Study of selenium enrichment metabolomics in Bacillus subtilis BSN313 via transcriptome analysis.

In this study, the transcriptome analysis was practiced to identify potential genes of probiotic Bacillus subtilis BSN313 involved in selenium (Se) enrichment metabolism. The transcriptomic variation of the strain was deliberated in presence of three different sodium selenite concentrations (0, 3, and 20 µg/mL). The samples were taken at 1 and 13 h subsequent to inoculation of selenite and gene expression profiles in Se metabolism were analyzed through RNA sequencing. The gene expression levels of the pre log phase were lower than the stationary phase. It is because, the bacteria has maximum grown with high concentration of Se (enriched with organic Se), at stationary phase. Bacterial culture containing 3 µg/mL concentration of inorganic Se (sodium selenite) has shown highest gene expression as compared to no or high concentration of Se. This concentration (3 µg/mL) of sodium selenite (as Se) in the medium promoted the upregulation of thioredoxin reductase expression, whereas its higher Se concentration inhibited the formation of selenomethionine (SeMet). The result of 5 L bioreactor fermentation showed that SeMet was also detected in the fermentation supernatant as the growth entered in the late stationary phase and reached up to 857.3 ng/mL. The overall intracellular SeMet enriched content in BSN313 was extended up to 23.4 µg/g dry cell weight. The other two selenoamino acids (Se-AAs), methyl-selenocysteine, and selenocysteine were hardly detected in medium supernatant. From this study, it was concluded that SeMet was the highest content of organic Se byproduct biosynthesized by B. subtilis BSN313 strain in Se-enriched medium during stationary phase. Thus, B. subtilis BSN313 can be considered a commercial probiotic strain that can be used in the food and pharmaceutical industries. This is because it can meet the commercial demand for Se-AAs (SeMet) in both industries.

Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria.

In the present study, lysozyme was purified by the following multi-step methodology: salt (ammonium sulfate) precipitation, dialysis, and ultrafiltration. The lysozyme potential was measured by enzymatic activity after each purification step. However, after ultrafiltration, the resulting material was considered extra purified. It was concentrated in an ultrafiltration centrifuge tube, and the resulting protein/lysozyme was used to determine its bactericidal potential against five bacterial strains, including three gram-positive (Bacillus subtilis 168, Micrococcus luteus, and Bacillus cereus) and two gram-negative (Salmonella typhimurium and Pseudomonas aeruginosa) strains. The results of ZOI and MIC/MBC showed that lysozyme had a higher antimicrobial activity against gram-positive than gram-negative bacterial strains. The results of the antibacterial activity of lysozyme were compared with those of ciprofloxacin (antibiotic). For this purpose, two indices were applied in the present study: antimicrobial index (AMI) and percent activity index (PAI). It was found that the purified lysozyme had a higher antibacterial activity against Bacillus cereus (AMI/PAI; 1.01/101) and Bacillus subtilis 168 (AMI/PAI; 1.03/103), compared to the antibiotic (ciprofloxacin) used in this study. Atomic force microscopy (AFM) was used to determine the bactericidal action of the lysozyme on the bacterial cell. The purified protein was further processed by gel column chromatography and the eluate was collected, its enzymatic activity was 21.93 U/mL, while the eluate was processed by native-PAGE. By this analysis, the un-denatured protein with enzymatic activity of 40.9 U/mL was obtained. This step shows that the protein (lysozyme) has an even higher enzymatic potential. To determine the specific peptides (in lysozyme) that may cause the bactericidal potential and cell lytic/enzymatic activity, the isolated protein (lysozyme) was further processed by the SDS-PAGE technique. SDS-PAGE analysis revealed different bands with sizes of 34 kDa, 24 kDa, and 10 kDa, respectively. To determine the chemical composition of the peptides, the bands (from SDS-PAGE) were cut, enzymatically digested, desalted, and analyzed by LC-MS (liquid chromatography-mass spectrometry). LC-MS analysis showed that the purified lysozyme had the following composition: the number of proteins in the sample was 56, the number of peptides was 124, and the number of PSMs (peptide spectrum matches) was 309. Among them, two peptides related to lysozyme and bactericidal activities were identified as: A0A1Q9G213 (N-acetylmuramoyl-L-alanine amidase) and A0A1Q9FRD3 (D-alanyl-D-alanine carboxypeptidase). The corresponding protein sequence and nucleic acid sequence were determined by comparison with the database.

Expression of BSN314 lysozyme genes in Escherichia coli BL21: a study to demonstrate microbicidal and disintegarting potential of the cloned lysozyme.

This study is an extension of our previous studies in which the lysozyme was isolated and purified from Bacillus subtilis BSN314 (Naveed et al., 2022; Naveed et al., 2023). In this study, the lysozyme genes were cloned into the E. coli BL21. For the expression of lysozyme in E. coli BL21, two target genes, Lyz-1 and Lyz-2, were ligated into the modified vector pET28a to generate pET28a-Lyz1 and pET28a-Lyz2, respectively. To increase the production rate of the enzyme, 0.5-mM concentration of IPTG was added to the culture media and incubated at 37 °C and 220 rpm for 24 h. Lyz1 was identified as N-acetylmuramoyl-L-alanine amidase and Lyz2 as D-alanyl-D-alanine carboxypeptidase. They were purified by multi-step methodology (ammonium sulfate, precipitation, dialysis, and ultrafiltration), and antimicrobial activity was determined. For Lyz1, the lowest MIC/MBC (0.25 µg/mL; with highest ZOI = 22 mm) were recorded against Micrococcus luteus, whereas the highest MIC/MBC with lowest ZOI were measured against Salmonella typhimurium (2.50 µg /mL; with ZOI = 10 mm). As compared with Aspergillus oryzae (MIC/MFC; 3.00 µg/mL), a higher concentration of lysozyme was required to control the growth of Saccharomyces cerevisiae (MIC/MFC; 50 µg/mL). Atomic force microscopy (AFM) was used to analyze the disintegrating effect of Lyz1 on the cells of selected Gram-positive bacteria, Gram-negative bacteria, and yeast. The AFM results showed that, as compared to Gram-negative bacteria, a lower concentration of lysozyme (Lyz1) was required to disintegrate the cell of Gram-positive bacteria.

Nutritional composition assessment and antimicrobial activity of Catostylus perezi, jellyfish blooms along the coast of Pakistan: an awareness to avoid food neophobia in Pakistan.

This study highlighted the nutritional importance of Catostylus perezi (an edible jellyfish) in Pakistan; a society where a large proportion of the population suffers from malnutrition, while C. perezi, a blessing of the sea, is wasted or exported. In the present study, the amino acid and fatty acid profiles of the oral arms and umbrella of C. perezi were determined by HPLC. The total amino acid concentration (ΣAA) in the oral arms was 151.19 mg/100g, while in the umbrella it was 100.17 mg/100g. The ratio of total essential amino acids to total non-essential amino acids (TEAA/TNEAA) was 0.72 in the oral arms, while it was 0.70 in the umbrella. Higher amount of ω-3 with lower ratio of ω-6/ω-3 in oral arms (0.52), rather umbrella (ω-6/ω-3 ratio; 0.62). The antimicrobial activity, MIC, MBC, and MFC of the whole body of the edible jellyfish were determined. On the basis of polarity, different solvents were used, e.g. water, methanol, dichloromethane, chloroform, and n-hexane. Among all the extracts, the water extract gave the highest ZOI against C. xerosis (29 mm), while the n-hexane extract gave the lowest ZOI against S. aureus (MRSA) ATCC 33591 (8.20 mm). The water extract of C. perezi had high potential against C. xerosis with the highest AMI and PAI (1.53 and 153, respectively), while the same extract had the highest TAI against E. coli (81.43 mL/g). For fungi/yeast, the methanolic extract had the highest ZOI (29.70 mm) against S. cerevisiae and the lowest MIC/MFC (2.40 µg/mL) against the same pathogen. The n-Hexane extract gave the lowest ZOI (11.10 mm) against P. variotii and the highest MIC/MFC (31.60 µg/mL) against Penicillium sp. Atomic force microscopy (AFM) was used to analyse the disintegrating effect of the extracts (with the highest antimicrobial effect) on the cells of selected Gram-positive, Gram-negative and yeast species. The amino acid and fatty acid profiles and antimicrobial assessment showed that C. perezi has great nutritional importance, so the use of C. perezi as food is highly recommended for the Pakistani community.

Banned Sudan dyes in spices available at markets in Karachi, Pakistan.

Sudan dyes were investigated in branded and non-branded spices, commonly available in the markets of Karachi, Pakistan. High performance liquid chromatography (HPLC) with a variable wavelength detector (VWD) was applied to determine Sudan dyes I-IV. The non-branded samples had higher concentrations of Sudan dyes than the maximum limits of 0.1 mg/kg. The highest concentration of Sudan dye (I) was found in turmeric powder (8460 mg/kg) and the lowest concentration (1.50 mg/kg) of Sudan (IV) in Chaat Masala. This indicates that the use of non-branded spices is not safe, whereas no Sudan dye was found in the branded spice samples. Further studies regarding the higher carcinogenic risk posed by Sudan dye adulterated spices in Pakistan is strongly advised.

Biogenic Selenium Nanoparticles and Their Anticancer Effects Pertaining to Probiotic Bacteria-A Review.

Selenium nanoparticles (SeNPs) can be produced by biogenic, physical, and chemical processes. The physical and chemical processes have hazardous effects. However, biogenic synthesis (by microorganisms) is an eco-friendly and economical technique that is non-toxic to human and animal health. The mechanism for biogenic SeNPs from microorganisms is still not well understood. Over the past two decades, extensive research has been conducted on the nutritional and therapeutic applications of biogenic SeNPs. The research revealed that biogenic SeNPs are considered novel competitors in the pharmaceutical and food industries, as they have been shown to be virtually non-toxic when used in medical practice and as dietary supplements and release only trace amounts of Se ions when ingested. Various pathogenic and probiotic/nonpathogenic bacteria are used for the biogenic synthesis of SeNPs. However, in the case of biosynthesis by pathogenic bacteria, extraction and purification techniques are required for further useful applications of these biogenic SeNPs. This review focuses on the applications of SeNPs (derived from probiotic/nonpathogenic organisms) as promising anticancer agents. This review describes that SeNPs derived from probiotic/nonpathogenic organisms are considered safe for human consumption. These biogenic SeNPs reduce oxidative stress in the human body and have also been shown to be effective against breast, prostate, lung, liver, and colon cancers. This review provides helpful information on the safe use of biogenic SeNPs and their economic importance for dietary and therapeutic purposes, especially as anticancer agents.

A comprehensive assessment of environmental pollution by means of heavy metal analysis for oysters' reefs at Hab River Delta, Balochistan, Pakistan.

The heavy metal pollution status of oyster reefs has been assessed with respect to ten metals pollutants in seawater, sediments, and tissues of above two oysters (soft tissues and shells) for assessing the pollution status in a short food chain in Hab River Delta. The results showed that heavy metals accumulated in M. bilineata were higher than those in M. cuttackensis. Simultaneously, the population of M. bilineata species has been ironically decreasing as a results of high pollution. The determined concentrations revealed a significant differences in their profiles among sediments, seawater and bioaccumulation in tissues and shells of two native oysters. The present study also compared these metal concentrations with national and international database by applying different pollution indices. Heavy metals in all samples were above the national environmental quality standards (NEQS-Pakistan). High level of pollution with an alarming condition of Hab River Delta need more attention for coastal management.

Systematic revision of the genus Peronia Fleming, 1822 (Gastropoda, Euthyneura, Pulmonata, Onchidiidae).

The genus Peronia Fleming, 1822 includes all the onchidiid slugs with dorsal gills. Its taxonomy is revised for the first time based on a large collection of fresh material from the entire Indo-West Pacific, from South Africa to Hawaii. Nine species are supported by mitochondrial (COI and 16S) and nuclear (ITS2 and 28S) sequences as well as comparative anatomy. All types available were examined and the nomenclatural status of each existing name in the genus is addressed. Of 31 Peronia species-group names available, 27 are regarded as invalid (twenty-one synonyms, sixteen of which are new, five nomina dubia, and one homonym), and four as valid: Peronia peronii (Cuvier, 1804), Peronia verruculata (Cuvier, 1830), Peronia platei (Hoffmann, 1928), and Peronia madagascariensis (Labbé, 1934a). Five new species names are created: P. griffithsi Dayrat & Goulding, sp. nov., P. okinawensis Dayrat & Goulding, sp. nov., P. setoensis Dayrat & Goulding, sp. nov., P. sydneyensis Dayrat & Goulding, sp. nov., and P. willani Dayrat & Goulding, sp. nov. Peronia species are cryptic externally but can be distinguished using internal characters, with the exception of P. platei and P. setoensis. The anatomy of most species is described in detail here for the first time. All the secondary literature is commented on and historical specimens from museum collections were also examined to better establish species distributions. The genus Peronia includes two species that are widespread across the Indo-West Pacific (P. verruculata and P. peronii) as well as endemic species: P. okinawensis and P. setoensis are endemic to Japan, and P. willani is endemic to Northern Territory, Australia. Many new geographical records are provided, as well as a key to the species using morphological traits.