Forty-two healthy adults (female = 30, male = 12; mean age ± SD = 23.2 ± 5.5 years) participated in the study. Participants were recruited from the community and the University of Florida campus in Gainesville, FL, USA in accordance with an Institutional Review Board (IRB) approved study at the University of Florida. Exclusion criteria included gastrointestinal disease (gastric ulcers, Crohn’s, ulcerative colitis, etc.), chronic disease such as diabetes, kidney disease, and heart disease; current or past treatment for immune-compromising diseases or conditions; currently working or living with an immunocompromised person; currently taking medications for constipation, diarrhea, or a psychological disorder (depression, anxiety, insomnia, etc.); antibiotics within the past 4 weeks prior to randomization; currently taking a probiotic supplement and unwilling to discontinue a minimum of 2 weeks prior to the study start; current smoker; pregnant or lactating or a female who plans to become pregnant in the next 6 months; and a known allergy to milk. Inclusion criteria included men and women 18–50 years of age and approval to participate following screening blood work and physical examination by the advising physician.

In a double-blinded study, healthy volunteers were randomly assigned to one of two treatments, L. johnsonii N6.2 at 5 × 10⁸ colony-forming units (CFU) per capsule or placebo (skim milk) capsule for 8 weeks in a parallel design. Prior to treatment, there was a 1-week pre-baseline period, and treatment was followed by a 4-week washout period. One week prior to randomization, consented participants underwent a physical examination and were screened via a comprehensive metabolic panel (CMP), and females received a pregnancy test. During pre-baseline, intervention and washout periods, participants completed a daily online questionnaire, reporting on: study supplement intake, hours of sleep, bowel movement frequency, gastrointestinal symptoms, general wellness, and medication use. In addition, participants completed the gastrointestinal symptom rating scale (GSRS), and quality of life was assessed with the quality of life questionnaire, SF-36v2^® on a weekly basis. At the randomization appointment, height, weight, vitals (blood pressure, heart rate), and demographic information were obtained. CMP and complete blood count were assessed at baseline, during weeks 2, 4, and 8 of the study intervention, and during washout. An additional pregnancy test was given during week 4 of the intervention phase. At a final appointment, participants returned any unconsumed supplements.

Lactobacillus johnsonii N6.2 was grown in modified MRS medium (FGM-LJ2). The media contained peptone 10 g, meat powder 10 g, yeast peptone 5 g, table sugar 20 g, K₂HPO₄ 2 g, sodium acetate 5 g, ammonium citrate tribasic 2 g, MgSO₄⋅7H₂O 0.2 g, MnSO₄⋅H₂O 0.05 g, tween 80 1 g; final volume of 1 L with DI water. L. johnsonii N6.2 was incubated at 37°C for 16 h under microaerophilic conditions. Cells were pelleted by centrifugation at 6,000 g for 20 min at 4°C and washed twice with BAM R61 0.02 M phosphate buffer pH 7.3 (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). The cell pellet was resuspended in sterile reconstituted food grade skim milk at 100 g/L (Real Food, IL, USA), transferred to sterile bags (Whirl-Pak, USA), and frozen at −80°C for at least 2 h. The frozen samples were freeze dried (LabConco FreeZone, LabConco Corp., MO, USA) for 48 h. The dried powder was saved at 4°C until capsule filling. Acid resistant capsules (AR Caps, Size #1, CapsCanada, Pompano Beach, FL, USA) were filled using a sterilized Profiller (Torpac^®, NJ, USA) with the freeze-dried preparation of L. johnsonii N6.2 in skim milk (Real Food, IL, USA). Lyophilized skim milk in identical capsules was used as the placebo. The study capsules were provided in bottles labeled with treatment codes by a study collaborator who did not have contact with study participants.

Single stools were collected using a commode specimen collection system (Fisher Scientific, Pittsburgh, PA, USA) during the last 2 days of pre-baseline, during weeks 2, 4, and 8 of the intervention, and during washout. Participants were instructed to place the stool containers on ice immediately after defecation and deliver samples to study personnel within 4 h of defecation. Samples were homogenized and fractionated on sterile vials (approximately 1.0 g/vial) and saved at −80°C until use. Fresh samples (approximately 1 g) were immediately diluted (1/10 w/v) in phosphate buffer solution (pH: 7.4), and serial dilutions were made and plated on MRS agar media (pH; 5.5 ± 0.1). Plates were incubated for 48 h under microanaerobic conditions. Values were referred as CFU per wet gram stool (CFU/g). The identity of L. johnsonii N6.2 was confirmed by PCR amplification of the strain-specific gene T285_00345 gene (26).

From fasting blood samples, serum (Red top Tube, BD, USA) and plasma (EDTA Purple top Tube, BD, USA) were collected. The CMP was obtained from serum samples evaluating glycemia (glucose level), kidney function (creatinine and urea level), and liver function (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and bilirubin level). The analysis was performed by Vista Clinical Diagnostics, Clermont, FL, USA. Additionally, plasma and serum samples were aliquoted, flash frozen in liquid nitrogen, and stored at −80°C for further assays. ELISA assays were used to quantify serum insulin levels (eBioscience, CA, USA) and C-reactive protein (CRP) (Cayman Chemical, MI, USA).

Quantification of tryptophan catabolites and other metabolites in blood plasma samples (i.e., tryptophan, kynurenine, kynurenic acid, xanthurenic acid, serotonin, and anthranilic acid) were performed using global high-performance liquid chromatography and mass spectrometry (LC-HRMS/HRMS) at the Southeast Center for Integrated Metabolomics of the University of Florida.

The following ELISA kits were used: IL-2, IL-6, and TNF-α from eBioscience (CA, USA); IFN-γ and IFN-α from Abcam (MA, USA); IL-2SsRα from BD (NJ, USA) following the manufacturers’ instructions.

Direct immunofluorescence surface staining of whole blood samples with six antibody panels was performed to provide a detailed assessment of immune cell subsets. Five of the panels and gating strategies are emulated from Maecker (27) and consist of a B cell subset panel (CD3, CD19, CD20, CD24, CD27, CD38, and IgD), innate cell panel (CD3, CD11c, CD14, CD16, CD19, CD20, CD56, CD123, and HLA-DR), T cell naïve and memory panel (CD3, CD4, CD8, CD38, CD45RA, CD197, and HLA-DR), T cell effector subset panel (CD3, CD4, CD38, CD45RO, CD183, CD196, and HLA-DR), and a Treg panel (CD3, CD4, CD25, CD45RO, CD127, CD194, and HLA-DR). A sixth follicular helper T (Tfh) panel (CD3, CD4, CD45RA, CD183, CD196, CD197, and CD279) was designed to assess precursor Tfh (28) and memory Tfh (29). Antibodies against the following antigens were used: CD3 (SK7), CD8 (SK1), CD19 (HIB19), CD20 (2H7), and CD45RO (UCHL1) from BD Biosciences, HLA-DR (LN243), IgD (IA6-2), CD3 (UCHT1), CD4 (RPA-T4), CD11c (Bu15), CD14 (M5E2), CD16 (3G8), CD24 (ML5), CD25 (BC96), CD27 (O323), CD38 (HB7), CD45RA (HI100), CD56 (HCD56), CD123 (6H6), CD127 (A019D5), CD183 (G025H7), CXCR3 (G025H7), CD185 (J252D4), CD194 (L291H4), CD196 (G034E3), and CD197 (G043H7) from BioLegend (USA), and CD279 (eBioJ105) from eBiosciences (USA). Whole blood (200 μL/stain) was incubated for 30 min at room temperature and protected from light. Afterward, 2 mL of Fix/lyse 1× (eBioscience, USA) was added and incubated at room temperature for 5 min. Successive washing/centrifugation (5 min, 450 g) steps were performed until hemolysis color completely faded. The samples were acquired on a BD Fortessa cytometer, and data were analyzed by FlowJo software.

DNA was extracted from fecal samples and preserved at −80°C using the PowerFecal^® DNA isolation kit (MoBio Lab, Inc., USA) with the following modification. 250 mg of fecal sample were homogenized in 750 µL of bead solution and 100 µL of Protease from Streptomyces griseus 20 mg/mL (Sigma-Aldrich, Steinheim, Germany) were added. The mixture was incubated at 37°C for 15 min and samples were processed according to the manufacturers’ protocol. In the elution step, the DNA was collected in 70 µL of water and quantified. The DNA concentration was standardized to 1 ng/µL before the amplification of the V4 region using primers for paired-end sequencing on the Illumina MiSeq platform as described earlier (30).

Clustering of operational taxonomic units (OTUs) at 97% similarity was performed with the subsampled open-reference OTU picking method (31), with no removal of singletons. The Greengenes reference dataset version 13.8 (32) was used as the reference for OTU picking and for taxonomy assignment with uclust (33). OTUs identified as mitochondrial DNA or as chloroplasts were removed from further analyses. Parsed raw sequencing reads are publicly available through NCBI’s Sequence Read Archive under the BioProject accession number PRJNA378749.

Unless otherwise noted, statistical analysis was performed using JMP Pro software (SAS Institute, Cary, NC, USA). Multivariate analysis was performed by two-way analysis of variance (ANOVA) with a post hoc Tukey’s honestly significant difference test. Bivariate analysis was performed using Student’s t-tests. Numerical data are summarized as mean ± SE. Significance was defined as p < 0.05.

The study was approved by the IRB (# 201400370) at the University of Florida and conducted according to guidelines established by the Declaration of Helsinki. Participants were informed of the aims, requirements, and risk/benefits of the study, and written informed consent indicating their full knowledge of the study protocol was received from participants prior to study enrollment. In addition, an Investigational New Drug (IND#016829) has been filed with the Food and Drug Administration of the United States of America.

Of the 92 individuals initially screened and assessed for eligibility, 50 were consented, and 42 randomized to the treatment groups (Figure 1) in a double-blind parallel study design. Table 1 summarizes the characteristics and compliance of the subjects that participated in the study.

The analysis of the hemogram and CMP data showed no statistically significant differences between the L. johnsonii N6.2 and placebo groups (Table S1 in Supplementary Material). The only parameter that showed statistical significance after 8 weeks of treatment was total bilirubin values (placebo = 0.54 ± 0.05 versus L. johnsonii N6.2 = 0.70 ± 0.05, p < 0.05); however, both groups were within the reference range of 0.2–1.9 mg/dL (38). These differences were not observed after the washout period (12 weeks). No statistically significant differences were observed in either the control or treatment cohorts with regards to kidney and liver function (Table S1 in Supplementary Material). As expected, no alterations in the circulating levels of insulin and CRP were observed in the L. johnsonii N6.2 group at 8 weeks or 12 weeks compared to placebo. Based on these results, the consumption of L. johnsonii N6.2 was well tolerated without apparent risks or deviations from the reference ranges for standard clinical assessments.

We next assessed general digestive health over the course of the trial. During pre-baseline, intervention, and washout periods, participants completed a daily online questionnaire reporting study supplement intake, hours of the sleep, bowel movement frequency, gastrointestinal symptoms, general wellness, and medication use. Of the five domains of the weekly GSRS questionnaire, indigestion (p < 0.05) and abdominal pain (p < 0.05) were significantly lower among L. johnsonii N6.2 treated subjects during treatment weeks 2 to 8, and during washout weeks 1–4 compared to placebo (Table 2). Syndrome scores from the daily questionnaire indicated that cephalic syndrome, including the symptoms of headache and dizziness, was significantly lower (p < 0.05) for L. johnsonii N6.2 versus placebo for treatment weeks 2 and 4–8. The gastrointestinal distress syndrome was lower (p < 0.05) for L. johnsonii N6.2 versus placebo during most treatment weeks and neared significance at baseline (p = 0.05). Interestingly, the probiotic group showed a significant decrease (p < 0.05) in the epidermal syndrome at the end of the washout period (Table 3). Stomach ache or pain as an individual symptom was significantly lower in the group receiving L. johnsonii N6.2 during treatment weeks 1–8 compared to the placebo group, with a similar trend at baseline (p = 0.06) (Table S2 in Supplementary Material). Bloating as an individual symptom was lower (p < 0.01) in L. johnsonii N6.2 versus placebo at baseline and during most treatment and washout weeks (Table S2 in Supplementary Material). Administration of L. johnsonii N6.2 also resulted in lower individual daily symptoms of cramping, abdominal noises, and headache for most treatment weeks. Furthermore, a significant decrease in the anxiety symptom after the washout period in the probiotic group was observed (Table S2 in Supplementary Material). The anxiety changes may have affected the psychology syndromes where a trend to decrease (p < 0.1) during the washout was observed (Table 3).

Gastrointestinal transit survival of L. johnsonii N6.2 was evaluated by following the total CFU/g of stools counts of lactic acid bacteria (LAB) in fresh fecal samples. Additionally, the presence of L. johnsonii N6.2 was confirmed by RT-PCR of the T285_00345 gene.

Overall, a large variability in the amount of the total CFU of LABs was observed between subjects (from 10² to 10⁸ CFU/g stools) at time 0, and no significant changes were observed over time either in the placebo or in the L. johnsonii N6.2 treatment group (Figure 2A). However, it was possible to observe three groups of subjects within each treatment: (a) subjects with high counts of LAB throughout the study (>10⁵ CFU/g), (b) a group that at time 0 showed low concentrations of LAB that increased over time (from 10⁴ to 10⁸ CFU/g), and (c) subjects with low counts of LAB throughout the study (<10⁵ CFU/g). To quantify the variation of the LAB population, the log CFU/g values for time 0 was subtracted at each time point and expressed as relative change (log CFU/g_{at each time point}/log CFU/g_{at time 0}). For subjects with consistently high or low LAB populations (groups a and c), the relative fold change in LAB was 1.0 ± 0.11 and 0.8 ± 0.02, respectively, in the probiotic group (Figure 2B). Similar results were obtained in the placebo group (Figure 2C). As expected, subjects in group b that received L. johnsonii N6.2 capsules displayed the highest relative change in LAB counts (Figure 2B). After the washout period, the LAB counts appeared to return to baseline levels (Figures 2B,C). These results suggest that L. johnsonii N6.2 survived the transit through the gastrointestinal system and potentially may not colonize the gut.

To verify this hypothesis, the presence of L. johnsonii N6.2 was confirmed using specific RT-PCR of the T285_00345 gene and expressed as genomic equivalents/100 ng of DNA (Figure 2D). This gene was found in the genome of L. johnsonii N6.2 but not in others in the NCBI database. It was found that the T285_00345 gene gave background amplification on the placebo-treated group (Figure 2F), while a significant increase in genomic equivalents over time (p < 0.05) were observed in the L. johnsonii N6.2 group (Figure 2E). Interestingly, the presence of L. johnsonii N6.2 was confirmed in all the subgroups (a, b, and c) independently of the total LAB counts (see Figures 2E,F). However, after washout, the genomic equivalents in L. johnsonii N6.2 group were similar to time 0 or below the detection limit.

We previously reported that the administration of L. johnsonii N6.2 to BB-DP rats resulted in decreased expression of IDO and, consequently, changes in the kynurenine:tryptophan (K:T) ratios in peripheral serum (25). Here, the impact of L. johnsonii N6.2 on IDO activity was evaluated by quantifying plasma levels of the following metabolic intermediates in the tryptophan pathway: tryptophan, kynurenine, serotonin, xanthurenic acid, anthranilic acid, and kynurenic acid. Samples taken at different time points (0, 8, and 12 weeks) were quantified using liquid chromatography-mass spectrometry (LC-HRMS/HRMS) (Table S3 in Supplementary Material). Based on our findings in rodent studies, it was expected that a decrease in IDO activity or expression would increase the concentration of tryptophan, while decreasing the concentrations of kynurenine, xanthurenic acid, anthranilic acid, and kynurenic acid associated also with a possible increase in serotonin levels (25). For each of the metabolites, we observed no significant differences between the treatment groups during the treatment period (Table S3 in Supplementary Material). Similarly, the K:T ratio was not affected, being similar for both groups during treatment with placebo or L. johnsonii N6.2 (Table S3 in Supplementary Material).

Similar statistical analyses of the metabolic intermediates were also conducted considering the LAB counts (groups a, b, or c as described earlier). After 8 and 12 weeks, the kynurenine values did not change significantly (p > 0.1) in the different subgroups (Figure 3A). After 8 weeks (last day of treatment), a slight increase in the tryptophan concentration was observed, which correlated with a decrease in the K:T ratio in the L. johnsonii N6.2 versus placebo-treated group b subjects (low to high LAB counts), although statistical significance was not reached (p = 0.17 and p = 0.13, respectively) (Figures 3B,C). Interestingly, at 12 weeks (after 4 weeks of washout), the changes in tryptophan and K:T ratio reached statistical significance with p < 0.01 and p < 0.05, respectively (Figures 3B,C). These results suggest that the effects of L. johnsonii N6.2 supplementation may take longer than 8 weeks to be quantified.

The fact that the expected modulation of the tryptophan pathway was only observed in one group of subjects (low to high LAB counts), suggests that the effects of L. johnsonii N6.2 supplementation may require an intestinal environment that is permissive to microbe colonization over time. These results indicate that the counts of LAB during baseline may also be used as biomarkers to predict responders from non-responders in a heterogeneous population, although this will require confirmation.

The impact of L. johnsonii supplementation on the immune system was evaluated by flow cytometry of PBMCs as described by Maecker (27). The identification of immune cell subsets was performed by eight-color antibody staining at time 0, after 8 weeks of treatment or after the washout period (12 weeks). Six antibody staining panels were used to differentiate the following immune cell subsets of the innate and adaptive arms of the immune system: (i) B cells, (ii) natural killers (NKs), monocytes, and DCs, (iii) naïve and memory T cells, (iv) Tfh cells, (v) differentiated effector T cells (Teff), and (vi) regulatory T cells (Tregs).

Based on the results obtained by immunophenotyping, we determined the levels of the following serum-soluble cytokines and immune markers: IL-6, TNF-α, IFN-γ, IFN-α, IL-2, soluble CD25 (IL-2Rα), and IgA by ELISA. IL-2 and IFN-α were below the detection limit and were not further analyzed. No statistical differences were obtained between the treatment groups or the time points for IL-6, TNF-α, IFN-γ, TNFα, and soluble CD25 (p > 0.1). A significant (p < 0.05) increase in the concentration of IgA was observed during the washout period in the L. johnsonii treatment group, while no differences observed in the placebo group (Figure 8).

Based on the observations that many significant changes in the immune cell populations and IgA levels were observed 4 weeks after finalizing the L. johnsonii N6.2 treatment (washout period), we investigated whether L. johnsonii N6.2 induced changes in the microbiota.

The microbiome was analyzed at time 0 and after 8 or 12 weeks of administration of the placebo or L. johnsonii N6.2. DNA was extracted from all stool samples, and the microbial communities were characterized by sequencing the V4 region of the 16S rDNA with Illumina MiSeq. An average of 54,743 ± 15,800 sequencing reads per sample were obtained. Approximately 380,933 OTUs were detected, representing 173 families. The 10 most abundant families detected were Bacteroidaceae, Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Paraprevotellaceae, an unclassified Clostridiales family, Bifidobacteriaceae, Desulfovibrionaceae, Porphyromonadaceae, and Veillonellaceae (Figure S5 in Supplementary Material), which was consistent throughout the time course of the study, although variation between individuals was observed. Bacterial communities clustered only by individual (ANOSIM R = 0.921, p < 0.01), and the community structure did not differ significantly by treatment (ANOSIM R = 0.011, p = 0.05) or over time (ANOSIM R = −0.017, p > 0.99). In addition, community structure was not correlated with the combined effects of treatment and time (PERMANOVA R² = 0.002, p = 1.00) (Figure S6 in Supplementary Material). The statistical analysis showed that the relative abundance of genera or families was not significantly different between treatment groups or time points.

Due to the high variability observed in the microbiome among the subjects, we tested if the microbiome of each subject could be used to determine changes in each individual over time. Using this normalization approach, it was found that of the 173 families in the dataset, only 34 changed in relative abundance between weeks 0 and 8. The change per family was compared between the two treatment groups by Welch’s two-sample t-test. Although no significant differences at p < 0.05 were obtained, two families, Prevotellaceae and Ruminococcaceae showed trends with p < 0.1 (Table S6 in Supplementary Material) while families Lactobacillaceae, Erysipelotrichaceae, and Odoribacteraceae showed values of p = 0.17.

After the washout period, the observed microbial changes induced by L. johnsonii N6.2 supplementation (i.e., increase in Ruminococcaceae, Lactobacillaceae, and Erysipelotrichaceae; or decrease in Prevotellaceae and Odoribacteraceae) were reverted such that the families returned to their initial abundancies. Interestingly, one family, Christensenellaceae significantly increased in concentration in the L. johnsonii N6.2 treated group after 12 weeks (after the washout period) (p < 0.05), while the Clostridiaceae and Bacteroidaceae families showed trends to increase or decrease, respectively (p = 0.06 and p = 0.09, respectively). However, when this normalization method was tested at the genus level, no statistical differences were observed.