Nefise Akkoç

Isolations of α-glucosidase-producing thermophilic bacilli from hot springs of Turkey

From 42 different hot springs in six provinces belonging to distinct geographical regions of Turkey, 451 thermophilic bacilli were isolated and 67 isolates with a high amylase activity were selected to determine the α-glucosidase production capacities by using pNPG as a substrate. α-Glucosidase production capacities of the isolates varied within the range from 77.18 to 0.001 U/g. Eleven of our thermophilic bacilli produced α-glucosidase at significant levels comparable with that of the reference strains tested; thus, five strains, F84b (77.18 U/g), A333 (48.64 U/g), F84a (36.64 U/g), E134 (32.09 U/g), and A343 (10.79 U/g), were selected for further experiments. 16S rDNA sequence analysis revealed that these selected isolates all belonged to thermophilic bacilli 16S rDNA genetic group 5, four of them representing the genus Geobacillus, while strain A343 had an uncultured bacterium as the closest relative. Changes in α-glucosidase levels in the intracellular and extracellular fractions were determined during 48-h cultivation of A333, A343, F84a, F84b, E134, and the reference strain G. stearothermophilus ATCC 12980. According to α-glucosidase production type and enzyme levels in intracellular and extracellular fractions, Geobacillus spp. A333, F84a, and F84b were defined as extracellular enzyme producers, whereas the thermophilic bacterium A343 was found to be an intracellular α-glucosidase producer, similar to ATCC 12980 strain. Geobacillus sp. E134 differed in α-glucosidase production type from all tested isolates and the reference strain; it was described as a membrane-associated cell-bound enzyme producer. In this study, apart from screening a great number of new thermophilic bacilli from the hot springs of Turkey, which have not yet been thoroughly studied, five new thermostable α-1,4-glucosidase-producing bacilli that have biotechnological potential with α-glucosidases located at different cell positions were obtained.

Influence of growth conditions on the nisin production of bioengineered Lactococcus lactis strains

Nisin production of three bioengineered strains, (LAC338, LAC339 and LAC340) with immunity (nisFEG) and/or regulation (nisRK) genes of nisin biosynthesis on plasmids in the Lactococcus lactis LL27 nisin producer, was evaluated under pH-controlled and pH-uncontrolled batch fermentations. Optimization studies showed that fructose and yeast extract yielded the highest nisin activity. The strains LAC338, LAC339, and LAC340 produced 24, 45, and 44% more nisin, respectively, than wild-type L. lactis LL27 after 12-h incubation. However, sharp decreases in the yield of nisin were observed at the late phase of fermentation with LAC339 and LL27 in contrast to LAC340 and LAC338 strains for which the high level of nisin could be maintained longer. Obviously, increasing the copy number of the regulation genes together with immunity genes in the nisin producers retarded the loss of nisin in the late phase of the fermentation.

In vitro characterization of probiotic properties ofPediococcus pentosaceus BH105 isolated from human faeces

In this study, a new bacteriocin-producing strainPediococcus pentosaceus BH105 was isolated from human faeces and subsequentlyin vitro probiotic and antagonistic properties were investigated. This strain exhibited high viability at pH 3.0 and in the presence of pepsin, pancreatin and bile salts (0.3%). BH105 was sensitive to 19 of 25 antibiotics and showed no haemolytic activity. The bacteriocin produced by BH105 was active at wide pH range of 2.0 to 11.0 and was heat stable at 80, 90 and 100°C for 15 min. Bacteriocin activity was inhibited by pepsin, a-chymotrypsin, and proteinase K but not by trypsin,a-amylase, catalase, lysozyme and lipase. The Tricine-SDS-PAGE analysis allowed an approximation of bacteriocin BH105 size about 5 kDa.Pediococcus pentosaceus BH105 was able to adhere to Caco-2 cells (10.12±2.40%) and inhibited the adhesion ofEscherichia coli LMG3083 (88.72±5.53%) andSalmonella typhimurium SL1344 (60.64±10.97%).

Characterization of antibiotic resistance in Salmonella enterica isolates determined from ready-to-eat (RTE) salad vegetables

In the last decade, ready-to-eat (RTE) salad vegetables are gaining increasing importance in human diet. However, since they are consumed fresh, inadequate washing during processing can bring on some foodborne illnesses, like salmonellosis, since these food items have natural contamination from soil and water. During 2009–2010, a total of 81 samples were purchased arbitrarily from local markets in Ankara, and were examined for Salmonella contamination. Salmonella screening was performed by using anti-Salmonella magnetic beads system and polymerase chain reaction (PCR) identification of the suspected colonies. Then, the antibiotic resistance profiles of four Salmonella strains identified (strains RTE-1, RTE-2, RTE-3, and RTE-4) were also investigated, since the mechanism by which Salmonella spp. have accumulated antibiotic resistance genes is of interest. All strains showed resistance against sulfonamides (MIC > 128 mg/L). Further results suggested that associated sulfonamide resistance genes were encoded by the 55.0 kb plasmid of strain RTE-1 that involves no integrons. As a result of using two primers (P1254 and P1283) in randomly amplified polymorphic DNA-PCR (RAPD-PCR) analysis, two common amplicons (364 bp and 1065 bp) were determined. The findings of this study provide support to the adoption of guidelines for the prudent use of antibiotics in order to reduce the number of pathogens present on vegetable and fruit farms. Besides, since it is shown that these bacteria started to gain resistance to antibiotics, it is necessary to further investigate the prevalence of them in foods.

Partial characterization of bacteriocins produced by two Lactobacilus strains with probiotic properties

The probiotic characteristics of Lactobacillus brevis BG18 and Lb. plantarum BG33, isolated from traditional Turkish Tulum cheese were assessed. These two bacteriocinproducer strains exhibited good probiotic characteristics such as resistance in media containing 0.3% bile salt, pepsin (3 mg mL⁻¹), and pancreatine (1 mg mL⁻¹) as well as acid resistance at pH 2. They were also adhered to Caco-2 epithelial cells in a manner comparable to Escherichia coli LMG3083 (ETEC) and Salmonella Typhimurium SL1344. The strains produced a heat-stable antimicrobial compound that was shown to be proteinaceous in nature, and therefore, referred to as bacteriocins. The bacteriocins were able to inhibit growth of a number grampositive bacteria such as Listeria monocytogenes, Clostridium botulinum, Staphylococcus aureus and Bacillus cereus. Tricine-SDS-PAGE of the active fraction resulted in single bands with estimated molecular masses of 2.5 kDA and 2.7 kDA for Lb. brevis BG18 and Lb. plantarum BG33 bacteriocins, respectively.

Stress Response Kinetics of Two Nisin Producer Strains of Lactococcus lactis spp. lactis

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.

The role of stj fimbrial operon in the intestinal persistence of Salmonella Typhimurium in mice

Salmonella Typhimurium contains 13 operons coding for fimbriae with unique binding specificities to host epithelial surfaces. stj operon is only detected in S. Typhimurium genome suggesting that Stj fimbria may effect serovarspecific virulence characteristics. In this study, the role of stj fimbrial operon in the long-term persistence of S. Typhimurium was identified by competitive infection experiment in genetically resistant mouse (CBA) model system. Knock-out mutation of stjA (major subunit of the Stj fimbria) gene reduced recovery of S. Typhimurium from fecal samples and its colonization to spleen, cecum and mesenteric lymph nodes over a 34-day time period (p < 0.05). This data indicate that stj fimbrial operon has a role in long-term intestinal persistence of S. Typhimurium in CBA mice.

Thermal inactivation kinetics of Lactococcus lactis subsp. lactis bacteriophage PLL98-22

Survival curves of Lactococcus lactis subsp. lactis bacteriophage pll98 inactivated by heat were obtained at seven temperature values (50-80 degrees C) in M17 broth and skim milk. Deviations from first-order kinetics in both media were observed as sigmoidal shapes in the survival curves of pll98. An empirical model with four parameters was used to define the thermal inactivation. Number of parameters of the model was reduced from four to two in order to increase the robustness of the model. The reduced model produced comparable fits to the full model. Both the survival data and the calculations done using the reduced model (time necessary to reduce the number of phage pll98 six- or seven- log10) indicated that skim milk is a more protective medium than M17 broth within the assayed temperature range.