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Brain signalling pathways involved in subclinical anxiety and depressed mood can be modulated via the gut brain axis (GBA), providing the potential for diet and dietary components to affect mood. We investigated behavioural, physiological and gut microbiome responses to the Lacticaseibacillus rhamnosus strain HN001 (LactoB HN001™), which has been shown to reduce postpartum anxiety and depression, and a milk fat globule membrane-enriched product, Lipid 70 (SurestartTM MFGM Lipid 70), which has been implicated in memory in stress-susceptible Wistar Kyoto rats. We examined behaviour in the open field, elevated plus maze and novel object recognition tests in conjunction with the expression of host genes in neuro-signalling pathways, and we also assessed brain lipidomics. Treatment-induced alterations in the caecal microbiome and short-chain fatty acid (SCFA) profiles were also assessed. Neither ingredient induced behavioural changes or altered the brain lipidome (separately or when combined). However, with regard to brain gene expression, the L. rhamnosus HN001 + Lipid 70 combination produced a synergistic effect, reducing GABAA subunit expression in the amygdala (Gabre, Gat3, Gabrg1) and hippocampus (Gabrd). Treatment with L. rhamnosus HN001 alone altered expression of the metabotropic glutamate receptor (Grm4) in the amygdala but produced only minor changes in gut microbiota composition. In contrast, Lipid 70 alone did not alter brain gene expression but produced a significant shift in the gut microbiota profile. Under the conditions used, there was no observed effect on rat behaviour for the ingredient combination. However, the enhancement of brain gene expression by L. rhamnosus HN001 + Lipid 70 implicates synergistic actions on region-specific neural pathways associated with fear, anxiety, depression and memory. A significant shift in the gut microbiota profile also occurred that was mainly attributable to Lipid 70.

This study explores how slowed gastrointestinal transit (constipation) induced by the opioid agonist loperamide (imodium) alters the microbiota composition such that the richness and diversity of the bacterial communities is lowered. Notably Bacteroides were relatively abundant in the loperamide treated group suggesting that slowed colonic transit facilitates the amplification and colonization of genera such as Bacteroides that adapt well in slow moving and competitive environments with limited resources.

Background/Aims: Gastrointestinal(GI) motility is an integral part of digestive function. The enteric nervous system(ENS) is the primary controller of GI motility. Dysfunction of the ENS can cause impaired GI motility as seen in the case of constipation. Animal models mimicking symptoms of constipation have been developed by way of pharmacological manipulations. We have previously shown that loperamide, an opioid agonist that works by inhibiting enteric neuronal activity, reduces propulsion and delays GI transit, inducing constipation in aged rats. Previous studies have reported an association between altered GI motility and gut microbial population. Little is known about the changes in gut microbiota profile resulting from pharmacologically induced constipation in rats. The aim of this study was to examine how constipation induced by a pharmacological drug loperamide, affects the gut microbiota composition. Methods: Caecal digesta samples were collected from adult male Sprague Dawley rats that were administered with 1 mg/kg/day loperamide(in 100%DMSO) or DMSO vehicle only(Control) for seven days via a subcutaneous 2 mL capacity slow release osmotic mini pump. Caecal microbial composition was determined by 16S rRNA gene amplicon sequencing. Results: Significant differences in caecal microbial communities were observed between loperamide treated and control groups. At the phylum level Proteobacteria and Bacteroidetes were relatively more abundant in loperamide treated vs control(PConclusion: Constipation induced by loperamide affects the diversity and abundance of gut microbiota. Identifying bacterial species that are associated with slowed GI transit may help in developing prebiotic and probiotic treatments for the management of constipation.

Background: The enteric nervous system (ENS) is an important pathway through which the gut communicates with the brain. However, there is little information on how modulating ENS activity influences higher cognitive function. The objective of this experiment was to investigate if modulating the ENS affects anxiety related behaviour in rats. We used loperamide, a μ-opioid receptor agonist that does not cross the blood brain barrier, to manipulate ENS function and assess whether any behavioural effects are associated with changes in gut and brain gene expression, and microbiota profile. Methods: Male and female Sprague Dawley rats were treated with an acute dose of loperamide, or control solution and anxiety related behaviour assessed using the Open Field and Elevated-Plus maze tests. Gene expression in the caecum, prefrontal cortex, hippocampus, and amygdala was assessed by RNAseq, and caecal microbiota composition determined by shotgun metagenome sequencing. Results: In female rats, loperamide treatment significantly decreased total distance moved (P=0.04) and frequency of supported rearing (P=0.03) compared to controls, which indicated a decrease in exploratory behaviour and increased anxiety. However, loperamide did not significantly alter behaviour in male rats. Similarly, loperamide did not alter gene expression in any brain region in male rats. However, in female rats, loperamide significantly altered expression of 10 genes in the hippocampus (FDRConclusion: The ability of loperamide to modulate behaviour and brain gene expression indicates that gut to brain signaling involved μ-opioid receptors. Because loperamide slows motility, the changes in the microbiome are likely a consequence of this.

The probiotic Lacticaseibacillus rhamnosus strain HN001 has been shown to have several beneficial health effects for both pediatric and maternal groups, including reduced risk of eczema in infants and gestational diabetes and postnatal depression in mothers. While L. rhamnosus HN001 appears to modify immune and gut barrier biomarkers, its mode of action remains to be fully elucidated. To gain insights into the role of HN001 on the infant microbiome, the impacts of L. rhamnosus HN001 supplementation was studied in 10-day old male piglets that were fed either infant formula, or infant formula with L. rhamnosus HN001 at a low (1.3 × 105 CFU/ml) or high dose (7.9 × 106 CFU/ml) daily for 24 days. The cecal and fecal microbial communities were assessed by shotgun metagenome sequencing and host gene expression in the cecum and colon tissue was assessed by RNA-seq. Piglet fecal samples showed only modest differences between controls and those receiving dietary L. rhamnosus HN001. However, striking differences between the three groups were observed for cecal samples. While total lactobacilli were significantly increased only in the high dose L. rhamnosus HN001 group, both high and low dose groups showed an up to twofold reduction across the Firmicutes phylum and up to fourfold increase in Prevotella compared to controls. Methanobrevibacter was also decreased in HN001 fed piglets. Microbial genes involved in carbohydrate and vitamin metabolism were among those that differed in relative abundance between those with and without L. rhamnosus HN001. Changes in the cecal microbiome were accompanied by increased expression of tight junction pathway genes and decreased autophagy pathway genes in the cecal tissue of piglets fed the higher dose of L. rhamnosus HN001. Our findings showed supplementation with L. rhamnosus HN001 caused substantial changes in the cecal microbiome with likely consequences for key microbial metabolic pathways. Host gene expression changes in the cecum support previous research showing L. rhamnosus HN001 beneficially impacts intestinal barrier function. We show that fecal samples may not adequately reflect microbiome composition higher in the gastrointestinal tract, with the implication that effects of probiotic consumption may be missed by examining only the fecal microbiome.

While infant formula is usually bovine milk-based, interest in other ruminant milk-based formulas is growing. However, whether different ruminant milk treatments with varying nutrient compositions influence the infant’s brain development remains unknown. The aim was to determine the effects of consuming bovine, caprine, or ovine milk on brain gene expression in the early postnatal period using a pig model of the human infant. Starting at postnatal day 7 or 8, pigs were exclusively fed bovine, ovine, or caprine milk for 15 days. The mRNA abundance of 77 genes in the prefrontal cortex, hippocampus, and striatum regions was measured at postnatal day 21 or 22 using NanoString. The expression level of two hippocampal and nine striatal genes was most affected by milk treatments, particularly ovine milk. These modulatory genes are involved in glutamate, gamma-aminobutyric acid, serotonin, adrenaline and neurotrophin signaling and the synaptic vesicle cycle. The expression level of genes involved in gamma-aminobutyric acid signaling was associated with pigs’ lactose intake. In contrast, milk treatments did not affect the mRNA abundance of the genes in the prefrontal cortex. This study provides the first evidence of the association of different ruminant milk treatments with brain gene expression related to cognitive function in the first 3 months of postnatal life.

A final report covering aspects of Objective 1 of the WRONZ-MBIE New Materials from New Zealand Wool Partnership.

Additional file 1: Fig. S1. Data collection outline of 40 infants participating in the "Nourish to Flourish" infant complementary feeding pilot feasibility study (2018-2019). Table S1. Development of a tree-based analysis for foods consumed by infants participating in the “Nourish to Flourish” infant complementary feeding pilot feasibility study (2018-2019).

Additional file 2: Fig. S1. PCoA of calf faecal bacterial communities in relation to farm source and sampling date. Fig. S2. Variation in calf milk replacer intakes. Fig. S3. Correlation heatmap between bacterial community composition and calf dietary intakes. Fig. S4. Correlation heatmap between bacterial community composition and SCFA profiles. Fig. S5. Correlation heatmap between bacterial community composition and calf performance data. Fig. S6. Correlation heatmap between SCFA profiles and calf dietary intakes. Fig. S7. Correlation heatmap between SCFA profiles and calf performance data. Table S3. Nutritional composition of milk replacer, pelleted calf starter and ryegrass hay.