Prebiotics for Lactose Intolerance: Variability in Galacto-Oligosaccharide Utilization by Intestinal Lactobacillus rhamnosus

Abstract

Lactose intolerance, characterized by a decrease in host lactase expression, affects approximately 75% of the world population. Galacto-oligosaccharides (GOS) are prebiotics that have been shown to alleviate symptoms of lactose intolerance and to modulate the intestinal microbiota, promoting the growth of beneficial microorganisms. We hypothesized that mechanisms of GOS utilization by intestinal bacteria are variable, impacting efficacy and response, with differences occurring at the strain level. This study aimed to determine the mechanisms by which human-derived Lactobacillus rhamnosus strains metabolize GOS. Genomic comparisons between strains revealed differences in carbohydrate utilization components, including transporters, enzymes for degradation, and transcriptional regulation, despite a high overall sequence identity (>95%) between strains. Physiological and transcriptomics analyses showed distinct differences in carbohydrate metabolism profiles and GOS utilization between strains. A putative operon responsible for GOS utilization was identified and characterized by genetic disruption of the 6-phospho-β-galactosidase, which had a critical role in GOS utilization. Our findings highlight the importance of strain-specific bacterial metabolism in the selection of probiotics and synbiotics to alleviate symptoms of gastrointestinal disorders including lactose intolerance.

Keywords: Lactobacillus; galacto-oligosaccharides; prebiotics for lactose intolerance; probiotics; β-galactosidases.

Figure 1

Comparative genomic of lactose metabolism operons in Lactobacillus rhamnosus. Genomes were analyzed using Geneious software, comparing operon organization and nucleotide identity between the human isolates AMC010, AMC143, LGG, and the dairy isolate Lc705. Promoters were identified using the BPROM (Softberry, Bangkok, Thailand) promoter prediction tool [48]. Terminators were identified using the ARNold terminator prediction tool [49]. * Genes showed >60% identity to homologs in lac2 operon; bgl: beta glucoside antiterminator; PTS: phosphotransferase system; NA: not applicable.

Figure 2

Specific growth rates of L. rhamnosus strains. Growth rates were calculated for each strain in MRS containing 1% glucose, 1% GOS, 0.1% lactose (the lactose component of GOS formulation), and in MRS without carbon and energy sources. Eight biological replicates, each in triplicate (three technical replications) were included in each experiment. Letters (A, B, C) represent statistical differences between treatments in each strain (p < 0.05).

Figure 3

GOS utilization and generation of secondary metabolites. Minimal media containing 1% GOS as a sole carbohydrate source were analyzed by HPLC to determine residual carbohydrates after incubation with each strain to early logarithm, late logarithm, and stationary growth phase. Three biological replicates, each in triplicate (three technical replications) were included in each experiment. The utilization of lactose and GOS varied between strains, and the production of lactic acid correlated with utilization.

Figure 4

Transcriptomics analysis. Heat maps for AMC143, AMC010, and Lc705 were generated from sequencing reads mapped to their respective genome sequence and plotted to show fold changes between cultures in glucose compared to GOS. Boxed genes represent potential operons, and grayed genes represent genes absent in the associated strain. Three biological replicates were included in each experiment.

Figure 5

Expression of lac operons in GOS and lactose. Heat maps were generated from mRNA sequencing data for the identified lac operons in AMC143, AMC010, and Lc705, significantly differentially regulated by treatment with either GOS or lactose (p < 0.05 after Bonferroni correction). The fold change for each gene was determined by comparing expression data from lactose or GOS versus glucose. Gray boxes represent genes not identified in the corresponding genome. PTS: Phosphotransferase System.

Figure 6

Growth and fermentation profiles of AMC143::pβgal_lac3. (A) Maximum specific growth rates for AMC143 and AMC143::βgal_lac3 were determined in MRS containing 1% glucose, 1% GOS, 0.1% lactose, 1% cellobiose, and 0% glucose. Letters represent statistical differences between strain culture in each media type (n = 8, p < 0.05). (B) Carbohydrate utilization profiles were generated by API 50CH, revealing that the mutant was unable to ferment GOS and lactose after 24 h.

Figure 6

Growth and fermentation profiles of AMC143::pβgal_lac3. (A) Maximum specific growth rates for AMC143 and AMC143::βgal_lac3 were determined in MRS containing 1% glucose, 1% GOS, 0.1% lactose, 1% cellobiose, and 0% glucose. Letters represent statistical differences between strain culture in each media type (n = 8, p < 0.05). (B) Carbohydrate utilization profiles were generated by API 50CH, revealing that the mutant was unable to ferment GOS and lactose after 24 h.