Individuals with coeliac disease (CeD) often experience gastrointestinal symptoms despite adherence to a gluten-free diet (GFD). While we recently showed that a diet low in fermentable oligo-, di-, monosaccharides and polyols (FODMAP) successfully provided symptom relief in GFD-treated CeD patients, there have been concerns that the low FODMAP diet (LFD) could adversely affect the gut microbiota. Our main objective was therefore to investigate whether the LFD affects the faecal microbiota and related variables of gut health. In a randomised controlled trial GFD-treated CeD adults, having persistent gastrointestinal symptoms, were randomised to either consume a combined LFD and GFD (n 39) for 4 weeks or continue with GFD (controls, n 36). Compared with the control group, the LFD group displayed greater changes in the overall faecal microbiota profile (16S rRNA gene sequencing) from baseline to follow-up (within-subject β-diversity, P < 0·001), characterised by lower and higher follow-up abundances (%) of genus Anaerostipes (Pgroup < 0·001) and class Erysipelotrichia (Pgroup = 0·02), respectively. Compared with the control group, the LFD led to lower follow-up concentrations of faecal propionic and valeric acid (GC-FID) in participants with high concentrations at baseline (Pinteraction ≤ 0·009). No differences were found in faecal bacterial α-diversity (Pgroup ≥ 0·20) or in faecal neutrophil gelatinase-associated lipocalin (ELISA), a biomarker of gut integrity and inflammation (Pgroup = 0·74), between the groups at follow-up. The modest effects of the LFD on the gut microbiota and related variables in the CeD patients of the present study are encouraging given the beneficial effects of the LFD strategy to treat functional GI symptoms (Registered at clinicaltrials.gov as NCT03678935).

Environmental DNA is a powerful tool for monitoring biodiversity. Although environmental DNA surveys have successfully been implemented in various environments, protocol choice has been shown to affect results and inferences. Thus far, few method comparison studies for soil have been undertaken. Here, we optimized the workflow for soil metabarcoding through a comparative study encompassing variation in sampling strategy (individual and combined samples), DNA extraction (PowerSoil®, NucleoSpin® Soil, PowerSoil® + phosphate buffer and NucleoSpin® Soil + phosphate buffer) and library preparation (one-step and two-step quantitative polymerase chain reaction methods). Using a partial 18S rRNA marker, a total of 309 eukaryotic taxa across 21 phyla were identified from Antarctic soil from one site in the Larsemann Hills. Our optimized workflow was effective with no notable reduction in data quality for a considerable increase in time and cost efficiency. The NucleoSpin® Soil + phosphate buffer was the best-performing extraction method. Compared to similar studies in other regions, we obtained low taxonomic coverage, perhaps because of the paucity of Antarctic terrestrial organisms in genetic reference databases. Our findings provide useful methodological insights for maximizing efficiency in soil metabarcoding studies in Antarctica and other low-biomass environments.

Inulin affects nutrition and metabolism in many animals. Although inulin is widely used in the diet of teleosts, its mechanism of action is unknown. Here, we investigated the effect of inulin (2 %) on the intestinal microbiome and metabolism in rainbow trout (Oncorhynchus mykiss) selected for growth and survival when fed a 100 % plant-based diet (suave) and a control line (temoin). Metabolic responses to the two factors (line and inulin) in liver, intestine, muscle and adipose were tissue-specific, with line and interaction between the two factors influencing overall expression in liver. In the intestine, inulin and line and in muscle, line influenced the expression of metabolic genes. Microbiota between the mucus and digestive contents was significantly different, with genera from Proteobacteria being more abundant in the mucus, whereas genera from the Firmicutes and Planctomycetes being more abundant in contents. Effect of inulin and interaction between factors on the microbiome was evident in contents. The significant taxa of control and inulin-fed groups differed greatly with Streptococcus and Weissella being significantly abundant in the inulin-fed group. There was a general trend showing higher levels of all SCFA in temoin group with propionic acid levels being significantly higher. An operational taxonomic unit (OTU) belonging to the Ruminococcaceae was significantly abundant in suave. The tissue-specific correlations between OTU and gene expression may indicate the link between microbiome and metabolism. Together, these results suggest that line and inulin impact the gene expression in a tissue-specific manner, possibly driven by specific OTUs enriched in inulin-fed groups and suave.

For the omnivorous Cherax quadricarinatus crayfish, plant raw materials can be good alternatives to dietary fish meal (FM). A 56-d feeding trial was conducted in C. quadricarinatus (11·70 (se 0·13) g). Diet with 100 % FM as the protein source was the control. Seven experimental diets were formulated by replacing 75 or 100 % of FM with soyabean meal (SM75, SM100) or cottonseed meal (CM75 and CM100), and a mixture of SM and CM (protein content is 1:1) replacing 50, 75 or 100 % of FM (SC50, SC75 and SC100). Crayfish fed the CM100 and SC100 showed significantly lower weight gain (WG), specific growth rate, trypsin and pepsin activities compared with the control diet. Crayfish in CM100 group showed significantly higher GPx, alanine aminotransferase, aspartate aminotransferase activities and malondialdehyde content than the control. SM100 and CM100 diets can cause slight separation of the peritrophic membrane from the intestinal folds. The pepsin activity of crayfish in SC50 was significantly higher than those in other experimental diets. The highest WG and muscle arginine content were also found in crayfish fed SC50. The relative abundance of Proteobacteria, Unclassified Enterobacteriaceae and Candidatus Bacilloplasma was significantly higher, but Actinobacteriota was significantly lower in SM100, CM100 and SC100 than in control. Microbiota functional prediction indicated that the relative abundance of ‘cell motility’ pathway in crayfish fed CM100 was significantly decreased compared with the control. In conclusion, only half of the FM can be effectively substituted with a mixture of SM and CM (protein content is 1:1) for C. quadricarinatus.

Vitamin D (VD) plays a vital role in various physiological processes in addition to its classic functions on maintaining the balance of Ca and P metabolism. However, there still are gaps to understand in depth the issues on the precise requirement, metabolic processes and physiological functions of VD in fish. In this study, we investigated the effects of VD on the growth, intestinal health, host immunity and metabolism in turbot (Scophthalmus maximus L.), one important commercial carnivorous fish in aquaculture, through the supplementation of different doses of dietary VD3 (0, 200, 400, 800 and 1600 μg VD3/kg diet). According to our results, the optimal VD3 level in the feed for turbot growth was estimated to be around 400 μg/kg, whereas VD3 deficiency or overdose in diets induced the intestinal inflammation, lowered the diversity of gut microbiota and impaired the host resistance to bacterial infection in turbot. Moreover, the level of 1α,25(OH)2D3, the active metabolite of VD3, reached a peak value in the turbot serum in the 400 μg group, although the concentrations of Ca and phosphate in the turbot were stable in all groups. Finally, the deficiency of dietary VD3 disturbed the nutritional metabolism in turbot, especially the metabolism of lipids and glucose. In conclusion, this study evaluated the optimal dose of dietary VD3 for turbot and provided the evidence that VD has a significant impact on intestinal health, host immunity and nutritional metabolism in fish, which deepened our understanding on the physiological functions and metabolism of VD3 in fish.

Poor utilisation efficiency of carbohydrate always leads to metabolic phenotypes in fish. The intestinal microbiota plays an important role in carbohydrate degradation. Whether the intestinal bacteria could alleviate high-carbohydrate diet (HCD)-induced metabolic phenotypes in fish remains unknown. Here, a strain affiliated to Bacillus amyloliquefaciens was isolated from the intestine of Nile tilapia. A basal diet (CON), HCD or HCD supplemented with B. amy SS1 (HCB) was used to feed fish for 10 weeks. The beneficial effects of B. amy SS1 on weight gain and protein accumulation were observed. Fasting glucose and lipid deposition were decreased in the HCB group compared with the HCD group. High-throughput sequencing showed that the abundance of acetate-producing bacteria was increased in the HCB group relative to the HCD group. Gas chromatographic analysis indicated that the concentration of intestinal acetate was increased dramatically in the HCB group compared with that in the HCD group. Glucagon-like peptide-1 was also increased in the intestine and serum of the HCB group. Thus, fish were fed with HCD, HCD supplemented with sodium acetate at 900 mg/kg (HLA), 1800 mg/kg (HMA) or 3600 mg/kg (HHA) diet for 8 weeks, and the HMA and HHA groups mirrored the effects of B. amy SS1. This study revealed that B. amy SS1 could alleviate the metabolic phenotypes caused by HCD by enriching acetate-producing bacteria in fish intestines. Regulating the intestinal microbiota and their metabolites might represent a powerful strategy for fish nutrition modulation and health maintenance in future.

Muramidases constitute a superfamily of enzymes that hydrolyse peptidoglycan (PGN) from bacterial cell walls. Recently, a fungal muramidase derived from Acremonium alcalophilum has been shown to increase broiler performance when added as a feed additive. However, the underlying mechanisms of action are not yet identified. Here, we investigated the hypothesis that this muramidase can cleave PGN to muramyl dipeptide (MDP), activating nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) receptors in eukaryotic cells, potentially inducing anti-inflammatory host responses. Using Micrococcus luteus as a test bacterium, it was shown that muramidase from A. alcalophilum did not display antimicrobial activity, while it could cleave fluorescently labelled PGN. It was shown that the muramidase could degrade PGN down to its minimal bioactive structure MDP by using UPLC-MS/MS. Using HEK-Blue™-hNOD2 reporter cells, it was shown that the muramidase-treated PGN degradation mixture could activate NOD2. Muramidase supplementation to broiler feed increased the duodenal goblet cell and intraepithelial lymphocyte abundance while reducing duodenal wall CD3+ T lymphocyte levels. Muramidase supplementation to broiler feed only had moderate effects on the duodenal, ileal and caecal microbiome. It was shown that the newly discovered muramidase hydrolysed PGN, resulting in MDP that activates NOD2, potentially steering the host response for improved intestinal health.

A high-carbohydrate diet could achieve a protein-sparing effect, but it may cause negative impacts on the growth condition of fish due to their poor utilisation ability of carbohydrate. How to reduce the adverse effects caused by a high-carbohydrate diet is important for the development of aquaculture. In the present study, we aimed to identify whether inulin could attenuate the metabolic syndrome caused by a high-carbohydrate diet in fish. Nile tilapia (Oreochromis niloticus) (1·19 (sd 0·01) g) were supplied with 35 % carbohydrate (CON), 45 % carbohydrate (HC) and 45 % carbohydrate + 5 g/kg inulin (HCI) diets for 10 weeks. The results showed that addition of inulin improved the survival rate when fish were challenged with Aeromonas hydrophila, indicating that inulin had an immunostimulatory effect. Compared with the HC group, the HCI group had lower lipid accumulation in liver and the gene expression analyses indicated that addition of inulin down-regulated genes related to lipogenesis and up-regulated genes relevant to β-oxidation significantly (P < 0·05). Higher liver glycogen and glucose tolerance were found in the HCI group compared with the HC group (P < 0·05). These results indicated that inulin could alleviate the metabolic syndrome induced by a high-carbohydrate diet. Furthermore, addition of inulin to a high-carbohydrate diet changed the intestinal bacterial composition and significantly increased the concentration of acetic acid and propionic acid in fish gut which have the potential to increase pathogen resistance and regulate metabolic characteristics in fish. Collectively, our results demonstrated a possible causal role for the gut microbiome in metabolic improvements induced by inulin in fish.

There is increasing evidence linking the gut microbiota to various aspects of human health. Nuts are a food rich in prebiotic fibre and polyphenols, food components which have been shown to have beneficial effects on the gut microbiota. This systematic review aimed to synthesise the evidence regarding the effect of nut consumption on the human gut microbiota. A systematic search of the databases MEDLINE, PubMed, Cochrane CENTRAL and CINAHL was performed until 28 November 2019. Eligible studies were those that investigated the effects of nut consumption in humans (aged over 3 years old), utilising next-generation sequencing technology. Primary outcome measures were between-group differences in α- and β-diversity metrics and gut microbial composition. A total of eight studies were included in the review. Included studies assessed the effects of either almonds, walnuts, hazelnuts or pistachios on the gut microbiota. Overall, nut consumption had a modest impact on gut microbiota diversity, with two studies reporting a significant shift in α-diversity and four reporting a significant shift in β-diversity. Walnuts, in particular, appeared to more frequently explain shifts in β-diversity, which may be a result of their unique nutritional composition. Some shifts in bacterial composition (including an increase in genera capable of producing SCFA: Clostridium, Roseburia, Lachnospira and Dialister) were reported following the consumption of nuts. Nut intake may yield a modulatory effect on the gut microbiota; however, results were inconsistent across studies, which may be explained by variations in trial design, methodological limitations and inter-individual microbiota.

The gut microbiota is directly influenced by dietary components, and it plays critical roles in chronic diseases. Excessive consumption of trans-fatty acids (TFA) is associated with obesity induced by alterations in gut microbiota, but the links between obesity and gut microbiota remain unclear. Therefore, studies examining the impact of TFA on intestinal microflora are essential. In our study, we performed 16S ribosomal RNA gene sequencing on faecal samples from Sprague–Dawley rats fed a basal diet (control (CON) group), high-fat (HF) diet (diet-induced obesity (DIO) group) or TFA diets (1 % TFA group and 8 % TFA group) for 8 weeks to investigate the effects of TFA/HF diets on obesity and gut microbiota composition. We found that the TFA/HF diets significantly induced obesity and changes in blood and brain physiological parameters of the rats. The relative abundance of the phyla Firmicutes and Bacteroidetes was inversely altered in the three test groups compared with the CON group. Proteobacteria increased slightly in the DIO, 1 % TFA and 8 % TFA groups. The genus Bacteroides increased in the DIO and 1 % TFA groups, but Muribaculaceae decreased in all experimental groups compared with the CON group. Moreover, significant differences were observed among clusters of orthologous group functional categories of the four dietary groups. Our observations suggested that the TFA/HF diets induced obesity and dysfunction of gut microbiota. Gut dysbiosis might mediate the obesity effects of TFA/HF diets.

The effect of holly polyphenols (HP) on intestinal inflammation and microbiota composition was evaluated in a piglet model of lipopolysaccharide (LPS)-induced intestinal injury. A total of twenty-four piglets were used in a 2 × 2 factorial design including diet type and LPS challenge. After 16 d of feeding with a basal diet supplemented with or without 250 mg/kg HP, pigs were challenged with LPS (100 μg/kg body weight) or an equal volume of saline for 4 h, followed by analysis of disaccharidase activities, gene expression levels of several representative tight junction proteins and inflammatory mediators, the SCFA concentrations and microbiota composition in intestinal contents as well as proinflammatory cytokine levels in plasma. Our results indicated that HP enhanced intestinal disaccharidase activities and reduced plasma proinflammatory cytokines including TNF-α and IL-6 in LPS-challenged piglets. Moreover, HP up-regulated mRNA expression of intestinal tight junction proteins such as claudin-1 and occludin. In addition, bacterial 16S rRNA gene sequencing showed that HP altered hindgut microbiota composition by enriching Prevotella and enhancing SCFA production following LPS challenge. These results collectively suggest that HP is capable of alleviating LPS-triggered intestinal injury by improving intestinal disaccharidase activities, barrier function and SCFA production, while reducing intestinal inflammation.

Recent studies have demonstrated that the nutritional properties of peanut meal (PM) can be improved after being fermented. The assessment of fermented PM has been reported to be limited to various physical and chemical evaluations in vitro. In the present study, PM was fermented by Bacillus natto to explore the effects of fermented PM extract (FE) on growth performance, learning and memory ability and intestinal microflora in mice. Ninety newly weaned male Kunming (KM) mice were randomly divided into seven groups: normal group (n 20), low-dose FE group (n 10), middle-dose FE group (MFE) (n 10), high-dose FE group (HFE) (n 20), unfermented extraction group (n 10), model group (10) and natural recovery group (10). Learning and memory skills were performed by the Morris water maze (MWM) test, and the variation in gut microbiota (GM) composition was assessed by 16S rDNA amplicon sequencing. The results show that HFE remarkably improved the growth performance in mice. In the MWM test, escape latency was shortened in both MFE and HFE groups, while the percentage of time, distance in target quadrant and the number crossing over the platform were significantly increased in the HFE group. Moreover, the FE played a preventive role in the dysbacteriosis of mice induced by antibiotic and increased the richness and species evenness of GM in mice.

Wholegrain oats are known to modulate the human gut microbiota and have prebiotic properties (increase the growth of some health-promoting bacterial genera within the colon). Research to date mainly attributes these effects to the fibre content; however, oat is also a rich dietary source of polyphenols, which may contribute to the positive modulation of gut microbiota. In vitro anaerobic batch-culture experiments were performed over 24 h to evaluate the impact of two different doses (1 and 3 % (w/v)) of oat bran, matched concentrations of β-glucan extract or polyphenol mix, on the human faecal microbiota composition using 16S RNA gene sequencing and SCFA analysis. Supplementation with oats increased the abundance of Proteobacteria (P <0·01) at 10 h, Bacteroidetes (P <0·05) at 24 h and concentrations of acetic and propionic acid increased at 10 and 24 h compared with the NC. Fermentation of the 1 % (w/v) oat bran resulted in significant increase in SCFA production at 24 h (86 (sd 27) v. 28 (sd 5) mm; P <0·05) and a bifidogenic effect, increasing the relative abundance of Bifidobacterium unassigned at 10 h and Bifidobacterium adolescentis (P <0·05) at 10 and 24 h compared with NC. Considering the β-glucan treatment induced an increase in the phylum Bacteroidetes at 24 h, it explains the Bacteriodetes effects of oats as a food matrix. The polyphenol mix induced an increase in Enterobacteriaceae family at 24 h. In conclusion, in this study, we found that oats increased bifidobacteria, acetic acid and propionic acid, and this is mediated by the synergy of all oat compounds within the complex food matrix, rather than its main bioactive β-glucan or polyphenols. Thus, oats as a whole food led to the greatest impact on the microbiota.

Working canines are deployed by the Federal Emergency Management Agency (FEMA), as part of a National Disaster Response Plan. Stress associated with helicopter flight and the resulting physical effects on the dog are unknown. Our objective was to test the hypotheses that (1) helicopter travel affects the physiology and faecal microbiota of working canines, but that (2) physiological consequences of helicopter travel will not negatively affect their work performance. A total of nine FEMA canines were loaded onto helicopters and flown for 30 min in July 2015. Rectal temperature, behavioural stress indicators and saliva swabs (for cortisol) were collected at baseline, loading, mid-flight and post-flight. After flight, canines completed a standardised search exercise to monitor work performance. Faecal samples were collected for microbial DNA extraction and Illumina sequencing. All canines were on a standardised diet (CANIDAE® Grain Free PURE Land®) for 3 weeks prior to the study. Visible indicators of stress were observed at loading and at mid-flight and corresponded with an increase (P < 0·05) in salivary cortisol from 5·4 µg/l (baseline) to 6·4 µg/l (loading). Additionally, rectal temperature increased (P < 0·05) from 38·61°C (baseline) to 39·33°C (mid-flight) and 39·72°C (post-flight). Helicopter travel did not affect search performance (P > 0·05). We found that α- and β-diversity measures of faecal microbiota were not affected (P > 0·05). Our data suggest that although helicopter travel may cause physiological changes that have been associated with stress in working dogs, it does not make an impact on their search performance or the stability of faecal microbiota.

Probiotic yogurt and milk supplemented with probiotics have been investigated for their role in ‘low-grade’ inflammation but evidence for their efficacy is inconclusive. This study explores the impact of probiotic yogurt on metabolic and inflammatory biomarkers, with a parallel study of gut microbiota dynamics. The randomised cross-over study was conducted in fourteen healthy, young men to test probiotic yogurt compared with milk acidified with 2 % d-(+)-glucono-δ-lactone during a 2-week intervention (400 g/d). Fasting assessments, a high-fat meal test (HFM) and microbiota analyses were used to assess the intervention effects. Baseline assessments for the HFM were carried out after a run-in during which normal milk was provided. No significant differences in the inflammatory response to the HFM were observed after probiotic yogurt compared with acidified milk intake; however, both products were associated with significant reductions in the inflammatory response to the HFM compared with the baseline tests (assessed by IL6, TNFα and chemokine ligand 5) (P<0·001). These observations were accompanied by significant changes in microbiota taxa, including decreased abundance of Bilophila wadsworthia after acidified milk (log 2-fold-change (FC)=–1·5, Padj=0·05) and probiotic yogurt intake (FC=–1·3, Padj=0·03), increased abundance of Bifidobacterium species after acidified milk intake (FC=1·4, Padj=0·04) and detection of Lactobacillus delbrueckii spp. bulgaricus (FC=7·0, Padj<0·01) and Streptococcus salivarius spp. thermophilus (FC=6·0, Padj<0·01) after probiotic yogurt intake. Probiotic yogurt and acidified milk similarly reduce postprandial inflammation that is associated with a HFM while inducing distinct changes in the gut microbiota of healthy men. These observations could be relevant for dietary treatments that target ‘low-grade’ inflammation.

Polymannuronic acid (PM), one of numerous alginates isolated from brown seaweeds, is known to possess antioxidant activities. In this study, we examined its potential role in reducing body weight gain and attenuating inflammation induced by a high-fat and high-sucrose diet (HFD) as well as its effect on modulating the gut microbiome in mice. A 30-d PM treatment significantly reduced the diet-induced body weight gain and blood TAG levels (P<0·05) and improved glucose tolerance in male C57BL/6J mice. PM decreased lipopolysaccharides in blood and ameliorated local inflammation in the colon and the epididymal adipose tissue. Compared with low-fat and low-sucrose diet (LFD), HFD significantly reduced the mean number of species-level operational taxonomic units (OTU) per sample as well as species richness (P<0·05) but did not appear to affect other microbial diversity indices. Moreover, compared with LFD, HFD altered the abundance of approximately 23 % of the OTU detected (log10 linear discriminant analysis (LDA) score>2·0). PM also had a profound impact on the microbial composition in the gut microbiome and resulted in a distinct microbiome structure. For example, PM significantly increased the abundance of a probiotic bacterium, Lactobacillus reuteri (log10 LDA score>2·0). Together, our results suggest that PM may exert its immunoregulatory effects by enhancing proliferation of several species with probiotic activities while repressing the abundance of the microbial taxa that harbor potential pathogens. Our findings should facilitate mechanistic studies on PM as a potential bioactive compound to alleviate obesity and the metabolic syndrome.

Mechanistic research suggests a unique evolutionary relationship between complex milk oligosaccharides and cognate bifidobacteria enriched in breast-fed infants. Bovine milk oligosaccharides (BMO) were recently identified as structurally and functionally similar to human milk oligosaccharides. The present single-blind three-way crossover study is the first to determine the safety and tolerability of BMO consumption by healthy human participants (n 12) and its effects on faecal microbiota and microbial metabolism. Participants consumed each supplement (placebo-control; low- and high-BMO doses) for eleven consecutive days, followed by a 2-week washout period prior to initiating the next supplement arm. Low and high BMO doses were consumed as 25 and 35 % of each individual's daily fibre intake, respectively. Safety and tolerability were measured using standardised questionnaires on gut and stomach discomfort and stool consistency. Faecal extracts were profiled for bacterial populations by next-generation sequencing (NGS) and bifidobacteria presence was confirmed using quantitative PCR. Urine was analysed for changes in microbial metabolism using nuclear magnetic resonance spectroscopy (1H-NMR). Consumption of both the low and high BMO doses was well tolerated and did not change stool consistency from baseline. Multivariate analysis of the NGS results demonstrated no change in faecal microbiota phyla among the placebo-control and BMO supplement groups. In conclusion, BMO supplementation was well tolerated in healthy adults and has the potential to shift faecal microbiota toward beneficial strains as part of a synbiotic treatment with probiotic cultures that selectively metabolise oligosaccharides.

Human milk decreases the risk of necrotising enterocolitis (NEC), a severe gastrointestinal disease that occurs in 5–10 % of preterm infants. The prebiotic and immune-modulatory effects of milk oligosaccharides may contribute to this protection. Preterm pigs were used to test whether infant formula enriched with α1,2-fucosyllactose (2'-FL, the most abundant oligosaccharide in human milk) would benefit gut microbial colonisation and NEC resistance after preterm birth. Caesarean-delivered preterm pigs were fed formula (Controls, n 17) or formula with 5 g/l 2'-FL (2'-FL, n 16) for 5 d; eight 2'-FL pigs (50 %) and twelve Controls (71 %) developed NEC, with no difference in lesion scores (P=0·35); 2'-FL pigs tended to have less anaerobic bacteria in caecal contents (P=0·22), but no difference in gut microbiota between groups were observed by fluorescence in situ hybridisation and 454 pyrosequencing. Abundant α1,2-fucose was detected in the intestine with no difference between groups, and intestinal structure (villus height, permeability) and digestive function (hexose absorption, brush border enzyme activities) were not affected by 2'-FL. Formula enrichment with 2'-FL does not affect gut microbiology, digestive function or NEC sensitivity in pigs within the first few days after preterm birth. Milk 2'-FL may not be critical in the immediate postnatal period of preterm neonates when gut colonisation and intestinal immunity are still immature.