www.nature.com/scientificreports OPEN Impact of synbiotics on gut microbiota during early life: a randomized, double‑blind study Nopaorn Phavichitr1, Shugui Wang3, Sirinuch Chomto4, Ruangvith Tantibhaedhyangkul1, Alexia Kakourou2, Sukkrawan Intarakhao5, Sungkom Jongpiputvanich5, COLOR Study Group2*, Guus Roeselers2* & Jan Knol2,6 Human milk is considered the optimal nutrition for infants and found to contain signifcant numbers of viable bacteria. The aim of the study was to assess the efects of a specifc synbiotic combination at doses closer to the bacterial cells present in human milk, on intestinal bifdobacteria proportions (relative abundance), reduction of potential pathogens and gut physiological conditions. A clinical study was conducted in 290 healthy infants aged from 6 to 19 weeks. Infants received either a control infant formula or one of the two investigational infant formulas (control formula with 0.8 g/100 ml scGOS/lcFOS and Bifdobacterium breve M‑16V at either 1 × 104 cfu/ml or 1 × 106 cfu/ml). Exclusively breastfed infants were included as a reference. Analyses were performed on intention‑ to‑treat groups and all‑subjects‑treated groups. After 6 weeks of intervention, the synbiotics at two diferent doses signifcantly increased the bifdobacteria proportions in healthy infants. The synbiotic supplementation also decreased the prevalence (infants with detectable levels) and the abundance of C. difcile. Closer to the levels in the breastfed reference group, fecal pH was signifcantly lower while l‑lactate concentrations and acetate proportions were signifcantly higher in the synbiotic groups. All formulas were well tolerated and all groups showed a comparable safety profle based on the number and severity of adverse events and growth. In healthy infants, supplementation of infant‑type bifdobacterial strain B. breve M‑16V, at a dose close to bacterial numbers found in human milk, with scGOS/lcFOS (9:1) created a gut environment closer to the breastfed reference group. This specifc synbiotic mixture may also support gut microbiota resilience during early life. Clinical Trial Registration This clinical study named Color Synbiotics Study, was registered in ClinicalTrials.gov on 18 March 2013. Registration number is NCT01813175. https ://clini caltr ials.gov/ ct2/show/NCT01 81317 5. Abbreviations EPEC Enteropathogenic Escherichia coli EAEC Enteroaggregative Escherichia coli HMOs Human milk oligosaccharides scGOS/lcFOS Short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides CFU Colony forming unit Syn4 Control formula supplemented with 0.8 g/100 ml scGOS/lcFOS and B. breve M-16V at a dose of 1 × 104 cfu/ml Syn6 Control formula supplemented with 0.8 g/100 ml scGOS/lcFOS and B. breve M-16V at a dose of 1 × 106 cfu/ml FISH Fluorescent in situ hybridization SCFA Short chain fatty acid ITT Intention-to-treat AST All-subjects-treated 1Department of Paediatrics, Phramongkutklao Hospital, Bangkok, Thailand. 2Danone Nutricia Research, Utrecht, The Netherlands. 3Danone Nutricia Research, Singapore, Singapore. 4Nutritional Unit, Department of Pediatrics, King Chulalongkorn Memorial Hospital, Chulalongkorn University, Bangkok, Thailand. 5Department of Pediatrics, Thammasat Hospital, Faculty of Medicine, Thammasat University, Bangkok, Thailand. 6Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands. *A list of authors and their afliations appears at the end of the paper. *email: [email protected] Scientifc Reports | (2021) 11:3534 | https://doi.org/10.1038/s41598-021-83009-2 1 Vol.:(0123456789) www.nature.com/scientificreports/ MMRM Mixed-efect model for repeated measures ANCOVA Analysis of covariance GLMM Generalized linear mixed model RDA Redundancy analysis TRT​ Treatment WHO World Health Organization In early life, the infant’s gut microbiota of healthy breastfed infants is normally dominated by infant-type bif- dobacteria such as Bifdobacterium breve, Bifdobacterium bifdum and Bifdobacterium longum subsp. infantis. Tese members of the gut microbiota make the infant gut more resistant to pathogen colonization1, improve certain vaccination responses 2, support immune maturation and support gut barrier development 3. However, not all infants’ gut microbiotas are dominated by Bifdobacterium species and some are even devoid of them4,5. Environmental factors such as mode of delivery, antibiotics and feeding patterns infuence bifdobacterial colo- nization of the infant’s gut. Given the major role of infant-type bifdobacteria in structuring the gut microbiome in early life, it is important to support the colonization by relevant Bifdobacterium species6. Opportunistic pathogens such as Clostridium difcile, Clostridium perfringens, enteropathogenic Escherichia coli (EPEC) and enteroaggregative Escherichia coli (EAEC) are ofen found in infants’ guts. C. difcile colonizes 10–70% of infants below 1 year of age 7. C. difcile infections during infancy may not only cause diarrhoea but are also associated with higher risk of allergic diseases during early life8. Breastfeeding, known to reduce the prevalence of C. difcile in infants compared to formula feeding (14% vs 30%, respectively)9, also helps in pre- vention of infections and allergic diseases during early life10,9. Human milk is considered the optimal nutrition for infants and contains a signifcant number of viable bacteria, which are an important source for vertical microbial transmission from mother to infant11–13. If this colonization route is disrupted, early life microbiota development may be impaired. Human milk is estimated to contain about 103–105 bacterial cells/ml based on fow cytometry and quantitative polymerase chain reaction (q-PCR) methods12,14–18. Te human milk microbiota is a taxonomically diverse com- munity, to which, bifdobacteria contribute up to 104 cells/ml17,19. B. breve is the most commonly isolated infant- type Bifdobacterium species from human milk 20. It is one of the dominant members of the infant’s gut micro- biota, involved in the metabolism of human milk oligosaccharides (HMOs) and the production of vitamins21,22. Non-infant-type bifdobacteria such as B. animalis subsp. lactis, isolated from diverse mammalian hosts, and B. adolescentis, normally found in the adult human gut, are genetically less equipped to metabolize HMOs 22. Synbiotics, a combination of probiotics and prebiotics that confers health benefts to the host 23,24, ofer an efcient way to mimic milk driven colonisation and formation of a Bifdobacterium dominated ecosystem in the infant gut25. Synbiotics containing short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (scGOS/lcFOS) with a 9:1 ratio and B. breve M-16V, has been shown to restore the delayed bifdobacteria colo- nization in caesarean section (C-section) born infants25 and to improve the symptoms of IgE-associated atopic dermatitis26. In addition, there is long and comprehensive tolerance and safety track record for the use of B. breve M-16V as a probiotic for infants, including infants with a very low birth weight. Te efects of probiotics or synbiotics are dose and strain dependent. Te doses of probiotics and synbiotic used in previous studies in infants and children range from 108 to 1011 cfu/day27. As human milk contains relatively low numbers of viable bacteria (ranging from 10 3 to 10 5 cfu cfu/ml, about 106–108 cfu/day)12, it is important to understand the efects of diferent doses of synbiotics on the infant’s gut microbiota. Te primary objective of this study was to evaluate the bifdogenic efect of an infant formula containing synbiotics with two doses of B. breve M-16V (either 1 × 104 cfu/ml or 1 × 106 cfu/ml), in combination with scGOS/ lcFOS (9:1) in healthy infants aged 6–19 weeks. Te study also explored the efects of this specifc synbiotics on pathogen reduction and gut physiological conditions in early life. Patients and methods Tis was an exploratory, randomized, double-blind, controlled study conducted between May 2013 and Septem- ber 2015 in Tailand. Te protocol and all accompanying material provided to the subjects, such as information sheets or description of the study used to obtain informed consent, were submitted to the following ethics review committees: Institutional Review Board, Chulalongkorn University; Institutional Ethics Review Committee, Royal Tai Army Medical Department; Human Research Ethics Committee, Tammasat University. Approval from the three Ethics Committees was obtained before start of the study, and was documented in a letter to the investigators specifying the date on which the committee met and granted the approval. Written informed consent was obtained from all parents/caregivers before inclusion in the study. Te study was registered in ClinicalTrials.gov (March 18, 2013; #NCT01813175). Te study was conducted according to ICH-GCP principles, and in compliance with the principles of the ‘Declaration of Helsinki’ (59th WMA General Assembly, Seoul, October 2008) and with the Tai laws and regulations. Inclusion criteria were a gestational age between 37 and 42 weeks, infant age 43–65 days, and exclusive formula feeding for at least 1 week (except for the breastfed reference group). Exclusion criteria were, malnutrition, weaned before inclusion, malformations, use of systemic antibiotics or anti-mycotic drugs within 4 weeks prior to study entry, gastroenteritis or diarrhoea in the last 2 weeks prior to study entry. Sample size calculation methods and randomisation and unblinding procedures are reported in detail in the