Genomic Stability and Phenotypic Characteristics of Industrially Produced Lacticaseibacillus rhamnosus GG in a Yogurt Matrix

Abstract

Lacticaseibacillus rhamnosus GG is a widely marketed probiotic with well-documented probiotic properties. Previously, deletion of the mucus-adhesive spaCBA-srtC1 genes in dairy isolates was reported. In this study, we examined the genome preservation of industrially produced L. rhamnosus GG (DSM 33156) cofermented in yogurts. In total, DNA of 66 samples, including 60 isolates, was sequenced. Population samples and 59 isolates exhibited an intact genome. One isolate exhibited loss of spaCBA-srtC1. In addition, we examined phenotypes related to the probiotic properties of L. rhamnosus GG either from frozen pellets or cofermented in yogurt. L. rhamnosus GG from frozen pellets induced a response in intestinal barrier function in vitro, in contrast to frozen pellets of the starter culture. Yogurt matrix, containing only the starter culture, induced a response, but cofermentation with L. rhamnosus GG induced a higher response. Conversely, only the starter culture stimulated cytokine secretion in dendritic cells, and it was observed that the addition of L. rhamnosus GG to the starter culture reduced the response. We conclude that the L. rhamnosus GG genome is preserved in yogurt and that common in vitro probiotic effects of L. rhamnosus GG are observed when examined in the yogurt matrix. IMPORTANCELacticaseibacillus rhamnosus GG is a well-documented probiotic strain recognized for its high acid and bile tolerance and properties of adhesion to enterocytes and mucus. The strain exhibits SpaCBA pili, which have been demonstrated to play an important role in adhesion and therefore are relevant for persistence in the gastrointestinal tract. Recently we demonstrated that the genome and phenotypes of L. rhamnosus GG are preserved throughout an industrial production pipeline. However, as gene deletions in L. rhamnosus GG were previously reported for isolates from dairy products, a key question on the genomic stability of L. rhamnosus GG in a yogurt matrix remained. The aim of this study was to analyze genome stability and phenotypic characteristics of L. rhamnosus GG in yogurt. We found that the genome of L. rhamnosus GG is well conserved when the organism is cofermented in yogurt. Some phenotypic characteristics are consistent in all product matrixes, while other characteristics are modulated.

Keywords: Lacticaseibacillus rhamnosus GG; SpaCBA; genome stability; industrial fermentation; probiotics; yogurt.

FIG 1

Overview of samples included in genome sequencing. Samples of frozen pellets of L. rhamnosus GG were collected from start of pelletizing (F_Start) and end of pelletizing (F_end) in a production batch and included in yogurt fermentation. Samples of yogurt were collected on the day of production (Y1) and after 28 days of refrigerated storage (Y28). Full-genome sequencing was performed on an overnight culture (ON) of the frozen pellets and the four samples of yogurt (Y1_F_Start, Y1_F_end, Y28_F_start, and Y28_F_end). Ten isolates were purified from each of the six full population samples. Sample identification and colors are defined in the key.

FIG 2

Genome similarity between sequenced samples of L. rhamnosus GG. Three publicly available reference genomes for L. rhamnosus GG on NCBI (ASM2650v1, ASM335345v1, and ASM1104v1) and five previously published genomes of L. rhamnosus GG in which the spaCBA-srtC1 gene cluster was lost (SRR645838, SRR3098054, SRR645868, SRR3096056, and SRR3096092 in NCBI Sequence Read Archive [SRA]) are included. Hierarchical clusters of genomes are based on gene similarities. Sample identifiers and colors are the same as in Fig. 1.

FIG 3

Circular representation of alignments of DNA sequence contigs derived from the 66 samples sequenced. Each circle indicates the sequence from one sample, color-coded as defined in the key. For comparison, contigs from five sequences with gene deletions reported previously by Sybesma et al. (17) and Douillard et al. (14) are included. The three outer circles represent GC content and annotated genes on both strands, as indicated in the key.

FIG 4

Validation of the gene deletion. (A) Proportion of genomes containing the spaCBA-srtC1 gene cluster presented as the ratio of coding sequence (CDS) coverages of the genes deleted in isolate 9 and the remaining genes. The samples included all sequences of overnight cultures (_ON) and Y1_F_start_I9. Ratios of CDS are given on the y axis. (B) qPCR on the spaA gene representing the spaCBA-srtC1 gene cluster and 16S rRNA included as a control of DNA. Samples included Y1_F_Start_I4 (positive control), Y1_F_Start_ON (population from which Y1_F_Start_I9 was purified), L. rhamnosus LC705 (strain without the spaCBA-srtC1 gene cluster as a negative control), and Y1_F_Start_I9 where the spaCBA-srtC1 gene cluster was lost. C_T values are given on the y axis.

FIG 5

Tolerance to gastric acid and bile of L. rhamnosus GG. Samples included L. rhamnosus GG as frozen product (Frozen Lr GG [F_start]) and in yogurt. Yogurt samples included frozen L. rhamnosus GG added to yogurt immediately before treatment (Frozen Lr GG added to Yogurt) and yogurt cofermented with L. rhamnosus GG, both from the day of production (Yogurt with Lr GG - Day 1) and at the end of shelf life (Yogurt with Lr GG - Day 28) (x axis). Results are shown as CFU per milliliter after 1 h of incubation in gastric acid solution at pH 2.0 (A) and 1% porcine bile solution (B). For each treatment a control is included, shown as CFU per milliliter after 1 h of incubation in 10% MRS broth (y axis). Data points represent the means for technical duplicates, and horizontal lines represent the means from three experiments. One-way ANOVA followed by Tukey’s test comparing the different sample sets was performed on treatment normalized to control.

FIG 6

Transepithelial electrical resistance (TEER) of L. rhamnosus GG and the starter culture as frozen product and in yogurt. (A) TEER of Caco-2 cell monolayers stimulated with frozen pellets of the starter culture (Frozen St + Lb), frozen pellets of L. rhamnosus GG (Frozen Lr GG [F_start]), or a combination of the two samples (Frozen St + Lb + Lr GG). In addition, yogurt containing only the starter culture (Yogurt St + Lb) and yogurt cofermented with L. rhamnosus GG on the day of production (Yogurt Day 1 St + Lb + Lr GG [Y-1_F_start]) and by end of shelf life (Yogurt Day 28 St + Lb + Lr GG [Y28_F_start]) were included in the experiment. The final concentrations used for all samples were 2 × 10⁸ CFU/ml and 4 × 10⁷ CFU/ml for the starter culture and L. rhamnosus GG, respectively. (A) The y axis shows the percent change in TEER relative to the baseline, which is the TEER reading just prior to adding the bacterial strains (time = 0 h). The x axis shows the time in hours. Each data point represents the mean ± SD of results from technical triplicates. (B) Area under the curve (AUC) of TEER measurements from 0 to 20 h relative to the baseline (100%) (y axis) for the measurement presented in panel A. Each bar represents the mean ± SD for technical triplicates. One-way ANOVA followed by Tukey’s test was performed, and statistically significant differences are shown.

FIG 7

Cytokine response to L. rhamnosus GG and starter culture as frozen product and in yogurt. Cytokine concentrations (y axis) of IL-10 (A), IL-8 (B), IL-6 (C), TNF-α (D), IL-12p70 (E), and IL-1β (F) secreted by monocyte-derived human dendritic cells derived from three different donors. Dendritic cells were stimulated with the following samples: frozen pellets of L. rhamnosus GG (Frozen Lr GG [F_start]), frozen pellets of the starter culture (Frozen St + Lb), a combination of the frozen samples (Frozen St + Lb + Lr GG), yogurt containing only the starter culture (Yogurt St + Lb), and yogurt cofermented with L. rhamnosus GG [Yogurt St + Lb + Lr GG (F_start)] (x axis). The final concentrations used for all samples were 5 × 10⁷ CFU/ml and 1.5 × 10⁷ CFU/ml for the starter culture and L. rhamnosus GG, respectively. Negative controls were incubated in cell culture medium only. Each data point represents the mean of technical replicate measurements, and biological triplicates were assessed for each donor. Black bars represent the mean concentrations for the three donors.