Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Effects of NaCl on Antioxidant, Antifungal, and Antibacterial Activities in Safflower Essential Oils

The present study aims to evaluate the antioxidant and antimicrobial activity of essential oils (EO) extracted from safflower plants grown in the absence and presence of NaCl, 50 mM. Plants treated with 50 mM of NaCl showed decreases in root, stem, and leaf dry weight. Results of the essential oils showed that roots have a higher EO yield than leaves and stems. Salinity caused a decrease in this yield in roots and leaves but not in stems. The compounds identified in the EO extracted from these organs belong to seven chemical classes of which the dominant class is the sesquiterpene hydrocarbons. The chemotype of C. tinctorius EO is variable depending on the organ and the treatment. The safflower essential oils showed low antioxidant, antiradical, and iron-reducing activities compared to those of the positive control (BHT). In an antifungal activity test, only two strains, Aspergillus niger and Candida albicans, were found to be highly sensitive to these oils as they showed almost total inhibition of their growth. For antibacterial activity, safflower EOs showed significant antimicrobial activity against Bacillus subtilis, Bacillus cereus, and Xanthomonas campestris in both control and NaCl-treated plants: for these three strains, total inhibition of growth was noted at 50,000 ppm of EO in leaves and roots; whereas for stems, total inhibition was noted only for the third strain (Xanthomonas campestris). For other strains, this inhibition was variable and weak. Salt was found to have no effect on the activities of safflower EOs.

Neutrophil NET Formation with Microbial Stimuli Requires Late Stage NADPH Oxidase Activity

Neutrophils respond to a range of stimuli by releasing extracellular traps (NETs), a mesh consisting of chromatin plus granule and cytoplasmic proteins. We have investigated NET release in response to phorbol myristate acetate (PMA), Pseudomonas aeruginosa (PAO1), Staphylococcus aureus and Candida albicans, and the involvement of NADPH oxidase (NOX2) and myeloperoxidase (MPO) activities. An oxidative mechanism was involved with each stimulus, and the NOX2 inhibitor diphenylene iodonium (DPI) gave almost total inhibition. Notably, DPI added up to 60–90 min after stimulation still gave significant inhibition of subsequent NET formation. As most of the NOX2 activity had already occurred by that time, this indicates a requirement for late-stage low-level oxidant production. Inhibition of histone citrullination did not suppress NET formation, indicating that this was not the essential oxidant-dependent step. With PMA and P. aeruginosa PAO1, MPO activity played an important role in the induction of NETs and MPO inhibitors added up to 30–90 min after stimulation suppressed NET formation. NET formation with S. aureus and C. albicans was insensitive to MPO inhibition. Thus, MPO products are important with some stimuli but not others. Our results extend earlier observations with PMA and show that induction of NETs by microbial stimuli requires late stage oxidant production. Others have shown that NET formation involves NOX2-dependent elastase release from granules. As this is an early event, we conclude from our results that there is more than one oxidant-dependent step.

Effect of α-Terpineol on Chicken Meat Quality during Refrigerated Conditions

The present study was designed to evaluate the in vitro antimicrobial properties of nine bioactive compounds (BACs). Applying the disc paper and minimum inhibitory concentration (MIC) assays, we found that the BACs with the widest spectrum of in vitro antibacterial activity against the studied bacteria were carvacrol and α-terpineol (αTPN). Subsequently, αTPN was selected and applied at different concentrations into the fresh minced chicken meat. The meat was then vacuum packaged and stored for 14 days at 4 °C. Physicochemical properties, lipid oxidation (thiobarbituric acid reactive substances, TBARS), electronic-nose-based smell detection, and microbiological characteristics were monitored. At day 14, meat treated with higher concentrations of αTPN (MIC-2 and MIC-4) exhibited a significantly increased pH and lightness (L*), increased yellowness (b*), decreased redness (a*), caused a significant decrease in water holding capacity (WHC), and decreased lipid oxidation by keeping TBARS scores lower than the control. Although αTPN showed perceptibly of overlapped aroma profiles, the E-nose was able to distinguish the odor accumulation of αTPN between the different meat groups. During the 2-week storage period, αTPN, particularly MIC-4, showed 5.3 log CFU/g reduction in aerobic mesophilic counts, causing total inhibition to the Pseudomonas lundessis, Listeria monocytogenes, and Salmonella Typhimurium. These promising results highlight that αTPN is exploitable to improve the shelf life and enhance the safety of meat and meat products.

Two Novel Mutations Associated with Polymyxin-B Resistance in a Pandemic Lineage of Uropathogenic Escherichia coli of the Sequence Type 69

<b><i>Background:</i></b> Uropathogenic <i>Escherichia coli</i> (UPEC) are frequent pathogens worldwide, impacting on the morbidity and economic costs associated with antimicrobial treatment. <b><i>Objectives:</i></b> We report two novel mutations associated with polymyxin-B resistance in an UPEC isolate collected in 2019. <b><i>Methods:</i></b> Isolate was submitted to antimicrobial susceptibility testing including broth microdilution for polymyxin B. Whole genome was sequenced and analyzed. <b><i>Results:</i></b> Polymyxin-B total inhibition occurred at 16 mg/L (resistant). UPEC isolate was assigned to the phylogroup D, serotype O117:H4, and Sequence Type 69. <i>mcr</i> genes were not detected, but two novel mutations in the <i>pmrA/basS</i> (A80S) and <i>pmrB/</i>basR (D149N) genes were identified. <b><i>Conclusions:</i></b> The occurrence of non-<i>mcr</i> polymyxin resistance in <i>E. coli</i> from extraintestinal infections underscores the need of a continuous surveillance of this evolving pathogen.

Combined laser and ozone therapy for onychomycosis in an in vitro and ex vivo model

In order to develop a fast combined method for onychomycosis treatment using an in vitro and an ex vivo models, a combination of two dual-diode lasers at 405 nm and 639 nm wavelengths, in a continuous manner, together with different ozone concentrations (until 80 ppm), was used for performing the experiments on fungal strains growing on PDA agar medium or on pig’s hooves samples. In the in vitro model experiments, with 30 min combined treatment, all species are inhibited at 40 ppm ozone concentration, except S. brevicaulis, which didn’t show an inhibition in comparison with only ozone treatment. In the ex vivo model experiments, with the same duration and ozone concentration, A. chrysogenum and E. floccosum showed total inhibition; T. mentagrophytes and T. rubrum showed a 75% growth inhibition; M. canis showed a delay in sporulation; and S. brevicaulis and A. terreus did not show growth inhibition. This combined laser and ozone treatment may be developed as a fast therapy for human onychomycosis, as a potential alternative to the use of antifungal drugs with potential side effects and long duration treatments.

Viability and phenotypic heterogeneity of Rhodococcus Rhodochrous CNMN-AC-05 in the presence of fullerene C60

Introduction. In recent years, due to wide applications of nanotechnologies in various fields, the safety of nanomaterials has become a pressing issue. Fullerene C60 is not an exception. Research on the activity of microorganisms and their interaction with nanoparticles is of major importance, both for microorganisms and for the ecosystem as a whole. Material and methods. Fullerene C60 powder was purchased from Sigma-Aldrich. The object of study was R. rhodochrous CNMN-Ac-05 strain. The number of viable bacterial cells was estimated by colony-forming units (CFU). The morphological features of the rhodococci colonies have been described according to the usual microbiological method. Results. It was established that fullerene C60 in concentrations of 1-25 mg/L fullerene C60 stimulated the growth of R. rhodochrous by 2.4-2.8 times. As the concentration of fullerene C60 increased up to 50-100 mg/L, the multiplication and growth of rhodococci decreased by 29.5% and 38% respectively. In the presence of 1-10 mg/L fullerene C60 the rhodococci population remained homogeneous, being composed of 100% S type colonies. The increase of fullerene C60 concentration led both to the decrease in the CFU number and to the appearance of R type colonies, up to 1.3% of population. Conclusions. Fullerene C60 in concentrations 1-100 mg/L had no obvious toxic effect on the rhodococci strain. The optimum concentration is 10 mg/L. The concentrations higher than 25 mg/L led to the dissociation of rhodococcal population and diminution in the CFU counts, but not to the total inhibition.

Effect of a Monascus sp. Red Yeast Rice Extract on Germination of Bacterial Spores

The pink-red color of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant color of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungus Monascus is known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for coloring foods, including meat products. Although, different biological activities of Monascus pigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, rubropunctatin, rubropunctamine complexes and monascuspiloin as the main pigments, on germination of Clostridium and Bacillus spores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination of C. beijerinckii spores but had no effect on B. subtilis spores. In contrast, total suppression of C. beijerinckii spore germination was reached after addition of RYR extract to the medium at a concentration of 2% v/v. For B. subtilis, total inhibition of spore germination was observed only after addition of 4% v/v RYR extract to the medium containing 1.3% w/w NaCl.

Antifungal Activity of Annona muricata Seed Extracts Against Cercospora malayensis, Causal Agent of Cercospora Leaf Spot Disease of Okra (Abelmoschus esculentus L.)

Background: Cercospora leaf spot disease of okra whose pathogen is Cercospora malayensis causes yield losses of up to 60% in plantations. To limit productivity losses, fungicides are commonly used, but are expensive and degrade the environment. Aims: This study aims to test in vitro efficacy of Annona muricata seed extracts against Cercospora malayensis. Study Design: Four extracts were used in this study (the ethyl acetate, acetone, methanol and aqueous extract of A. muricata seeds at the concentrations C1 = 7.5 μl/ml, C2 = 15 μl/ml, C3 = 30 μl/ml and C4 = 60 μl/ml as well as the synthetic fungicide at the concentration of 3.33 g/l) in triplicate. The phytochemical screening of the extracts was performed, the radial growth of pure explants (7 mm diameter) of C. malayensis deposited in sterile Petri dishes containing the PDA medium supplemented with the different concentrations of extracts and incubated at 23 ± 1°C for 6 days were evaluated. Minimum inhibitory concentrations (MIC50, MIC90) were calculated. Results: The extracts of A. muricata seeds are rich in tannins, flavonoids, terpenoids and phenols. The ethyl acetate extract at the concentration C3 resulted in 100% total inhibition of growth of C. malayensis in the Petri dishes. The other extracts resulted in total inhibition of the growth of C. malayensis at C4. The low MIC50 values (12.9 and 21 μl/ml) were obtained with the ethyl acetate and acetone extract, respectively. The ethyl acetate and aqueous extract at the C4 concentration were found to be fungicidal. Conclusion: The extracts were found to be potential fungicide against the C. malayensis strain and might be an alternative in the fight against fungal diseases of okra as their activity was comparable to that of the synthetic fungicide Monchamp 72 WP.

Novel Prosopis juliflora leaf ethanolic extract as natural antimicrobial agent against food spoiling microorganisms

Fresh produces spoilage is a worldwide concern that accompany the global increase in food demand. Adverse human health and environmental effects of commercial spoilage control agents are major public concern. In this study, Prosopis juliflora leaves and fruit extracts had their antimicrobial activities evaluated against the growth of selected bacteria and yeast, and against mycelial growth and conidial germination of selected mycotoxins-producing fungi. P. juliflora water-soluble leaf ethanolic (PJ-WS-LE) extract with its novel extraction method showed the strongest antibacterial activity. Antimicrobial tests showed total inhibition of Botrytis cinerea, Alternaria alternata, Bacillus subtilis, Staphylococcus aureus and Candida albicans with MICs ranging between 0.125 and 1 mg/ml. Percent inhibition of mycelial growth (PIMG) of the extract was also determined against seven other fungal strains with highest value against Geotrichum candidum (66.2%). Even the least affected fungal strain showed alterations in their hyphae and spores exposed to PJ-WS-LE extract when observed using scanning electron microscope (SEM), alterations include exfoliated flakes, pores, vacuolation and applanation. Small-scale fruit bioassays controlled experiment showed high efficacy of the extract in protecting inoculated cherry tomato samples from B. cinerea and A. alternata infections. In conclusion, PJ-WS-LE extract is a feasible, natural antifungal agent that can replace common anti-spoiling chemicals.