Expression and purification of epinecidin-1 variant (Ac-Var-1) by acid cleavage.

The demand for massive quantities of therapeutic active antimicrobial peptides (AMPs) is high due to their potential as alternatives to antibiotics. However, each antimicrobial peptide has unique properties, necessitating distinct synthesis and purification strategies for their large-scale production. In this study, we bio-synthesized and purified a functional enhanced variant of the AMP epinecidin-1, known as Ac-Var-1 (acid-cleavable variant-1). To generate the active peptide, we cloned the gene for Ac-Var-1 with acid-cleavable site (aspartic acid-proline) into the pET-32a expression vector, purified the fusion protein by His tag enrichment chromatography, and performed acid cleavage to release the active Ac-Var-1 peptide. After acid cleavage, the active Ac-Var-1 was purified and characterized by SDS-PAGE and mass spectrometry. The results from both techniques provided confirmation of the intactness of the purified Ac-Var-1. The Ac-Var-1 inhibited the growth of pathogenic Escherichia coli and Staphylococcus aureus. KEY POINTS : • Epinecidin-1 is a well-known antimicrobial peptide having multipotential bioactivities. • Epinecidin-1 variant is developed via the site-directed mutagenesis method to improve its structural stability and bioactivity. • AC-Var-1 development is an economical and easy method to remove peptide from tag protein.

Analysis of the main bioactive compounds from Ocimum basilicum for their antimicrobial and antioxidant activity.

The interest in bioactives especially from botanicals to treat vancomycin-resistant enterococcal (VRE) infections is increased. Many species of Ocimum have a long history in folk medicinal and food industries. Nevertheless, their bioactive compounds remain unexplored. This study is aimed to assess the antimicrobial and antioxidant activity of basil leaf extract prepared using ethanol, methanol, and water. The ethanol and methanol extract have all the phytochemicals (alkaloids, flavonoids, phenolic compounds, tannins, saponins, quinones, carbohydrates, and proteins) except steroids and terpenoids. In addition to steroids and terpenoids, tannin was also absent in the aqueous extract. Total phenolic and flavonoid content was high in ethanol and followed by methanol and aqueous extract. Similarly, ethanol and methanol extract showed strong antimicrobial activity against VRE and MTCC strains at a concentration of 20 mg/mL than aqueous extract. Among the 10 indicators, Staphylococcus aureus is highly susceptible to ethanol extract at a concentration of 8 mg/mL and followed by other MTCC strains. Vancomycin-resistant enterococci pathogens were inhibited at the minimum inhibitory concentration of 14, 16, and 20 mg/mL of ethanol, methanol, and aqueous extract. Further, on the basis of determining the absorbing material (nucleic acid and protein) at 260 nm and scanning electron microscopic, it was confirmed that the loss of cell membrane integrity and cell membrane damage were the effective mechanisms of plant extract antimicrobial activity. All three solvents have shown remarkable antioxidant activity. Gas chromatography-mass spectrometry analysis of basil leaves ethanol extract identified 19 compounds with various therapeutic and food applications.

Bactericidal and non-cytotoxic activity of bacteriocin produced by Lacticaseibacillus paracasei F9-02 and evaluation of its tolerance to various physico-chemical conditions.

This study aims to explore novel lactic acid bacteria (LAB) from breast-fed infants' faeces towards characterizing their antimicrobial compound, bacteriocin. The LAB, Lacticaseibacillus paracasei F9-02 showed strong antimicrobial activity against clinical pathogens. Their proteinaceous nature was confirmed as the activity was completely abolished when treated with proteinaceous enzymes and retained during neutral pH and catalase treatment. The purified bacteriocin showed antimicrobial activity at the minimum inhibitory concentration (MIC) value of 7.56 µg/ml against vancomycin-resistant Enterococcus sp. [vancomycin-resistant enterococcal (VRE)], and methicillin-resistant Staphylococcus aureus (MRSA), 15.13 µg/ml against Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhi and 30.25 µg/ml against Shigella flexneri. Present study also proved the bactericidal, non-cytotoxic and non-hemolytic nature of bacteriocin. Additionally, bacteriocin retained their stability under various physico-chemical conditions, broad range of pH (2-10), temperature (40-121°C), enzymes (amylase, lipase and lysozyme), surfactants [Tween-20, 80, 100 and sodium dodecyl sulfate (SDS)], metal ions (CaCl2 , FeSO4 , ZnSO4 , MgSO4 , MnSO4 , CuCl2 ) and NaCl (2%-8%). The molecular weight of bacteriocin (~28 kDa) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), functional and active groups were assessed by Fourier Transform-Infrared (FT-IR). To our knowledge, this is the first study reporting L. paracasei from breast-fed infants' faeces and assessing their antimicrobial compound, bacteriocin. The study results furnish the essential features to confirm the therapeutic potential of L. paracasei F9-02 bacteriocin.

Phenotypic Assessment of Probiotic and Bacteriocinogenic Efficacy of Indigenous LAB Strains from Human Breast Milk.

Breast milk is the combination of bioactive compounds and microflora that promote newborn's proper growth, gut flora, and immunity. Thus, it is always considered the perfect food for newborns. Amongst their bioactives, probiotic communities-especially lactic acid bacteria (LAB)-are characterized from breast milk over the first month of parturition. In this study, seven LAB were characterized phenotypically and genotypically as Levilactobacillus brevis BDUMBT08 (MT673657), L. gastricus BDUMBT09 (MT774596), L. paracasei BDUMBT10 (MT775430), L. brevis BDUMBT11 (MW785062), L. casei BDUMBT12 (MW785063), L. casei BDUMBT13 (MW785178), and Brevibacillus brevis M2403 (MK371781) from human breast milk. Their tolerance to lysozyme, acid, bile, gastric juice, pancreatic juice, and NaCl and potential for mucoadhesion, auto-aggregation, and co-aggregation with pathogens are of great prominence in forecasting their gut colonizing ability. They proved their safety aspects as they were negative for virulence determinants such as hemolysis and biofilm production. Antibiogram of LAB showed their sensitivity to more than 90% of the antibiotics tested. Amongst seven LAB, three isolates (L. brevis BDUMBT08 and BDUMBT11, and L. gatricus BDUMBT09) proved their bacteriocin producing propensity. Although the seven LAB isolates differed in their behavior, their substantial probiotic properties with safety could be taken as promising probiotics for further studies to prove their in vivo effects, such as health benefits, in humans.

Bacteriocin-a potential antimicrobial peptide towards disrupting and preventing biofilm formation in the clinical and environmental locales.

Biofilm, a consortium of microbial cells, protected by extracellular polymeric matrix, is considered a global challenge due to the inherent antibiotic resistance conferred by its lifestyle. Besides, it poses environmental threats causing huge damage in food industries, fisheries, refineries, water systems, pharmaceutical industries, medical industries, etc. Living in a community of microbial populations is most critical in the clinical field, making it responsible for about 80% of severe and chronic microbial diseases. The necessity to find an alternative approach is the need of the hour to solve these crises. So far, many approaches have been attempted to disrupt the initial stage of biofilm formation, including adherence and maturation. Bacteriocins are a group of antimicrobial peptides, produced by bacteria having the potential to disrupt biofilm either by itself or in combination with other drugs than antibiotic counterparts. A clear understanding on mechanisms of bacterial biofilm formation, progression, and resistance will surely lead to the development of innovative, effective biofilm control strategies in pharmaceutical, health care industries and environmental locales.