Huijin Jeong

Probiotic candidates require rigorous strain-level safety assessments prior to functional food applications because safety-relevant properties are not universally conserved across strains within the same species. The aim of this study was to evaluate the safety of Levilactobacillus brevis KU15151 in accordance with Food and Agriculture Organization/World Health Organization, European Food Safety Authority (EFSA), and Korean Ministry of Food and Drug Safety guidelines, through phenotypic assays and whole-genome-based analyses. The minimum inhibitory concentrations for all eight evaluated antibiotics were within the EFSA-established cut-off values, and no acquired resistance genes were detected using ResFinder. Phenotypic assessments confirmed the absence of β-hemolytic activity, mucin degradation, gelatin liquefaction, urease activity, indole production, β-glucuronidase activity, bile salt deconjugation, and cytotoxicity towards Caco-2 intestinal epithelial cells at a multiplicity of infection of 250. D- and L-lactic acids were produced at approximately equimolar concentrations, which is consistent with heterofermentative metabolism. Among the eight assessed biogenic amines, only tyramine was detected (79.26 ppm), at less than the published food safety thresholds. VirulenceFinder did not identify any virulence-associated genes. Collectively, these findings suggest that L. brevis KU15151 satisfies the principal safety criteria under the examined conditions, supporting its potential as a probiotic candidate for in vivo validation.

Lactococcus lactis subsp. lactis strains have been widely recognized for their probiotic potential, including their immunomodulatory and anti-obesity effects. This study aims to determine the genomic basis of the health-promoting properties of L. lactis subsp. lactis CAB701 and WiKim0124 using whole-genome sequencing and comparative genomic analysis. Taxonomic classification using average nucleotide identity and digital DNA-DNA hybridization confirms both strains as L. lactis subsp. lactis. Genome alignment reveals high synteny among CAB701, WiKim0124, and the reference strains, with minor structural variations. Functional annotation identifies genes associated with carbohydrate metabolism, biofilm formation, and immune modulation. Pan-genome analysis shows a substantial proportion of strain-specific genes, suggesting niche-specific adaptation. Notably, L. lactis subsp. lactis CAB701 and WiKim0124 harbor unique biosynthetic gene clusters for nisin Z and sactipeptides, which may contribute to their antimicrobial and immunomodulatory functions. Additionally, genes involved in short-chain fatty acid metabolism are identified, highlighting their potential role in metabolic regulation. These findings provide valuable insights into the functional genetics of L. lactis subsp. lactis and their applicability as probiotics for immune modulation and obesity management. KEY POINTS: • Comparative genomics revealed strain-specific immune- and metabolism-linked genes • CAB701 and WiKim0124 uniquely harbor nisin Z and sactipeptide biosynthetic clusters • Genomic features support L. lactis application as functional probiotics.

The presence of antibiotic resistance in probiotic bacteria has raised concerns about the safety of the strains used in food systems. This study elucidated the molecular mechanisms underlying vancomycin resistance in Enterococcus faecium KU22001 using integrated genomic and physiological analyses. The minimum inhibitory concentration (MIC) for vancomycin was determined using the broth microdilution method and whole-genome sequencing was performed to identify resistance-associated genes. The E. faecium KU22001 had a very high MIC for vancomycin (1,280 mg/L); genome annotation identified vanZ and several multidrug transporter genes, but did not detect canonical van operon genes; and the high-performance liquid chromatography analysis suggested that there was no enzymatic degradation of vancomycin in E. faecium KU22001 under the test conditions. The physiological assays indicated a partial modulation of cell wall permeability, whereas cell membrane permeability remained largely unchanged. In contrast, the inhibition of efflux pumps after adding reserpine reduced the vancomycin MIC by approximately 1,024-fold. These results suggested that efflux pump–driven antibiotic export plays a major role in vancomycin resistance in E. faecium KU22001. Cell wall–associated adaptive responses also contributed to vancomycin resistance.

Advanced glycation end products (AGEs) contribute to chronic inflammation and metabolic dysregulation and are closely associated with obesity-related disorders. Probiotics have emerged as promising functional agents capable of modulating immune and metabolic responses. In this study, the anti-inflammatory and anti-adipogenic effects of Pediococcus pentosaceus TAP041 were investigated using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and 3T3-L1 preadipocytes, with a particular focus on AGE-associated adipogenesis. Treatment with P. pentosaceus TAP041 attenuated LPS-induced responses in RAW264.7 macrophages, as evidenced by reduced nitric oxide production, downregulation of inducible nitric oxide synthase, cyclooxygenase-2, and interleukin-6, and concomitant upregulation of the anti-inflammatory cytokine, interleukin-10. These effects were associated with selective inhibition of p38 mitogen-activated protein kinase phosphorylation, whereas ERK and JNK signaling remained largely unaffected. In 3T3-L1 adipocytes, P. pentosaceus TAP041 suppressed adipogenic differentiation and lipid accumulation, without inducing cytotoxicity. Co-treatment with P. pentosaceus TAP041 markedly attenuated N^ε-(carboxymethyl)lysine-induced lipid accumulation. This anti-adipogenic effect was accompanied by coordinated downregulation of key adipogenesis- and lipogenesis-related genes, including sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor-δ, adiponectin, acetyl-CoA carboxylase, fatty acid synthase, and lipoprotein lipase. Collectively, these findings demonstrate that P. pentosaceus TAP041 exerts dual regulatory effects on inflammatory activation and adipogenic differentiation by modulating macrophage inflammatory signaling and adipocyte lipid metabolism. This study provides mechanistic evidence supporting the potential of P. pentosaceus TAP041 as a functional probiotic candidate for mitigating inflammation- and AGE-associated metabolic disturbances.

Neurodegenerative diseases are increasingly associated with the convergence of oxidative, inflammatory, and glycation-associated stress pathways. The present study evaluated the stress-modulatory activity and probiotic-related characteristics of Pediococcus pentosaceus TAP041, a lactic acid bacterium isolated from citrus fruit. cell-free supernatant from P. pentosaceus TAP041 (TAP041 CFS) was assessed in in vitro models of neuronal and glial stress. In SH-SY5Y neuroblastoma cells, TAP041 CFS was associated with improved cell viability, reduced intracellular ROS accumulation, a lower Bax/Bcl-2 mRNA ratio, and increased BDNF and TH mRNA expression under methylglyoxal (MGO)- and lipopolysaccharide (LPS)-induced stress. The protective effects under N ^ε -(carboxymethyl)-lysine (CML) exposure were comparatively limited, with partial modulation of apoptosis-related markers in the absence of significant viability recovery. In primary rat glial cells, TAP041 CFS attenuated LPS-induced GFAP immunoreactivity and suppressed IL-6 and TNF-α expression at the transcriptional level. TAP041 CFS also exhibited ABTS radical scavenging activity, providing direct biochemical evidence for secreted antioxidant constituents. The strain demonstrated gastrointestinal stress tolerance, intestinal adhesion capacity, and a favourable safety profile, including non-hemolytic and non-cytotoxic phenotypes, absence of transferable antibiotic resistance determinants, and no known virulence factors. whole-genome sequencing identified predicted pathways for glyoxalase-mediated detoxification, antioxidant defence, and B-vitamin biosynthesis, consistent with but not directly establishing the observed phenotypes. These findings identify P. pentosaceus TAP041 as a candidate strain warranting further investigation and provide a basis for future mechanistic and in vivo studies on probiotic-derived metabolites in neurodegeneration-related stress. Key points: • TAP041 CFS reduced ROS, normalized Bax/Bcl-2, and suppressed glial inflammation. • TAP041 exhibits probiotic potential with GI tolerance, adhesion, and a safe profile. • Genome predicts glyoxalase and antioxidant pathways; ABTS confirms CFS antioxidants.

Based on previously established probiotic function of Lactiplantibacillus pentosus SC65, this study aimed to produce synbiotic yogurt using L. pentosus SC65 as a probiotic and fructooligosaccharide (FOS) as a prebiotic. Functional properties of the resulting yogurt were evaluated after incubating for 21 days. The pH of the yogurt slightly decreased with the increase in incubation time, whereas titratable acidity and lactic acid production increased. Syneresis degree in the FOS-containing yogurt decreased as the water-holding capacity increased. Moreover, the antioxidant effect of probiotic-supplemented yogurt was higher than that of nonprobiotic-supplemented yogurt. In addition, RAW 264.7 macrophages showed higher nitric oxide production when treated with probiotic or synbiotic yogurt than when treated with probiotic alone, suggesting that probiotic or synbiotic yogurt is beneficial to human health. Therefore, L. pentosus SC65 shows promise as a candidate ingredient for synbiotic yogurt.

The lactic acid bacteria, Lactiplantibacillus plantarum KU15149 and Levilactobacillus brevis KU15176 were verified through phenotypic and genotypic analyses. Safety evaluation was conducted using multiple assays, including minimum inhibitory concentration assay for nine antibiotics, hemolytic activity, mucin degradation, gelatin liquefaction, urease activity, indole production, β-glucuronidase activity, bile salt deconjugation, cell cytotoxicity, D-/L-lactic acid production, and biogenic amine formation. Genotypically L. plantarum KU15149 and L. brevis KU15176 lacked all virulence and antibiotic resistance genes investigated. Consistent with these results, phenotypic assays showed that both strains were susceptible according to EFSA cut-off values and tested negative for hemolysis, mucin degradation, gelatin liquefaction, urease activity, indole production, β-glucuronidase activity, and bile salt deconjugation. Furthermore, neither strain showed cytotoxicity toward Caco-2 cells at a multiplicity of infection of 250. Production of D-lactic acid and biogenic amines was negligible in both bacteria. Overall, L. plantarum KU15149 and L. brevis KU15176 demonstrated safety and beneficial characteristics and therefore could serve as probiotic strains.

The safety profile of Lentilactobacillus buchneri KU200793, which has neuroprotective effects, was comprehensively evaluated through both phenotypic and genotypic analyses. Phenotypically, the strain exhibited no β-hemolysis, mucin degradation, indole production, gelatin liquefaction, urease activity, or β-glucuronidase activity. Additionally, it did not produce D-lactate, and only trace amounts of spermidine were detected among the biogenic amines. Furthermore, L. buchneri KU200793 did not exhibit bile salt deconjugation, further supporting its safety profile. However, its tetracycline resistance exceeded the threshold set by the European Food Safety Authority. Genotypic analysis using the HGTree program identified tetA(58) and nalD genes with sequence similarities of 33.64% and 30.17%, respectively, indicating a low level of homology. These findings suggest that tetracycline resistance in L. buchneri KU200793 is unlikely to have been acquired through horizontal gene transfer, thereby minimizing the risk of resistance gene dissemination. This study underscores the importance of comprehensive safety assessments to evaluate the suitability of L. buchneri KU200793 for probiotic applications.

Alzheimer’s disease is characterized by progressive cognitive decline associated with oxidative stress, neuroinflammation, and impaired neurotrophic signaling. Sulforaphane, a bioactive compound found in broccoli, has demonstrated neuroprotective effects by activating NRF2 and inhibiting NF-κB. However, the efficacy of whole-food-derived sulforaphane remains unclear. This study evaluated the neuroprotective potential of broccoli sprout extract using a scopolamine-induced mouse model of memory impairment. Mice were orally administered broccoli sprout extract once daily at doses of 100 mg/kg or 200 mg/kg for four weeks prior to behavioral and biochemical assessments. Treatment with broccoli sprout extract significantly improved scopolamine-induced deficits in long-term memory, as determined by the passive avoidance test. The spatial working memory remained unaffected. High doses of broccoli sprout extract restored hippocampal brain-derived neurotrophic factor levels and reduced cortical lipid peroxidation, suggesting antioxidant and neurotrophic benefits. Additionally, the low dose preserved striatal choline acetyltransferase expression and reduced systemic tumor necrosis factor-alpha and hippocampal cyclooxygenase-2 levels, indicating its anti-inflammatory and cholinergic protective effects. No significant changes in acetylcholinesterase activity or glutathione levels were observed. Overall, these results imply that broccoli sprout extract has multi-targeted neuroprotective effects, possibly involving redox and inflammatory regulation. Therefore, it may be a safe dietary strategy to support cognition in neurodegenerative conditions.