A putative cytotoxic serine protease from Salmonella typhimurium UcB5 recovered from undercooked burger.

A putative virulence exoprotease designated as UcB5 was successfully purified from the bacterium Salmonella typhimurium to the electrophoretic homogeneity with 13.2-fold and 17.1% recovery by hydrophobic, ion-exchange, and gel permeation chromatography using Phenyl-Sepharose 6FF, DEAE-Sepharose CL-6B, and Sephadex G-75, respectively. By applying SDS-PAGE, the molecular weight was confirmed at 35 kDa. The optimal temperature, pH, and isoelectric point were 35 °C, 8.0, 5.6 ± 0.2, respectively. UcB5 was found to have a broad substrate specificity against almost all the tested chromogenic substrates with maximal affinity against N-Succ-Ala-Ala-Pro-Phe-pNA achieving Km of 0.16 mM, Kcat/Km of 3.01 × 105 S-1 M-1, and amidolytic activity of 28.9 µmol min-1 L-1. It was drastically inhibited by TLCK, PMSF, SBTI, and aprotinin while, DTT, ß-mercaptoethanol, 2,2'-bipyridine, o-phenanthroline, EDTA, and EGTA had no effect, which suggested a serine protease-type. Also, it has shown a broad substrate specificity against a broad range of natural proteins including serum proteins. A cytotoxicity and electron microscopy study revealed that UcB5 could cause subcellular proteolysis that finally led to liver necrosis. For this, future research should focus on using a combination of external antiproteases and antimicrobial agents for the treatment of microbial diseases instead of using drugs alone.

Evaluation of the Impacts of Potassium Bicarbonate, Moringa oleifera Seed Extract, and Bacillus subtilis on Sugar Beet Powdery Mildew.

Powdery mildew disease, caused by Erysiphe betae, is one of the most threatening diseases on sugar beet plants worldwide. It causes a great loss in the root yield, sugar percentage, and quality of produced sugar. In the current study, we aimed to evaluate the susceptibility of 25 sugar beet cultivars to infection with powdery mildew disease under Egyptian conditions. Moreover, we evaluated the impacts of three eco-friendly materials, including potassium bicarbonate (KHCO3; at 5 and 10 g L-1), Moringa oleifera seed extract (25 and 50 g L-1), and the biocontrol agent, Bacillus subtilis (108 cell suspension) against E. betae in two successive seasons 2020 and 2021. Our findings showed that there were significant differences between these 25 cultivars in their susceptibility to the disease under study. Using the detached leaves technique in vitro, B. subtilis showed strong antifungal activity against E. betae. Moreover, both concentrations of KHCO3 and moringa seed extract significantly reduced the disease severity. Under field conditions, tested treatments significantly reduced the severity of powdery mildew disease and prevented E. betae from producing its conidiophores and conidia. Scanning electron microscope examination of treated leaves demonstrated the presence of the decomposition of fungal hyphae, conidiophores, conidia, and the occurrence of plasmolysis to fungal cells and spores on the surface of the leaves. Furthermore, these treatments greatly improved the percent of sucrose and soluble solids content, as well as the enzymatic activity of peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase. It is noteworthy that treatment with moringa seed extract gave the best results, followed by potassium bicarbonate, then B. subtilis cell suspension. Generally, it is recommended to use the substances used in this research to combat powdery mildew to minimize or prevent the use of chemical fungicides harmful to public health and the environment.

Efficacy of Bacillus subtilis, Moringa oleifera seeds extract and potassium bicarbonate on Cercospora leaf spot on sugar beet.

Cercospora leaf spot caused by Cercospora beticola are among the most dangerous plant diseases on sugar beet plants. It causes heavy economic losses, whether on the yield of roots, the percentage of sugar in them, or the quality of sugar produced. In addition to the economic cost caused by chemical control, these chemical pesticides cause an imbalance in the ecosystem and harm the health of humans and animals. In an attempt to search for a safer method than pesticides and environmentally friendly, an evaluation of using biocontrol agents, Bacillus subtilis as cell suspension (108 cell/ml), was conducted in this study. Seeds extract of Moringa oleifera with two concentrations (25 and 50 g/L) and potassium bicarbonate at (5 and10 g/L (compared to fungicide Montoro 30% EC (Propiconazole 15% + Difenoconazole 15%). The evaluation results for twenty-five sugar beet varieties showed a significant discrepancy between these varieties in the extent of their susceptibility to infection with the disease under investigation. In-Vitro, B. subtilis induced an antagonist to C. beticola, and both M. oleifera seeds extract and potassium bicarbonate significantly reduced the linear growth of this pathogen. Under field conditions, the treatments used have given positive results in controlling Cercospora leaf spots. They significantly decreased the severity of disease and prevented C. beticola from creating conidiophores and conidiospores, along with examining their cell walls with the formation of plasmolysis of the fungus cells and reducing both the number and diameter of the spots on the surface leaves; this was demonstrated using a scanning electron microscope (SEM). It is worth noting that the best results obtained were most often when treated with M. oleifera seeds extract, followed by potassium bicarbonate, then cell suspension of B. subtilis. In addition, the percentage of the content of beet roots from total soluble solids and sucrose has improved significantly due to spraying sugar beet plants with the substances mentioned earlier. These treatments also contributed to a significant improvement in the enzymes polyphenol oxidase, peroxidase, and phenylalanine ammonia-lyase.

Binding and removal of polycyclic aromatic hydrocarbons in cold smoked sausage and beef using probiotic strains.

The aim of this study is to bio-monitor the levels of 16 polycyclic aromatic hydrocarbons (PAHs) in cold smoked beef and sausages. The ability of probiotics to remove PAHs was also investigated as function of the cell viability (viable, non-viable and acid-treated cells), bacterial counts (107, 108, and 109 CFU/mL), pH (3, 5, and 7), and incubation time (6, 12, and 24 h). The results indicated a significant difference (p < 0.05) among the analyzed sausages and beef samples for the PAHs concentration. Non-viable probiotics achieved the highest PAHs reduction rates. Limosilactobacillus fermentum EMCC 1346 presented the lowest binding activity value (i.e. 41.10-56.80 %) for all PAHs, followed by Lacticaseibacillus rhamnosus EMCC 1105 with binding percentage of 50.40-65.80 %. On the other hand, the highest removal for all PAHs was achieved by Lactobacillus bulgaricus EMCC 1102 with binding rate of 60.50-76.80 %, at 109 CFU/mL, pH 7, after incubation for 24 h. The fortified sausages results revealed that L. bulgaricus EMCC 1102 cultures exhibited the maximum and significant reduction (p < 0.05) of PAHs with values of 44.71 µg/kg for the center part, compared to control non treated sausages (82.65 µg/kg). Regarding the sensorial profile, treated samples with probiotics led to a preference from the panelists, compared to control. Consequently, the results confirm that fermented probiotic suspension is a feasible future strategy to control PAHs levels in cold smoked meat stuffs.

Evaluation of the Effectiveness of Charcoal, Lactobacillus rhamnosus, and Saccharomyces cerevisiae as Aflatoxin Adsorbents in Chocolate.

The high incidence of aflatoxins (AFs) in chocolates suggests the necessity to create a practical and cost-effective processing strategy for eliminating mycotoxins. The present study aimed to assess the adsorption abilities of activated charcoal (A. charcoal), yeast (Saccharomyces cerevisiae), and the probiotic Lactobacillus rhamnosus as AFs adsorbents in three forms-sole, di- and tri-mix-in phosphate-buffered saline (PBS) through an in vitro approach, simulated to mimic the conditions present in the gastrointestinal tract (GIT) based on pH, time and AFs concentration. In addition, the novel fortification of chocolate with A. charcoal, probiotic, and yeast (tri-mix adsorbents) was evaluated for its effects on the sensory properties. Using HPLC, 60 samples of dark, milk, bitter, couverture, powder, and wafer chocolates were examined for the presence of AFs. Results showed that all the examined samples contained AFs, with maximum concentrations of 2.32, 1.81, and 1.66 µg/kg for powder, milk, and dark chocolates, respectively. The combined treatment demonstrated the highest adsorption efficiency (96.8%) among all tested compounds. Scanning electron microscope (SEM) analysis revealed the tested adsorbents to be effective AF-binding agents. Moreover, the novel combination of tri-mix fortified chocolate had a minor cytotoxicity impact on the adsorptive abilities, with the highest binding at pH 6.8 for 4 h, in addition to inducing an insignificant effect on the sensory attributes of dark chocolate. Tri-mix is thus recommended in the manufacturing of dark chocolate in order to enhance the safety of the newly developed product.

Detoxification of Aflatoxin B1 and Ochratoxin A Using Salvia farinacea and Azadirachta indica Water Extract and Application in Meat Products.

Seventy-five samples of selected meat products, including luncheon, beef burger, sausage, basterma, and kofta, were collected from Alexandria and New Borg El-Arab cities (Egypt). The samples were subjected to mycological examination as well as for detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA) residues. Besides, the study evaluated the effect of aqueous leaf extracts from mealycup sage (Salvia farinacea) and neem (Azadirachta indica), individually and in combination, on the growth of human pathogens Aspergillus parasiticus and Aspergillus flavus producing AFB1, as well as Aspergillus ochraceus and Aspergillus niger which produce OTA. The obtained results revealed that sausage samples had the highest mould count with a mean value of 13.20×102/g, followed by basterma samples 12.05×102/g, then beef burger 7.39×102/g. In contrast, luncheon and kofta samples had the lowest count with a mean value of 5.51×102/g and 2.82×102/g. The findings revealed the antifungal potential of tested extracts. The total inhibition of A. parasitcus and A. niger growth was observed at 2 mg/mL of the combined extract. Salvia farinacea extract had the highest total phenolic content and total flavonoid content with a value of 174.1 and 52.6 mg g-1, respectively. Rutin was the major phenolic component in neem and combined extracts, accounting for 19123 and 8882 µg/g, respectively. Besides, the study investigated detoxification of AFB1 and OTA using combined extract in albino rats. The results confirmed the convenient and safe use of Salvia farinacea and Azadirachta indica extract and their combination as natural antifungal and antioxidant agents. The combined extract could be used as a natural preservative in food processing to control or prevent contamination.