Rika Indri Astuti

Limosilactobacillus fermentum 5.1.2, a tempeh-derived probiotic candidate, was investigated through an integrated genome-to-cell approach to evaluate its antioxidant capacity. Whole-genome analysis using GhostKOALA (KEGG) and antiSMASH 7.0 identified a terpene precursor biosynthetic gene cluster and key genes involved in terpenoid backbone (idi, GGPS) and terpenoid-quinone biosynthesis (menA, menB, menC, menE, ubiE). LC-QTOF-MS of the cell-free supernatant extract putatively identified terpenoid and terpenoid-quinone metabolites, including linalyl propionate, geranylbenzoquinone, geranylhydroquinone, theasapogenol A, and momordol. This is consistent with the predicted biosynthetic pathways. The cell-free supernatant exhibited strong DPPH radical-scavenging activity (84.85–88.99% inhibition), while the extract showed antioxidant activity against DPPH and ABTS+ radicals with IC₅₀ values of 660.5647 ± 35.67 µg/mL and 333.886 ± 13.13 µg/mL, respectively. Extract conferred cellular protection to Schizosaccharomyces pombe under H₂O₂-induced oxidative stress, comparable to calorie-restriction and ascorbic acid controls. These findings establish L. fermentum 5.1.2 as a multifunctional probiotic candidate with genomically supported antioxidant capacity.

Background and Aim: After more than three decades of freedom from foot-and-mouth disease (FMD), Indonesia experienced widespread outbreaks in 2022–2023, raising major concerns regarding viral evolution and vaccine effectiveness. Foot-and-mouth disease virus (FMDV) serotype O remains the predominant circulating serotype in the region. However, the immunological and structural consequences of recent genetic variation have not been comprehensively evaluated. This study aimed to integrate molecular, immunoinformatic, and structural analyses to characterize FMDV serotype O isolates from West Java and South Sumatra and to assess their implications for antigenicity, immune recognition, and vaccine matching. Materials and Methods: Clinical epithelial samples were collected from naturally infected cattle during outbreaks in West Java and South Sumatra. Viral RNA was extracted, and the capsid genes VP1, VP2, and VP3 were amplified and sequenced. Phylogenetic relationships were inferred using VP1 nucleotide and amino acid sequences. Immunoinformatic analyses were conducted to predict VP1-derived T-cell (BoLA-restricted) and B-cell epitopes, followed by in silico evaluation of antigenicity, allergenicity, and toxicity. Structural analyses included prediction of VP1 ligand-binding pockets and molecular docking between VP1 and Toll-like receptor 7 (TLR7) to explore innate immune recognition. Results: Sequence comparison revealed reduced identity of VP1 (98.26–99.05%) and VP3 (as low as 98.48%) relative to the 2022 Indonesian reference strain. Phylogenetic analysis identified three nucleotide-based clusters and two amino acid–based clusters, indicating intra-country diversification and the emergence of potential micro-lineages. Several amino acid substitutions occurred near known immunogenic regions of VP1, resulting in altered T- and B-cell epitope binding profiles in selected isolates. Predicted epitopes were predominantly antigenic and non-toxic, although some showed potential allergenicity. Structural modeling demonstrated variability in VP1 binding-pocket composition among isolates. Docking analysis revealed favorable VP1–TLR7 interactions, particularly in selected South Sumatra isolates, suggesting strong innate immune engagement. Conclusion: This integrated molecular–immunoinformatic–structural analysis demonstrates that newly circulating Indonesian FMDV serotype O isolates exhibit genetic, antigenic, and structural divergence that may reduce current vaccine matching. Continuous molecular surveillance and regionally adapted vaccine design are therefore essential to maintain effective FMD control in Indonesia. Keywords: foot-and-mouth disease virus, immunoinformatics, Indonesia, molecular docking, phylogenetic analysis, serotype O, TLR7 interaction, VP1 gene.

The growing global prevalence of diabetes mellitus has sharply increased the demand for insulin and its analogues. Pichia pastoris is a well-established system for recombinant pro-insulin and its analogues production. However, conventional gene constructs often include additional sequences, such as Glu-Ala repeats, spacer peptides, and c-peptides that complicate downstream processing and reduce efficiency. This study aimed to construct and express a proglargine (PG) gene cassette lacking the Glu-Ala repeats, spacer, and c-peptide in P. pastoris GS115 to obtain a uniform PG protein. The recombinant vector propagated in Escherichia coli TOP10F’, then expressed in P. pastorisGS115. Selected transformants were cultivated in YPG medium, then induced with 1% and 2% methanol daily in BMMY. The optimum methanol concentration further evaluated in ½ BSM induction medium. The result demonstrated that optimal PG expression was achieved with 2% methanol induction in BMMY, producing higher levels than those with ½ BSM. Among the transformants, PG.c2 produced the highest PG protein in BMMY medium induced with 2% methanol. Dot-blot analysis confirmed the secretion of PG, and LC-HRMS analysis demonstrated 100% amino acid sequence coverage of PG, confirming the integrity and completeness of the expressed protein. This study presented a newly modified PG gene cassette, inserted into pPICZαA vector, to express uniform secreted PG in P. pastoris GS115. By simplifying the precursor structure, a more homogeneous precursor product can be obtained, which is expected to simplify purification and also the downstream enzymatic process of PG into mature insulin glargine.

Pathogenic bacterial infection is still a major health problem that causes death. Endophytic bacteria in medicinal plants have the potential as antibacterial agents. Thus, endophytic bacteria in the medicinal plant, clove (Syzygium aromaticum), allow it to produce the same bioactive compounds as those contained in its host. This study aimed to analyze the antibacterial activity of five endophytic bacterial isolates from clove leaves. Antibacterial activity was evaluated using disc diffusion assay. Based on the results, isolate Niallia nealsonii DCL1 have strong antibacterial activity against Escherichia coli ATCC 8739, while Bacillus paranthracis DCL3 isolate has intermediate antibacterial activity against Pseudomonas aeruginosa ATCC 15442. In addition, extracellular metabolites of these two isolates of DCL1 and DCL3 which harvested at stationary phase showed antibacterial activity against and E. coli ATCC 8739 and P. aeruginosa ATCC 15442. These data indicate that the potential antibacterial compounds from DCL1 and DCL3 are considered extracellular secondary metabolites. Further analysis on the mode of antibacterial action should be carried out for further medical application.

Aflatoxin B1 (AFB1) is a potent mycotoxin that threatens food safety and human health. This study investigated the capacity of yeast isolates to tolerate and detoxify AFB1 and performed genomic characterization of the most active strain. Among several Pichia and Wickerhamomyces isolates, Pichia kudriavzevii RWT exhibited the strongest tolerance, maintaining growth at AFB1 concentrations up to 200 ppb and decontaminating 26.5% of AFB1 within six hours, more than twice the reduction achieved by the next best isolate. In contrast, heat-killed yeast cells showed no significant reduction activity. This suggests that cell wall adsorption alone is insufficient for meaningful AFB1 reduction in this strain and highlights the importance of enzymatic or transporter-mediated mechanisms that require live, metabolically active cells. Whole-genome sequencing of RWT yielded a 10.86 Mb assembly with 5,445 predicted proteins grouped into 4,274 orthologous clusters, sharing 2,448 core clusters with related yeasts but retaining 953 clusters unique to Pichia species. Functional annotation highlighted genes potentially involved in AFB1 detoxification, including predicted cytochrome P450, epoxide hydrolase (LAP2), glutathione-S-transferase (URE2), and the ABC transporter (YCF1), suggesting pathways for AFB1 activation, glutathione conjugation, and vacuolar sequestration. AFB1 treatments were found to increase the gene expression of those key genes involved in AFB1 detoxification. Comparative genomics confirmed that RWT’s genome size and core gene content are typical of P. kudriavzevii, while its unique clusters are enriched in membrane transport, stress response, and metal-ion binding functions. These findings position P. kudriavzevii RWT as a promising candidate for biological AFB1 mitigation, providing a genetic basis for its robust detoxification capacity.