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. 2022 May 18:13:863228.
doi: 10.3389/fmicb.2022.863228. eCollection 2022.

Metabolomic Analysis of Lactobacillus acidophilus, L. gasseri, L. crispatus, and Lacticaseibacillus rhamnosus Strains in the Presence of Pomegranate Extract

MaryClaire Chamberlain  1 , Sarah O'Flaherty  1 , Natalia Cobián  1 , Rodolphe Barrangou  1
Affiliations

Metabolomic Analysis of Lactobacillus acidophilus, L. gasseri, L. crispatus, and Lacticaseibacillus rhamnosus Strains in the Presence of Pomegranate Extract

MaryClaire Chamberlain et al. Front Microbiol. .

Abstract

Lactobacillus species are prominent inhabitants of the human gastrointestinal tract that contribute to maintaining a balanced microbial environment that positively influences host health. These bacterial populations can be altered through use of probiotic supplements or via dietary changes which in turn affect the host health. Utilizing polyphenolic compounds to selectively stimulate the growth of commensal bacteria can have a positive effect on the host through the production of numerous metabolites that are biologically active. Four Lactobacillus strains were grown in the presence of pomegranate (POM) extract. Two strains, namely, L. acidophilus NCFM and L. rhamnosus GG, are commonly used probiotics, while the other two strains, namely, L. crispatus NCK1351 and L. gasseri NCK1342, exhibit probiotic potential. To compare and contrast the impact of POM on the strains' metabolic capacity, we investigated the growth of the strains with and without the presence of POM and identified their carbohydrate utilization and enzyme activity profiles. To further investigate the differences between strains, an un_targeted metabolomic approach was utilized to quantitatively and qualitatively define the metabolite profiles of these strains. Several metabolites were produced significantly and/or exclusively in some of the strains, including mevalonate, glutamine, 5-aminoimidazole-4-carboxamide, phenyllactate, and fumarate. The production of numerous discrete compounds illustrates the unique characteristics of and diversity between strains. Unraveling these differences is essential to understand the probiotic function and help inform strain selection for commercial product formulation.

Keywords: Lactobacillus; metabolomics; pomegranate extract; prebiotic; probiotic.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Growth of Lactobacillus strains with and without the POM extract (400 μg/ml). Growth assays were performed over a 24-h period with the L. acidophilus NCFM, L. crispatus NCK1351, L. gasseri NCK1342, and L. rhamnosus GG grown in SDM with 0.5% glucose and no POM extract (green curve, No POM) or in SDM with 0.5% glucose and 400 μg/ml of POM extract (red curve, POM). Graphs on the left-hand side show OD 600 nm values, and log CFU/ml growth curves are shown on the right-hand side. Error bars represent the standard deviation of three biological replicates, and * indicates p ≤ 0.05.
Figure 2
Figure 2
Carbohydrate and enzyme profiles of Lactobacillus strains. Carbohydrate utilization and enzymatic activity were determined using API assays for the four strains, namely, L. acidophilus NCFM (Lac), L. crispatus NCK1351 (Lcr), L. gasseri NCK1342 (Lga), and L. rhamnosus GG (Lrh). The heat maps show the mean values of two biological replicates with the values scored according to the manufacturer's instructions. The values range from 0 to 5 and are assigned to the standard color where zero represents no reaction and 5 represents a reaction of maximum intensity. The relative activity may be estimated from color strength.
Figure 3
Figure 3
Global metabolomic analysis overview. (A) Hierarchical cluster analysis (HCA) of log2 transformed fold change values of the metabolomic data for the cell-free supernatants of the Lactobacillus strains and controls. The entire metabolite profile (left-hand side) and top 50 significant (p ≤ 0.05) highest produced metabolites (right-hand side) were performed for each strain. The legend shows the log2 transformed fold change values. (B) Principal component analysis (PCA) and (C) partial least-squares discriminant analysis (PLS_DA) of the global metabolite profile for the four Lactobacillus strains and controls. L. acidophilus NCFM (Lac), L. crispatus NCK1351 (Lcr), L. gasseri NCK1342 (Lga), and L. rhamnosus GG (Lrh), SDM media with POM 400 μg/ml at T0 (POM 0 h) and T16 (POM 16 h).
Figure 4
Figure 4
Representation of the global metabolic profile of Lactobacillus strains based on super pathway analysis. Significant (p ≤ 0.05) log2 transformed fold change values for each metabolite were used for mapping to nine biochemical pathways. Distinct colors and grids indicate the overarching biochemical pathway for the detected metabolites. Outlined circles (black and blue) indicate highly detected and unique metabolites for each strain. L. acidophilus NCFM (Lac), L. crispatus NCK1351 (Lcr), L. gasseri NCK1342 (Lga), and L. rhamnosus GG (Lrh).
Figure 5
Figure 5
Boxplots of specific metabolites. The data points are from the metabolomic data that were normalized and scaled to the median value for each compound. Boxplots for mevalonate, glutamine, 5-aminoimidazole-4-carboxamide, fumarate, malate, and succinate are shown. L. acidophilus NCFM (Lac), L. crispatus NCK1351 (Lcr), L. gasseri NCK1342 (Lga), and L. rhamnosus GG (Lrh), SDM media alone (ctrl), SDM media with POM 400 μg/ml at T0 (pom0) and T16 (pom16). p-values are indicated as follows: *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, and they were determined by comparison to the control.
Figure 6
Figure 6
Loci comparison across strains and transcriptional profiles of L. acidophilus NCFM for defined loci in metabolite pathways. Conserved loci and amino acid sequence identities relative to L. acidophilus NCFM are shown in select Lactobacillus strains for (A) mevalonate (B) fumarate (C) glutamine, and (D) 5-aminoimidazole-4-carboxamide. The gray and white arrows represent genes that are not required in the specific metabolic pathway but are located within the operon. The vertical solid black line distinguishes between operons but loci are adjacent to one another on the chromosome. Dashed black lines distinguish between operons on separate parts of the chromosome.
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