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Fulltext Protocol for isolation, fractionation, and system biology-based profiling of gastrointestinal digested dairy colostrum and milk proteome

Patil SM, Ramu R, Goh KW

STAR Protoc, 2025 Mar 21;6(1):103657.
PMID: 40073020 DOI: 10.1016/j.xpro.2025.103657

Colostrum and milk from dairy sources consist of whey, casein, and fat, which have notable pharmacological properties due to their proteins and peptides. Here, we present a protocol for isolating, simulating in vitro gastrointestinal digestion, and fractionating colostrum and milk hydrolysates from any dairy source. We also describe steps for nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS) identification of proteins and peptides and in silico system biology-based profiling of the proteins and peptides present in the hydrolysates.
Fulltext Investigating the antidiabetic efficacy of dairy-derived Lacticaseibacillus paracasei probiotic strains: modulating α-amylase and α-glucosidase enzyme functions

Huligere SS, Kumari V B C, Desai SM, Wong LS, Firdose N, Ramu R

Front Microbiol, 2023;14:1288487.
PMID: 38111646 DOI: 10.3389/fmicb.2023.1288487

The current study aims to evaluate and characterize the probiotic andantidiabetic properties of lactic acid bacteria (LAB) obtained from milk and other dairy-based products. The strains were tested physiologically, biochemically, and molecularly. Based on biochemical tests and 16S rRNA gene amplification and sequencing, all three isolates RAMULAB18, RAMULAB19, and RAMULAB53 were identified as Lacticaseibacillus paracasei with homology similarity of more than 98%. The inhibitory potential of each isolate against carbohydrate hydrolysis enzymes (α-amylase and α-glucosidase) was assessed using three different preparations of RAMULAB (RL) isolates: the supernatant (RL-CS), intact cells (RL-IC), and cell-free extraction (RL-CE). Additionally, the isolate was evaluated for its antioxidant activity against free radicals (DPPH and ABTS). The strain's RL-CS, RL-CE, and RL-IC inhibited α-amylase (17.25 to 55.42%), α-glucosidase (15.08-59.55%), DPPH (56.42-87.45%), and ABTS (46.35-78.45%) enzymes differently. With the highest survival rate (>98%) toward tolerance to gastrointestinal conditions, hydrophobicity (>42.18%), aggregation (>74.21%), as well as attachment to an individual's colorectal cancer cell line (HT-29) (>64.98%), human buccal and chicken crop epithelial cells, all three isolates exhibited extensive results. All three isolates exhibited high resistance toward antibiotics (methicillin, kanamycin, cefixime, and vancomycin), and other assays such as antibacterial, DNase, hemolytic, and gelatinase were performed for safety assessment. Results suggest that the LAB described are valuable candidates for their significant health benefits and that they can also be utilized as a beginning or bio-preservative tradition in the food, agriculture, and pharmaceutical sectors. The LAB isolates are excellent in vitro probiotic applicants and yet additional in vivo testing is required.
Fulltext Cell-free supernatant of Levilactobacillus brevis (RAMULAB51) from coconut inflorescence sap (Neera) enhances glucose uptake and PPAR-γ in 3T3-L1 adipocytes and inhibits α-glucosidase and α-amylase

Kumari V B C, Ramu R, Shirahatti PS, Alam P, Wong LS

Front Microbiol, 2024;15:1497023.
PMID: 39764448 DOI: 10.3389/fmicb.2024.1497023

INTRODUCTION: Lactic acid bacteria are prized for their probiotic benefits and gut health improvements. This study assessed five LAB isolates from Neera, with RAMULAB51 (Levilactobacillus brevis, GenBank ON171686.1) standing out for its high hydrophobicity, auto-aggregation, antimicrobial activity, and enzyme inhibition. It evaluated RAMULAB51's potential in probiotics and diabetes management, focusing on its effects on digestive enzyme inhibition, glucose uptake, and adipocyte function.
METHODS: Isolates were characterized by Gram staining, catalase reaction, growth at 37°C, and tolerance to phenol, pH, and gastrointestinal conditions. Molecular identification using 16S rRNA sequencing. Evaluations included hydrophobicity, auto-aggregation, HT-29 cell line adhesion, antimicrobial activity, and antibiotic susceptibility. Enzyme inhibition was measured for α-glucosidase and α-amylase using cell-free supernatant, cell extract, and intact cells. Adipogenesis was assessed through Oil-Red O staining, gene expression analysis (PPAR-γ, C/EBPα, Adiponectin, Glut-4, FAS), and glucose uptake assays on 3T3-L1 cells.
RESULTS: All isolates showed good tolerance to pH (≤9.15 CFU/ml), phenol (≤9.90 CFU/ml), hydrophobicity (≤78.14%), and auto-aggregation (≤92.23%). RAMULAB51 demonstrated the highest tolerance, hydrophobicity, and auto-aggregation. It strongly co-aggregated with Micrococcus luteus and Bacillus subtilis, showing significant antimicrobial activity with a 24 mm inhibition zone against Micrococcus luteus. All isolates were sensitive to Ampicillin, Azithromycin, Streptomycin, and Tetracycline, but resistant to Methicillin and Vancomycin. RAMULAB51 demonstrated the highest enzyme inhibition: α-glucosidase (68.45% CFS, 60.18% CE, 42.15% IC) and α-amylase (80.74% CFS, 61.23% CE, 35.12% IC). By inhibiting these digestive enzymes, RAMULAB51 reduces the conversion of carbohydrates into glucose, thereby decreasing blood glucose levels. This reduction in circulating glucose subsequently influences adipocyte function, as evidenced by the enhanced glucose uptake (1000 µg/mL) and upregulation of PPAR-γ, C/EBPα, Adiponectin, and Glut-4, alongside the downregulation of FAS.
CONCLUSION: The study highlights RAMULAB51's potential for improving glucose and lipid metabolism. Further, in vivo research is needed to explore its full therapeutic benefits. These findings confirm RAMULAB51's significant probiotic potential and its promise for diabetes management, warranting further clinical investigation.
Fulltext A protocol for assessing the adhesion potential of lactic acid bacteria on various cell types

Jayaram L, Ramu R, Puttegowda D, M K J, Kumari V B C, Goh KW

STAR Protoc, 2025 Mar 21;6(1):103619.
PMID: 39913288 DOI: 10.1016/j.xpro.2025.103619

Lactic acid bacteria (LAB) are a large group produced during carbohydrate fermentation, resulting in lactic acid. Here, we present a protocol for probiotic adhesion and interaction into buccal epithelial cells, chicken epithelial cells, and HT-29 cells, as well as for autoaggregation and coaggregation. We describe steps for visualizing LAB adhesion by imaging cell adhesion using a light microscope. We then detail procedures for performing an adhesion assay. For complete details on the use and execution of this protocol, please refer to Kumari et al.1.
Fulltext Characterization of Lactobacillus spp. as Probiotic and Antidiabetic Potential Isolated from Boza, Traditional Fermented Beverage in Turkey

Kumari V B C, Huligere S, M K J, Goh KW, Desai SM, H L K, et al.

Int J Microbiol, 2024;2024:2148676.
PMID: 38962395 DOI: 10.1155/2024/2148676

Boza, a cereal-based beverage popular in southeast Europe, is fortified with probiotics and is believed to positively impact the composition of the gut microflora. This investigation focused on fermented cereal-based beverage boza to identify strains of probiotic Lactobacillus spp. capable of inhibiting carbohydrate-hydrolysing enzymes α-glucosidase (AG) and α-amylase (AA). The isolated bacterial strains underwent a comprehensive assessment, including biochemical, molecular, and probiotic trait analyses such as tolerance survivability, adhesion, safety, and health-promoting attributes. We evaluated the inhibitory potential of the supernatant, cell lysate, and intact cells of Lactobacillus spp. Molecular analysis has revealed that isolates RAMULAB30 and RAMULAB29 exhibit a significant genetic similarity (>97%) to Lacticaseibacillus paracasei and Limosilactobacillus fermentum, respectively. These findings are documented in the NCBI database. They exhibited significant resistance to gastrointestinal and intestinal fluids, also indicating their potential for adhesion. Additionally, the isolates showed a significant antibacterial activity, particularly against Micrococcus luteus. They showed resistance to vancomycin and methicillin antibiotics but were more susceptible to streptomycin and ampicillin. Furthermore, the strains demonstrated antioxidant properties. To ensure their safety, a haemolytic assay was conducted despite their general recognition as safe (GRAS) status. The study primarily aimed to evaluate the inhibitory effects of the extract on enzymes AG and AA. Bacterial isolates demonstrated a significant inhibitory activity against both enzyme AG (32%-67% inhibition) and enzyme AA (18%-46% inhibition) in different forms, including supernatant (CS), lysed extract (CE), and intact cell (IC). These findings underscore the potential of bacterial isolates to inhibit the enzyme activity effectively. Furthermore, the L. fermentum RAMULAB29 and L. paracasei RAMULAB30 strains exhibit remarkable antidiabetic potential. Food products incorporating these strains have promising prospects as nutraceuticals, providing improved health benefits.
Fulltext Sauerkraut-derived LAB strains as potential probiotic candidates for modulating carbohydrate digestion attributing bacterial organic acid profiling to antidiabetic activity

Huligere SS, Kumari V B C, Patil SM, M K J, Wong LS, Kijsomporn J, et al.

Food Sci Nutr, 2024 Nov;12(11):9682-9701.
PMID: 39620004 DOI: 10.1002/fsn3.4444

Sauerkraut-derived lactic acid bacterial (LAB) strains have gained attention due to their potential health benefits. This study focuses on evaluating seven Sauerkraut-derived RAMULAB strains isolated from sauerkraut, aiming to identify promising candidates for modulating α-glucosidase (AG) and α-amylase (AM) enzymatic functions. RAMULAB strains with remarkable probiotic potential can contribute to the digestive health and manage conditions like diabetes. Identifying robust candidates from sauerkraut, a fermented food, holds promise for natural and cost-effective probiotic sources. The RAMULAB strains underwent extensive characterization, including identification through 16S ribosomal RNA (rRNA) sequencing. Their tolerance to harsh conditions, adherence properties, antimicrobial activity, antioxidant potential, and inhibition of AG and AM were assessed. In silico analyses explored their molecular interactions, particularly with hydroxycitric acid, a potential antidiabetic compound. Among the RAMULAB strains, RAMULAB48 emerged as a standout candidate. It displayed exceptional resilience to acidic bile (≥97%), and simulated gastrointestinal conditions (≥95%), highlighting its suitability for probiotic applications. RAMULAB48 exhibited robust adherence properties, including cell-surface hydrophobicity (80%), autoaggregation (42%), coaggregation with pathogens (≥33%), and adhesion to epithelial cells. Additionally, all seven isolates demonstrated gamma-hemolysis and resistance to antibiotics (Kanamycin, Methicillin, and Vancomycin), while displaying strong antibacterial properties against foodborne pathogens. These RAMULAB strains also exhibited varying degrees of antioxidant activity, with RAMULAB48 displaying the highest potential (≥41%). In terms of antidiabetic activity, cell-free supernatant (CS) obtained from RAMULAB48 expressed the highest inhibition levels, notably inhibiting yeast AG by an impressive 59.55% and AM being by a remarkable 67.42%. RAMULAB48 produced organic acids, including hydroxycitric acid (28.024 mg/mL), which showed promising antidiabetic properties through in silico analyses, indicating favorable interactions with the target enzymes. This study identifies Lacticaseibacillus paracasei RAMULAB48, a Sauerkraut-derived RAMULAB strain, as a promising probiotic candidate with exceptional tolerance, adherence properties, antimicrobial activity, antioxidant potential, and antidiabetic effects. The presence of hydroxycitric acid further underscores its potential in managing diabetes.
Fulltext Fermented sugarcane juice-derived probiotic Levilactobacillus brevis RAMULAB54 enhances lipid metabolism and glucose homeostasis through PPAR-γ activation

Kumari VBC, Ramu R, Huligere SS, Patil SM, Nayakvadi S, Bijoor S, et al.

Front Microbiol, 2024;15:1502751.
PMID: 39943964 DOI: 10.3389/fmicb.2024.1502751

The gut microbiota plays a significant role in metabolic disorders such as diabetes and obesity, with the peroxisome proliferator-activated receptor gamma (PPAR-γ) being a key regulator in adipogenesis and glucose metabolism. This study is a novel approach that explores the antihyperglycemic and antihyperlipidemic effects of Levilactobacillus brevis RAMULAB54 (LB13243), isolated from fermented sugarcane juice. LB13243 was cultured for SEM imaging, and its supernatant (LBR54) was analyzed. Organic acid interactions with PPAR-γ were evaluated via molecular docking, while cytotoxicity and adipocyte differentiation in 3T3-L1 cells were tested using MTT assays, Oil Red O staining, triglyceride quantification, and qRT-PCR. In vivo, male Wistar rats in hyperlipidemic and streptozotocin-induced hyperglycemic models were treated with LB13243 for 4 weeks, followed by analysis of food intake, body weight, serum glucose, lipids, and histopathology. LB13243 inhibited carbohydrate-hydrolyzing enzymes and showed an organic acid profile. In silico, hydroxycitric acid had similar binding to PPARγ as rosiglitazone (binding energy:-8.4 kcal/mol vs.-8.3 kcal/mol), with greater stability (RMSD: 1.2 Å vs. 1.7 Å). Pharmacokinetics indicated moderate GI absorption (20%) and low toxicity for hydroxycitric acid. LBR54 did not affect 3T3-L1 cell viability but reduced lipid accumulation by 13% and triglycerides by ≤ 44 mg/dL. qRT-PCR revealed upregulation of PPAR-γ and C/EBP-α, and downregulation of FAS, suggesting modulation of adipogenesis. In vivo, LB13243 reduced food intake, weight gain, and normalized organ weights in hyperlipidemic rats, while improving glucose levels and lipid profiles in hyperglycemic models. Histopathology showed improved tissue structure, indicating LB13243's potential to reduce hyperglycemia and hyperlipidemia by modulating lipid metabolism and inflammation. LB13243's modulation of PPAR-γ suggests it as a promising natural option for managing diabetes and hyperlipidemia. This study also highlights LB13243's distinctive capability to modulate PPAR-γ through its organic acids, particularly hydroxycitric acid, providing novel insights into its therapeutic potential.
Fulltext Novel Benzimidazole-Endowed Chalcones as α-Glucosidase and α-Amylase Inhibitors: An Insight into Structural and Computational Studies

Rai PV, Ramu R, Akhileshwari P, Prabhu S, Prabhune NM, Deepthi PV, et al.

Molecules, 2024 Nov 27;29(23).
PMID: 39683757 DOI: 10.3390/molecules29235599

In search of novel antidiabetic agents, we synthesized a new series of chalcones with benzimidazole scaffolds by an efficient 'one-pot' nitro reductive cyclization method and evaluated their α-glucosidase and α-amylase inhibition studies. The 'one-pot' nitro reductive cyclization method offered a simple route for the preparation of benzimidazoles with excellent yield and higher purity compared to the other conventional acid- or base-catalyzed cyclization methods. 1H, 13C NMR, IR, and mass spectrum data were used to characterize the compounds. Single-crystal XRD data confirmed the 3D structure of compound 7c, which was crystalized in the P1¯ space group of the triclinic crystal system. Hirshfeld surface analysis validates the presence of O-H..O, O-H…N, and C-H…O intermolecular hydrogen bonds. From the DFT calculations, the energy gap between the frontier molecular orbitals in 7c was found to be 3.791 eV. From the series, compound 7l emerged as a potent antidiabetic agent with IC50 = 22.45 ± 0.36 µg/mL and 20.47 ± 0.60 µg/mL against α-glucosidase and α-amylase enzymes, respectively. The in silico molecular docking studies revealed that compound 7l has strong binding interactions with α-glucosidase and α-amylase proteins. Molecular dynamics studies also revealed the stability of compound 7l with α-glucosidase and α-amylase proteins.