Table 5.

Individual publication results of changes in faecal microbiota profile among infants receiving AAF-Syn vs. AAF.

Lead Author (Date)	Outcome Measures	Comparison of Findings in AAF-Syn vs. AAF Groups	Statistical Comparison	Conclusions
Burks (2015) [39]	Secondary outcome was change in proportion of faecal BSp, CH & ER/CC	Mean AAF-Syn vs. AAF. Baseline proportions similar in both groups -BSp: 14.3% vs. 16.7%, p = 0.60 NS -CH: 9.3% vs. 9.1%, p = 0.93 NS -ER/CC: 23.8% vs. 20.1%, p = 0.30 NS At 16 weeks significant differences observed: -BSp: 51.2% vs. 13.0%, p < 0.001 -CH: 7.1% vs. 17.6%, p < 0.001 -ER/CC: 13.8% vs. 35.5%, p < 0.001	All differences between groups at 16 weeks statistically significant	“The indigenous gut microbiota of [CMPA] infants receiving an AAF can be influenced by synbiotics. As expected, synbiotics in the test formula increased Bifidobacterium, a genus typically predominant in the GI tract of breastfed infants” “…It can therefore be hypothesized that abolishing this gut microbiota dysbiosis may decrease [CMPA] risk or [CMPA] persistence…”
Candy (2018) [32] ASSIGN study	Primary outcome was change in proportion of faecal BSp & ER/CC Baseline measures were used as covariates for ANCOVA	Median, AAF-Syn vs. AAF. Baseline proportions not given At 8 weeks -BSp: 35.4% vs. 9.7%, p < 0.001 -ER/CC: 9.5% vs. 24.2%, p < 0.001 In the healthy breastfed comparison group: -BSp: 55.0%; -ER/CC: 6.5%	Between groups comparison for both BSp and ER/CC were statistically significant at 8 weeks	“The primary objective of modifying gut microbiota using an AAF including [synbiotics] for 8 weeks in subjects with suspected non-IgE [CMPA] was achieved.” “…The current study showed that microbial composition of infants with suspected non-IgE [CMPA] who received the test formula was closer to the profile of the HBR group than those infants receiving control formula.”
Fox (2019) [33] ASSIGN study Subset of infants who continued intervention for 26 weeks	26-week extension study of Candy (2018) [32]	The between-group differences in microbiota composition seen at week 8 (primary trial endpoint) were maintained with longer study follow-up. At weeks 12 and 26, the AAF-Syn group had a higher percentage of BSp and a lower percentage of ER/CC compared with the AAF group. Mean AAF-Syn vs. AAF: At baseline (0 weeks) -BSp 26.6% vs. 17.7%, p = 0.23 NS -ER/CC 23.6% vs. 13.4%, p = 0.07 NS At 8 weeks -BSp 38.4% vs. 13.1%, p = 0.002 -ER/CC 6.3% vs. 25.5%, p < 0.001 At 12 weeks -BSp 49.7% vs. 17.1%, p = 0.002 -ER/CC 7.5% vs. 32.1%, p = 0.002 At 26 weeks -BSp 48.8% vs. 15.1%, p < 0.001 -ER/CC 13.1% vs. 33.9%, p < 0.001	Between groups comparison for both BSp and ER/CC were statistically significant at 26 weeks	“…In conclusion, use of the AAF including specific synbiotics investigated in this study resulted in a sustained improvement in gut microbiota composition over 26 weeks…” “…it may suggest that the effects on gut microbiota by AAF including synbiotics can even be maintained in a [CMPA] population receiving systemic antibiotics.”
Wopereis (2019) [40] ASSIGN study	Detailed genomic characterisation of faecal microbiota, population from Candy (2018) [32] and Fox (2019) [33]. Primary outcome was the assessment of bacterial species diversity over time.	Diversity in faecal microbiota increased over time in both groups. The effect was less pronounced in the AAF-Syn group. Mean difference per week from week 0 to 26 -PD: −0.022 units, p = 0.069 NS -SI: −0.026 units, p = 0.005 Estimated average difference AAF-Syn vs. AAF was significant At 12 weeks: -PD: −0.349 units, p = 0.031; SI: −0.236 units, p= 0.049 At 26 weeks: -PD: − 0.653 units, p = 0.012; SI: −0.596 units, p = 0.002	Significant improvement in faecal microbial diversity	“…AAF including the specific synbiotics offers an effective nutritional strategy to modulate the gut microbiota of infants with suspected non-IgE mediated [CMPA] closer to a healthy breastfed profile…” “The AAF including synbiotics compared to the AAF without synbiotics showed a more gradual increment over time of bacterial diversity, which is also typically observed in longitudinal studies investigating early life gut microbiota development of breastfed infants as compared to formula-fed infants.”
Wopereis (2020) [30] PRESTO study	Detailed genomic characterisation of faecal microbiota; abundances of BSp, LSp and adult-type genera; faecal bacterial species diversity	At 6 and 12 months, compared to AAF, AAF-Syn was associated with: -Increased relative abundances of Bifidobacterium and Lactobacillus and decreased relative abundances of adult-type genera (Blautia, Tyzzerella and Romboutsia) -Lower overall bacterial diversity -Higher BSp diversity Data not given.	p-values not reported	“The predominant abundance of Bifidobacterium in subjects receiving [AAF-Syn] was reflected in lower overall diversity at 6 and 12 months.” “…Subjects receiving [AAF-Syn] showed increased diversity of species within the genus Bifidobacterium compared to AAF at 6 and 12 months.”

CMPA: cow’s milk protein allergy; AAF-Syn: amino acid formula with synbiotics; AAF: amino acid formula; BSp: Bifidobacterium; CH: Clostridium histolyticum; ER/CC: Eubacterium rectale/Clostridium coccoides; GI: Gastro-intestinal; PD: Phylogenetic diversity; SI: Shannon index; LSp: Lactobacillus; GI: gastrointestinal; HBR: healthy breastfed reference.