枯草芽孢桿菌作為新的益生菌演講稿

1、,,,,,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 microorgani

2、smspositive effect on diverse functions of an organismprevent 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

3、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

4、 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

5、 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 a

6、nd B. coagulans were shown to have a growth-stimulating and prophylactic effect on broiler chickens,1,INTRODUCTION,標(biāo)題,,,,The goal of the present work was to characterize the properties of these new strains and to asses

7、s 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

8、 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.,

9、02,MATERIALS AND METHODS,,Nutrient media and cultivation conditions. The following media were used for cultivation. These were:(1) LB (Luria–Bertoni) medium (g/L): tryptone, 10.0; yeast extract, 5.0; NaCl, 5.0; (2) LA

10、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;

11、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 37°C and using an IKA®KS 4000 incuba

12、tor shaker (Germany) at 37°C 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 dyna

13、mics 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 (37°C, 200 rpm). Bacterial culture samples were collected

14、 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 chamb

15、er (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

16、ambient pH wasstudied in 250-mL Erlenmeyer flasks containing 50 mL of the LB medium (pH 2–10). Bacteria were cultivated using the incubator shaker (37°C, 200 rpm, aeration). Optical density of the cultures was dete

17、rmined after 24 h of growth.,,Resistance of the spores to pH and spore capacityfor in vitro germination were studied according to themethod described (Haller et al., 2001). Spores of bacilli were obtained by heating a

18、2-day-old bacterial culture for 90 min at 60°C 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

19、 at 40°C. 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 medi

20、um (pH 3.0) and incubated for 90 min. An aliquot of the suspension (0.1 mL) was taken and heated for 90 min at 60°C. A series of dilutions was prepared and plated to form a lawn on the LA medium for the subsequent e

21、numeration 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 60°C and used to de

22、termine 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 s

23、pores 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 μm). The spores we

24、re 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 40°C and used to determine CFU/mL.,02,MATERIALS AND METHODS,,Proteolytic

25、 activity in the bacterial liquid culture was determined by digestion of azocasein (Sigma, United States) (Demidyuk et al., 2006). Inhibitoryanalysis was performed in the presence of 1,10- phenanthroline (Serva, Germany

26、), 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 tempera

27、ture, 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-hydroly

28、zing activity was determined using a selective nutrient medium for phytate-degrading bacteria, phytase screening medium (PSM), according to Mittal’s technique (Mittal et al., 2011). Ability to hydrolyze sodium phytate wa

29、s 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 Laborat

30、ory 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 stand

31、ard 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 agai

32、nst 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

33、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 .,Resistan

34、ce to the GIT acids and bile,Ability of spores to germinate at different pH values in vitro,Proteolytic activity of the strains was determined byhydrolysis of azocasein,Capacity for phytate use,Antagonistic activity,03,

35、RESULTS AND DISCUSSION,03,課題研究過(guò)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 fr

36、action 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 lipopep

37、tides 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 acti

38、vity against all test cultures studied. However, the inhibitory effect was higher in case of the strain B. subtilis GM5, indicating its higher antibacterial and antifungal activity. Thus, B. subtilis strains GM2 and GM

39、5 isolated from the rhizosphere are characterized by antagonistic properties against phytopathogenic micromycetes and pathogenic and opportunistic enterobacteria, which may be due to the effect of lipopeptides. These

40、 metabolites of bacilli can suppress pathogenic and opportunistic microflora. Spores of the bacteria studied were resistant to bile and a wide range of the ambient pH. Vegetative cells of the bacilli were able to synth

41、esize proteolytic and phytate-hydrolyzing enzymes, which is important for improving digestive functions by adding the strains to feeds. The results obtained suggest that B. subtilis GM2 and GM5 have prospective use as