The effect of gestational and lactational age on the human milk metabolome

Human milk is the ideal nutrition source for healthy infants during the first six months of life and a detailed characterisation of the composition of milk from mothers that deliver prematurely (<37 weeks gestation), and of how human milk changes during lactation, would benefit our understanding of the nutritional requirements of premature infants. Individual milk samples from mothers delivering prematurely and at term were collected. The human milk metabolome, established by (NMR) spectroscopy, was influenced by gestational and lactation age. Metabolite profiling identified that levels of valine, leucine, betaine, and creatinine were increased in colostrum from term mothers compared with mature milk, while those of glutamate, caprylate, and caprate were increased in mature term milk compared with colostrum. Levels of oligosaccharides, citrate, and creatinine were increased in pre-term colostrum, while those of caprylate, caprate, valine, leucine, glutamate, and pantothenate increased with time postpartum. There were differences between pre-term and full-term milk in the levels of carnitine, caprylate, caprate, pantothenate, urea, lactose, oligosaccharides, citrate, phosphocholine, choline, and formate. These findings suggest that the metabolome of pre-term milk changes within 5-7 weeks postpartum to resemble that of term milk, independent of time of gestation at pre-mature delivery.

Bifidobacterium breve UCC2003 metabolises the human milk oligosaccharides lacto-N-tetraose and lacto-N-neo-tetraose through overlapping, yet distinct pathways

In this study, we demonstrate that the prototype B. breve strain UCC2003 possesses specific metabolic pathways for the utilisation of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), which represent the central moieties of Type I and Type II human milk oligosaccharides (HMOs), respectively. Using a combination of experimental approaches, the enzymatic machinery involved in the metabolism of LNT and LNnT was identified and characterised. Homologs of the key genetic loci involved in the utilisation of these HMO substrates were identified in B. breve, B. bifidum, B. longum subsp. infantis and B. longum subsp. longum using bioinformatic analyses, and were shown to be variably present among other members of the Bifidobacterium genus, with a distinct pattern of conservation among human-associated bifidobacterial species.

Studies on equine milk and comparative studies on equine and bovine milk systems

The composition of equine milk differs considerably from that of the milk of the principal dairying species, i.e., the cow, buffalo, goat and sheep. Because equine milk resembles human milk in many respects and is claimed to have special therapeutic properties, it is becoming increasingly popular in Western Europe, where it is produced on large farms in several countries. Equine milk is considered to be highly digestible, rich in essential nutrients and to possess an optimum whey protein:casein ratio, making it very suitable as a substitute for bovine milk in paediatric dietetics. There is some scientific basis for the special nutritional and health-giving properties of equine milk but this study provides a comprehensive analysis of the composition and physico-chemical properties of equine milk which is required to fully exploit its potential in human nutrition. Quantification and distribution of the nitrogenous components and principal salts of equine milk are reported. The effects of the high concentration of ionic calcium, large casein micelles (~ 260 nm), low protein, lack of a sulphydryl group in equine β-lactoglobulin and a very low level of κ-casein on the physico-chemical properties of equine milk are reported. This thesis provides an insight into the stability of equine casein micelles to heat, ethanol, high pressure, rennet or acid. Differences in rennet- and acid-induced coagulation between equine and bovine milk are attributed not only to the low casein content of equine milk but also to differences in the mechanism by which the respective micelles are stabilized. It has been reported that β-casein plays a role in the stabilization of equine casein micelles and proteomic techniques support this view. In this study, equine κ-casein appeared to be resistant to hydrolysis by calf chymosin but equine β-casein was readily hydrolysed. Resolution of equine milk proteins by urea-PAGE showed the multi-phosphorylated isoforms of equine αs- and β-caseins and capillary zone electrophoresis showed 3 to 7 phosphorylated residues in equine β-casein. In vitro digestion of equine β-casein by pepsin and Corolase PP™ did not produce casomorphins BCM-5 or BCM-7, believed to be harmful to human health. Electron microscopy provided very clear, detailed images of equine casein micelles in their native state and when renneted or acidified. Equine milk formed flocs rather then a gel when renneted or acidified which is supported by dynamic oscillatory analysis. The results presented in this thesis will assist in the development of new products from equine milk for human consumption which will retain some of its unique compositional and health-giving properties.

Vitamin D status and requirements during pregnancy and lactation

Vitamin D deficiency during pregnancy, lactation, and early infancy has been widely reported. Current understanding of vitamin D metabolism during pregnancy and lactation is incomplete, and to date, experimental data to support vitamin D requirements for these life stages are scarce. There is a shortage of nationally representative data and appropriate reference ranges for serum 25-hydroxyvitamin D (25OHD) during pregnancy, lactation and infancy, including in umbilical cord blood. This thesis described concentrations of total 25OHD and individual metabolites including 25OHD3, 25OHD2, and 3-epi-25OHD3 at 15 weeks’ gestation in a large seasonally balanced pregnancy cohort study (n 1768), carried out in Cork, Ireland (52oN). The prevalence of low 25OHD concentrations in pregnant women was higher than published reports in other Caucasian women, and was highest among non-users of vitamin D-containing supplements during winter. A longitudinal pregnancy study was included which suggested gestational stages had an impact on the total serum 25OHD concentration. This thesis incorporated a randomized controlled trial carried out among 100 women across 3 intervention groups using 20 μg/day of vitamin D3 with or without 500 mg calcium, or placebo, over 12-weeks of lactation to investigate the vitamin D requirement for lactating mothers and the vitamin D content of human milk. A daily intake of 25 μg/day was suggested to meet the requirement of lactating women to maintain a 25OHD levels above 50 nmol/L in 97.5% of the population at 52oN all year around. However, vitamin D content in human milk did not increase in response to supplementation. Serum 25OHD concentration has been used as a predictor of a number of health outcomes. This thesis reported large differences in serum 25OHD concentrations using different methods in 86 umbilical cord samples. The need for international standardization of serum 25OHD measurements was re-emphasized in this thesis.

Metabolism of host-derived carbohydrates by Bifidobacterium breve UCC2003

Bifidobacteria are Gram positive, anaerobic, typically Y-shaped bacteria which are naturally found in the digestive tract of certain mammals, birds and insects. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. The prototypical B. breve strain UCC2003 has previously been shown to utilise numerous carbohydrates of plant origin. Various aspects of host-derived carbohydrate metabolism occurring in this bacterium will be described in this thesis. Chapter II describes B. breve UCC2003 utilisation of sialic acid, a nine-carbon monosaccharide, which is found in human milk oligosaccharides (HMOs) and the mucin glycoprotein. B. breve UCC2003 was also shown to cross-feed on sialic acid released from 3’ sialyllactose, a prominent HMO, by the extracellular sialidase activity of Bifidobacterium bifidum PRL2010. Chapter III reports on the transcriptional regulation of sialic acid metabolism in B. breve UCC2003 by a transcriptional repressor encoded by the nanR gene. NanR belongs to the GntR-family of transcriptional regulators and represents the first bifidobacterial member of this family to be characterised. Chapter IV investigates B. breve UCC2003 utilisation of mucin. B. breve UCC2003 was shown to be incapable of degrading mucin; however when grown in co-culture with B. bifidum PRL2010 it exhibits enhanced growth and survival properties. A number of methods were used to investigate and identify the mucin components supporting this enhanced growth/viability phenotype. Chapter V describes the characterisation of two sulfatase-encoding gene clusters from B. breve UCC2003. The transcriptional regulation of both sulfatase-encoding gene clusters was also investigated. The work presented in this thesis represents new information on the metabolism of host-derived carbohydrates in bifidobacteria, thus increasing our understanding of how these gut commensals are able to colonise and persist in the gastrointestinal tract.

Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms

Infant milk formula manufacture: process and compositional interactions in high dry matter wet-mixes

Infant milk formula (IMF) is fortified milk with composition based on the nutrient content in human mother's milk, 0 to 6 months postpartum. Extensive medical and clinical research has led to advances in the nutritional quality of infant formula; however, relatively few studies have focused on interactions between nutrients and the manufacturing process. The objective of this research was to investigate the impact of composition and processing parameters on physical behaviour of high dry matter (DM) IMF systems with a view to designing more sustainable manufacturing processes. The study showed that commercial IMF, with similar compositions, manufactured by different processes, had markedly different physical properties in dehydrated or reconstituted state. Commercial products made with hydrolysed protein were more heat stable compared to products made with intact protein, however, emulsion quality was compromised. Heat-induced denaturation of whey proteins resulted in increased viscosity of wet-mixes, an effect that was dependant on both whey concentration and interactions with lactose and caseins. Expanding on fundamental laboratory studies, a novel high velocity steam injection process was developed whereby high DM (60%) wet-mixes with lower denaturation/viscosity compared to conventional processes could be achieved; powders produced using this process were of similar quality to those manufactured conventionally. Hydrolysed proteins were also shown to be an effective way of reducing viscosity in heat-treated high DM wet-mixes. In particular, using a whey protein concentrate whereby β-Lactoglobulin was selectively hydrolysed, i.e., α-Lactalbumin remained intact, reduced viscosity of wet-mixes during processing while still providing good emulsification. The thesis provides new insights into interactions between nutrients and/or processing which influence physical stability of IMF both in concentrated liquid and powdered form. The outcomes of the work have applications in such areas as; increasing the DM content of spray drier feeds in order to save energy, and, controlling final powder quality.

Microbially derived bioactive peptides to improve human health

This thesis describes a study of various methods to produce bioactive peptides. Initially, the generation of anti-Cronobacter spp. peptides by fermentation of milk protein is described. Lactobacillus johnsonii DPC6026 was used to generate two previously described antimicrobial peptides. Phenotypic analysis indicated unsatisfactory casein hydrolysis. The genome of the strain was sequenced and annotated. Results showed a number of unique features present, most notably a large symmetrical inversion of approximately 750kb in comparison with the human isolate L. johnsonii NCC 533. The data suggest significant genetic diversity and intra-species genomic rearrangements within the L. johnsonii spp.. Cronobacter spp. have emerged as pathogens of concern to the powdered infant formula industry. Chapters 3 and 4 of this thesis describe novel methods to generate two antimicrobial peptides, Caseicin A and B. In Chapter 3 a bank of Bacillus strains was generated and investigated for caseicin production. Following casein hydrolysis by specific B. cereus and B. thuringiensis strains the peptides of interest were generated. Chapter 4 describes a sterile enzymatic method to generate peptides from casein. Bioinformatic tools were used to predict enzymes capable of liberating caseicin peptides from casein. Hydrolysates were generated using suitable enzymes, examined and some were found to produce peptides with activity against Cronobacter spp.. This study establishes a potential industrial-grade method to generate antimicrobial peptides. Administration of GLP-1 leads to improved glycaemic control in diabetes patients. Generation of a recombinant lactic acid bacteria capable of producing a GLP-1 analogue is described in Chapter 5. In-vivo analysis confirmed insulinotropic activity. The results illustrate a method using bacteriocin producing cellular machinery to generate bioactive peptides. This thesis describes the generation of bioactive peptides by bacterial fermentation, tailored enzymatic hydrolysis and recombinant bacterial methods. The techniques described contribute to bioactive peptide research with regards novel methods of production and industrial scale-up.

Evaluation of physicochemical and glycaemic properties of commercial plant-based milk substitutes

The market for plant-based dairy-type products is growing as consumers replace bovine milk in their diet, for medical reasons or as a lifestyle choice. A screening of 17 different commercial plant-based milk substitutes based on different cereals, nuts and legumes was performed, including the evaluation of physicochemical and glycaemic properties. Half of the analysed samples had low or no protein contents (<0.5 %). Only samples based on soya showed considerable high protein contents, matching the value of cow’s milk (3.7 %). An in-vitro method was used to predict the glycaemic index. In general, the glycaemic index values ranged from 47 for bovine milk to 64 (almond-based) and up to 100 for rice-based samples. Most of the plant-based milk substitutes were highly unstable with separation rates up to 54.39 %/h. This study demonstrated that nutritional and physicochemical properties of plant-based milk substitutes are strongly dependent on the plant source, processing and fortification. Most products showed low nutritional qualities. Therefore, consumer awareness is important when plant-based milk substitutes are used as an alternative to cow’s milk in the diet.