Pre-clinical safety and toxicity assessment of Limosilactobacillus fermentum NCDC 400 in murine model.

Comprehensive safety assessment of potential probiotic strains is crucial in the selection of risk-free strains for clinical translation. This study aimed to evaluate the biosafety of Limosilactobacillus fermentum NCDC 400, a potential probiotic strain, using oral toxicity tests in a Swiss albino mouse model. Mice were orally gavaged with low (108 CFU/mouse/day) and high (1010 CFU/mouse/day) doses of NCDC 400 for 14 (acute), 28 (subacute), and 90 (subchronic) days to assess behavioral, hematological, biochemical, immunological, and histological effects. The administration of NCDC 400 did not result in any observable adverse effects on general health parameters, including body weight, feed and water intake, and organ indices. Hematological and biochemical parameters, such as glucose, serum enzymes, urea, creatinine, serum minerals, total serum proteins, and lipid profile, remained largely unaffected by the test strain. Notably, NCDC 400 administration led to a significant reduction in harmful intestinal enzymes and improvement in gut health indices, as indicated by fecal pH, lactate, ammonia, and short-chain fatty acids. There were no instances of bacterial translocation of NCDC 400 to blood or extra-intestinal organs. Immune homeostasis was not adversely affected by repeated exposure to NCDC 400 in all three oral toxicity studies. Histopathological examination revealed no strain-related changes in various tissues. Based on these findings, a dose of 1010 CFU/mouse/day was considered as the No Observable Effect Level (NOEL) in healthy mice. In conclusion, this study demonstrates the safe and non-toxic behavior of L. fermentum NCDC 400. The results support and ensure the safety and suitability for clinical trials and eventual translation into clinical practice as potential probiotic.

Influence of exopolysaccharide EPSKar1-iron complexation on iron bioavailability and alleviating iron deficiency anaemia in Wistar rats.

The prevalence of iron deficiency anaemia is a significant issue worldwide, affecting individuals of all ages and often associated with inadequate iron bioavailability. Despite the use of ferrous salt supplements to address anaemia, their limited bioaccessibility and bioavailability in human GIT and adverse impact on food properties remain significant challenges. Hence, this study aims to explore the iron chelation mechanism of an exopolysaccharide EPSKar1 to enhance iron bioaccessibility, bioavailability, and anti-anaemic effects using cell culture and an anaemic rat model. EPSKar1 was extracted from Lacticaseibacillus rhamnosus Kar1 and complexed with FeSO4 to form "EPSKar1-iron". This novel complex, besides being bio-accessible after in vitro gastric digestion, demonstrated 61.27 ± 1.96% iron bioavailability to the Caco-2 cells. In line with these in vitro findings, intragastric administration of the EPSKar1-iron complex to anaemic Wistar rats at 25 and 50 mg per kg body weight significantly restored blood haemoglobin levels and re-established the morphological features of red blood cells. Furthermore, the apparent digestibility co-efficient and iron uptake improved significantly without adversely affecting the serum biochemical parameters in these anaemic rats. The levels of iron-transport proteins including serum transferrin and ferritin in tissue and plasma have increased remarkably upon oral administration of EPSKar1-iron at a higher dose of 50 mg per kg body weight. Oral supplementation of EPSKar1-iron did not foster adverse histological changes in the liver, kidneys, and spleen. In fact, the treatment with the EPSKar1-iron complex had a restitution effect on the tissue architecture, thereby ameliorating the tissue lesions. These findings collectively indicate that the EPSKar1-iron complex shows nutraceutical potential in enhancing the bioavailability of iron and could be a promising approach to tackle iron deficiency anaemia.

Physicochemical and rheological characterizations of a novel exopolysaccharide EPSKar1 and its iron complex EPSKar1-Fe: Towards potential iron-fortification applications.

Iron is a micronutrient essential for human health and physiology. Iron-deficiency anemia, the most common form of anemia, may occur from an iron homeostasis imbalance. Iron fortification is a promising and most sustainable and affordable solution to tackle the global prevalence of this anemia. Herein, we investigate physicochemical, rheological and stability characteristics of a novel exopolysaccharide 'EPSKar1' (derived from Lacticaseibacillus rhamnosus strain Kar1) and its iron complex 'EPSKar1-Fe (II)'. Our findings demonstrate that EPSKar1 is a high molecular-weight (7.8 × 105 Da) branched-chain heteropolysaccharide composed of galactose, N-acetylglucosamine, and mannose in a molar ratio of 8:4:1, respectively, and exhibits strong emulsifying and water-holding capacities. We find that EPSKar1 forms strong complexes with Fe, wherein the interactions between EPSKar1-Fe (II) complexes are mediated by sulfate, carboxyl, and hydroxyl groups. The rheological analyses reveal that the EPSKar1 and EPSKar1-Fe (II) complexes exhibited shear thickening and thinning properties in skim milk and water, respectively; however, the suspension of EPSKar1 in skim milk is viscoelastic with predominantly elastic response (G'>G" and tan Î´ < 1). In comparison, EPSKar1-Fe (II) complex exhibits remarkable stability under various processing conditions, highlighting its usefulness for the development of fortified dairy products. Together, these findings underpin considerable prospects of EPSKar1-Fe (II) complex as a novel iron-fortifier possessing multifarious rheological benefits for food applications.

Extractable surface proteins of indigenous probiotic strains confer anti-adhesion knack and protect against methicillin-resistant Staphylococcus aureus induced epithelial hyperpermeability in HT-29 cell line.

Probiotic intervention has been long believed to have beneficial effects on human health by curbing the intestinal colonization of pathogens. However, the application of live probiotics therapy may not be an ideal approach to circumvent the infections of superbug origin due to the risk of horizontal antibiotic resistance genes transfer. In this study, the anti-adhesion ability of extractable cell surface proteins from two indigenous potential probiotic strains (Lactiplantibacillus plantarum A5 and Limosilactobacillus fermentum Lf1) and two standard reference strains (Lactobacillus acidophilus NCFM and Lacticaseibacillus rhamnosus LGG) was evaluated against clinical isolates of Methicillin-Resistant Staphylococcus aureus (MRSA) on porcine gastric mucin and HT-29 cells. The surface proteins from the probiotic strains were extracted by treatment with 5 M lithium chloride. The surface protein quantification and SDS-PAGE profiling indicated that the yield and protein patterns were strain-specific. Surface proteins significantly hampered the mucoadhesion of MRSA isolates via protective, competitive, and displacement. Similarly, the treatment with surface proteins probiotic strains displayed anti-adhesion against MRSA isolates on HT-29 cells without affecting the viability of the cell line. Surface proteins treatment to the confluent monolayer of HT-29 cells maintained the epithelial integrity; however, MRSA isolates (109 cells/mL) showed considerable alteration in the epithelial integrity by exacerbating the FITC-dextran transflux. Contrarily, the co-treatment with surface proteins with MRSA isolates significantly lowered the FITC-dextran transflux across the differentiated HT-29 monolayer. Overall, the findings of this study suggest that probiotic-derived surface proteins could be the novel biotherapeutics to combat the MRSA colonization and their concomitant intestinal infections.

Characterization of Antibiotic Resistance and Virulence Traits Present in Clinical Methicillin-Resistant Staphylococcus aureus Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) is a notorious superbug which poses serious health threats to humanity. The severity of the infections depends on the prevalence of virulence factors and antibiotic resistance. In this study, attempts have been made to nominate the two most virulent and multidrug-resistant MRSA isolates demonstrating the preliminary features of intestinal adhesion for the futuristic applications of probiotics and postbiotics as antagonists to combat MRSA infections. In this context, six clinical isolates of MRSA were polyphasically characterized for their identity, multidrug resistance, and few selected virulence determinates such as hemolytic activity and production of coagulase, nuclease, and capsule. The gut colonizing ability of MRSA isolates was assessed by mucoadhesion, auto-aggregation, and cell surface hydrophobicity. An antibiogram of MRSA isolates suggested the resistance towards several antibiotics with multiple antibiotic resistance (MAR) index >0.5 (12/241, 12/206, and 5/255) as well as their genome portraying mecA mediated methicillin resistance. Besides exhibiting strong biofilm formation ability, all the isolates exhibited positive responses towards tested virulence assays coupled with their genome displaying Coa, NucA, and CapE genes. On the other hand, isolates exhibited different levels of auto-aggregation (37.90 ± 1.8 to 51.53 ± 3.1%) and mucin adhesion ability (68.93 ± 0.61% to 86.62 ± 1.96%) with a significant (P ≤ 0.05) variation in adhesion to different hydrocarbons. Finally, multivariate Principal Component Analysis and Hierarchical Cluster Analysis (HCA) heatmap using Euclidean distance measurement indicated MRSA 12/206 and 5/255 as most resistant and virulent isolates with the potential to adhere to the hydrophobic gut niche.