Characterization of Lactobacillus spp. as Probiotic and Antidiabetic Potential Isolated from Boza, Traditional Fermented Beverage in Turkey

Abstract

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.

Figure 1

The phylogenetic tree provides information on the evolutionary relationships of RAMULAB29 and RAMULAB30 strains from boza samples based on maximum likelihood bootstrap analysis and provides information on the evolutionary history and relatedness of different bacterial strains based on their 16S rRNA sequences comparative with reference strains.

Figure 2

The autoaggregation and coaggregation data of the LAB strains were presented as mean ± SD. (a) Percentage of aggregation of RAMULAB29 and RAMULAB30 strains that autoaggregate over time at room temperature (28°C) and (b) aggregation percentage of RAMULAB29 and RAMULAB30 strains that coaggregate after 2 h at 28°C. DMRT was used to compare between means in the aggregation, and alphabetic superscripts (A–E) designate statistically significant differences (p < 0.05).

Figure 3

Survival rates of isolates at acidic pH 2 and different bile salt concentrations. The experiment was carried out by incubating the strains for 2 and 4 h (37°C) under 0.3% and 1% bile salt concentrations. Data are presented as mean ± SD, and means were compared using the DMRT with superscripts denoting significant differences (p < 0.05).

Figure 4

The results of the survival rates of the isolates in gastric and intestine juices are presented as mean ± SD. The means of the values obtained expressed the survival rates for different time intervals (1, 3, 5, and 8 h) and compared using DMRT, and the statistically significant differences were represented with different superscripts (a–c) (p < 0.05).

Figure 5

Results of the isolate's scavenging activity of DPPH and ABTS radicals. (a) ABTS scavenging activity and (b) DPPH scavenging activity. Data are expressed as mean ± SD. DMRT was used to compare the mean ± SD of the scavenging activity at various CFU/ml, and those with distinct superscripts (A–C) are significantly different (p < 0.05).

Figure 6

The inhibitory activity of the two isolates against the enzymes AG and AA was tested using IC, C, and CE. The results showed the isolate extract's ability to hinder the enzymes AG (a) and AA (b). Data are presented as mean ± SD, and the DMRT was used to determine the significance of means of the inhibitory activity of the isolate extracts with different alphabetic superscripts (A–C) indicating a significant difference (p < 0.05).