Probiotic Potential Lacticaseibacillus casei and Limosilactobacillus fermentum Strains Isolated from Dosa Batter Inhibit α-Glucosidase and α-Amylase Enzymes

Abstract

Fermented food plays a major role in gastrointestinal health, as well as possesses other health benefits, such as beneficiary effects in the management of diabetes. Probiotics are thought to be viable sources for enhancing the microbiome of the human gut. In the present study, using biochemical, physiological, and molecular approaches, the isolated Lactobacillus spp. from dosa batter were identified. The cell-free supernatant (CS), cell-free extract (CE), and intact cells (IC) were evaluated for their inhibitory potential against the carbohydrate hydrolyzing enzymes α-glucosidase and α-amylase. Then, 16S rDNA amplification and sequencing were used to identify the species. A homology search in NCBI database was performed that suggests the isolates are >95% similar to Limosilactobacillus fermentum and Lacticaseibacillus casei. Different standard parameters were used to evaluate the probiotic potential of strains RAMULAB07, RAMULAB08, RAMULAB09, RAMULAB10, RAMULAB11, and RAMULAB12. The strains expressed a significant tolerance to the gastric and intestinal juices with a higher survival rate (>98%). A high adhesion capability was observed by the isolates exhibited through hydrophobicity (>65%), aggregation assays (>75%), and adherence assay on HT-29 cells (>82%) and buccal epithelial cells. In addition, the isolates expressed antibacterial and antibiotic properties. Safety assessments (DNase and hemolytic assay) revealed that the isolates could be classified as safe. α-glucosidase and α-amylase inhibition of the isolates for CS, CE, and IC ranged from 7.50% to 65.01% and 20.21% to 56.91%, respectively. The results suggest that these species have exceptional antidiabetic potential, which may be explained by their use as foods that can have health-enhancing effects beyond basic nutrition.

Keywords: dosa batter; lactic acid bacteria; probiotics; α-amylase; α-glucosidase.

Figure 1

The (A) autoaggregation (%) strains at different time interval at room temperature and (B) coaggregation (%) of LAB strains after incubation of 2 h at room temperature. Data are expressed as the mean ± SD. Means in aggregation for 2 h with distinct superscripts (a–e) are significantly different (p ≤ 0.05), as separated by Duncan multiple range test.

Figure 2

LAB strains adhesion to buccal epithelial cells observed under a light microscope. (A) Buccal epithelial cells (control). (B) The adhesion of isolate: RAMULAB07 (B), RAMULAB08 (C), RAMULAB09 (D), RAMULAB10 (E), RAMULAB11 (F), and RAMULAB12 (G) to buccal epithelial cells. Note: the black arrow shows the LAB strains attached to the epithelial cells.

Figure 3

Acid and bile survival rate of LAB strains isolated from dosa batter sample with an acidic pH 2 value and under different bile salt conditions: (A) 0.3%, (B) 1% bile salt concentration conditions for 2 and 4 h at 37 °C in MRS agar plates. Data are expressed as the mean ± SD. Means in survival rate with time interval of 2 h with superscripts (#) are significantly different (p ≤ 0.05), as separated by Duncan multiple range test.

Figure 4

Survival rate of isolates in gastric (A) and intestinal (B) juice. Data are expressed as the mean ± SD. Means in survival rate for time interval (1 h, 3 h, 5 h, and 8 h) with distinct superscripts (a–c) are significantly different (p ≤ 0.05), as separated by Duncan multiple range test.

Figure 5

Phylogenetic tree of LAB isolates (RAMULAB07–12) from dosa batter samples based on maximum likelihood bootstrap analysis of 16S rDNA.

Figure 6

The (A) scavenging activity of ABTS radicals and (B) DPPH free radical scavenging activity of the isolates. Data are expressed as the mean ± SD. Means in scavenging activity of different CFU/mL with distinct superscripts (a–c) are significantly different (p ≤ 0.05), as separated by Duncan multiple range test.

Figure 7

α-glucosidase (A) and α-alpha amylase (B) inhibitory activity of the isolates. Data are expressed as the mean ± SD. Means in inhibition activity of CS, CE, and IC with distinct superscripts (a–c) are significantly different (p ≤ 0.05), as separated by Duncan multiple range test.