1、New Bacillus subtilis Strains as Promising ProbioticsFRONTIERS IN IMMUNOLOGYCONTENTS01020304INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONINTRODUCTIONProbiotics:live microorganismspositive effect on diverse functions of an organismprevent the invasion of various pathogens Experime
2、nts 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 bacte
3、ria 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 lip
4、opeptides, 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 chickens1 INTRODUCTION标题The goal of the p
5、resent work was to characterize the properties of these new strains and to assess their potential use as probiotics. 01 INTRODUCTIONMATERIALS AND METHODSThe subjects of the study were B. subtilis strains GM2 and GM5 with high antimicrobial activity (Mardanova et al., 2017). Opportunistic coliform ba
6、cteria, 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 METHODSNutrient media and cultivation conditions. The following me
7、dia 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
8、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
9、 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 METHODSThe dynamics of bacterial growth and spore formation were studied on the LB medium and the medium no. 1. Bacteria were cultivated for 48 h usi
10、ng 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 chamb
11、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 METHODSAbility to grow at various values of ambient pH wasstudied in 250-mL Erlenmeyer flasks containing 50 mL of the LB mediu
12、m (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 capacityfor in vitro germination were studied according to themethod described (Haller et al., 2001).
13、 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 fo
14、r 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 dilut
15、ions 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
