枯草芽孢杆菌作为新的益生菌演讲稿.ppt

,New Bacillus subtilis Strains as Promising Probiotics,FRONTIERS IN IMMUNOLOGY,CONTENTS,01,02,03,04,INTRODUCTION,MATERIALS AND METHODS,RESULTS AND DISCUSSION,CONCLUSION,INTRODUCTION,Probiotics: live microorganisms positive effect on diverse functions of an organism prevent the invasion of various pathogens,Experiments with mice proved that B. subtilis spores were able to germinate, and the cells could proliferate and form the spores again in intestines of animals,Autochthonous lactic acid bacteria of the genera Lactobacillus and Bifidobacterium are usually used as probiotics; gram positive spore-forming bacteria of thegenus Bacillus are used as probiotics as well,Studies have revealed that in addition to the resistance of the probiotic Bacillus strains to bile acids, they were capable of immune stimulation in case of gastrointestinal disorders. Ability to synthesize antibiotics, bacteriocins, cyclic lipopeptides, and lytic enzymes with antimicrobial activity provides probiotic activity for bacteria of the genus Bacillus . Being probiotics, the strains of B. subtilis and B. coagulans were shown to have a growth-stimulating and prophylactic effect on broiler chickens,1,INTRODUCTION,标题,The goal of the present work was to characterize the properties of these new strains and to assess their potential use as probiotics.,01,INTRODUCTION,MATERIALS AND METHODS,The subjects of the study were B. subtilis strains GM2 and GM5 with high antimicrobial activity (Mardanova et al., 2017). Opportunistic coliform bacteria, Salmonella enterica serotype Typhimurium ATCC14028s, Klebsiella oxytoca, and Escherichia coli, as well as micromycetes Fusarium avenaceum, F. oxysporum, F. redolens, and F. solani, were used as test cultures.,02,MATERIALS AND METHODS,Nutrient media and cultivation conditions. The following media were used for cultivation. These were: (1) LB (LuriaBertoni) medium (g/L): tryptone, 10.0; yeast extract, 5.0; NaCl, 5.0; (2) LA medium(g/L): tryptone, 10.0; yeast extract, 5.0; NaCl, 5.0; agar, 20.0; (3) medium no. 1 (g/L): maltose, 20.0; peptone, 10.0; CaCl2, 1.0; (4) medium no. 2 (g/L): soybean meal, 30.0; NaNO3, 3.0; K2HPO4, 1.0; MgSO4, 0.2; KCl, 0.2; (5) medium no. 3 (g/L): corn extract, 20.0; lactose, 10.0; (6) deoxycholate citrate agar for salmonella cultivation. Bacteria were cultivated in a thermostat at 37C and using an IKAKS 4000 incubator shaker (Germany) at 37C and rotation speed of 200 rpm. Optical density of the culture was measured using a Bio-Rad spectrophotometer (United States) at a wavelength of 590 nm.,02,MATERIALS AND METHODS,The dynamics of bacterial growth and spore formation were studied on the LB medium and the medium no. 1. Bacteria were cultivated for 48 h using the incubator shaker (37C, 200 rpm). Bacterial culture samples were collected every 2 h in order to measure optical density (at the wavelength of 590 nm) using a spectrophotometer and to determine the extracellular proteolytic activity. The numbers of spores formed were counted in a Goryaev chamber (Optical Market, Ukraine), starting after 10 h of cultivation of bacteria. The bacteria were examined under a MICROS AUSTRIA MC 300 microscope (Austria).,02,MATERIALS AND METHODS,Ability to grow at various values of ambient pH was studied in 250-mL Erlenmeyer flasks containing 50 mL of the LB medium (pH 210). Bacteria were cultivated using the incubator shaker (37C, 200 rpm, aeration). Optical density of the cultures was determined after 24 h of growth.,Resistance of the spores to pH and spore capacity for in vitro germination were studied according to the method described (Haller et al., 2001). Spores of bacilli were obtained by heating a 2-day-old bacterial culture for 90 min at 60C to eliminate the vegetative cells. The initial concentration of the spores was 4 107 CFU/mL. The spore suspension in LB medium was incubated at various pH values at 40C. The spores were incubated at pH 5.0 for 60 min and concentrated by centrifugation (6000 rpm, 10 min). The supernatant was removed; the spore pellet was transferred into the same volume of the fresh LB medium (pH 3.0) and incubated for 90 min. An aliquot of the suspension (0.1 mL) was taken and heated for 90 min at 60C. A series of dilutions was prepared and plated to form a lawn on the LA medium for the subsequent enumeration of CFU/mL. The remaining spore suspension was concentrated by centrifugation, transferred to LB medium (pH 7.0), and incubated for 150 min. An aliquot (0.1 mL) was heated for 90 min at 60C and used to determine CFU/mL. To enumerate the vegetative cells after spore germination from the unheated suspension, a series of dilutions was prepared and plated onto the LA medium,02,MATERIALS AND METHODS,Resistance of bacterial spores to bile was investigated in vitro using the method described by Cenci et al. (2006). Bile of broiler chickens was used, which was sterilized by filtration through a sterile Millipore filter (0.22 m). The spores were introduced into the LB medium after addition of bile (1 and 10%) and adjusting pH to 7.5. The suspension of spores was incubated for 6 h at 40C and used to determine CFU/mL.,02,MATERIALS AND METHODS,Proteolytic activity in the bacterial liquid culture was determined by digestion of azocasein (Sigma, United States) (Demidyuk et al., 2006). Inhibitory analysis was performed in the presence of 1,10- phenanthroline (Serva, Germany), a specific inhibitor of metalloproteinases, and PMSF (Serva, Germany), a specific inhibitor of serine proteinases. The inhibitors (1.5 mM) were added to the liquid culture of bacilli, incubated for 1 h at room temperature, and residual activity was determined by hydrolysis of azocasein. The activity was expressed as a percentage relative to the control (a similar activity without inhibitors).,02,MATERIALS AND METHODS,Phytate-hydrolyzing activity was determined using a selective nutrient medium for phytate-degrading bacteria, phytase screening medium (PSM), according to Mittals technique (Mittal et al., 2011). Ability to hydrolyze sodium phytate was assessed by the presence of zones of hydrolysis around bacterial colonies.,Determination of virulence, toxicity, and toxigenicity of bacteria was carried out in mice of both sexes of the ICR (CD-1) line at the Laboratory of Chemical and Biological Research of the Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences. Mice were housed under the standard vivarium conditions; the standard granulated combined feed was used. For each experimental group, six mice of the same age weighing 16 0.5 g were selected.,02,MATERIALS AND METHODS,Antagonistic activity of B. subtilis strains GM2 and GM5 against test cultures was determined by the methods of wells, double-layer agar, and blocks (Egorov, 2004). The level of antagonistic activity of bacteria and exometabolites was judged by the diameter of zones of inhibition of the test culture growth around the wells containing the liquid culture or the colonies of antagonistic bacteria.,RESULTS AND DISCUSSION,Dynamics of bacterial growth, proteinase biosynthesis, and sporulation .,Resistance to the GIT acids and bile,Ability of spores to germinate at different pH values in vitro,Proteolytic activity of the strains was determined by hydrolysis of azocasein,Capacity for phytate use,Antagonistic activity,03,RESULTS AND DISCUSSION,03,课题研究过程展示,03,RESULTS AND DISCUSSION,03,RESULTS AND DISCUSSION,03,RESULTS AND DISCUSSION,03,RESULTS AND DISCUSSION,03,RESULTS AND DISCUSSION,CONCLUSION,Antibacterial activity of the lipopeptide fraction of B. subtilis GM2 and GM5 was studied. The lipopeptide fraction was isolated from the liquid culture of bacteria grown after 72 h on the medium containing maltose (medium no. 1). Inhibitory activity of lipopeptides was determined by the disk diffusion method against different species of enterobacteria, Salmonella typhimurium, E. coli, and K. oxytoca (Table 5). Lipopeptides of B. subtilis GM2 and GM5 showed antagonistic activity agai