping and Clifton 2001). The most complex microbial populations and highest hydrolytic activities have been found in the rumen ecosystem. The isolates belonging to the genus Butyrivibrio represent a significant proportion (10– 30 %) of culturable rumen bacteria in domestic and wild ruminants (Stewart et al. 1997). Phylogenetically they represent a very heterogeneous group and strong indications for the reclassification were presented (e.g., Mrázek and Kopečný 2001; for review see Čepeljnik and Marinšek-Logar 2002). Recently, two new species were established – Butyrivibrio hungatei and Pseudobutyrivibrio xylanivorans (Kopečný et al. 2001, 2003). The Butyrivibrio strains possess many traits that could potentially have beneficial effects in the gut environment. They produce butyrate on different substrates and synthesize many hydrolytic enzymes (e.g., xylanases; Zorec et al. 2001) important for efficient fiber digestion (Stewart et al. 1997). Less favored fibrous and waste plant biomass could be fed if highly effective fibrolytic bacteria were present in GIT or if isolated enzymes were added to the forage (Bedford 2000). Bacteria of Butyrivibrio and Pseudobutyrivibrio *Corresponding author.
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genera produce bacteriocins. Use of these substances has become common in extending shelf life as well as for controlling pathogenic bacteria (Kalmokoff and Teather 1997). It is believed that Butyrivibrio strains are among the few rumen bacteria capable of forming conjugated linoleic acid (CLA) during the biohydrogenation of linolenic acid (Kepler et al. 1966). CLA exhibits anticancerogenic effects, reduces the development of atherosclerosis and may regulate energy metabolism and nutrient partitioning resulting in reduction of body fat and thus enhancing lean body mass (Pariza et al. 1999). Here we report on in vitro investigation of the probiotic traits of our isolate Pseudobutyrivibrio xylanivorans Mz5T.
MATERIALS AND METHODS Pseudobutyrivibrio xylanivorans strain Mz5T (DSM 14809; Fig. 1) was isolated from the rumen of Holstein-Friesian cow fed 2 kg compound food (180 g crude protein per kg; Mešalnica Homec, Slovenia) twice a day and meadow hay ad libitum. During the isolation procedure the selection for active fiber degrading bacteria was accomplished with the addition of oat spelt xylan (Sigma) in the growth medium as the main carbon source. Bacteria were grown at 37 °C under anaerobic conditions in a medium without rumen fluid (Deutsche Sammlung von Mikroorganismen und Zellkulturen 2002a; DSMZ medium 330) where all saccharides were replaced by oat spelt xylan at a final concentration of 0.5 % (or 0.5 % glucose for the induction studies). In order to follow enzyme activity and product concentration, samples were taken from cultures at defined time intervals. Cells were harvested by centrifugation (50 Hz, 10 min, 4 °C). Pellets were washed twice in sodium phosphate buffer (50 mmol/L; pH 6.5). To test the ability of this strain to grow at “semianaerobic” conditions, the media were not reduced completely. Quantitative xylanolytic activity was determined according to Lever (1977) and expressed as µkat/g (µmol reducing sugars released per s per g protein). Comparative activity testing was done by the most active xylandegrading rumen bacteria – Prevotella bryantii, Butyrivibrio fibrisolvens, Fibrobacter succinogenes and Ruminococcus albus (Marinšek-Logar et al. 2000). Xylanases were detected on zymograms (xylanograms) after SDS-PAGE (Laemmli Fig. 1. Pseudobutyrivibrio xylanivorans strain Mz5T; on a slightly 1970), where xylan (0.2 %) was incorporated in curved rod is seen one subpolar flagellum; ×30 150. the electrophoresis gel. Following electrophoresis, gels were renatured, incubated for 4 h at 37 °C and finally stained with Congo Red (Béguin 1983). Xylanases were visualized after destaining as clearing zones. Molar mass (M) markers (SDS-6H; Sigma) were stained with Coomassie Brilliant Blue R-250 (Wong et al. 2000). Xylanases were isolated from cell extract according to Čepeljnik et al. (2002). Excreted xylanases were isolated from 1.6 L of cell-free spent culture medium after 2 d of incubation. The first isolation step was fractional precipitation (60–80 % saturation) with diammonium sulfate followed by ultrafiltration through a 30-kDa cut-off ultrafilter (Sartorius, Germany) and hydrophobic interaction chromatography on HiLoad 16/10 Phenyl Sepharose column (Pharmacia, Sweden) by linear gradient from 1.7 mol/L to zero concentration of diammonium sulfate in 50 mmol/L sodium phosphate buffer (pH 6.5). Fermentation products (SCFA) were analyzed by GC following double diethyl ether extraction of culture supernatants (Holdeman et al. 1977) on Shimadzu GC-14A chromatograph equipped with FID detector and Supelcowax 10 capillary column (Supelco). Six-h cultures of strain Mz5T were screened for bacteriocin-like inhibition of different bacteria by agar-well diffusion method using 10 µL of culture extract (Klaenhammer 1988). The inhibitory effects against anaerobic rumen bacteria (Prevotella bryantii B14, P. brevis GA33, Butyrivibrio fibrisolvens ATCC 19171, Fibrobacter succinogenes S85 and Selenomonas ruminantium DSM 2150) were tested in anaerobic glove
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box (Scholzen Technik, Germany) at 37 °C. The inhibitory effects against selected strains of Salmonella (30/39 VF, S-200/90 VF, S-22-95 VF) and E. coli (II/6, III/5) were tested aerobically at temperatures suitable for the indicator strain. In both cases the isolates were from poultry meat (gift from Veterinary Faculty, University of Ljubljana, Slovenia). The DNAase nature of Mz5T bactericidal activity was tested on salmon sperm DNA plates (Flint and Thomson 1990). Production of cis-9,trans-11 CLA isomer was determined in 1-d cultures grown in M2 medium (Hobson 1969). Cultures were concentrated by centrifugation (50 Hz, 10 min) and subjected to methylation and extraction into hexane following the procedure of Park and Goins (1994). Extracts were analyzed by the HP-6890 GC system equipped with capillary column Supelco 2-4152 (Omegawax 320, fused silica column) and FID. Helium was used as carrier gas (flow 1.5 mL/min); hydrogen (30 mL/min) and air (400 mL/min) were used as detector gases, temperature program 170–215 °C being applied. The cis-9,trans11 CLA isomer was detected and quantified according to the standard solution of CLA isomers CLA 9c,11t (no. 4036; Natural ASA, Norway) and using the internal standard method. Adhesion of P. xylanivorans Mz5T was studied on fully differentiated model epithelial Caco-2 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen 2002b; ACC 169). Caco-2 cells were cultured in 24-well tissue culture plates, with average number 4.8 × 105 cells per well, and then an adhesion test was performed in an anaerobic glove box. Approximately 140 cells in mid-exponential growth phase were added directly into 24-cell culture wells per 1 Caco-2 cell. After a 30-min incubation, Caco-2 cells were lysed with 50 ppm Triton X-100 for 10 min. The attached bacteria were quantified by direct counting in a Petroff– Hausser counting chamber (EMS, USA).
RESULTS AND DISCUSSION P. xylanivorans Mz5T showed good growth in xylan-containing medium under anaerobic conditions. The culture reached the mid-exponential phase after 10 h and entered the stationary phase after 20 h (Fig. 2). It was believed that Butyrivibrio and Pseudobutyrivibrio strains were strict anaerobes but our observations revealed that this strain was capable of growing in a medium where the redox potential was above –100 mV. The growth and clarification of culture medium, which is the result of xylan degradation, was observed also in not fully reduced growth medium (qualitative observation; data not shown). This would be very important when feeding the strain Mz5T to the animal, because it will retain viability after exposure to air. We propose the encapsulation of the microbial culture in mid-exponential phase (see below) for feeding purposes (cf. Mattila-Sandholm et al. 2002, who suggested similarly for lactic acid bacteria). Fig. 2. Growth of strain Mz5T in xylan-containing medium; circles – protein concentration (g/L), triangles – xylanolytic activity (µkat/g).
Table I. Inductiona of xylanolytic activity (µkat/g)
Time, h
Xylanases of strain Mz5T showed to be highly inducible by oat spelt xylan (Table I) which is in agreement with the fact that the majority of xylanases are substrate-inducible (Williams and Whiters 1992). The inducibility was most strongly expressed after 12 h of growth (140-fold higher xylanolytic activity of the culture grown on xylan compared with glucose-repressed expression of xylanases).
10 12 24 72
Xylanolytic activity Xln
Glc
36.5 33.4 19.4 6.35
0.31 0.24 0.20 0.13
Xln/Glc
118.8 140.0 95.3 48.2
aThe factor of induction (Xln/Glc) was calculated as
a ratio between specific xylanolytic activity measured in cells grown on xylan (Xln) and on glucose (Glc) as the main carbon source, respectively.
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The high xylanolytic activity of strain Mz5T was detected already when it was isolated and was shown to be at least 1.7 times higher than of any other tested rumen xylanolytic bacterium (Marinšek-Logar et al. 2000). The xylanolytic activity of strain Mz5T was time- and substrate concentration-dependent which could have functional implications. Emami et al. (2002) found out that the five xylanase genes from Pseudomonas cellulosa were temporally expressed, according to the function of a particular enzyme. Esterases removing acetate from the xylanase backbone were transcribed before other xylanolytical enzymes that attacked deacetylated xylan. In our case the overall peak activity (61.1 µkat/g) was reached after 10 h (in the midexponential growth phase) and gradually decreased (30.5 µkat/g) until the beginning of the stationary phase. It was still detectable after 9 d (3.31 µkat/g). These results suggest the existence of highly resistant xylanases to low pH and/or proteinases that occur in the later growth phases. Resistance of the enzymes to harsh conditions during the passage through GIT is advantageous when they are used as feed additives (Inborr and Grönlund 1993). Strain Mz5T possesses at least 7 cell-bound xylanolytic enzymes; some of them were also secreted into the growth medium. In the cell-associated fraction often up to 14 clearing zones were detected on the xylanograms (Fig. 3) which confirms a high xylanolytic potential of the isolate. The molar mass of major xylanases were calculated to be approximately 145, 126, 111, 100, 77, 51 and 27 kDa. Multiple xylanases appear to be a common trait of ruminal bacteria (Malburg et al. 1993; Lin and Thompson 1991a). Previously we showed that the smallest, 27-kDa xylanase was the most stable and retained its activity in the later stationary phase (Zorec et al. 2000) when the pH decreased from 6.7 to 5.7. The 51- and 58-kDa xylanases were successfully isolated from a cell extract (Čepeljnik et al. 2002). Purification from the culture supernatant resulted in the isolation of 27-kDa xylanase with purification fold equal to 26.4 and yield of 0.21 % (Table II). Fig. 3. Multiple xylanases in culture broth of PseudoOnly two cloned xylanases had been isolated from butyrivibrio xylanivorans strain Mz5T after a 1- and 2-d bacteria of the genera Pseudobutyrivibrio and Butyriincubation; gels show clearing zones; left: cell-bound xylanvibrio (Lin and Thompson 1991b; Mannarelli et al. ases; right: excreted xylanases; M – protein molar mass markers (29–205 kDa). 1990). These results implicate the possible scale-up for xylanase purification and future application as feed additive, especially in the nutrition of monogastric animals like pigs and poultry. Xylanolytic enzymes, or microorganisms producing them, added to fodder like barley, oats and rye, reduce the viscosity of digesta (caused by high xylan content) and, in this way, could prevent malabsorption, diarrhea and other health problems. Up to date some isolated polysaccharidases have already found their use as feed additives for pigs and poultry resulting in better forage digestion and animal performance (Bedford 2000). For enhancing ruminal fiber digestion by microbial inoculation has been suggested to isolate native strains with exceptional fibrolytic ability (Krause et al. 1999); we assume Table II. Steps in artial purification of xylanases from cell extract (51 and 58 kDa) and supernatant (27 kDa) of strain Mz5T Purification step Cell extract Anion-exchange chromatography Gel filtration (51 kDa) Gel filtration (58 kDa) Supernatant Fractional precipitation Hydrophobic-interaction chromatography (27 kDa)
Specific activity µkat/g
Yield %
Purification factor
21.6 21.0 57.8 10.5
100 15.8 1.16 0.23
1.0 1.0 2.7 0.5
10.4 87.9 275
100 14.1 0.21
1.0 8.4 26.4
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that our isolate could be one of them. Further characterization of isolated xylanases (their stability, pH and temperature optimum) is to be determined. The production of SCFA in strain Mz5T summed up in net production of the most favorable compounds, butyrate and lactate, which rendered our bacterial isolate yet another promising probiotic potential. The strain Mz5T produced after a 1-d growth relatively high levels of butyrate (9 mmol/L) and lactate (3.6 mmol/L) (Fig. 4). During growth the concentration was further increased and reached a plateau after 6 d (butyrate 16.5 mmol/L, lactate 7.5 mmol/L). The strain utilized acetate and no production of succinate and propionate was detected. Butyrate has an important role in the metabolism and normal development of colonocytes and has also been implicated in protection against cancer and other intestinal diseases (Engelhart et al. 1998). The major part of intestinal butyrate is provided by butyrogenic microflora that can be selectively enriched with the use of prebiotics or introduced with appropriate probiotics (Jacobasch et al. 1999). P. xylanivorans Mz5Tshowed bacteriocin-like inhibition of rumen bacteria P. bryantii B14, P. brevis GA33 and B. fibrisolvens ATCC 19171T. Inhibition zones on agar plates covered by these bacteria were clearly detected. Bacteriocins are frequently produced by closely related Butyrivibrio strains (Kalmokoff and Teather 1997). P. brevis is Fig. 4. Butyrate (circles) and lactate (triangles) production a highly active proteolytic rumen species and hin(both mmol/L) by cultures of the strain Mz5T. dering its growth and proteolysis with the use of the strain Mz5T is an option. The growth of F. succinogenes S85 and S. ruminantium DSM 2150 was not affected by the presence of the strain Mz5T or its extracts. The most promising fact was the weak bacteriocin-like inhibition of several isolates of Salmonella enteritidis and E. coli (in both cases isolates from poultry meat). The DNAase nature of bacteriocinlike activity was clearly proved on salmon sperm agar plates, other possible mechanisms have not yet been tested. Favorable bacteriocin-like activity is another positive attribute of our probiotic candidate for its use as feed additive, as they also assure its competitive fitness (Riley and Gordon 1999). We were able to detect the production of 0.5 to 3 ng/mL of cis-9,trans-11 CLA in a 2-d culture. This result is encouraging as feeding such probiotic preparation to monogastric animals could result in meat (or egg) quality and diversity of available meat with CLA. Naturally CLA is only present in ruminant derived meat and products (Bauman et al. 2000). Adhesion of probiotic strains to the intestinal surface and the subsequent colonization of the gastrointestinal tract has been suggested as an important prerequisite for probiotic action. We investigated the in vitro adherence capacities of P. xylanivorans Mz5T using human cells Caco-2, a model cell line for human intestinal epithelium. Humans and pigs share close similarities in metabolic and digestive properties of GIT; hence the pig is a suitable model (Bogovič Matijašić et al. 2003). Cells of P. xylanivorans Mz5T attached well to Caco-2 cells in PBS at pH 7 in ratio 2.5 : 1. Under the same conditions the ratio between attached cells of positive control strain Lactobacillus rhamnosus GG (one of the best adhering Lactobacillus strain; Tuomola and Salminen 1998) to Caco-2 cells was 8.7 : 1. Although these in vitro results cannot be directly related to in vivo situation, there is evidence relating adhesion of some bifidobacteria strains to Caco-2 cells to the temporary colonization of human intestinal tract (Crociani et al. 1995). We suggest encapsulation for feeding purposes in order to protect it from harsh condition in the stomach. This manipulation could be satisfactorily easy, as we demonstrated that the strain Mz5T is not highly sensitive to oxygen. Our results demonstrate some extraordinary properties (high xylanolytic activity; butyrate, bacteriocin and CLA production) of the strain Mz5T that favor its use as probiotic bacterium. Isolated xylanases from Mz5T could be used as feed additive for more effective forage digestion and reducing health problems in monogastric animals. We are thankful to Ms. A. Levart for her kind help with CLA analysis. This work was supported by Ministry of Education, Science and Sport of the Republic of Slovenia and Municipality of Domžale (Slovenia).
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