Applied Microbiology

Unsustainable agricultural practices and overreliance on chemical fertilizers have led to severe environmental issues, such as soil and water pollution, loss of biodiversity, and risks to human and animal health. Moreover, plant diseases continuously decrease crop productivity and threaten global food security. Therefore, there is a strong need to focus on sustainable agricultural practices. Microbial biofertilizers emerge as environment-friendly alternatives to chemical fertilizers that help in nutrient solubilization and availability, soil fertility, and plant growth promotion, in addition to curbing the application of chemical fertilizers. Microbial inoculants enhance agricultural yield by performing complementary roles, such as facilitating nutrient uptake through biological nitrogen fixation and phosphate solubilization, promoting plant growth via phytohormone synthesis, and mitigating diseases by activating plant defense responses. A 2025 meta-analysis of 107 field studies in China reported mean yield increases of 22.3% in wheat, 13.6% in rice, 12.8% in maize, and 65.4% in millet, while a field study in saline soil reported a 25% reduction in NPK fertilizer use in barley without reducing the grain yield. This review provides an overview of the major types of microbial biofertilizers, their modes of action, and their use in important cropping systems. Special emphasis is placed on microbial consortia that can enhance nutrient cycling, plant productivity, and tolerance to abiotic stress factors. The application of nanotechnology, genetically engineered microorganisms, and combinations of microbial inoculants with organic waste are some strategies that could be adopted for next-generation biofertilizer development. The review also addresses the major hurdles in the formulation, field performance, and commercialization of microbial biofertilizers. Future perspectives revolve around optimizing microbial formulations, applying advanced biotechnological tools, and developing enabling policies for the rapid adoption of microbial biofertilizers for sustainable agriculture. Full article

Bacteria sense, respond, and adapt to rapidly changing environments through highly sensitive intracellular networks. Among them, cyclic-di-GMP (c-di-GMP) is a well-characterized second messenger controlling the transition from planktonic to sessile lifestyles, making it a key determinant of bacterial adaptation and survival. Two main approaches have been developed to investigate c-di-GMP dynamics. The first uses biological biosensors for live-cell monitoring of intracellular c-di-GMP through transcriptional reporters, RNA-based sensors, or protein-based sensors. Their main advantage is real-time, spatiotemporal analysis in living cells, although they generally do not provide absolute quantification. The second approach relies on analytical chemistry, particularly liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), which enables sensitive and accurate quantification of intracellular c-di-GMP across diverse bacterial species. However, the lack of standardized extraction protocols and the need for cell lysis prevent real-time measurements, providing only a snapshot of the total c-di-GMP pool. These approaches are complementary: biosensors reveal dynamic, single-cell responses, whereas LC-MS/MS provides precise global quantification. Nevertheless, most studies rely on only one method, limiting a comprehensive understanding of c-di-GMP biology. Combining both approaches would provide a more integrated view of c-di-GMP signaling and its role in bacterial physiology. Full article

This study aimed to investigate the microbiome profile of Culex pipiens, the most abundant mosquito and pathogen vector in the Palearctic, and to find microbiome region and ecotype specificity. The taxon Cx. pipiens includes two ecotypes—pipiens and molestus—and their hybrids, which differ in their epidemiological role. Microbiomes were extracted using bioinformatic analysis from whole-genome datasets of 47 individual Cx. pipiens (pipiens and molestus forms and their hybrids) collected from seven geographically and climatically distinct Russian regions. The dominant phyla and genera were identified, along with a fourfold decrease in microbiome diversity during the transition from larva to adult, and a negative effect of Wolbachia on Cx. pipiens microflora diversity. Microbiome community composition differed significantly between the majority of geographical groups. Analysis of microbial community composition points to anthropogenically altered ecosystems in mosquito habitats. Microbes specific to certain geographical locations were identified. Both forms, pipiens and molestus, share a phylogenetically similar core of dominant taxa but differ in their abundances. A shared core microbiome, present across all samples, is most likely essential for the normal development and survival of Cx. pipiens, and may serve as a target for pathogen-blocking paratransgenesis and region-specific control strategies. Full article

The emergence of antimicrobial resistance (AMR) has created a need for new, targeted alternatives to conventional antibiotics. Bacteriocins and other targeted antimicrobial peptides (AMPs) are emerging therapeutic approaches that have attracted attention for their potent antimicrobial properties and potential for more specific effects on microbial communities. The review describes the development of probiotics from lactic acid bacteria (LAB) to next-generation probiotics (NGPs), incorporating genomics, metagenomics, and synthetic biology to develop and engineer antimicrobial-producing microbial platforms. A comparative analysis of the bacteriocin profiles of conventional LAB and NGPs is presented, highlighting differences in diversity, specificity, and therapeutic potential. Additionally, recent advances in large-scale bacteriocin production systems, including recombinant expression and bioengineering methods, are discussed. Issues related to delivery systems, stability, host interactions, and targeted release are discussed. Most evidence comes from in vitro and animal studies, with limited clinical data on bacteriocin-producing probiotics and NGPs. There is also a significant gap in direct experimental proof of bacteriocin production by many proposed NGPs, hindering their development as targeted antimicrobials. Regulatory, scale-up, and manufacturing challenges remain major barriers to commercialization and broad therapeutic use. Target pathogen prioritization is a final step highlighted in the review that will help in therapeutic precision and improve the outcome of treatment for multidrug-resistant pathogens. Overall, LAB-derived bacteriocins have the most substantial evidence for production, characterization, and safety, while NGP-associated systems are promising but mostly preclinical and need further structural and functional validation. Moving towards precision antimicrobial therapy will depend on developing standardized activity assays, evaluating microbiome interactions, monitoring resistance, ensuring strain safety, establishing scalable manufacturing processes, and conducting comprehensive human clinical trials. Full article

The gastrointestinal microbiome plays a central role in host physiology, and the predominance of beneficial microorganisms is associated with improved metabolic and immune functions. Enterococci are natural members of the gut microbiota and are known to produce enterocins with antimicrobial activity against various pathogenic bacteria, thereby modulating microbial community structure. This study investigates the metabolic potential of Enterococcus sp. SB12, including vitamin and amino acid profiles, as well as its effects on intestinal microbiome composition, blood biochemistry, and oxidative stress markers in mice. Vitamin and amino acid profiles were analyzed by HPLC in bacterial biomass, as well as in the growth medium before and after cultivation of Enterococcus sp. SB12. Twenty one-month-old female mice were divided into control and experimental groups (n = 10 per group). The experimental group received Enterococcus sp. SB12 daily in drinking water at a dose of 1 × 10⁸ CFU/g body weight for 29 days. Gut microbiota composition was assessed using 16S rRNA gene and ITS2 sequencing, and biochemical and oxidative stress parameters were determined in blood and tissues. HPLC analysis of bacterial biomass detected several biologically important vitamins, including vitamins B1, B3, B5, B6, and C, together with amino acids such as valine (9.27 mg/g), cysteine (5.80 mg/g), glutamine (5.34 mg/g), proline (4.47 mg/g), and taurine (2.82 mg/g). Metataxonomic analysis revealed an increased relative abundance of Enterococcus in the experimental group, which may be consistent with persistence of enterococci in the gastrointestinal tract. Administration of SB12 was associated with an increased relative abundance of Enterococcus and several Firmicutes-associated taxa, accompanied by a reduced relative abundance of members of the family Desulfovibrionaceae and Helicobacter-associated taxa compared with the control group. Mice receiving SB12 exhibited significantly higher body weight than controls (28.1 ± 1.05 vs. 27.2 ± 0.82 g; p < 0.05), whereas visceral fat mass, organ weights, blood glucose, total protein, HDL cholesterol, LDL cholesterol, and oxidative stress markers did not differ significantly between groups. Overall, administration of Enterococcus sp. SB12 was associated with compositional changes in the intestinal microbiome and did not induce detectable alterations in biochemical or oxidative stress parameters. These findings provide preliminary evidence supporting the probiotic potential and safety of Enterococcus sp. SB12 under the experimental conditions used; however, additional studies are required to confirm its long-term safety profile and functional effects on the host. Full article

Lactobacilli are Gram-positive fermentative bacteria with GRAS (“Generally Recognized As Safe”) status. Since 2017, it has been reported that many lactobacilli strains can form extracellular vesicles. Extracellular vesicles (EVs) are spherical bilayered membrane structures of nanometric size that carry cytoplasmic components. Bacterial EVs are considered important mediators between host and commensal bacteria, but their biological effects have not been widely studied. The purification of EVs involves methodological difficulties. Due to their nanometric size, EVs are prone to being co-purified with media compounds, which interfere with their characterization. The present work aimed to obtain high-purity EVs and to determine their physicochemical properties. To achieve the objectives, we developed a minimal synthetic medium (MSM) suitable for the growth of Lactiplantibacillus plantarum CIDCA 83114 and also designed a protocol to concentrate and obtain its EVs. The medium was free of high-molecular-weight compounds and surfactants, and the purification protocol included EV concentration via a tangential flow filtration step. The EVs obtained had an average size of 110 nm ± 40 nm and a negative zeta potential. They exhibited a mean protein content of 100 µg/mL with a clear protein profile absent in the culture media. EVs also contain RNA and scarce DNA. Our work contributed to improving the purification of lactobacilli EVs by replacing the traditional medium used in lactic acid bacteria growth (MRS) with a synthetic minimal medium (MSM). The design of the MSM allows for high-purity vesicles to be obtained and accurately characterized. Full article

Plants have coevolved with microbes for nearly 500 million years; however, their interrelationship is not well understood. Plant roots have an intricate relationship with soil microbes. Such relationships mold the growth, development, immunity and physiology of plants and thus are of immense interest to agriculture and the environment. Technological advancements in sequencing, imaging, omics, synthetic biology and artificial intelligence have allowed scientists to dissect such relationships to a higher resolution. Thus, these advances have facilitated a deeper understanding of plant–microbe interactions and their role in the life cycle of plants and the environment. However, factors such as microbial diversity, microbial abundance, heterogeneity of soil and plasticity of the environment have precluded a clear in situ understanding of microbial community structure. Thus, constructing synthetic microbial communities or SynComs and investigating their effect on plants in a controlled environment offers a reductionist and manageable approach to understand plant–microbe relationships. This approach reduces the confounding variables present in the natural environment and facilitates the understanding of such complex interactions. Members of such communities are identified using 16S rRNA sequencing and are constructed using few microorganisms; often fungal strains are added for cross-kingdom SynComs. Metabolic modeling, metabolic cross-feeding along with ecological and evolutionary principles, can be used while choosing candidates for SynComs. Scalability, transferability, reproducibility, predictability and stability are the major bottlenecks in SynCom research. This emerging area of science may have transformative impact in agriculture, environment and space colonization. In this article, we present our perspective on the latest advancements, challenges and future potential of this technology. Full article

Traditional fermented foods are important reservoirs of wild microorganisms with technological, sensory, protective, and functional potential. However, the performance of these isolates in controlled or compositionally different food matrices remains fragmented across microbial groups and food systems. This systematic review synthesized evidence on using wild microbial isolates from traditional fermented foods and beverages as starters or potential probiotic cultures. The conducted a systematic search exclusively in Scopus, following PRISMA 2020, to include original research articles published between 2021 and 2026, yielding 68 eligible studies. The included studies were analyzed by geographical origin, isolation source, recipient matrix, microbial group, and key physicochemical, technological, sensory, microbiological, nutritional, and functional outcomes. The evidence was organized into wild yeasts and filamentous fungi, lactic acid bacteria (LAB), Bacillus isolates, and defined mixed microbial cultures. Across food matrices, microbial incorporation frequently accelerated acidification, shortened fermentation time, modified volatile compound profiles, and altered texture, color, enzymatic activity, or substrate utilization. Sensory responses improved aroma, flavor, texture, and acceptance, whereas others produced profiles that deviated from the characteristic product and reduced overall liking. Functional effects included increases in phenolic compounds, antioxidant activity, GABA, folate, peptides, and resistant starch, along with reductions in phytates, nitrites, biogenic amines, aflatoxins, and nondigestible oligosaccharides. Researchers also reported antimicrobial, antifungal, protective, and preliminary probiotic properties. Defined mixed microbial cultures often provided complementary metabolic effects, although true synergistic interactions were demonstrated only sporadically. Overall, wild isolates from traditional fermentations represent promising resources for food bioprocessing; however, their performance varies widely across strains, recipient matrices, experimental conditions, and outcomes evaluated. Consequently, their application requires strain–matrix validation, comprehensive sensory assessment, safety characterization, and evaluation under processing and storage conditions relevant to industrial production. Full article

Farnesol is a small isoprenoid metabolite that has emerged as a key regulator of fungal biology beyond its original identification as a quorum-sensing molecule in Candida albicans. This review examines farnesol across pathogenic and non-pathogenic fungi, emphasizing its roles in morphogenesis, biofilm development, stress adaptation, membrane-associated physiology, and ecological interaction. In pathogenic fungi, farnesol modulates virulence-related traits, antifungal susceptibility, and host interaction, while in non-pathogenic systems, it influences growth, differentiation, and metabolic balance. Evidence from Saccharomyces cerevisiae, Trichoderma, Candidozyma auris (formerly known as Candida auris), and other fungi highlights the context-dependent nature of its effects. We also discuss farnesol biosynthesis, secretion, and the apparent absence of canonical salvage pathways in fungi. Together, these findings support a broader view of farnesol as a multifunctional fungal metabolite that links signaling with metabolism. Understanding its diverse biological roles may clarify fungal evolution and inform future strategies targeting fungal persistence, adaptation, and antifungal tolerance. Full article

Cocoa fermentation is a critical postharvest process that determines the physicochemical and sensory quality of cocoa beans through complex microbial and biochemical transformations. This study evaluated the effect of selected lactic acid bacteria (LAB), i.e., Weissella confusa and Lactiplantibacillus plantarum, and the fermentation system on the fermentation dynamics and final quality of two cocoa genotypes (i.e., Theobroma cacao L.; Nacional and CCN-51). A completely randomized 2 × 2 × 2 factorial design was applied, considering cocoa genotypes, LAB species, and fermentation system (i.e., laboratory fermentation and cascade-type box fermentation). During fermentation, pH, temperature, and total soluble solids (°Brix) were monitored at 0, 24, 48, and 72 h. In addition, cut test parameters and the physicochemical properties of the final cocoa paste were evaluated. The results showed a progressive decrease in pH (from 3.13 to 4.17), accompanied by a temperature increase up to 46.50 °C and a marked reduction in soluble solids during the final fermentation stages, reflecting intense microbial metabolism and substrate utilization. Treatments inoculated with L. plantarum achieved the highest proportion of well-fermented beans (up to 93.73%), indicating enhanced fermentation performance. Furthermore, the physicochemical properties of the final cocoa paste, including moisture, fat, ash, pH, and °Brix, were significantly influenced by the interaction among cocoa genotype, LAB species, and fermentation system. Overall, controlled fermentation using selected LAB species represents a promising biotechnological strategy for improving cocoa fermentation consistency and enhancing postharvest cocoa quality. Full article

Prebiotic potential refers to the stimulation of beneficial bacteria over pathogenic strains. This preliminary study evaluated the prebiotic potential of five agave-derived carbohydrate preparations using lactic acid bacteria (LAB) and Escherichia coli (pathogenic) strains. The tested substrates included commercial inulin, commercial agave syrup, artisanal maguey syrup, aqueous and cooked agave extract. Six Lactobacillus strains (Lactobacillus acidophilus, Lacticaseibacillus casei, Lactiplantibacillus plantarum ATCC 8014, Limosilactobacillus reuteri NRRL B-14171, Lactiplantibacillus plantarum NRRL B-4496, and Lacticaseibacillus rhamnosus) were evaluated against E. coli ATCC 43888 and E. coli clinical isolates. Carbohydrate characterization included analyses of total and reducing carbohydrates and thin-layer chromatography (TLC) to assess the degree of polymerization (DP). Prebiotic Index (PI) and Prebiotic Activity Score (PAS) were calculated to determine selective growth stimulation at 24 and 48 h. TLC analysis revealed two distinct groups: high-molecular-weight compounds (aqueous agave extract and commercial inulin), and low-molecular-weight compounds (cooked agave extract, commercial agave syrup, and artisanal maguey syrup). L. reuteri NRRL B-14171 exhibited the highest PI (approaching 2.0) with aqueous agave extract at 24 h. L. rhamnosus demonstrated superior PAS values across all prebiotic sources, while commercial inulin and artisanal maguey syrup showed the most favorable PAS values against clinical E. coli isolates, indicating preferential in vitro growth of LAB over the pathogen. These findings represent preliminary indications of selective carbohydrate utilization by LAB strains under controlled in vitro conditions. Further studies, incorporating digestion resistance, fermentation by complex microbiota, metabolite production, and host-related effects are required before confirming prebiotic functionality. Full article

Lactic acid bacteria (LAB) from traditional foods are a useful source of natural biopreservatives. This study isolated and characterized indigenous LAB from goat meat produced in the argan grove ecosystem of the Souss-Massa region, Morocco, to identify strains that could improve food safety and shelf life. LAB were isolated under anaerobic conditions and screened by Gram staining, catalase test, growth profiling, and biochemical assays. Proteolytic, gelatinase, and hemolytic activity were assessed, together with antimicrobial activity against foodborne pathogens by agar well diffusion. The most promising strains were identified by 16S rRNA gene sequencing. The selected isolates belonged to Latilactobacillus and Enterococcus. Several isolates strongly inhibited Salmonella enterica, Listeria monocytogenes and Staphylococcus aureus, and none were hemolytic or gelatinase-positive. The sustained inhibition even after neutralization of pH suggested that the antimicrobial effects might be mediated by bacteriocin-like compounds or other non-acidic metabolites. In the strains tested, L. sakei MG4013 gave the best results because of its wide range of antimicrobial activity and absence of hemolytic, gelatinase, proteolytic and lipolytic activities. Goat meat from the Souss-Massa region therefore harbours LAB with biopreservative potential, with potential for use as natural preservatives in traditional meat products or as starter cultures in fermented foods. However, before being used as natural preservatives or starter cultures, they need to be further validated in food matrices. Full article

This research focused on the plant growth-promoting properties of endophytic bacteria isolated from Morisonia scabrida, a tree adapted to heat stress. Sixteen bacterial strains were isolated; among them, S1R21 tolerated 50 °C; S1H21 inhibited the growth of Fusarium sp. FH at 25 and 30 °C; and S1R16 inhibited Alternaria sp. ATCC20084 and F. oxysporum CTLM12 at 25 and 30 °C. Regarding enzymatic activities, S1R21 exhibited amylase activity at 30 °C; S1T20 showed proteinase activity at 37 °C; S1T11 showed pectinase activity at 37 °C; S1R16 and S1R9 demonstrated lipase activity; and S1R20 showed CMCase activity at 30 °C. S1T1 solubilized tricalcium phosphate at 25 and 30 °C; S1H5 produced siderophores at 30 °C; and S1T11 produced IAA and fixed nitrogen at 30 and 37 °C. With respect to germination percentage of corn and alfalfa, the highest values were achieved with strain S1R21 at 30 and 37 °C. Seven strains were selected and identified by 16S rDNA analysis as Bacillus albus S1H21, Bacillus cereus S1R21, Lysinibacillus fusiformis S1R9, Enterobacter sp. (S1H5 and S1R8), Klebsiella sp. S1R16 and Stutzerimonas stutzeri S1T11. Most of these strains increased growth parameters of Zea mays and Medicago sativa, with Stutzerimonas stutzeri S1T11 standing out regarding high aerial and root length, as well as fresh and dry weight. This research study is one of the few studies on endophytic bacteria associated with the Morisonia scabrida tree. It demonstrates that its bacterial diversity could contribute to various biological roles, such as that of plant growth promoter under heat stress. Full article

High fructose intake rapidly induces protein glycation, oxidative stress, inflammation, and disturbances in the cultivable fraction of gut bacteria, contributing to early metabolic impairment. This study examined whether selected plant-derived bioactives and a prebiotic–probiotic formulation could mitigate fructose-induced biochemical and microbial alterations. Male Wistar rats were fed fructose for 45 days, and advanced glycation end product (AGE)-associated fluorescence, oxidative stress markers, and lipid peroxidation were measured to assess metabolic changes. Culture-dependent enumeration of intestinal and fecal bacteria was performed to evaluate shifts in cultivable aerobic and facultative bacterial counts, while limonene, eugenol, and emodin were tested for antibacterial activity against aerobic and facultative bacterial isolates obtained from fructose-fed rats during our previous study. The prebiotic–probiotic formulation was assessed alone and in combination with these bioactives. Fructose feeding increased protein glycation, oxidative stress, lipid peroxidation, and reduced counts of cultivable gut bacteria. Treatment with the bioactives and the formulation lowered glycation-related fluorescence, reduced oxidative stress, and decreased lipid peroxidation. The bioactives exhibited antioxidant and antiglycation activity and inhibited growth of selected cultivable bacterial isolates, including Corynebacterium stationis. While emodin contributed primarily through its known α-glucosidase inhibitory and antiglycation properties rather than antibacterial activity. Combined treatment partially restored cultivable bacterial counts and improved metabolic parameters. Overall, the interventions attenuated fructose-induced biochemical disturbances and modulated the cultivable gut bacterial counts, suggesting a complementary approach to managing early metabolic changes in rats associated with high fructose intake. Full article

Endophytic bacteria from medicinal plants are increasingly recognised as sources of antimicrobial metabolites. However, the endophytic bacterial community of Mitrephora heyneana remains poorly explored. In the present study, endophytic bacteria were isolated from the leaves of M. heyneana, collected from the Western Ghats of Tamil Nadu, India. Among seven isolates, the plant endophyte strain from leaf 6th strain (PEL6) was identified as Paenibacillus lactis through 16S rRNA gene sequencing. The ethyl acetate extract of PEL6 (EAE-PEL6) was subjected to gas chromatography–mass spectrometry (GC-MS) analysis, which putatively identified 32 metabolites based on GC-MS library matching, including pyrrolo [1,2-a] pyrazine-1,4-dione derivatives and triazole compounds as major constituents. The EAE-PEL6 demonstrated significant in vitro antibacterial activity against Staphylococcus aureus. In silico ADMET (absorption, distribution, metabolism, excretion, and toxicity), profiling predicted drug-likeness and pharmacokinetic properties of selected candidate compounds. Molecular docking suggested favourable binding of selected metabolites to S. aureus target proteins; however, these interactions require experimental validation. Density Functional Theory calculations indicated that CID 70504 had the lowest Highest Occupied Molecular Orbital (HOMO)–Lowest Unoccupied Molecular Orbital (LUMO) energy gap, reflecting higher electronic reactivity. Molecular electrostatic potential mapping further supported its enhanced binding propensity. Molecular dynamics simulations suggested structural stability, with Root Mean Square Deviation, Solvent Accessible Surface Area, radius of gyration, and hydrogen-bond analyses indicating stable interactions throughout the 100 ns trajectory. Overall, this study identifies P. lactis PEL6 as a promising endophytic source of anti-S. aureus metabolites and provides candidates for future purification, structural confirmation, and biological validation. Full article

This study investigates the impact of selected soil parameters and integrated fertilizer formulations on the productivity of three legume crops—mung bean, soybean, and cowpea—in order to identify the optimal strategies to maximize yields through reduced nitrogen and bio-augmentation. A five-year experiment composed of 2-year pot trials (2019–2020) and 3-year field trials (2022–2024) was conducted to assess the changes in soil parameters (N, P, K, OM, pH) and yield response of crops with varying amounts (1, 2, 4 kg) of Bradyrhizobium-based biofertilizer combined with a 25–50% reduction in mineral N fertilizer. The biofertilizer was composed of locally isolated strains, which were genetically identified in our previous reports as B. elkanii NE1-6, NE2-1, B. diazoefficiens NE1-65, Bradyrhizobium sp. NE1-19, NE1-34, and NE2-3. The results indicated that the amount of K strongly influenced yield increase for soybean (r = 0.85, p < 0.05) and mung bean (r = 0.67, p < 0.05), while the amount of N had the greatest influence on cowpea (r = 0.60, p < 0.05). Soybean yield was maximized with a 50% reduction in N fertilizer (20 kg N) combined with 2–4 kg biofertilizer, while cowpea and mung bean achieved increased yields at 25% reduced N fertilizer combined with 2–4 kg biofertilizer. This study confirms the viability of integrating Bradyrhizobium-based biofertilizer with a 25–50% reduction in mineral N fertilizer without yield loss by harnessing the efficient N-fixation ability of the strains in the consortium. Full article

The efficient extraction and purification of recombinant β-casein from Escherichia coli is a crucial step in bioprocess engineering. This study evaluates an adapted high-pressure homogenization (HPH) workflow for recombinant β-casein recovery and compares it with osmotic shock (OS) and standard HPH based on specific protein yield, estimated relative purity, and processing time. In contrast to standard HPH, which requires separate inclusion body recovery, washing, and subsequent denaturant-mediated solubilization, the adapted-HPH workflow integrates mechanical cell disruption with inclusion body recovery during homogenization. Standard HPH was evaluated in two downstream-processing runs, whereas adapted HPH and OS were each evaluated in three downstream-processing runs using separate aliquots from the same bioreactor biomass batch. Adapted HPH demonstrated higher specific yields (4.08 mg_casein/g_CDW) and required 40% less processing time compared to the OS and standard HPH methods. The specific yield obtained with adapted HPH was significantly higher than that obtained with standard HPH (p = 0.026), whereas the difference between adapted HPH and OS was not statistically significant (p = 0.086). Despite the inclusion body washing steps used for OS and standard HPH, adapted HPH achieved a slightly higher average purity than OS (63.46 ± 3.49% versus 59.38 ± 2.79%) and a purity comparable to standard HPH (68.10 ± 6.87%); however, these differences were not statistically significant. Overall, the comparative evaluation of these methods indicates that adapted HPH may provide a simplified alternative for recombinant β-casein recovery, although further validation with a larger number of independent experiments is required. Full article

Background: The fall armyworm, S. frugiperda, is a globally threatening insect pest of agricultural crops, including in Thailand. Symbiotic bacteria in the genera Xenorhabdus and Photorhabdus, which are derived from entomopathogenic nematodes, can produce diverse bioactive compounds with insecticidal activities. However, no study on Thai isolates of these symbiotic bacteria against S. frugiperda has been reported, leading to a significant gap in local biopesticide development. Methods: Two symbiotic bacterial isolates, X. miraniensis (bMH16.1_TH) and P. akhurstii (bSBR11.1_TH), were isolated from Thai entomopathogenic nematodes and cultured in LB broth at 10⁸ CFU/mL. Three bacterial fractions, i.e., whole-cell suspension, cell supernatant, and cell pellet, were prepared from each isolate. Insecticidal bioassays were conducted by topical application on second- and fifth-instar larvae of S. frugiperda (10 larvae × 5 replicates per treatment, CRD). Larval mortality was recorded daily for seven days, and median lethal time (LT₅₀) was calculated. Data were analyzed by two-way ANOVA with Duncan’s Multiple Range Test (p < 0.05). Results: The results indicated that both bacterial isolates caused significantly higher larval mortality than controls across all treatments (p < 0.05). X. miraniensis (bMH16.1_TH) showed higher virulence, achieving ~80% mortality of second-instar larvae within 24 h and 100% by day three. Whole-cell suspensions consistently produced the fastest and highest mortality with the lowest LT₅₀ values in both larval stages. Second-instar larvae were significantly more susceptible than fifth-instar larvae. The shortest LT₅₀ was recorded in second-instar larvae treated with X. miraniensis supernatant at 0.616 days, while fifth-instar larvae generally showed higher LT₅₀ values across all treatments. Conclusions: Our comparative study demonstrated that the insecticidal efficacy varied between the two symbiotic bacterial isolates and among their fractions. Whole-cell suspensions of X. miraniensis (bMH16.1_TH) produced the highest larval mortality and the smallest LT₅₀ values, particularly against early-instar larvae. These findings demonstrated that local symbiotic bacteria are promising candidates to be developed as sources of biopesticides against this insect pest. In addition, characterizing active insecticidal compounds, evaluating field efficacy, and assessing safety to non-target organisms need to be further studied to support integration of these biopesticides in sustainable pest management programs. Full article

Lactic acid bacteria (LAB) are promising oral drug delivery systems due to their probiotic properties, safety, and ability to stimulate mucosal immunity. In this study, we characterized five LAB strains, Lacticaseibacillus casei ATCC 393, Lacticaseibacillus paracasei UST1611, Lactiplantibacillus plantarum UST1611, Limosilactobacillus reuteri LR08, and Lacticaseibacillus rhamnosus GG, to assess their potential as a microbial chassis for oral drug delivery. We tested competence for transformation, plasmid stability, GFP expression under a constitutive promoter, survival under simulated gastrointestinal conditions, and survival in the gut of Danio rerio larvae. Successful transformation with the pTRKH3-ermGFP plasmid was achieved in L. casei ATCC 393, L. paracasei UST1611, and L. reuteri LR08. We also showed that the L. casei ATCC 393 strain had significantly higher protein expression than its L. paracasei UST1611 and L. reuteri LR08 counterparts. In the absence of selection, L. casei ATCC 393 and L. paracasei UST1611 retained their plasmid for 48 h. L. reuteri LR08 retained it for up to 72 h. The three transformed strains showed comparable tolerance to simulated gastrointestinal pH, though L. casei and L. reuteri were more resistant to 0.3% greater bile. In vivo testing showed the highest survival for L. casei ATCC 393 and L. paracasei UST1611 in the gut of zebrafish (Danio rerio) larvae. Overall, our results suggest that, among the five strains tested, L. casei ATCC 393 is the most promising candidate for an LAB-based microbial system for oral drug delivery. Full article