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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,New Bacillus subtilis Strains as Promising Probiotics,FRONTIERS IN IMMUNOLOGY,New Bacillus subtilis Strains,1,CONTENTS,01,02,03,04,INTRODUCTION,MATERIALS AND METHODS,RESULTS AND DISCUSSION,CONCLUSION,CONTENTS01020304INTRODUCTIONMA,2,INTRODUCTION,INTRODUCTION,3,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,Probiotics:Experiments with mi,4,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,标题01INTRODUCTION,5,MATERIALS AND METHODS,MATERIALS AND METHODS,6,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,The subjects of the study were,7,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,Nutrient media and cultivation,8,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.,The dynamics of bacterial grow,9,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 the spores to pH,10,Resistance of bacterial spores to,bile,was investigated in vitro using 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