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Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (∼6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)-depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and α

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5-90°C for 0.5-30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.

Background Human health depends on a population of microorganisms that inhabit the gut and contribute to homeostasis of the host, including nutrition, immunity and metabolism. Many of the organisms are interactive and mutually dependent, where the end-products for one organism become the fuel for another through substrate and metabolic cross-feeding. To optimise the gut microbiota using diet, the composition and functionality of the gut microbiota, including these interacting networks, must be understood. Microbial composition and functionality is affected by the structure of the energy input, which is primarily dietary fibre for the gut microbiota. The structure of dietary fibre has been reviewed by carbohydrate chemists, but knowledge of how dietary fibre structure affects the gut microbiota is limited. Scope and approach The hierarchical structures of dietary fibre are reviewed, encompassing macrostructure, mesostructure and molecular structure, and how they are affected by food processing and digestion. These factors are considered in relation to their affects on microbial composition and functionality, to provide insight on the interactions between diet, the microbiota, and human health. Key findings and conclusions Food processing and digestion affect food structure, primarily through the removal of some soluble fractions and increased solubilisation of insoluble fractions. The provision of insoluble carbohydrates to the colon appears important for the sustenance of ‘keystone’ species that play a crucial role in stabilising the gut community. Further work is needed at the microbial strain level to understand the impact of increasing fibre solubility. This should be done in studies using well-characterised carbohydrates that consider the impact of food processing and digestion.

This study aimed to examine the changes in lipid and metabolite profiles of ovariectomized (OVX) rats with diet-induced metabolic syndrome-associated osteoarthritis (MetOA) after supplementation with greenshell mussel (GSM) using an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics approach. Ninety-six rats were fed with one of four diets: control, control supplemented with GSM + GSM, high fat/high sugar (HFHS), or high fat/high sugar enriched with GSM (HFHS + GSM). After 8 weeks on experimental diets, half of the rats in each group underwent OVX and the other half were sham operated. After being fed for an additional 28 weeks, blood samples were collected for the metabolomics analysis. Lipid and polar metabolites were extracted from plasma and analysed by LC-MS. We identified 29 lipid species from four lipid subclasses (phosphatidylcholine, lysophosphatidylcholine, diacylglycerol, and triacylglycerol) and a set of eight metabolites involved in amino acid metabolism (serine, threonine, lysine, valine, histidine, pipecolic acid, 3-methylcytidine, and cholic acid) as potential biomarkers for the effect of HFHS diet and GSM supplementation. GSM incorporation more specifically in the control diet generated significant alterations in the levels of several lipids and metabolites. Further studies are required to validate these findings that identify potential biomarkers to follow OA progression and to monitor the impact of GSM supplementation.

Milk is commonly exposed to processing including homogenization and thermal treatment before consumption, and this processing could have an impact on its digestion behavior in the stomach. In this study, we investigated the in vitro gastric digestion behavior of differently processed sheep milks. The samples were raw, pasteurized (75 °C/15 s), homogenized (200/20 bar at 65 °C)–pasteurized, and homogenized–heated (95 °C/5 min) milks. The digestion was performed using a dynamic in vitro gastric digestion system, the human gastric simulator with simulated gastric fluid without gastric lipase. The pH, structure, and composition of the milks in the stomach and the emptied digesta, and the rate of protein hydrolysis were examined. Curds formed from homogenized and heated milk had much looser and more fragmented structures than those formed from unhomogenized milk, this accelerated the curd breakdown, protein digestion and promoted the release of protein, fat, and calcium from the curds into the digesta. Coalescence and flocculation of fat globules were observed during gastric digestion, and most of the fat globules were incorporated into the emptied protein/peptide particles in the homogenized milks. The study provides a better understanding of the gastric emptying and digestion of processed sheep milk under in vitro gastric conditions.

Human breast milk is the optimal source of nutrients for growing infants. However, many circumstances can arise which preclude breast milk feeding, leading to the use of infant formula, including during the weaning period. Many diet-related effects are modulated by the gut microbiome. Therefore, we investigated the effect of human milk (HM) or infant formula (IF) on the gut microbiota in weanling rats. The gut microbiota of weanling male Sprague–Dawley rats fed HM or IF for 28 days was analysed by shotgun metagenome sequencing. Caecal contents were analysed by liquid chromatography–mass spectrometry metabolomics. Numerous genera within the Proteobacteria phylum were relatively more abundant in the ileum, caecum, and colon of rats fed HM, including ileal Escherichia (HM = 9.6% ± 4.3 SEM; IF = 0.9% ± 0.3 SEM; P = 0.03). Other taxa that differed between HM- and IF-fed rats included Prevotella and Ruminococcus. Overall, more differences were observed in the ileum than the caecum and colon between rats fed HM and IF. For the rats fed IF, in the ileum, the relative abundance of Bifidobacterium was higher (HM = 1.7% ± 0.7 SEM; IF = 5.0% ± 1.5 SEM; P = 0.04) with gene functions related to carbohydrate and amino acid metabolism also decreased. In the caecum, metabolic features such as bile acids were elevated while amino sugars were also decreased. Our results show that HM and IF composition differences are reflected in the gut microbiome composition and function in both the small and large intestines.

Red meat restriction in the diet is increasingly recommended, with vegetarian-based diets being promoted. [...]

High protein diets may improve the maintenance of skeletal muscle mass in the elderly, although it remains less clear what broader impact such diets have on whole body metabolic regulation in the elderly. Non-targeted polar metabolomics analysis using HILIC HPLC-MS was used to profile the circulating plasma metabolome of elderly men (n = 31; 74.7 ± 4.0 years) who were randomized to consume for 10 weeks a diet designed to achieve either protein (RDA; 0.8·g−1·kg−1) or that doubled this recommend intake (2RDA; 1.6.g.kg−1). A limited number of plasma metabolites (n = 24) were significantly differentially regulated by the diet. These included markers of protein anabolism, which increased by the 2RDA diet, including; urea, creatine, and glutarylcarnitine. Whilst in response to the RDA diet; glutamine, glutamic acid, and proline were increased, relative to the 2RDA diet (p < 0.05). Metaboanalyst identified six major metabolic pathways to be influenced by the quantity of protein intake, most notably the arginine and proline pathways. Doubling of the recommended protein intake in older males over 10 weeks exerted only a limited impact on circulating metabolites, as determined by LC-MS. This metabolomic response was almost entirely due to increased circulating abundances of metabolites potentially indicative of altered protein anabolism, without evidence of impact on pathways for metabolic health.Trial Registration: This trial was registered on 3rd March 2016 at the Australia New Zealand Clinical Trial Registry (www.anzctr.org.au) at ACTRN 12616000310460.

Milk fat globule membrane (MFGM) is a glycosylated, protein-embedded, phospholipid fraction that surrounds triglycerides in milk. Commercial bovine sources have recently come to the market as a novel food ingredient and have been added to various products, including infant formula. Considering that MFGM is a heterogeneous mixture of fat, protein, and carbohydrate, it can be expected that variations among MFGM products exist. For this reason, our aim was to characterize the composition of commercial MFGM samples through a combination of proteomic and lipidomic analyses. Six bovine milk fractions, represented as MFGM fractions or phospholipid fractions, were obtained from various commercial sources. Additionally, the MFGM samples were compared with 2 infant formulas, a standard formula as well as a premium formula containing MFGM. For proteomic analysis, bottom-up data-dependent liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed on each MFGM fraction, and nearly a thousand proteins were identified across all samples, with 364 of them having different abundance across the samples tested. One hundred twelve proteins differed by a fold-change of 10 or greater, 14 by a fold-change of 50, and 2 by a fold-change of 100 in at least 1 pair, suggesting large differences in the proteins present in these fractions. Even though the classical MFGM proteins were enriched in the MFGM fractions, the relative protein composition varied considerably, and all contain an abundance of milk (casein and whey) proteins. Lipidomic analysis identified a total of 393 lipid species across both positive and negative ionization modes, with the major classes detected being triglycerides, sphingomyelins, and several phospholipids. Across all samples, triglycerides comprised at least 50% of total lipids, with phosphatidylcholine and sphingomyelin being the second and third most abundant lipid classes, respectively. These findings demonstrate the heterogeneous nature of various bovine commercial MFGM fractions. This variation must be considered when evaluating and describing potential functional benefits of these products shown in clinical trials.

OBJECTIVES: Metabolomic profiling of plasma provides a biochemical fingerprint of the circulating metabolites from the host and microbiome, and may assist with understanding mechanisms underlying functional gastrointestinal (GI) disorders such as Irritable Bowel Syndrome (IBS). In a case-control study, we aimed to identify microbial and host factors in plasma to provide mechanistic insights into functional GI disorders and increase the predictability of phenotypes for use in nutrition intervention studies. METHODS: Plasma samples from 246 individuals with functional GI disorders (cases) or asymptomatic (controls), consisting of 93 healthy controls, 54 with functional constipation (C) or IBS-C, 60 with functional diarrhoea (D) or IBS-D, and 39 diagnosed as IBS-mixed or awaiting diagnosis. Plasma was subjected to biphasic extraction and global metabolite profiling was performed using three separate untargeted liquid chromatography high resolution mass spectrometry analysis (LC-MS) methods (polar, semi-polar and non-polar chromatography). RESULTS: Plasma metabolomic profiles differed considerably between the IBS phenotypes and those of the control subjects. Multivariate partial least squares discriminant analysis of 428 annotated lipid species highlighted significant differences (P

OBJECTIVES: Nutrition modulates the interactions between the host and the gut microbiota. Changes in metabolites resulting from this interaction in a healthy compared to a perturbed gut is a critical approach to identify putative biomarkers that can be used to assess how diet effects gut function. Bile acids (BAs) are metabolites produced from cholesterol and the microbial-conversion of BAs play an important role in digestion and metabolism. Modification of BA metabolism has been reported in participants with Irritable Bowel Syndrome (IBS), which is characterised by abdominal bloating, pain, dysfunction, and a perturbed gut microbiota. One aim of the clinical nutrition study was to characterise BA profiles of healthy control and IBS participants. This work is part of a wider study investigating gut health and how food can be used to alleviate symptoms. METHODS: Healthy control and IBS participants were recruited and IBS subtypes determined (n = 337) (Rome Criteria IV). Biological samples and dietary questionnaire information were collected. 97 plasma samples (n = 42 healthy control, n = 25 IBS-diarrhoea, n = 17 IBS-constipation, n = 13 IBS-mixed) were quantitatively analysed for 13 BAs using a SCIEX 6500 + QTrap liquid chromatography-mass spectrometer in multiple reaction monitoring mode. Partial least squares discriminant analysis (SIMCA 14.1) and analysis of variance (Metaboanalyst 4.0) were used to perform multivariate and univariate analyses respectively. Ethical approval was obtained from the University of Otago Human Ethics Committee (Health) (Reference H16/094). RESULTS: Univariate analysis of the 13 BAs showed no significant difference within or between IBS subtypes or healthy controls (P > 0.05). However, multivariate analysis of 13 BAs showed separation of the data between IBS subtypes and healthy controls indicating that while individual BA concentrations did not vary significantly, differences in profiles existed in IBS participants. CONCLUSIONS: Data from a subset of an IBS cohort shows BA metabolism is likely perturbed in IBS. Further analysis of faecal samples will provide additional information that could confirm the relationships to IBS subtypes, and provide putative biomarkers for food interventions targeting improvement of gut comfort and function. FUNDING SOURCES: High-Value Nutrition National Science Challenge, New Zealand.

OBJECTIVES: The links between food, gastrointestinal (GI) function and comfort, and mental well-being are at the forefront of nutritional research. Irritable Bowel Syndrome (IBS) is a functional GI disorder and the combination of detailed patient reported outcomes (PROs) combined with omic data could better define these disorders. METHODS: The Christchurch COMFORT cohort is a case-control study: 337 participants with functional GI disorders (functional constipation FC, functional diarrhoea FD, IBS constipation IBS-C, IBS diarrhoea IBS-D) and asymptomatic controls. Demographics, symptom scores, psychological scores and dietary intake were recorded using the ‘Modified Hunter New England’ (Rome IV diagnostic criteria questionnaires; Medical History Questions; Hospital Anxiety and Depression Scale, HADS). Demographics included Structured Assessment of Gastrointestinal Symptoms (SAGIS); and Diet Diary and Live Symptoms Score (FAST) along with PRO Measurement Information System (PROMIS). Biological samples collected for an untargeted LCMS analysis of plasma and shot-gun metagenome analysis of faecal DNA. Ethical approval was obtained from the University of Otago Human Ethics Committee (H16/094). RESULTS: Symptom questionnaires were able to cluster 287 subjects into: IBS (42%), functional disorders (18%) and healthy controls (40%). Within IBS, 46% were IBS-D, 23% were IBS-C and 31% were IBS-M. Severity scores were higher for all IBS cases with PROMIS-GI scales. A higher score of health worry was reported in IBS-C than other IBS subtypes. HADS anxiety and depression scales were higher in IBS cases (vs. healthy controls). The FAST diary symptoms correlated with PROMIS GI scales (exception for constipation). Metabolomic analyses detected differential plasma metabolites and pathways (bile acids, lipids, specific amino acids) affected between IBS-C + FC vs IBS-D + FD, healthy vs IBS-D and healthy vs IBS-C. Metagenomics suggests that carbohydrate, methane, and sulfur metabolism may be important in IBS. CONCLUSIONS: These data will allow us to apply a systems biology approach to identify key pathways and correlate them with the questionnaire data to better understand functional GI disorders. FUNDING SOURCES: Funded by the NZ National Science Challenge High-Value Nutrition programme.

Complementary feeding transitions infants from a milk‐based diet to solid foods, providing essential nutrients to the infant and the developing gut microbiome while influencing immune development. Some of the earliest microbial colonisers readily ferment select oligosaccharides, influencing the ongoing establishment of the microbiome. Non‐digestible oligosaccharides in prebiotic‐supplemented formula and human milk oligosaccharides promote commensal immune‐modulating bacteria such as Bifidobacterium, which decrease in abundance during weaning. Incorporating complex, bifidogenic, non‐digestible carbohydrates during the transition to solid foods may present an opportunity to feed commensal bacteria and promote balanced concentrations of beneficial short chain fatty acid concentrations and vitamins that support gut barrier maturation and immunity throughout the complementary feeding window.

Seasonal variation of milk composition poses a challenge for maintaining dairy product quality. Multivariate statistical techniques were used here to identify seasonal patterns in mineral levels, fatty acid profiles and selected metabolites for New Zealand raw milk collected from 24 individual cows in early-season (November), mid-season (January/February) and late-season (March). Partial least-squares discriminant analysis (PLS-DA) showed clear seasonal differences in mineral and fatty acid profiles that may have been related to supplementary feeding, dilution effects (higher total solids in late-season milk) and the lactation cycle. Fatty acid profiles showed an inverse relationship between the C6–C12 fatty acids and the branched- or long-chain fatty acids, which was thought to be related to feeding of maize in mid- and late-season. Targeted metabolite analysis suggested elevated phosphatase activity in March, and this warrants further investigation as an indicator of metabolic changes related to declining milk quality at the end of the milking season.

Meat colour is one of the cues available to the consumer to gauge overall meat quality and wholesomeness. Colour stability of meat is determined by several factors both inherent to the animal and post-slaughter conditions, including ageing, storage/packaging and display times. A hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) based metabolomics study was undertaken to identify and compare polar metabolites between ovine meat samples that were exposed to different durations of ageing, storage conditions, and display times. Primary metabolites comprising amino acids, sugars, nucleotides, nucleosides, organic acids and their breakdown products were mainly identified as discriminating factors. For the first time, boron complexes of sugar and malic acid were also tentatively identified. As expected, most compounds identified were related to myoglobin chemistry, and compounds with antioxidant properties were found in higher levels in colour stable samples. Supplementary studies identifying semi-polar, non-polar and volatile compounds will provide a holistic understanding of the chemical basis of colour stability in ovine meat.

OBJECTIVES: The rapid diversification of foods introduced during complementary feeding coincides with profound changes in the infant gut microbiome, with potential effects on immediate and long-term health outcomes. The objective of this study is to elucidate dietary modulators of the infant gut microbiome by characterising dietary intake by food groups, nutrient composition, and nutrient source, followed by multivariate integration with faecal microbiome data. METHODS: Three-day food records were collected from a cohort of 40 infants recruited from the Auckland metropolitan area in New Zealand, who participated in a 6-month dietary intervention clinical pilot study. Thirty infants were provided kumara as an additional food to include in the daily diet, while 10 infants were provided commercially available probiotic drops (Bifidobacterium lactis) as a daily supplement. Faecal samples were collected on the final day of the 3-day food record at approximately 6, 9, and 12 months of age, and analysed using shotgun metagenomics and global metabolite profiling. Multivariate approaches have been used to integrate diet, microbiome, and metabolomic data. RESULTS: Preliminary results indicated that at 9 months of age some B-vitamins and minerals from meat products were strongly correlated (coefficient of ≥.6 using canonical correlations) with increased microbiome amino acid metabolism and biosynthesis, whereas a strong inverse correlation (≤–.6) was observed between breastmilk and dietary fibre consumption. At 12 months of age, the significance of associations throughout the network were diminished, and food intake data suggested that legumes, eggs, dairy products, breastmilk, and infant formula were the predominant modulators of metabolic pathways. CONCLUSIONS: While specific nutrients and food groups correlate independently with significant alterations in the microbiome, merging these metrics to encompass nutrient source provides a more complete understanding of dietary modulators from the perspective of the gut microbiome. The immature gut microbiome of younger infants may be more profoundly influenced by dietary factors than increasingly mature microbiomes. FUNDING SOURCES: This study was funded by the New Zealand Ministry of Business, Innovation, & Employment High-Value Nutrition National Science Challenge.

OBJECTIVES: Dietary fibre supplementation is recognised as important for functional gastrointestinal disorders (FGID). The exact role of the microbiome in this relationship remains unclear. We explored differences in dietary fibre intake, GI symptoms and the fecal microbiome in those with FGID. METHODS: The COMFORT cohort is an observational case control study examining FGID, particularly irritable bowel syndrome (IBS) aetiology (Ethics 16/NTA/21). Participants prospectively completed a food and GI symptoms diary over 3 days. Severity of GI symptoms and mood disorders were assessed using clinical questionnaires; SAGIS and PROMIS for GI symptoms and HADS for anxiety and depression. Fecal samples were analysed by shotgun sequencing; 95 healthy controls (HC), 22 constipation IBS (IBS-C) and 50 diarrhea IBS (IBS-D). Taxonomic classifications were assigned by aligning sequences against the NCBI non-redundant database using DIAMOND. RESULTS: Diet diaries were completed by 292 participants (176 cases, 71.2% female). Average daily fibre intake was higher in HC compared to FGID (23.99g, 95% CI = –2.06-0.55; 20.28g, 95% CI = -1.96-0.45; P |0.5|). At the genus level, Blautia were correlated with diarrhea and faecal incontinence (canonical r > 0.5). CONCLUSIONS: Higher fibre consumption was associated with decreased bloating, anxiety and depression in different FGID subgroups. IBS was also associated with altered fecal microbiome composition including some taxa linked to carbohydrate and hydrogen metabolism. FUNDING SOURCES: Funded by the Ministry of Business, Innovation and Employment through the High-Value Nutrition National Science Challenge.

OBJECTIVES: Vitamin deficiencies are prevalent in the elderly. Common diseases of aging are characterized by alterations of one-carbon (1C) metabolites, namely homocysteine. Despite the importance of diet-derived nutrients and interconversion to active coenzymes for regulation of 1C metabolism, little is known about age-related alterations in 1C metabolism in response to meals. This study aimed to identify whether postprandial 1C metabolite regulation is impaired in older adults, and determine the influence of meal type. We hypothesized that older adults would have altered 1C metabolite responses, and that these would differ between energy dense and nutrient dense meals. METHODS: In a double-blinded randomized crossover design, healthy younger (n = 15) and older (n = 15) adults consumed mixed-meal breakfasts on separate occasions: an energy-dense (ED) meal (sausage and egg sandwich) and a nutrient-dense (ND) meal (oats, toast, cottage cheese and fruit). Plasma collected at fasting and hourly for 5 h was analysed for 12 1C metabolites using high-performance liquid chromatography with tandem mass spectrometry. RESULTS: Postprandial concentrations of five 1C metabolites differed between older and younger subjects, dependent on meal type. Unlike younger subjects, older subjects had similar meal responses for betaine, choline, taurine, and glycine concentrations (meal × age interactions P 0.05 each, respectively). Younger subjects had higher homocysteine concentrations than older after the ED meal (meal × age interaction P

OBJECTIVES: The nutritional composition of human milk is affected by many factors, including stage of lactation and time of day. Metabolomic profiling of milk provides a biochemical fingerprint of hundreds of metabolites being consumed by the infant, which may help to understand potential factors affecting infant health, growth and nutritional status. We hypothesized that frequency of daily lactations would alter the profile of metabolites and lipids in the milk. METHODS: Human milk samples from 630 individuals [378 mothers exclusively breast feeding and 252 mothers partially breast feeding (i.e., infant supplemented with formula feeding)] from a single sample (infant aged 3 months) were subjected to biphasic extraction and metabolite profiling performed using two separate untargeted liquid chromatography high resolution mass spectrometry analysis methods (polar metabolites and the lipidome). RESULTS: Univariate statistical analysis of the lipidome data matrix revealed considerable differences in lipid concentrations between partial or exclusive feeding. Of the 241 lipids measured in the non-polar extracts, 203 were significantly different between the two groups after multiple testing correction (FDR corrected p-value

Fortified milk drinks are predominantly manufactured from bovine (cow) sources. Alternative formulations include those prepared with hydrolysed bovine milk proteins or from alternate bovidae species, such as caprine (goat) milk. Currently, there is little data on protein digestive and metabolic responses following ingestion of fortified milk drinks. To examine the digestive and metabolic responses to commercially-available fortified milks, young adults (n = 15 males: 15 females), in a randomised sequence, ingested isonitrogenous quantities of whole cow-protein (WC), whole goat-protein (WG), or partially-hydrolysed whey cow-protein (HC), commercial fortified milks. Plasma amino acid (AA) and hormonal responses were measured at baseline and again at 5 h after ingestion. Paracetamol recovery, breath hydrogen, and subjective digestive responses were also measured. Postprandial plasma AA was similar between WC and WG, while AA appearance was suppressed with HC. Following HC, there was a negative incremental AUC in plasma branched-chain AAs. Further, HC had delayed gastric emptying, increased transit time, and led to exaggerated insulin and GLP-1 responses, in comparison to whole protein formulas. Overall, WC and WG had similar protein and digestive responses with no differences in digestive comfort. Contrastingly, HC led to delayed gastric emptying, attenuated AA appearance, and a heightened circulating insulin response.

Wagyu beef products are marketed as luxury goods to discerning consumers and the lipid content and composition are important drivers of wagyu product value. Wagyu beef is an extensively marbled meat product, well characterised for its tenderness and flavour. In New Zealand, pasture-fed Wagyu-dairy beef production is increasing to meet demand for ultra-premium meat products. Important for these characteristics is the composition of lipid species and their distribution across the carcass. The aim of this study was to analyse the distribution of fatty acids and phospholipids in 26 table cuts, nine co-products and three fat deposits of carcasses from New Zealand pasture-fed Wagyu-dairy cross beef carcasses (n = 5). Phospholipid and fatty acid levels varied across different cuts of the carcass, but typically cuts with high levels of phospholipids also had high levels of omega-3 fatty acids and low levels of saturated fatty acids. This work will be used in the future to examine the potential health aspects of pasture-fed Wagyu beef.

Tea is the second most consumed beverage in the world after water and there are numerous reported health benefits as a result of consuming tea, such as reducing the risk of cardiovascular disease and many types of cancer. Thus, there is much interest in the chemical composition of teas, for example; defining components responsible for contributing to reported health benefits; defining quality characteristics such as product flavor; and monitoring for pesticide residues to comply with food safety import/export requirements. Covered in this review are some of the latest developments in mass spectrometry-based analytical techniques for measuring and characterizing low molecular weight components of tea, in particular primary and secondary metabolites. The methodology; more specifically the chromatography and detection mechanisms used in both targeted and non-targeted studies, and their main advantages and disadvantages are discussed. Finally, we comment on the latest techniques that are likely to have significant benefit to analysts in the future, not merely in the area of tea research, but in the analytical chemistry of low molecular weight compounds in general.

OBJECTIVES: Irritable Bowel Syndrome (IBS) is a functional gastrointestinal (GI) disorder featuring chronic or recurrent abdominal discomfort, usually with changes in GI habit. To improve our understanding of links between the microbiome and IBS, and how these links can be manipulated through diet, we undertook shotgun metagenomic sequencing of fecal samples from a case-control study. METHODS: Fecal samples from 172 individuals were analyzed by shotgun sequencing using the Illumina NextSeq platform. Of these, 77 were classified as controls, 16 were constipation-predominant IBS (IBS-C), 39 were diarrhea-predominant IBS (IBS-D), 29 were diagnosed with functional constipation (FC), and 11 had functional diarrhea (FD). Taxonomic classifications were determined using Metaxa2 and the SILVA 128 database. Gene functions were assigned by alignment of sequences against a protein reference database using DIAMOND. Mean relative abundance of bacterial taxa and functional genes were compared using permutation ANOVA. Ethical approval was obtained from the University of Otago Human Ethics Committee (Health) (Reference H16/094). RESULTS: Bacterial genera that discriminated case-controls (P

OBJECTIVES: Plasma levels of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) phenylalanine (phe) and tyrosine (tyr) have been associated with obesity, insulin resistance and risk of type 2 diabetes. This study aimed to investigate the response of circulating plasma and tissue levels of BCAA and AAA to weight loss, and to correlate the level of these metabolites in plasma and tissue in obese women. METHODS: 28 obese (mean BMI 46.2 kg/m(2)) women underwent low energy diet (LED)-induced weight loss (−9.2 ± 4.2 kg) followed by bariatric surgery-induced weight loss (−23.6 ± 2.5 kg). Plasma at baseline (t0), post-LED/pre-surgery (t1) and 6-month post-surgery (t2) as well as biopsies of subcutaneous abdomen adipose tissue (SAfat), superficial thigh adipose tissue (Tfat) and vastus lateralis thigh muscle (Tmuscle) at both t1 and t2 were collected, and profiled using mass spectrometry-based metabolomics approach. Paired t-tests were applied to assess between-timepoint differences, and Pearson correlation used to calculate correlation coefficient of metabolite levels between plasma and tissue. RESULTS: Plasma BCAA and AAA were all significantly reduced post-LED at t1 (fold-change of 0.76–0.85 for val, leu, ile, tyr and phe, P

Tea is an infusion made from the dried leaves of Camellia sinensis L. and is the second most consumed beverage in the world. It has been shown that factors such as fermentation methods, cultivar, geographical origin and season can affect the biochemical composition of tea. In this study, the biochemical composition of green, oolong and black commercial tea samples from around the world was studied using a non-targeted method utilising reversed phase ultra high pressure liquid chromatography (UHPLC) and high resolution mass spectrometry. Principal component analysis of green, oolong and black tea extracts clearly showed that fermented tea can be resolved from non-fermented tea. When the non-targeted data were combined with the supervised multivariate technique, partial least squares discriminant analysis, the method was able to clearly distinguish ‘country of origin’ within green tea and to a lesser extent within a black tea sample set, plus provide indicative marker ions for the country of origin. Many of the significant components detected in this study are unknowns, emphasising the importance of un-biased non-targeted analytical techniques. This study highlights the potential efficacy of non-targeted UHPLC–mass spectrometry when combined with multivariate statistics to differentiate fermented from non-fermented tea and provide potential indicators of provenance of tea samples for further examination.

Digestion resistant carbohydrates (DRC) are complex carbohydrates that resist digestion and absorption in the small bowel. Diets high in DRC can have wide ranging impacts on the health of the host, which include changes to immunity and allergy, incidence of cardiovascular disease, and obesity. The aim of this study was to characterise the effects of DRC (inulin, konjac or resistant starch) on large intestinal short-chain fatty acid (SCFA) concentrations and serum metabolite and lipid profiles. A rat model was used to compare the effects of feeding a basal diet or the basal diet containing 5 % inulin, konjac or resistant starch for 14 days. Of the three DRC, inulin had the greatest effect; ten serum phospholipids differed significantly in abundance between inulin-treated and control rats. In particular phosphatidylcholines and lysophosphatidylcholines containing fatty acyl chains 22:5, 22:4, 20:4, 18:0 and 16:0 were increased in the inulin-fed group, whereas phosphocholines containing fatty acyls 20:5 and 22:6 were decreased. These results indicated an impact on both n-3 and n-6 fatty acid metabolism as a result of inulin dietary intake. Increased intestinal concentrations of SCFA were detected in rats fed DRC, but only inulin caused appreciable changes to serum lipid profiles.

Tea is the second most consumed beverage in the world and its consumption has been associated with numerous potential health benefits. Factors such as fermentation methods, geographical origin and season can affect the primary and secondary metabolite composition of tea. In this study, a hydrophilic interaction liquid chromatography (HILIC) method coupled to high resolution mass spectrometry in both positive and negative ionisation modes was developed and optimised. The method when combined with principal component analysis to analyse three different types of tea, successfully distinguished samples into different categories, and provided evidence of the metabolites which differed between them. The accurate mass and high resolution attributes of the mass spectrometric data were utilised and relative quantification data were extracted post-data acquisition on 18 amino acids, showing significant differences in amino acid concentrations between tea types and countries. This study highlights the potential of HILIC chromatography combined with non-targeted mass spectrometric methods to provide a comprehensive understanding of polar metabolites in plant extracts.

Polyphenolics are an important class of biologically active compounds found in plants and are purported to have benefits for human health. To assess their significance in foods, improved methods are required for their detection and classification. In this paper, we describe a targeted MS2 approach using an ion-trap mass spectrometer for the analysis and classification of proanthocyanidins (PAs) in crude plant-derived extracts, which has been applied to compare green, oolong and black tea crude extracts and a grape seed extract. The method could separate, detect and provide qualitative information (full scan MS2 spectra) to enable chromatographic and mass spectral confirmation of PAs in the crude tea extracts. Using the developed methodology we report here for the first time the detection of PA tetramers in black tea, and PA trimers and tetramers in oolong tea.

Condensed tannin in the form of a grape seed extract (GSE) was dosed to weaned wether lambs fed white clover (WC) or perennial ryegrass (PRG) over a 9-week period to determine whether the ‘pastoral’ flavour and odour of meat could be altered. The concentrations of the pastoral flavour compounds indole and skatole were determined in the rumen fluid, blood plasma and intermuscular fat. The odour and flavour of fat and meat from the slaughtered lambs was assessed by a trained panel. The rumen fluid and blood plasma concentrations of indole and skatole were higher in those lambs fed WC compared to PRG (P < 0.05) and the overall meat flavour intensity was greater when feeding WC (P < 0.01). The observed concentration of indole and skatole in the fat between WC and PRG feeding treatments was not statistically different. Power analysis indicated that increasing the number of lambs per treatment group from 20 to 65 would result in a higher fat skatole concentration (P < 0.05) being detected in lambs fed WC compared to PRG. Dosing with GSE gave a small reduction in skatole concentration in the rumen fluid and reduced plasma concentration of indole and skatole (P < 0.001). Odour and flavour scores of the fat and meat samples were not particularly high however, dosing with GSE lowered the overall and sweet odour and the sheepy, camphor, faecal and barnyard flavour (P < 0.05). Although the plasma concentration of indole and skatole suggests that GSE reduced indole and skatole formation, the intermittent supply of the GSE to the rumen environment was not sufficient to reduce their concentration in the fat. Hence, the small difference in the scores for pastoral odour and flavour attributes associated with GSE treatment may arise from other unknown factors. From a primary investigation, there was no difference in the concentration of indole and skatole in fat samples collected from carcasses before and after chilling. Further investigations into meat pastoral flavour are warranted through feeding condensed tannin-containing forages. Copyright © 2007 Society of Chemical Industry

A comprehensive set of quality parameters has been recorded for meat and fat from groups of castrate and ram lambs raised on pasture and slaughtered in groups of six at 91‐day intervals from 122 to 668 days of age. These include mechanical evaluation of meat tenderness, sensory evaluations of meat flavour and fat odour, and measurements of fat colour, fatty acid composition, vitamins C and E, free glucose and iron in the meat, and of the concentrations of the flavour compounds indoles, phenols, and branched chain fatty acids in the fat. There was significant variation in many parameters between slaughter days, including markedly lower meat tenderness in one slaughter group, probably in response to unfavourable pasture conditions. Older ram lambs tended to be less tender than castrates of the same age. However, no longer‐term age‐related trends were observed in tenderness or flavour quality. Concentrations of free glucose in rigor muscle and vitamin C showed a declining trend with age. Concentrati...

A comparative study of cows grazing grasses differing in soluble sugar content was undertaken to explore whether sugar content improves ruminal plant protein utilisation and influences milk flavour chemistry. Lower concentrations of ammonia, and amino-acid degradation products including the flavour compounds skatole and indole were observed for rumen contents sampled from non-lactating fistulated cows grazing high sugar ryegrass relative to samples from cows grazing Italian or a standard perennial ryegrass, suggesting improved plant protein nitrogen utilisation in response to the high sugar grass diet. Concentrations of plant protein fermentation products in the rumen of the fistulated cows were significantly higher at noon, compared to morning and afternoon values. In milk collected twice daily from the lactating cows grazing the above ryegrass varieties, no differences in yields of milkfat or skatole were detected between cows fed the different grass varieties. However yields of skatole in milkfat were two times higher in afternoon than morning milk. The rumen response to morning feeding appears to influence the flavour chemistry of afternoon milk. Keywords: rumen metabolites, milkfat skatole, indole, perennial ryegrass, Italian ryegrass, water soluble carbohydrate

The proanthocyanidin polymer fractions of the leaves of the forage legume Dorycnium rectum were analysed by acid catalysis with benzyl mercaptan, NMR and ES-MS. The results showed that D. rectum differs from other temperate proanthocyanidin-containing forage legumes in that the range of polymers extends up to very high degrees of polymerisation. Three fractions were characterised as low, medium, and high molecular weight proanthocyanidin fractions with mean degree of polymerisations of 10.3, 41 and 127, respectively. Epigallocatechin was the most abundant extension unit and the terminating flavan-3-ols comprised largely catechin and gallocatechin units in equal proportions. Formation of thiolyated dimer products showed the interflavan-linkages of the lower molecular weight proanthocyanidins to be predominantly C4-->C8 with a small amount of C4-->C6. ES-MS spectra distinguished lower from higher polymeric proanthocyanidins from M2- to M8(2)-. The antibacterial activity of proanthocyanidin fractions against pure cultures of microbes selected from the ruminal population to represent fibre degrading, proteolytic and hyper ammonia producing bacteria in broth culture was evaluated. The activity of proanthocyanidin fractions against Clostridium aminophilum, Butyrivibrio fibrisolvens and Clostridium proteoclasticum was significantly dependent on their structure but not so against Ruminococcus albus and Peptostreptococcus anaerobius. The latter observation was unique in that they were sensitive to all proanthocyanidin fractions evaluated, even at the lowest concentration (100 microg/ml). The results suggest the effects of the extractable proanthocyanidins on rumen microbes should be considered when evaluating an alternative proanthocyanidin-containing forage source for ruminants, such as D. rectum.

Oolong tea is a semi-fermented tea that is partially oxidised during the manufacturing process to create a product unique in composition. In this study, we investigated the potential of non-targeted LC-MS with two complementary chromatographic modes to provide a "comprehensive and unbiased" view of biochemical compositional changes occurring during oolong tea manufacturing in New Zealand. Tea leaf samples from throughout the manufacturing/fermentation process during three different harvest periods (spring, summer and autumn) were analysed by four different LC-MS streams. Principal component analysis revealed the de-greening stage of the manufacturing process was responsible for major changes in the biochemical profile, with the methodology detecting changes in a wide range of metabolites of differing polarities, such as flavonoids, nucleosides and primeverosides. Changes during the fermentation phase of the manufacturing process were less marked, however significant increases in levels of free amino acids, a hydroxyjasmonic acid and related metabolites were observed.

Factors such as fermentation methods, geographical origin and season can affect the biochemical composition of tea leaves ( Camellia sinensis L.). In this study, the biochemical composition of oolong tea during the manufacturing and fermentation process was studied using a non-targeted method utilising ambient ionisation with a direct analysis in real time (DART) ion source and mass spectrometry (MS). Caffeine dominated the positive ionisation spectra throughout the manufacturing process, while the negative ion spectra collected during manufacturing were rich in ions likely to be surface lipids. Correlation analyses on the spectra revealed two volatile compounds tentatively identified as indole and geranic acid, along with ammonium and caffeine clusters/adducts with geranic acid that increased in concentration during the fermentation stages of the process. The tentative identifications were assigned using a combination of DART-ion-trap MS n and DART-accurate mass MS 1 and MS 2 on tea samples and standard compounds. This study highlights the potential of DART-MS to rapidly monitor the progress of complex manufacturing processes such as tea fermentation.

Lolium perenne cultivars differing in their capacity to accumulate water soluble carbohydrates (WSCs) were infected with three strains of fungal Neotyphodium lolii endophytes or left uninfected. The endophyte strains differed in their alkaloid profiles. Plants were grown at two different levels of nitrogen (N) supply in a controlled environment. Metabolic profiles of blades were analyzed using a variety of analytical methods. A total of 66 response variables were subjected to a principle components analysis and factor rotation. The first three rotated factors (46% of the total variance) were subsequently analyzed by analysis of variance. At high N supply nitrogenous compounds, organic acids and lipids were increased; WSCs, chlorogenic acid (CGA), and fibers were decreased. The high-sugar cultivar ‘AberDove’ had reduced levels of nitrate, most minor amino acids, sulfur, and fibers compared to the control cultivar ‘Fennema’, whereas WSCs, CGA, and methionine were increased. In plants infected with endophytes, nitrate, several amino acids, and, magnesium were decreased; WSCs, lipids, some organic acids, and CGA were increased. Regrowth of blades was stimulated at high N, and there was a significant endophyte × cultivar interaction on regrowth. Mannitol, a fungal specific sugar alcohol, was significantly correlated with fungal biomass. Our findings suggest that effects of endophytes on metabolic profiles of L. perenne can be considerable, depending on host plant characteristics and nutrient supply, and we propose that a shift in carbon/N ratios and in secondary metabolite production as seen in our study is likely to have impacts on herbivore responses.

An acetone:water (7:3) extract obtained from the leaves of Rumex obtusifolius was fractionated into procyanidin oligomer and polymer fractions using a linear gradient and a simple step method on Sephadex LH-20. The chemical characteristics of the procyanidin fractions were studied by 13C-NMR spectroscopy, acid-catalysed degradation in the presence of benzyl mercaptan, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS and electrospray ionisation (ESI) MS. The 13C-NMR showed that the polymer fraction consisted predominantly of procyanidin polymers, some with galloyl groups attached. The thiolysis reaction products indicated a mean degree of polymerisation (DP) of 4.3 for the step method, and a range of 2.3–8.2 mean DP for the gradient fractionation, with epicatechin as the most abundant flavan-3-ol extension unit, while the terminal units consisted of equal proportions of catechin, epicatechin and epicatechin gallate. Singly charged ions observed in MALDI-TOF/MS showed a range of oligomeric procyanidins and their polygalloyl derivatives. These species (in the range DP 2–7) were also observed by ESI/MS but the spectra were more complex due to overlapping multiply charged ions. Isolation of oligomers from the Sephadex LH-20 fraction by chromatography on polyamide and C18 yielded B1, B2, B3 and B7 dimers, an A-type trimer and a B2 3,3′-O-digallate. Copyright © 2007 John Wiley & Sons, Ltd.

Variations in diet, age and castration were employed to generate a range of flavours that were chemically analysed to find the cause of ‘pastoral’ flavour in sheepmeat and its relationship to species flavour. Lambs were raised on pasture (ram or castrate) or on a maize-based or lucerne-based concentrate diet (ram only). They were slaughtered at 132 and 232 days. Fat from animals raised on concentrates had lower proportions of fat-hardening stearic acid and higher proportions of oxidation-prone fat-softening oleic and linoleic acids. Concentrations of species-characterising short branched-chain fatty acids (BCFAs), typified by 4-methyloctanoic acid, were lower for pasture-fed lambs, particularly at 232 days, although between-animal variation was high. Castration did not statistically affect BCFA concentration at this age. Correlations between BCFAs and testes weight were not significant, suggesting that they were not acting as sex pheromones. Concentrations of 3-methylindole (skatole) in perirenal fat were higher for the pasture diet at both slaughter dates. Concentrations of 4-methylphenol in the fat were not affected by diet. However, 3-methylphenol was more abundant in pasture treatments. A sensory panel found that the intensity of ‘sheepmeat’ flavour was higher for pasture-raised animals, but that associations of ‘barnyard’ flavour (which has been linked to pastoral flavour) with diet were more complex. The issue was resolved by fat sniffing. Panel responses to heated subcutaneous fat were recorded as frequency of descriptive terms drawn from a limited lexicon. Volatiles from fats pooled by treatment were resolved on a gas chromatographic column whose effluent was monitored by odourport sniffing. Compounds were identified by parallel chromatographic/mass spectrometric runs. The headspace concentrations of these compounds were then measured for individual animals. These data were related to frequency data by the principal component method. ‘Mutton’ and ‘sheepmeat’ odour notes were clearly linked to indoles (skatole particularly) and, to a lesser extent, methylphenol, setting these notes apart from ‘lamb’, an odour note more associated with lucerne and maize diets through higher concentrations of BCFAs. It was concluded that 3-methylindole was the major cause of pastoral flavour in sheepmeat, and that fat oxidation products represented a background flavour that varied quantitatively but not qualitatively with fatty acid profile. © 2002 Society of Chemical Industry

Skatole and indole are flavour compounds formed in the rumen, which are key factors in the pastoral flavour of meat and milk products. A selection of indolic compounds has been screened for their potential to form skatole and indole on in vitro fermentation, with rumen inocula collected from New Zealand pasture-fed sheep. Typically, 8% (P < 0.005) of added tryptophan was converted to indole and 54% (P < 0.001) was converted to skatole via indole acetic acid (IAA). The addition to rumen inocula of a condensed tannin (CT) fraction isolated from the forage legume, Dorycnium rectum, reduced the conversion of plant protein to skatole and indole by 75% and specifically inhibited the transformation of IAA to skatole by 85% (P < 0.001). The conversion of tryptophan to indole or IAA was not affected by CTs. The inhibitory effect of CTs on the conversion of tryptophan to skatole was prevented by the addition of polyethylene glycol (PEG) to the rumen inocula prior to the addition of CTs, but not by the addition of PEG subsequent to CTs, demonstrating that interactions between CTs and microbes involved in skatole biosynthesis are not reversible by PEG. This study is the first to demonstrate that isolated CTs have an inhibitory effect on the conversion of protein to skatole and indole by rumen microbes, and that CTs specifically inhibit the transformation of IAA to skatole by rumen microbes.