Study design
The study was a single-center, randomized, double-blind, controlled study, stratified by sex in four parallel groups with a 1:1:1:1 allocation ratio: the Test 1, Control 1, Test 3 and Control 3 groups, receiving one (Test 1 and Control 1) or three (Test 3 and Control 3) bottles per day of the Test or the Control product. The study period was split into three subperiods (Fig. 1): a 2-week washout period (day 14 to day 0), a 4-week period of Test or Control product consumption (day 0 to day 28) and a 4-week follow-up period (day 28 to day 56). Dietary restrictions were imposed throughout the entire study period (from day 14 to day 56), with prohibition of the consumption of other fermented dairy products, probiotics, vitamins and mineral supplements, to limit potential interference with the evaluation of the Test product effects. Each subject attended five visits to a clinical unit (Harrison Clinical Research, Munich, Germany): inclusion visit (V1-day 14), randomization visit (V2-day 0), two evaluation visits (V3-day 14, V4-day 28), and an end-of-study evaluation visit (V5-day 56). Blood and stool samples were collected for assessments of eligibility and of the safety evaluation criteria at V1, 2, 3 and 4 (blood) and V2, 3, 4, and 5 (stool). Each visit had to take place within 2 days of the scheduled visit date (± 2 days) to ensure a consistent adequacy between the times of clinical and biological measures and the duration of each corresponding period of product intake or follow-up between subjects. This study was performed in accordance with the principles of the Declaration of Helsinki, the French Huriet law, and ICH-GCP recommendations, and was approved by the ethics committee of the Bavarian Medical Association, Munich, Germany. All volunteers provided written informed consent. This trial was registered on the ClinicalTrials.gov, with the registration number NCT01108419 (date of registration April 22, 2010). The study was funded by Danone Research (France).
Clinical study design.
Subject selection
Subjects were screened between March and April 2010, and the study lasted from March 29th 2010 (first subject included) to June 25th 2010 (last subject completed). The following eligibility criteria were assessed at subject inclusion (V1). The inclusion criteria were: male or female volunteers providing written informed consent, aged from 18 to 55 years, with a body mass index (BMI) of 18.5 to 30.0 kg/m2, free-living and considered to be in good health on the basis of a clinical examination, with a normal defecation pattern and either menopausal or with an approved method of contraception if female. Non-inclusion criteria were: any allergy, hypersensitivity to any component of the study product, including lactose, systemic or topical treatment (at the time of inclusion or in the previous 4 weeks) likely to interfere with the evaluation of the study parameters (antibiotics, intestinal or respiratory antiseptics, antirheumatic agents, anti-inflammatory drugs [except for aspirin or equivalent at doses preventing from platelet aggregation or blood clotting] and steroids prescribed for chronic inflammatory diseases), any symptoms of respiratory or gastrointestinal common infectious diseases, a history of chronic metabolic or gastrointestinal disease, abdominal pain or any other severe progressive or chronic disease (cardiac, respiratory, etc.), immunodeficiency, eating disorders or a medicated diet, pregnancy or breast-feeding. The following eligibility criteria were also assessed at the randomization visit (V2): compliance with the dietary and medication restriction (as defined in the non-inclusion criteria) between V1 and V2, negative pregnancy test and parameters within the normal range in the blood samples collected at V1, and absence of common infectious disease symptoms.
Product intervention
The Test product was a fermented dairy drink containing Lactobacillus paracasei CNCM I-1518, Lactobacillus paracasei CNCM I-3689 and Lactobacillus rhamnosus CNCM I-3690 strains, with 107 to 109 colony-forming units (CFU)/g of product, and four yogurt strains (Lactobacillus bulgaricus CNCM I-2787, Streptococcus thermophilus CNCM I-2773, Streptococcus thermophilus CNCM I-2835, Streptococcus thermophilus CNCM I-2778). Counts were measured for each of the bacterial strains present in the Test product, at the start and end of the authorized storage period (shelf life). Means and ranges of strains counts from the batches of product used in the study are provided in Supplementary Table S1. The Control product was a non-fermented dairy drink, acidified with lactic acid and containing pectin as a stabilizer. Both the Test and Control products were sweetened and multi-fruit flavored. Both products were similar in terms of their appearance, packaging, nutritional content (isocaloric) and taste, to ensure the maintenance of double-blinding (both the participants and key study personnel, including the outcome assessors) until the database was locked and the request by the statistician for unblinding (the only staff not blinded being those involved in the preparation of the study products). Products were manufactured in a pilot plant approved by the national health authorities for the production of dairy products for human consumption. They were supplied by Danone Research, France and stored at + 4 ± 2 °C, with a shelf life of 37 days. Analyses were performed to guarantee the absence of microbiological contaminants in all products. Subjects were randomly assigned to the Test or Control group according to a randomization list established before the start of the study by an external statistician. The randomization list contained balanced blocks, stratified by sex, with the allocation of an incremental number linked to product number given by an IWRS system, and was kept confidential at the sponsor’s premises in order to ensure allocation concealment. The subjects were then asked to ingest either one (100 g) or three (3 × 100 g) bottles of the Test or Control product daily, in accordance with their randomization group, for the entire 4-week product-consumption period (28 days). Subjects with three doses per day were recommended to consume no more than two doses at the same time. Compliance was evaluated by the investigator on the basis of the daily reporting of product consumption by each participant in a personal diary and a count of unused bottles.
Outcomes
The primary aim of the study was to compare product safety between the Test 1 and Control 1 groups over the 4-week period of product consumption. The safety evaluation was based on the following parameters: adverse events, physical examination, hematology, metabolism profile, markers of hepatic, kidney and thyroid function, inflammatory markers, bowel habits and frequency of digestive symptoms. Additional information about safety parameters is provided in Supporting Information.
As secondary criteria, safety parameters were also analyzed for the Test 3 and Control 3 groups, over the period of product consumption (V2 to V4), and for both 1 and 3 product doses during other periods: the follow-up period (V4–V5) and the whole experimental period (V2–V5). Stool samples were also subjected to testing to detect and quantify the strains present in the Test product and to analyze the microbiota, for both doses and different study periods (see details and methods below).
Procedure
At each visit, from V1 to V5, subjects underwent a physical examination and vital signs were recorded. Subjects completed a personal diary throughout the 10-week study period, which was collected and examined at each visit by the investigator. This diary included daily reports of study product consumption, the intake of unauthorized products, concomitant medication, symptoms, frequency and consistency of stool and a weekly scoring from the Frequency of Digestive Symptoms questionnaire. The physical activity and smoking habits of the subjects were recorded at each visit. Blood samples were collected for analyses after overnight fasting every two weeks from V1 to V4. The measure of calprotectin concentration, the detection and quantification of strains from the Test product, and the evaluation of the microbiota profile were performed on stool samples collected at each visit from V2 to V5. The study was performed in accordance with the protocol and the statistical analysis plan with no major change during the course of the trial.
Safety monitoring committee
A safety and monitoring committee (SMC), composed of three independent experts in internal medicine, hepato-gastro-enterology and pharmacology, performed an unblinded review of the subject withdrawals, the protocol deviations, the statistical analyses of study parameters and the individual data in the event of abnormal values for safety results. The statistical results were presented after the database lock by the study scientist and statistician to the SMC during two meetings. The SMC then presented its conclusions concerning the safety of the daily ingestion of the Test product at the two doses evaluated.
Stool collection, DNA extraction
We collected fecal samples from 90 subjects at four time points (Test 1 (N = 22), Test 3 (N = 23), Control 1 (N = 21), Control 3 (N = 24)) in RNAlater solution (Ambion, Courtaboeuf, France). Fecal DNA was extracted by mechanical lysis (FastprepFP120; ThermoSavant, Illkirch, France) followed by phenol/chloroform-based extraction, as previously described39. The DNA preparation was subjected to quality control by spectrophotometry on a NanoDrop 2000c spectrophotometer (Thermo Fisher). The DNA was analyzed by quantitative polymerase chain reaction (qPCR), 16S rRNA gene sequencing and whole-genome sequencing.
Quantitative PCR
Three strains, Lactobacillus paracasei subsp. paracasei CNCM I-1518, Lactobacillus paracasei subsp. paracasei CNCM I-3689 and Lactobacillus rhamnosus CNCM I-3690, were quantified by qPCR, as previously described39, with specific primers (Supplementary Table S2). Values were reported as median and interquartile range.
16S RNA gene sequencing, processing and analysis
16S RNA gene sequencing was performed as previously described18. Amplification was performed with the V3-V4 primers for the 16S rRNA (forward: CCTACGGGNGGCWGCAG, reverse: GACTACHVGGGTATCTAATCC). The samples were loaded into flow cells in an Illumina MiSeq 300PE Sequencing Platform, in accordance with the manufacturer’s instructions. Analyses were performed with QIIME (v. 19). The sequences were filtered for quality and a mean of 99,437 ± 36,973 reads per sample were retained. Reads were clustered into operational taxonomic units (OTUs; 97% identity threshold) with VSEARCH, and representative sequences for each OTU were aligned and taxonomically assigned with the SILVA database (v. 119). Alpha-diversity was assessed at genus level. Beta diversity was assessed with Bray–Curtis dissimilarity, Jensen-Shannon divergence, and weighted and unweighted UniFrac on genera and OTUs.
Metagenomic shotgun sequencing and preprocessing
Following standard DNA quality control and quantification, sequencing libraries were prepared with the Nextera XT DNA sample preparation kit in accordance with the manufacturer’s instructions. An overview of the bioinformatic pipeline used in this study is provided in Supplementary Fig. S1. We generated a mean of 35 million (± 8 million) paired-end reads per sample. Read cleaning, filtering and mapping were performed with NGLess version 0.740. An augmented catalog was built from the Integrated Gene Catalog (IGC)41 enriched with genes from the sequencing and de novo assembly of these 107 metagenomes and the seven bacterial genomes present in the Test product (Supplementary Fig. S2). Mapping and count matrix generation were also performed with NGLess. The taxonomic profile was extracted from the count matrix with the Metagenomic Species Pan-Genomes database42. For functional characterization, the catalog was annotated with functional data from the Kyoto encyclopedia of genes and genomes (KEGG, https://www.genome.jp/kegg/)43.
Functional contribution
Metagenomic gene count matrices were aggregated at KEGG orthologous (KO) levels, for the whole gene set and for genes from L. rhamnosus and L. paracasei from the Test product only. We estimated the contribution of the Test product to each KO, by dividing each KO relative abundance level for the Test product by the corresponding value for the whole gene set. A pseudocount of one was added. Corresponding KO relative abundances for the 31 universally distributed marker genes from Ciccarelli et al.44 were also obtained, to estimate the minimal functional contribution of each Test product gene. All KOs for the Test product with a contribution strictly higher than the minimal contribution, constituting a significant functional contribution of the Test product to the gut metagenome, were extracted for downstream analysis. KEGG BRITE and module annotations were used to explore this functional contribution, focusing on enzymes and transporters. We then assessed the extent to which this significant functional contribution set was shared by the other metagenomic species pan-genomes (MSPs).
Statistical analysis
Clinical parameters
No data on adverse events were available to assess the sample size required. The decision to include 24 subjects per group was thus made on the basis of previously published safety studies45,46. For assessment of the safety of consuming the Test product, in comparison to the Control product, adverse events were recorded (MedDRA version 13) and used to evaluate the number of subjects with at least one adverse event, and the total number of adverse events overall, and by relationship to the study product, intensity, seriousness, action taken, and subject outcome. Additional physical examination data, blood parameters, calprotectin concentration in feces, and questionnaires about bowel movements, stool consistency and the frequency of digestive symptoms were collected throughout the period of product consumption and were analyzed as raw data or in terms of clinical significance relative to the baseline value. No formal statistical tests has been performed to assess the safety and study conclusions were based on nominal statistics as described hereafter, on individual data and on overall agreement of the SMC. For quantitative variables, Cohen’s d was calculated for the change from baseline after 4-week product consumption in Test and Control groups as follows: Cohen’s d = (Average raw change from baseline in Test group − Average raw change from baseline in Control group)/Pooled standard deviation at baseline. Cohen’s d values around 0.50 are considered to be of medium magnitude, and those around or above 0.80 are considered to be large47,48. In this study, an absolute Cohen’s d value above 0.5 was considered to be large enough to detect a potential difference between the Test and Control groups. For qualitative binary parameters, the relative risk (RR) and its 95% confidence interval (CI) were calculated by the normal approximation method. Safety analyses were performed on all randomized subjects who had consumed the Test or Control product at least once, i.e. the full analysis set (FAS) population. Statistical analyses were performed with the Statistical Analysis Systems statistical software package version 9.1.3 (Windows XP Professional; SAS Institute, Cary, NC, USA).
Gut microbiota
We used non-parametric tests to analyze qPCR data, alpha and beta-diversity, gene and species richness within individuals, between groups, at baseline and over time. Differential analyses were performed with DESeq2 (version 1.14.1)49 and ZIBR50. For all tests, the alpha risk was set at 0.05 after FDR adjustment by the Benjamini–Hochberg procedure. Network analysis was performed with the SPIEC-EASI R package (version 1.0.751). All statistical analyses were performed, and graphs were plotted with R software (version 3.6.0). Details of the analyses and parameters are provided in Supporting Information.
Source: Ecology - nature.com
