Identification and Growth Characterization of a Novel Strain of Saccharomyces boulardii Isolated From Soya Paste.

The nonpathogenic yeast Saccharomyces boulardii (Sb) has beneficial effects on the human intestine, and thus has been prescribed as probiotics for the treatment of diarrhea and gastrointestinal diseases. This is the only commercialized yeast with the purpose of being used as human medicine. Currently, little is known about their multiple mechanisms of actions. The S. boulardii yeast strain is isolated and identified by using the BIOLOGTM microarray identification system and morphologically. To understand its functional roles, the present study investigates the ability of this yeast to tolerate different concentrations of bile salt up to 2.5%, cell hydrophobicity, antioxidants, autoaggregation activity, and simulated gastrointestinal digestion. The effect of temperatures (up to 50°C), pH (up to 8.0), and salinity (at best 7%) was also monitored on the growth and survival of the yeast cell. The physicochemical analyses revealed that S. boulardii could survive in stomach conditions at pH 2.5, temperature 37°C, and 2% bile salt. Antibiotic susceptibility of S. boulardii was carried out using commercial antibiotic discs. The antimicrobial activity of the isolated S. boulardii against bacterial pathogens related to diarrhea diseases was in-vitro determined by the Well Diffusion method. The biosafety assay findings also claimed S. boulardii could be a potential probiotic. The experimental findings suggest that the isolated S. boulardii possesses excellent probiotic capacities as a biotherapeutic agent for antidiarrheal and gastrointestinal disorders.

Evaluation of microplate immunocapture method for detection of Vibrio cholerae, Salmonella Typhi and Shigella flexneri from food.

BACKGROUND: Improved methods with better separation and concentration ability for detection of foodborne pathogens are in constant need. The aim of this study was to evaluate microplate immunocapture (IC) method for detection of Salmonella Typhi, Shigella flexneri and Vibrio cholerae from food samples to provide a better alternative to conventional culture based methods. RESULTS: The IC method was optimized for incubation time, bacterial concentration, and capture efficiency. 6 h incubation and log 6 CFU/ml cell concentration provided optimal results. The method was shown to be highly specific for the pathogens concerned. Capture efficiency (CE) was around 100% of the target pathogens, whereas CE was either zero or very low for non-target pathogens. The IC method also showed better pathogen detection ability at different concentrations of cells from artificially contaminated food samples in comparison with culture based methods. Performance parameter of the method was also comparable (Detection limit- 25 CFU/25 g; sensitivity 100%; specificity-96.8%; Accuracy-96.7%), even better than culture based methods (Detection limit- 125 CFU/25 g; sensitivity 95.9%; specificity-97%; Accuracy-96.2%). CONCLUSION: The IC method poses to be the potential to be used as a method of choice for detection of foodborne pathogens in routine laboratory practice after proper validation.

Antimicrobial and antioxidant activities of Saccharomyces cerevisiae IFST062013, a potential probiotic.

BACKGROUND: Probiotic yeast has become a field of interest to scientists in recent years. METHODS: Conventional cultural method was employed to isolate and identify yeast and standard methods were used to determine different probiotic attributes, antimicrobial and antioxidant properties. RESULTS: This study reports potential probiotic properties of a strain of S. cerevisiae IFST 062013 isolated from fruit. The isolate is tolerant to a wide range of temperature and pH, high concentration of bile salt and NaCl, gastric juice, intestinal environment, α-amylase, trypsin and lysozyme. It can produce organic acid and showed resistance against tetracycline, ampicillin, gentamycin, penicillin, polymixin B and nalidixic acid. It can assimilate cholesterol, can produce killer toxin, vitamin B12, glutathione, siderophore and strong biofilm. It showed moderate auto-aggregation ability and cell surface hydrophobicity. The isolate can produce enzymes such as amylase, protease, lipase, cellulose, but unable to produce galactosidase. The isolate can't produce gelatinase and DNase. The isolate showed moderate anti-microbial activity against bacteria and fungi and cell lysate showed better antimicrobial activity than whole cell and culture supernatant. Again, the isolate showed better anti-bacterial activity against gram negative bacteria than gram positive. The isolate showed strong antioxidant activity, reducing power, nitric oxide and hydroxyl radical scavenging activity, significant brine shrimp cytotoxicity and acute toxicity and metal ion chelating activity. The isolate did not induce any detectable change in general health of mice upon oral toxicity testing and found to be safe in mouse model. The isolate improve lymphocyte proliferation and cytokine production in treated mice. CONCLUSIONS: Such isolate could be potential as probiotic to be used therapeutically.

Characterization of aflatoxin producing Aspergillus flavus from food and feed samples.

Aspergillus flavus is one of the major producers of aflatoxin and can contaminate wide range of agricultural commodities either in field or in storage. 15 presumptive Aspergillus flavus has been isolated from 30 feed and grain samples. All the isolates were morphologically similar to Aspergillus flavus type strains. All the isolates were found to be aflatoxigenic. DNA sequencing of 5.8 s rDNA confirmed all of them to be Aspergillus flavus. Only 1 isolate possessed all the seven toxigenic gene (aflR, aflS, aflQ, aflP, aflD, aflM, and aflO) while aflP & aflQ were most prevalent in the isolates. All the isolates possessed at least three toxigenic genes. Toxin producing ability in solid culture media showed that 11 isolates isolates were able to produce both aflatoxin B1 & B2. More than 90% isolates produced aflatoxin B1 ranging 7-22 µg/g of agar. This study alarms us about the potential risks of Aspergillus flavus to public health if contaminate agricultural commodities such as grains or raw materials such as poultry feed. Proper harvest and storage management is required to reduce the risk of aflatoxin in feed and grains.

Stress tolerant virulent strains of Cronobacter sakazakii from food.

BACKGROUND: Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh. RESULT: Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress. CONCLUSION: Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Stress tolerant virulent strains of Cronobacter sakazakii from food

BACKGROUND: Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh. RESULT: Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress. CONCLUSION: Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Multiplex PCR (polymerase chain reaction) assay for detection of E. coli O157:H7, Salmonella sp., Vibrio cholerae and Vibrio parahaemolyticus in spiked shrimps (Penaeus monodon).

The coastal aquaculture mainly shrimps constitute major export sector in Bangladesh and is increasingly shaped by international trade conditions and by national responses to those stringent quality and safety standards. PCR based validated methods for detection of major bacterial pathogens in shrimp might be very useful tool for ensuring quality and safety standards of exportable shrimps. The objective of this study was to evaluate overall performance (sensitivity and specificity) of the multiplex PCR assay for detection of Vibrio cholerae, Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli O157:H7 from spiked shrimp samples. The targeted genes were ompW for V. cholerae, tdh for V. parahaemolyticus, sefA for Salmonella spp. and hlyEHEC for E. coli O157:H7. The genomic DNA was extracted by using standard method and amplified accordingly. Sensitivity of the assay was tested by inoculating the shrimp homogenate with viable cells of laboratory references strains (target pathogens). The genes were amplified individually both from culture homogenate and spiked samples. Twenty different uniplex and multiplex PCR assay were performed; the results showed that the sensitivity and specificity of multiplex PCR are comparable to that of the results of uniplex PCR for the samples. DNA extracted from shrimp samples spiked with non-target pathogen (Bacillus cereus, Shigella flexneri and Staphylococcus aureus) yielded negative results.

Influence of storage conditions of apples on growth and patulin production by Penicillium expansum.

Penicillium expansum causes blue mould rot, a serious post-harvest disease of apples, and is the main producer of the mycotoxin patulin. The present study aimed to determine the influence of storage conditions (i.e. temperature and O(2) level) on growth and patulin production by different P. expansum strains on a simulation medium and on apples. Growth was strongly influenced by the temperature, while the used atmosphere (20, 3, and 1% O(2); <1% CO(2)) had no effect. Optimal growth was observed at 25 degrees C for every strain tested. Patulin production was stimulated when the temperature decreased (from 20 to 10 or 4 degrees C), while a further decrease of the temperature to 1 degrees C caused a reduction in patulin production. The temperature at which the stimulation was changed into suppression was strain dependent. Similar results were observed for the O(2) level. A reduction of the O(2) level from 20 to 3% O(2) could stimulate or suppress patulin production depending on the strain, while a clear decrease of the patulin production was observed when the O(2) level was reduced from 3 to 1%. These results show that the induction of limited stress to the fungus, such as lowering the temperature or lowering the O(2) levels stimulates patulin production. However, the combination of different stress conditions (e.g. low temperature and low O(2)) will result in a reduced formation of the toxin. The combination of stress conditions, at which the transition from stimulation to suppression is observed, is strain dependent. Moreover, patulin production is characterized by a high natural variability. The presented results show that the temperature and O(2) level has to be as low as possible during the storage of apples in order to suppress patulin production and to guarantee food safety.

Modeling the effect of temperature on the growth rate and lag phase of Penicillium expansum in apples.

The objective of the present study was to develop validated models that describe the effect of storage temperature on the growth rate and lag phase of six Penicillium expansum strains. The growth of the selected strains was therefore studied on Apple Puree Agar Medium (APAM) at 30, 25, 16, 10, 4 and 2 degrees C. Growth rates and lag phases were estimated using linear regression. Several secondary models were evaluated and for the growth rate, a modification of the extended Ratkowsky model was selected. Regarding the lag phase, the Arrhenius-Davey model provided the best adjustment to the observed data. Model validation was performed in two steps. Firstly, the developed models were validated on APAM. The obtained bias factors (Bf) ranged from 0.91 to 1.14 and the accuracy factors (Af) were <1.2 for the validation performed on APAM, indicating that the models were good predictors of the true mean colony growth rate and lag phase. Afterwards, an external validation was carried out in apples. For the growth rate, Bf ranged from 0.64 to 0.81 and Af<1.39, indicating conservative predictions. On the contrary for the lag phase, a clear deviation was observed between predictions and observed values on apples (0.351.6). These results highlight that the use of simulation or synthetic media for the development of predictive models for the lag phase of moulds can lead to inadequate predictions and that a validation on the real food matrix is necessary. Application of the developed models is possible in the framework of Quantitative Risk Assessment to develop control strategies against blue mould rot in apple and enables the inclusion of strain variability. However, possible underestimation of the lag phase should be taken into account.