Subjects

This study was a randomized, double-blinded, placebo-controlled, parallel-arm clinical trial conducted at Rush University Medical Center (RUMC) in Chicago, USA. Women and men between 18 and 65 years with a BMI of 30.0–40.0 kg/m2 were recruited. Exclusion criteria included the following: (1) prior intestinal resection; (2) patient history of GI diseases except for hiatal hernia, gastroesophageal reflux disease (GERD), and hemorrhoids; (3) severe renal disease defined by creatinine more than twice normal; (4) markedly abnormal liver function defined by ALT/AST over four times normal levels or elevated bilirubin; (5) antibiotic use within the last 12 weeks prior to enrollment; (6) lean or overweight (BMI < 30.0 kg/m2), (7) intolerant to aspirin; (8) regular use of aspirin; (9) excessive alcohol intake (more than two drinks for men and one drink for women daily); (10) presence of chronic metabolic disease such as symptomatic cardiovascular disease, insulin-requiring or uncontrolled diabetes, current active treatment of cancer; (11) a plan to have a major change in dietary habit during the following 6 months; (12) consumption of probiotics, prebiotics, or synbiotics without an appropriate 2-week washout period; (13) self-reported lactose intolerance; (14) subjects younger than 18 or older than 65; and (15) unwillingness to consent to the study.

Study design

Four visits were required for each subject (Fig. 1a). At visit 1, potential subjects were screened for eligibility and provided written informed consent. Vitals and anthropometrics were completed, and blood was obtained for endotoxin and metabolic markers. Subjects were instructed to collect stool in anaerobic bags before visit 2 and deliver samples within 24 h if stored at − 20 °C or within 5 h if stored at room temperature. At visit 2, study subjects completed a 3-day food record and a standardized 34-item GI symptom questionnaire (GSSC; Gastrointestinal Symptom and Severity Checklist) to identify potential impacts of treatment on GI symptoms, including stool consistency, discomfort, flatulence, abdominal pain, and bloating, on a scale from 0 (best) to 10 (worst). Within 1 week, subjects returned for visit 3 to provide urine for baseline intestinal permeability measurement, completed a blood draw, and were randomized to one of the six treatments (see below). At the end of a 3-week treatment period, subjects returned to the clinic to provide stool and urine samples and to complete a 3-day food record to ensure consistency of dietary intake throughout the study. A blood draw, anthropometrics, and identical follow-up questionnaires were completed. This study was approved by the Institutional Review Board at RUMC in February of 2012, and all procedures were conducted according to the principles expressed in the Declaration of Helsinki. This trial was registered at clinicaltrials.gov, identifier NCT02355210.

Fig. 1 Experimental design and participant flow diagram. a Time line for the human trial. Four visits were required from each subject. At visit 1, potential subjects were screened for eligibility and provided with a 3-day food record, all supplies for stool and urine collection (stool kit, urine collection containers, sugar cocktail, and aspirin), and instructions for specimen handling and for completing these tasks before the next visit. Each subject collected stool before taking the sugar cocktail to avoid potential effects of sugar cocktail on microbiota composition. Details of the stool and urine collection are shown. GSSC, Gastrointestinal Symptom and Severity Checklist. b Participant flow diagram showing the progress through the phases of the randomized controlled trial (enrollment, intervention allocation, follow-up, and data analysis) Full size image

Treatments

The prebiotic, GOS, was obtained from FrieslandCampina, Amersfoort, the Netherlands (sold under the trade name, Vivinal GOS powder) and contained 72.5% GOS, 22.8% lactose, and 4.7% monosaccharides (galactose and glucose). It was previously established that a GOS dose of 5 g per day was sufficient to induce a bifidogenic response [39]. Therefore, the total amount of GOS powder was increased to 6.9 g to achieve a dose of 5 g GOS. An additional 0.1 g of lactose was added to achieve the same weight (7.0 g) as the other preparations.

The two probiotic strains used were B. adolescentis IVS-1 [61] and B. animalis subsp. lactis BB-12. Strain IVS-1 was produced from a contract manufacturer (Danwell Technology, Garden Grove, CA) and provided as a freeze-dried powder. Strain BB-12 was provided by Chr. Hansen (Hørsholm, Denmark), also as a high cell density freeze-dried powder. Its reported ability to ferment GOS was confirmed in preliminary experiments (data not shown). The probiotic powders were stored for up to 6 months at − 18 °C, and viable cell numbers, as determined by cultural enumeration, were shown to be stable throughout the entire study period (data not shown). Probiotic treatments were portioned into sachets, each containing 0.1 g of probiotic powder (1010 CFU/g), resulting in a daily dose of 1 × 109 CFU and 6.9 g of lactose as a carrier/control. Synbiotics contained 6.9 g of Vivinal and 0.1 g of probiotic (either B. adolescentis IVS-1 or B. animalis BB-12), for a total dose of 7.0 g. Placebo samples contained 7.0 g of lactose.

The material was packaged in sachets in the Food Processing Product Development Lab at the University of Nebraska. The sachet material was impermeable to oxygen and moisture. Subjects were provided with enough sachets for the entire length of the 3-week treatment period and were instructed to consume one sachet daily, with 2 h between treatment and food consumption in order to facilitate delivery of the probiotic/prebiotic compounds into the cecum/large intestine by rapid emptying of the compounds from the stomach and through small bowel. Subjects were instructed to mix the treatment with the provided bottled water only, as previous testing indicated reduction in bacterial count of IVS-1 with tap water (data not shown). All subjects were instructed to store treatments in their freezer (− 18 °C).

Analysis of fecal microbiota

Fecal samples were stored in aliquots at − 80 °C until further analysis. The DNA was extracted as previously described [35]. Amplicon sequencing (Illumina MiSeq platform v3 kit producing 300-bp paired-end sequences) was performed at the University of Minnesota Genomics Center, with all samples being included in a single run. The V5-V6 region of the 16S rRNA gene was amplified using primer pair 784F (5′-RGGATTAGATACCC-3′) and 1064R (5′-CGACRRCCATGCANCACCT-3′). These primers were chosen based on our previous findings that the V5-V6 region provided the best resolution among Bifidobacterium species [61, 63], which we considered the most important criteria for the aims of our study. The 25-μl PCR mixtures contained 5 μl of template DNA, 5 μl of 2× HotStarTaq PCR master mix, a final concentration of primers of 500 nM, and 0.025 U μl−1 HotStarTaq polymerase (Qiagen Inc.), as previously described [61]. The generated sequences were quality filtered with Illumina software at the University of Minnesota Genomics Center, resulting in more than 96% of the samples meeting all quality control criteria. Sequences that did not meet quality filtering criteria were removed from the analysis. All reads were trimmed to 240 base pairs using the FASTX-Toolkit. The reads were merged and analyzed for their sequencing depth. If a sample exceeded 37,000 reads, it was subsampled using Mothur v.1.31.162, while samples that had less than 37,000 sequences were left untreated. This normalization step was done as suggested by Weiss et al. to account for differences in the library size and minimize potential biases due to sequencing depth across samples [73]. Subsequently, reads were filtered by length with a minimum of 240 base pairs and a maximum of 260 base pairs, dereplicated, OTU clustered, chimeras removed, and taxonomically assigned as previously described [61]. After processing and quality control, samples contained an average of 22,487.59 ± 6683 sequences.

Quantitative real-time PCR (qPCR)

qPCR was performed by using a Mastercycler Realplex2 instrument (Eppendorf AG, Hamburg, Germany). Each PCR was performed with 25-μl volumes using real-time master mix containing SYBR (5 Prime Inc., Gaithersburg, MD) and either genus-specific primers for Bifidobacterium [39], or the strain-specific primers for B. adolescentis IVS-1, as described previously [61]. Strain-specific PCR for IVS-1 did not reveal products in baseline samples in subjects of groups IVS-1 or IVS-1 + GOS or in the baseline of another additional 20 randomly selected subjects (data not shown).

For strain-specific detection of B. animalis subsp. lactis BB-12, the PCR mixture contained 25 μl of PCR reaction mix (QuantiFast® Probe PCR Kit, QIAGEN, Hilden, Germany), 0.3 μmol of each primer (BAL-23S-F 5′-CAGGTGGTCTGGTAGAGTATACCG-3′ and BAL-23S-R 5′-ACGGCGACTTGCGTCTTG-3′), 0.25 μmol of probe (BAL-23S-P 5′-FAM-CGCCCACGACCCGCAAG-TAMRA-3′), and 5 μl DNA as previously described [74]. The target of these primers and probe is the elongation factor Tu (tuf) gene of BB-12. The specificity of the primers and probe for BB-12 was validated experimentally by qPCR using DNA from 11 different Bifidobacterium strains using the same approach as described previously for the IVS-1 strain-specific qPCR [61]. These strains included B. adolescentis IVS-1, B. adolescentis ATCC 15703, B. adolescentis L2-32, B. longum subsp. longum ATCC 15707, B. longum DJO10A, B. longum ATCC 15697, B. longum subsp. longum F8, B. longum subsp. longum JDM301, Bifidobacterium sp. strain 113, Bifidobacterium sp. strain 12_1_47BFAA, and Bifidobacterium sp. strain HMLN14. Furthermore, primers were tested against fecal DNA obtained from baseline fecal samples from subjects in BB-12 and BB-12 + GOS groups, and additional randomly selected samples from other subjects. Strain BB-12 was detected in three out of 51 tested subjects before the treatment was started, which may be due to accidental consumption of food products containing this strain.

Overall, we concluded that both qPCR assays were sufficiently specific and that the test strains were either absent or rare in the pre-treatment microbiota of the subjects. Absolute quantification of both strains was achieved through standard curves prepared by tenfold dilutions of DNA isolated from overnight cultures (14 h) for which cell numbers were determined by quantitative culture.

Intestinal permeability

Subjects ingested a sugar mixture containing 2 g mannitol, 7.5 g lactulose, 40 mg sucrose, and 2 g sucralose after a 12-h overnight fast (Fig. 1a). Subjects collected urine into three separate containers for 5, 7, and 12 h, for a total collection time of 24 h, in order to estimate gastroduodenal permeability (5 h urinary sucrose), proximal and distal small bowel permeability (5 h and first 12 h urinary mannitol and lactulose and lactulose:mannitol ratio), and total and primarily colonic permeability (24 h urinary sucralose level and sucralose/lactulose ratio) as we previously described [75]. Compared to normal-weight individuals, obese individuals are more likely to have a hyper-permeable intestine [76]. However, values vary greatly among individuals, and a treatment with aspirin has been shown to improve consistency in intestinal permeability measurements [76, 77]. Therefore, the subjects participated in an aspirin challenge consuming four 325-mg aspirin tablets both 12 h before and immediately before ingestion of the sugar mixture. Thus, urine was collected for two separate 24-h periods with and without aspirin treatment, and both before and after treatment. Urine was analyzed for concentrations of mannitol, lactulose, and sucralose using gas chromatography (GC). Intestinal permeability was expressed as a percent of the oral dose excreted in the urine.

Plasma and serum measures of endotoxin exposure

Serum endotoxin (LPS) and LPS-binding protein (LBP) were measured after the aspirin challenge both before and after treatment. Endotoxin was measured in serum by Limulus Amebocyte Lysate QCL-1000 (Lonza # 50-647U). Serum samples were diluted at a 1:5 ratio with LAL reagent water. LBP was measured in plasma using an ELISA kit from Cell Sciences Inc. (# HK315).

Serum metabolic markers

A complete metabolic panel and complete blood count were performed to allow an assessment of treatment safety. To assess impact of treatments on metabolic markers, a lipid panel was completed by Quest Diagnostics and included the following: total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides. Non-HDL was calculated by subtracting HDL-C from TC.

Vitals and anthropometrics

Blood pressure was measured using an automated cuff with the average of three assessments used for statistical comparisons. Body weight and waist circumference was assessed and BMI calculated before and after treatment.

Statistical analysis

All data presented was analyzed based on a per protocol analysis. Subjects were excluded from the analysis if the study protocol was not followed, including use of antibiotics during the treatment period, storage of the treatments at room temperature, and use of tap water to consume treatments. Data is presented as mean ± SEM for variables that were normally distributed, or median (IQR) for variables not normally distributed. Group means were compared by ANOVA and post hoc tests except when data were not normally distributed, in which case a nonparametric analysis of medians was performed using the Kruskal-Wallis test. Chi-square tests or Fisher’s exact tests were used for incidence data. If only two groups were compared, Student’s t tests were performed.

For the analysis of the gut microbiota, Mann-Whitney-Wilcoxon matched pair tests were used for pairwise comparisons between time points for the 16S rRNA gene sequencing and qPCR data, as data was normally distributed. Unpaired Mann-Whitney-Wilcoxon tests were used for pairwise comparisons between treatment groups. When more than two samples were compared, Kruskal-Wallis test was applied. P values were corrected for the total number of comparisons with the use of a false-discovery rate (FDR) method in R, whereby values were considered significant for FDR-adjusted P values (reported as Q values) of < 0.1.