Functional characterization of α-Gal producing lactic acid bacteria with potential probiotic properties

Abstract

The possibility of exploiting the human immune response to glycan α-Gal for the control of multiple infectious diseases has been the objective of recent investigations. In this field of research, the strain of Escherichia coli O86:B7 has been at the forefront, but this Gram-negative microorganism presents a safety concern and therefore cannot be considered as a probiotic. To address this challenge, this study explored the identification of novel lactic acid bacteria with a safe history of use, producing α-Gal and having probiotic potential. The lactic acid bacteria were isolated from different traditionally fermented foods (kununn-zaki, kindirmo, and pulque) and were screened for the production of α-Gal and some specific probiotic potential indicators. The results showed that Ten (10) out of forty (40) [25%] of the tested lactic acid bacteria (LAB) produced α-Gal and were identified as Limosilactobacillus fermentum, Levilactobacillus brevis, Agrilactobacillus composti, Lacticaseibacillus paracasei, Leuconostoc mesenteroides and Weissella confusa. Four (4) LAB strains with highest levels of α-Gal were further selected for in vivo study using a mouse model (α1,3GT KO mice) to elucidate the immunological response to α-Gal. The level of anti-α-Gal IgG observed were not significant while the level of anti-α-Gal IgM was lower in comparison to the level elicited by E. coli O86:B7. We concluded that the lactic acid bacteria in this study producing α-Gal have potential probiotic capacity and can be further explored in α-Gal-focused research for both the prevention and treatment of various infectious diseases and probiotic development.

Figure 1

The α-Gal level of selected lactic acid bacteria. The α-Gal levels in lactic acid bacteria isolated from traditionally fermented food is presented as the geo mean of the florescence intensity. Bacterial isolates are represented in bar chart to show the fluorescence intensity as against the negative control (bacteria with secondary antibody only) and E. coli O86:B7 served as the positive control. The LBH1148, LBH1073, LBH1074, LBH1066, YZ01, KZ06, YA03, KA20, KA27, KB30 represent lactic acid bacteria strains that were further identified molecularly.

Figure 2

Phylogenetic tree of lactic acid bacteria producing α-Gal based on the alignment of the partial 16S rRNA sequences of the LAB isolates from this study. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.95913905 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. All positions containing gaps and missing data were eliminated. E. coli ATCC_117775T was used as an out group while LAB producing α-Gal was annotated with pink and blue colored bullets. Evolutionary analyses were conducted using MEGA6.

Figure 3

Cell-Surface Hydrophobicity of the lactic acid bacteria. Ability of lactic acid bacteria to adhere to gastrointestinal wall was measured using the microbial adhesion to hydrocarbon method that measures the cell surface hydrophobicity (CSH). The CSH estimated in percentage gives the value of adhesion potential of each bacterial isolates presented in bar chart as a mean of triplicate values. Bars represent the standard deviation from the mean value. NB: Cell surface hydrophobicity of Isolates LBH1066, 1073, 1074, LBH1148 (data not available).

Figure 4

Oral administration of E. coli O86:B7 increased anti-Galα1-3Gal IgM antibody response in sera α1,3GT KO mice. Anti Galα1-3Gal IgM levels increased on α1,3GT KO mice more than the measured value in mice treated with L. brevis (LBH1073), L. composti (LBH1074), L. paracasei (LBH1146), and L. mesenteroides (LBH1148) by day 27. (A) No change was observed on IgG response against Galα1-3Gal antigen. (B) The levels of circulating anti-α-Gal IgM and IgG were measured by ELISA. Results shown are means and standard deviation values, IgM was significant at p < 0.05, p < 0.001, p < 0.0001; while IgG was not statistically significant, 2 experiments, n = 30 and three technical replicates per sample.