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.
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.
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.
Arterial/venous thrombosis is the major cardiovascular disorder accountable for substantial mortality; and the current demand for antithrombotic agents is extensive. Heparinases depolymerize unfractionated heparin (UFH) for the production of low molecular-weight heparins (LMWHs; used as anticoagulants against thrombosis). A microbial strain of Streptomyces sp. showing antithrombotic activity was isolated from the soil sample collected from north India. The strain was characterized by using 16S rRNA homology technique and identified as Streptomyces variabilis MTCC 12266 capable of producing heparinase enzyme. This is the very first communication reporting Streptomyces genus as the producer of heparinase. It was observed that the production of intracellular heparinase was [63.8 U/mg protein (specific activity)] 1.58 folds higher compared to extracellular heparinase [40.28 U/mg protein]. DEAE-Sephadex A-50 column followed by Sepharose-6B column purification of the crude protein resulted 19.18 folds purified heparinase. SDS-PAGE analysis of heparinase resulted an estimated molecular-weight of 42 kDa. It was also found that intracellular heparinase has the ability to depolymerize heparin to generate LMWHs. Further studies related to the mechanistic action, structural details, and genomics involved in heparinase production from Streptomyces variabilis are warranted for large scale production/purification optimization of heparinase for antithrombotic applications.