Heat Treatment and Homogenization Influence the Gastric Digestion of Bovine Milk Protein in Growing Pigs as an Adult Human Model.

BACKGROUND: Bovine milk processing influences the structure of the curd formed during gastric digestion, which may alter gastric protein hydrolysis and impact amino acid (AA) release into the small intestine. OBJECTIVES: This study aimed to determine the influence of heat treatment and homogenization on the gastric protein digestion and AA emptying of bovine milk. METHODS: Nine-wk-old pigs (n = 144) consumed either raw, pasteurized nonhomogenized (PNH), pasteurized homogenized (PH), or ultra-high-temperature homogenized (UHT) bovine milk for 10 d. On day 11, fasted pigs received the milk treatment (500 mL) before gastric contents were collected at 0, 20, 60, 120, 180, and 300 min postprandially. The apparent degree of gastric protein hydrolysis (based on the release of free amino groups), apparent gastric disappearance of individual proteins [based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel band intensity], and the gastric emptying of digested protein and AA were determined. RESULTS: During the first 60 min, the rate of apparent gastric protein hydrolysis was fastest in pigs fed UHT milk (0.29%/min compared with on average 0.07%/min in pigs fed raw, PNH, and PH milk). Differences in the apparent degree of gastric protein hydrolysis and emptying were reflected in the rate of digested protein entering the small intestine. The AA gastric emptying half-time was generally shorter in pigs fed PH and UHT milk than in pigs fed raw and PNH milk. For example, the gastric release of total essential AA was >2-fold faster (P < 0.01) in pigs fed PH or UHT milk than that in pigs fed raw or PNH milk (i.e., homogenized compared with nonhomogenized milk). CONCLUSIONS: Heat treatment and homogenization increased the apparent gastric degree of protein hydrolysis and the release of digested protein into the small intestine. However, the rate of AA entering the small intestine was mainly increased by homogenization.

Differences in small intestinal apparent amino acid digestibility of raw bovine, caprine, and ovine milk are explained by gastric amino acid retention in piglets as an infant model.

Background: The rate of stomach emptying of milk from different ruminant species differs, suggesting that the small intestinal digestibility of nutrients could also differ across these milk types. Objective: To determine the small intestinal amino acid (AA) digestibility of raw bovine, caprine, and ovine milk in the piglet as an animal model for the infant. Methods: Seven-day-old piglets (n = 12) consumed either bovine, caprine, or ovine milk diets for 15 days (n = 4 piglets/milk). On day 15, fasted piglets received a single meal of fresh raw milk normalized for protein content and containing the indigestible marker titanium dioxide. Entire gastrointestinal tract contents were collected at 210 min postprandially. Apparent AA digestibility (disappearance) in different regions of the small intestine was determined. Results: On average, 35% of the dietary AAs were apparently taken up in the small intestine during the first 210 min post-feeding, with 67% of the AA digestibility occurring in the first quarter (p ≤ 0.05) and 33% in the subsequent two quarters. Overall, except for isoleucine, valine, phenylalanine, and tyrosine, the small intestinal apparent digestibility of all AAs at 210 min postprandially in piglets fed ovine milk was, on average, 29% higher (p ≤ 0.05) than for those fed bovine milk. Except for lysine, there was no difference in the apparent digestibility (p > 0.05) of any AAs between piglets fed caprine milk or ovine milk. The apparent digestibility of alanine was higher (p ≤ 0.05) in piglets fed caprine milk than those fed bovine milk. When apparent digestibility was corrected for gastric AA retention, only small differences in the small intestinal apparent digestibility of AAs were observed across milk types. Conclusion: Bovine, caprine and ovine milk had different apparent small intestinal AA digestibility at 210 min postprandially. When corrected for gastric AA retention, the differences in apparent digestibility across species largely disappeared. The apparent AA digestibility differed across small intestinal locations.

The plasma amino acid response to blended protein beverages: a randomised crossover trial.

Soya-dairy protein blends can extend post-exercise muscle synthesis in young people more than whey protein control. Older adults differ metabolically from young people, and their ability to absorb amino acids from dietary protein is important for muscle function. The objective was to determine how protein source affects postprandial plasma amino acid response and/or metabolomic profile in older adults via a single-blind randomised crossover trial (n 16, males 50-70 years), using three nutritionally equivalent meal replacement drinks containing 30 g protein, from a 1:1 (mass ratio) soya:dairy blend, a 1:2 soya:dairy blend or whey protein. The outcome measures were plasma amino acid concentrations at 0-300 min postprandially and urine metabolomic fingerprint. Soya:dairy drinks gave similar amino acid response in plasma over time and similar urinary metabolite fingerprints. However, there were significant differences in plasma amino acid concentrations and AUC values for the soya:dairy drinks v. the whey protein drink. AUC for Leu, Trp and Lys was lower and AUC for Phe and Pro was higher for the soya:dairy drinks. Differences partly reflected the amino acid profiles of the drinks, but overall plasma amino acid response patterns were qualitatively unchanged. Plasma amino acid differences between the whey protein drink and the soya:dairy blends were reflected in urine metabolite patterns. In conclusion, postprandial plasma amino acid responses were broadly similar, irrespective of protein source (and soya:dairy ratio). There were significant differences for some plasma amino acid concentrations, reflecting different amino acid profiles of the protein source and influencing urine metabolite fingerprints.

Postprandial lipemic response in dairy-avoiding females following an equal volume of sheep milk relative to cow milk: A randomized controlled trial.

Background: Sheep milk (SM) is an alternate dairy source, which despite many similarities, has both compositional and structural differences in lipids compared to cow milk (CM). Studies are yet to examine the apparent digestibility of SM lipids, relative to CM, and the potential impact on the plasma lipidome. Objective: To determine the response of the circulatory lipidome to equal volume servings of SM and CM, in females who avoid dairy products. Method: In a double-blinded, randomized, cross-over trial, self-described dairy avoiding females (n = 30; 24.4 ± 1.1 years) drank SM or CM (650 mL; 33.4 vs. 21.3 g total lipid content; reconstituted from spray dried milk powders) following an overnight fast. Blood samples were collected at fasting and at regular intervals over 4 h after milk consumption. The plasma lipidome was analyzed by LC-MS and fatty acids were quantified by GC-FID. Results: The overall postprandial triglyceride (TG) response was similar between SM and CM. TG concentrations were comparable at fasting for both groups, however they were higher after CM consumption at 30 min (interaction milk × time p = 0.003), well before any postprandial lipemic response. This was despite greater quantities provided by SM. However, there were notable differences in the postprandial fatty acid response, with SM leading to an increase in short- and medium-chain fatty acids (MCFAs) (C6:0, C8:0, and C10:0) and several long-chain fatty acids (LCFAs) (C18:1 t11, c9, t11-CLA, and C20:0; interaction time × milk p < 0.05). This corresponded to a greater postprandial response for medium chain triglycerides (MCTs) C10:0, including TG(10:0/14:0/18:1), TG(16:0/10:0/12:0), and TG(16:0/10:0/14:0) (interaction time × milk p < 0.05). Conclusions: Despite a higher fat content, SM ingestion resulted in a greater circulating abundance of MCTs, without increasing total postprandial triglyceride response, when compared to CM. The greater abundance and postprandial appearance of MCTs may provide advantageous metabolic responses in children and adults. Unique identifier and registry: U1111-1209-7768; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=375324.

Applications of novel processing technologies to enhance the safety and bioactivity of milk.

Bioactive compounds in food can have high impacts on human health, such as antioxidant, antithrombotic, antitumor, and anti-inflammatory activities. However, many of them are sensitive to thermal treatments incurred during processing, which can reduce their availability and activity. Milk, including ovine, caprine, bovine, and human is a rich source of bioactive compounds, including immunoglobulins, vitamins, and amino acids. However, processing by various novel thermal and non-thermal technologies has different levels of impacts on these compounds, according to the studies reported in the literature, predominantly in the last 10 years. The reported effect of these technologies either covers microbial inactivation or the bioactive composition; however, there is a lack of comprehensive compilation of studies that compare the effect of these technologies on bioactive compounds in milk (especially, caprine and ovine) to microbial inactivation at similar settings. This research gap makes it challenging to conclude on the specific processing parameters that could be optimized to achieve targets of microbial safety and nutritional quality at the same time. This review covers the effect of a wide range of thermal and non-thermal processing technologies including high-pressure processing, pressure-assisted thermal sterilization, pulsed-electric field treatment, cold plasma, microwave-assisted thermal sterilization, ultra-high-pressure homogenization, ultrasonication, irradiation on the bioactive compounds as well as on microbial inactivation in milk. Although a combination of more than one technology could improve the reduction of bacterial contaminants to meet the required food safety standards and retain bioactive compounds, there is still scope for research on these hurdle approaches to simultaneously achieve food safety and bioactivity targets.

Using TD-NMR relaxometry and 1D 1H NMR spectroscopy to evaluate aging of Nellore beef.

The aim of this study was to explore the use of TD-NMR relaxometry and 1H NMR spectroscopy-based for detecting differences in meat quality attributes. There was limited association between various TD-NMR signals and any physicochemical parameters of fresh and aged meat differing in tenderness ratings. Samples were then divided into three groups based on statistical changes in metabolite concentration. Group A samples possessed near linear increases in metabolite concentration over aging time; whereas samples assigned to Groups B and C were characterized by increases in metabolites that peaked between 7 and 14 days, and up to 14 days aging, respectively. 1H NMR spectroscopy discriminated meat quality using changes in metabolites reflective of glycolysis, the citric acid cycle, protein degradation, amino acid generation and purine metabolisms. These data suggest segregation of meat quality is possible using both NMR technologies but additional work is necessary to understand fully their utility in a commercial industry setting.

Comparing Response of Sheep and Cow Milk on Acute Digestive Comfort and Lactose Malabsorption: A Randomized Controlled Trial in Female Dairy Avoiders.

Background: Sheep milk (SM) is a possible alternate dairy source for those who experience digestive symptoms with cow milk (CM). While both the milks contain lactose, one of the causes for self-reported intolerance to CM, the composition of SM and CM also differs across proteins and fats, which have been shown to impact digestive processes. Objective: To compare the acute digestive comfort and lactose malabsorption of SM to CM in female dairy avoiders. Method: In a double-blinded, randomized cross over trial, 30 dairy-avoiding females (aged 20-30 years) drank 650 mL of SM or CM (each reconstituted from spray dried powder) following an overnight fast, on two separate occasions at least 1 week apart. Blood samples were collected for glucose and insulin assessment, and single nucleotide polymorphisms of the lactase (LCT) gene (C/T13910 and G/A22018). Breath H2 and visual analog scale (VAS) digestive symptom scores were recorded at fasting and regular intervals over 4 h after ingestion. Results: Eighty percentage of study participants were lactase non-persistent (LNP; CC13910 and GG22018 genotype). Digestive symptoms, including abdominal cramps, distension, rumbling, bloating, belching, diarrhea, flatulence, vomiting, and nausea, were similar in response to SM and CM ingestion (milk × time, P > 0.05). Breath H2 was greater after CM than SM (72 ± 10 vs. 43 ± 6 ppm at 240 min, P < 0.001), which may be due to greater lactose content in CM (33 vs. 25 g). Accordingly, when corrected for the lactose content breath H2 did not differ between the two milks. The response remained similar when analyzed in the LNP subset alone (n = 20). Conclusions: Despite a higher energy and nutrient content, SM did not increase adverse digestive symptoms after ingestion, relative to CM, although there was a reduced breath H2 response, which could be attributed to the lower lactose content in SM. The tolerability of SM should be explored in populations without lactose intolerance for whom underlying trigger for intolerance is unknown.

Preparation of drip samples from leg of lamb with extended shelf life for nuclear magnetic resonance metabolomics studies.

Metabolomics has been used for the analysis of meat samples for different applications. Using drip as a proxy for meat could offer an easy and non-invasive way of sampling meat, yielding a homogenous liquid sample easy to prepare for metabolomics analysis. There is currently no standard method for the preparation of drip samples for quantitative metabolomics. The aim of this study was to evaluate six different sample preparation methods for quantitative Nuclear Magnetic Resonance (NMR) metabolomics analysis of drip from a lamb leg with extended shelf life: centrifugation, ultrafiltration, and solvent precipitation using four different solvents or solvent mixtures. The six methods were evaluated based on protein removal efficiency, ability to quantify metabolites, metabolite concentrations, reproducibility, speed and relative cost. Three methods (ultrafiltration, solvent precipitation with either acetonitrile/acetone/methanol or chloroform/methanol) resulted in excellent protein removal, high concentrations of metabolites and high reproducibility and are therefore recommended for preparation of extended shelf life lamb leg drip samples for NMR metabolomics.

Characterization of chicken muscle disorders through metabolomics, pathway analysis, and water relaxometry: a pilot study.

Metabolite profiles of chicken breast extracts and water mobility in breasts were studied using proton nuclear magnetic resonance (1H-NMR) spectroscopy and time-domain NMR (TD-NMR) relaxometry, respectively, using normal breast (NB), and wooden breast (WB) and white striping (WS) myopathies in broilers. One thousand eight hundred sixty broilers were raised to commercial standards, receiving the same diets that were formulated as per the different growth stages. At 49 D of age, 200 animals were slaughtered following routine commercial procedures, and at 4 h postmortem, the whole breast (pectoralis major muscle) was removed and visually inspected by an experienced meat inspector who selected NB (without myopathies) and samples with the presence of WS and WB myopathies. Fifteen breasts (5 each of NB, WS, and WB) were analyzed through TD-NMR relaxometry, and samples of approximately 20 g were taken from each breast and frozen at -80°C for metabolite profiling through 1H-NMR spectroscopy. Multivariate statistical analysis was used to evaluate the effect on water relaxometry and metabolite profile in accordance with the presence and type of myopathy in the breast. 1H-NMR data showed that the metabolite profiles in WS and WB breasts were different from each other and from NB. This pilot study shows that myopathies appear to be related to hypoxia, connective tissue deposition, lower mitochondrial function, and greater oxidative stress compared with NB. The longitudinal and transverse relaxation time of the breasts determined by TD-NMR relaxometry was shorter for NB than that for WS and WB, indicating greater water mobility in breasts affected by myopathies. 1H-NMR spectroscopy can be used to differentiate the metabolism of WS, WB, and NB, and TD-NMR has the potential to be a fast, simple, and noninvasive method to distinguish NB from WB and WS. As a practical application, the metabolomic profile as per the occurrence of breast myopathies may be used for a better understanding of these issues, which opens a gap to mitigate the incidence and severity of WS and WB. In addition, the present study brings an opportunity for the development of a new and objective tool to classify the incidence of breast myopathies through TD-NMR relaxometry.

Circulating Branched Chain Amino Acid Concentrations Are Higher in Dairy-Avoiding Females Following an Equal Volume of Sheep Milk Relative to Cow Milk: A Randomized Controlled Trial.

Background: Intolerances to bovine dairy are a motivating factor in consumers seeking alternate-or replacement-dairy beverages and foods. Sheep milk (SM) is an alternate dairy source, with greater protein, although similar amino acid composition compared to cow milk (CM). Studies are yet to address the appearance of circulating amino acids following consumption of SM, relative to CM, in humans. Objective: To clinically determine the appearance of branched chain amino acids, and other amino acids, in circulation in response to equal servings of SM and CM, in females who avoid dairy products. Design: In a double-blinded, randomized, cross-over trial, 30 self-described dairy avoiding females (20-40 years) drank 650 mL of SM or CM that were reconstituted from the spray dried powders (30 and 25 g in 180 mL water, respectively) on separate occasions, following an overnight fast. After reconstitution, the energy and protein provided by SM was higher than for CM (2,140 vs. 1,649 kJ; 29.9 vs. 19.4 g protein); content of branched chain amino acids (BCAAs) were 10.5 and 6.5 mg·mL-1, respectively. Blood samples were collected at fasting and at regular intervals over 5 h after milk consumption. Plasma amino acids were measured by HPLC. Results: 80% of subjects self-identified as lactose intolerant, and the majority (47%) "avoided drinking milk" "most of the time". SM resulted in greater plasma appearance of BCAAs at 60 min (641.1 ± 16.3 vs. 563.5 ± 14.4 µmol·L-1; p < 0.001) compared with CM. SM similarly resulted in elevated postprandial concentrations of the amino acids lysine, methionine, and proline, particularly at 240 min (time × milk interactions p = 0.011, 0.017, and p = 0.002, respectively). Postprandial increases in plasma alanine concentrations were sustained to 120 min after CM (time × milk interaction p = 0.001) but not after SM, despite greater quantities provided by SM. Conclusions: SM is a rich source of protein, and relative to CM, provides a greater quantity of BCAAs, with a corresponding elevation of the postprandial circulating BCAA response. SM is therefore a possible dairy alternative of benefit to those who need to increase total protein intake or for individuals with heightened protein requirements. Unique Identifier and Registry: https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=375324, identifier U1111-1209-7768.

In Vitro Fermentation of Sheep and Cow Milk Using Infant Fecal Bacteria.

While human milk is the optimal food for infants, formulas that contain ruminant milk can have an important role where breastfeeding is not possible. In this regard, cow milk is most commonly used. However, recent years have brought interest in other ruminant milk. While many similarities exist between ruminant milk, there are likely enough compositional differences to promote different effects in the infant. This may include effects on different bacteria in the large bowel, leading to different metabolites in the gut. In this study sheep and cow milk were digested using an in vitro infant digestive model, followed by fecal fermentation using cultures inoculated with fecal material from two infants of one month and five months of age. The effects of the cow and sheep milk on the fecal microbiota, short-chain fatty acids (SCFA), and other metabolites were investigated. Significant differences in microbial, SCFA, and metabolite composition were observed between fermentation of sheep and cow milk using fecal inoculum from a one-month-old infant, but comparatively minimal differences using fecal inoculum from a five-month-old infant. These results show that sheep milk and cow milk can have differential effects on the gut microbiota, while demonstrating the individuality of the gut microbiome.

Investigation of metabolites associated with confinement odour in chilled vacuum-packed lamb by proton nuclear magnetic resonance (1H NMR) spectroscopy.

This study aimed to determine the profiles of water-soluble metabolites in lamb drip and meat by Nuclear Magnetic Resonance (NMR) spectroscopy, in order to better understand the confinement odour (CO) phenomenon in lamb meat on a molecular level. Thirty-five lamb legs were obtained from two New Zealand meat processing plants and stored for 11 to 13 weeks at temperatures ranging from -1.5 °C to +4.0 °C. A sensorial test classified meat samples as having CO, no odour (NO) or persistent odour (PO). Sixty-three and sixty-two metabolites were identified and quantified in drip and meat samples, respectively. Partial least squares canonical analysis (PLS-CA) showed that CO was correlated with meat and drip metabolites tyramine, formate, alanine, carnosine, urea, proline, aspartate, glutathione and nicotinate. CO was also positively associated with appearance and bloom, but not directly associated with pH, size of the bacterial population or with processing plant. Metabolites associated with CO/PO are substrates or products of glucose fermentation and amino acid catabolism.

Factors affecting levels of volatile 4-alkyl branched-chain fatty acids in sheep milk from 2 contrasting farming systems in New Zealand.

Knowledge of factors influencing the levels of 4-alkyl branched-chain fatty acid (vBCFA), and consequently the "sheepy flavor" intensity of New Zealand sheep milk, is currently limited. In this study, we investigated the effects of 2 contrasting farming systems (fully housed/mid-lactation or pasture-grazed/late lactation) on the levels of vBCFA in sheep milk on a commercial farm in the North Island of New Zealand. Fully housed/mid-lactation ewes were housed 24 h/d and fed a total mixed ration. Pasture-grazed/late-lactation ewes were grazed 24 h/d and offered approximately 40% supplements because of poor pasture growth resulting from dry and hot climatic conditions. Any effects of genetics, age, lactation stage, feed composition, lambing date, or the environment in the housing barn or outdoors were confounded. The results obtained in this study were descriptive rather than definitive, because of the limitations of the experimental design. Levels of 4-methyloctanoic acid and 4-methylnonanoic acid in milk from fully housed/mid-lactation ewes increased during the trial period, but remained low in milk from pasture-grazed/late-lactation ewes. Levels of 4-ethyloctanoic acid in milk from the 2 groups of ewes were comparable throughout the trial. Increases in levels of 4-methyloctanoic acid and 4-methylnonanoic acid in sheep milk were associated with lactation stage and the proportion of lucerne silage fed to ewes. The level of free-form 4-ethyloctanoic acid was positively correlated with the proportion of soy meal in the diet and negatively correlated with the proportion of barley. Milk from fully housed/mid-lactation ewes had a higher flavor values than milk from pasture-grazed/late-lactation ewes because of its higher total amounts of vBCFA.

Effects of dry-aging on meat quality attributes and metabolite profiles of beef loins.

The objectives of this study were to evaluate different dry-aging regimes and their impacts on quality attributes and metabolite profiles of beef loins. Thirty loins (M. longissimus lumborum) from 15 beef carcasses at 2 days post-mortem were obtained. Each loin was cut in half yielding 60 sections, which were randomly assigned to six treatments including 4 dry-aging (2 temperatures (1 or 3°C) × 2 air-velocities (0.2 or 0.5 m/s)) and 2 wet-aging regimes for 3 weeks; n=10/treatment. The sensory panel found that dry-aged loins had better flavour and overall liking (P<0.05), but there were no differences in tenderness and juiciness. No differences in drip/cook-loss and colour were observed. Metabolite analysis showed that 7 metabolites, including several flavour precursors, were more abundant in the dry-aged beef compared to the wet-aged beef, which may contribute to the enhanced flavours of the dry-aged beef. Overall, dry-aging loins at 3°C with 0.2m/s resulted in the greatest improvement in beef palatability.

Human Breast Milk and Infant Formulas Differentially Modify the Intestinal Microbiota in Human Infants and Host Physiology in Rats.

BACKGROUND: In the absence of human breast milk, infant and follow-on formulas can still promote efficient growth and development. However, infant formulas can differ in their nutritional value. OBJECTIVE: The objective of this study was to compare the effects of human milk (HM) and infant formulas in human infants and a weanling rat model. METHODS: In a 3 wk clinical randomized controlled trial, babies (7- to 90-d-old, male-to-female ratio 1:1) were exclusively breastfed (BF), exclusively fed Synlait Pure Canterbury Stage 1 infant formula (SPCF), or fed assorted standard formulas (SFs) purchased by their parents. We also compared feeding HM or SPCF in weanling male Sprague-Dawley rats for 28 d. We examined the effects of HM and infant formulas on fecal short chain fatty acids (SCFAs) and bacterial composition in human infants, and intestinal SCFAs, the microbiota, and host physiology in weanling rats. RESULTS: Fecal Bifidobacterium concentrations (mean log copy number ± SEM) were higher (P = 0.003) in BF (8.17 ± 0.3) and SPCF-fed infants (8.29 ± 0.3) compared with those fed the SFs (6.94 ± 0.3). Fecal acetic acid (mean ± SEM) was also higher (P = 0.007) in the BF (5.5 ± 0.2 mg/g) and SPCF (5.3 ± 2.4 mg/g) groups compared with SF-fed babies (4.3 ± 0.2 mg/g). Colonic SCFAs did not differ between HM- and SPCF-fed rats. However, cecal acetic acid concentrations were higher (P = 0.001) in rats fed HM (42.6 ± 2.6 mg/g) than in those fed SPCF (30.6 ± 0.8 mg/g). Cecal transcriptome, proteome, and plasma metabolite analyses indicated that the growth and maturation of intestinal tissue was more highly promoted by HM than SPCF. CONCLUSIONS: Fecal bacterial composition and SCFA concentrations were similar in babies fed SPCF or HM. However, results from the rat study showed substantial differences in host physiology between rats fed HM and SPCF. This trial was registered at Shanghai Jiào tong University School of Medicine as XHEC-C-2012-024.

Aqueous and lipid nuclear magnetic resonance metabolomic profiles of the earthworm Aporrectodea caliginosa show potential as an indicator species for environmental metabolomics.

The common pasture earthworm Aporrectodea caliginosa has often been neglected in environmental metabolomics in favor of species easily bred in the laboratory. The present study assigns aqueous metabolites in A. caliginosa using high-resolution 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. In total, 51 aqueous metabolites were identified, including typical amino acids (alanine, leucine, asparagine, phenylalanine), sugars (maltose, glucose), the dominant earthworm-specific 2-hexyl-5-ethyl-furansulfonate, and several previously unreported metabolites (oxoglutarate, putrescine). Examining the lesser-known earthworm lipid metabolome showed various lipid fatty acyl chains, cholesterol, and phosphatidylcholine. To briefly test if the NMR metabolomic techniques could differentiate A. caliginosa from different sites, earthworms were collected from 2 adjacent farms. Orthogonal partial least squares discriminant analysis detected metabolomic differences, suggesting the worms from the 2 sites differed in their energy metabolism, as indicated by altered levels of alanine, glutamine, glutamate, malate, fumarate, and lipids. Evidence of greater utilization of lipid energy reserves and onset of protein catabolism was also present. While the precise cause of the metabolomic differences could not be determined, the results show the potential of this species for further environmental metabolomic studies.

The impact of temperature on the metabolome and endocrine metabolic signals in Atlantic salmon (Salmo salar).

The aim was to elucidate the effects of elevated temperature on growth performance, growth- and appetite-regulating hormones and metabolism in Atlantic salmon, Salmo salar. Post-smolts in seawater (average mass 175g) that had been reared at 12°C were kept at three temperatures (8, 12 and 18°C) and sampled after one and three months. After three months, the fish kept in 18°C had decreased growth rate and condition factor, and elevated plasma levels of growth hormone (GH) and leptin, compared with fish kept at the lower temperatures. Food conversion efficiency was also decreased at 18°C, while at the same time protein uptake was improved and thus was not a limiting mechanism for growth. Redistribution of energy stores in fish at the highest temperature is evident as a preference of maintaining length growth during times of limited energy availability. NMR-based metabolomics analyses of plasma revealed that several metabolites involved in energy metabolism were negatively affected by temperature in the upper temperature range of Atlantic salmon. Specifically, the high temperature induced a decline of several amino acids (glutamine, tyrosine and phenylalanine) and a shift in lipid metabolism. It appears likely that the decreased food intake at the highest temperature is linked to an anorexigenic function of leptin, but also that the decreased food intake, feed conversion efficiency and condition factor can be linked to changes in GH endocrinology.

Reproducible (1)H NMR-based metabolomic responses in fish exposed to different sewage effluents in two separate studies.

Treated sewage effluents contain complex mixtures of micropollutants, raising concerns about effects on aquatic organisms. The addition of advanced treatment steps has therefore been suggested. However, some of these could potentially produce effluents affecting exposed organisms by unknown modes of action. Here, (1)H NMR (proton nuclear magnetic resonance spectroscopy) metabolomics of fish blood plasma was used to explore potential responses not identified by more targeted (chemical or biological) assays. Rainbow trout was exposed in parallel to six differently treated effluents (e.g., conventional activated sludge, addition of sand filter, further addition of ozonation and/or a moving bed biofilm reactor or a separate membrane bioreactor line). Multivariate data analysis showed changes in the metabolome (HDL, LDL, VLDL and glycerol-containing lipids, cholesterol, glucose, phosphatidylcholine, glutamine, and alanine) between treatment groups. This formed the basis for postulating a hypothesis on how exposure to effluent treated by certain processes, including ozonation, would affect the metabolic profiles of exposed fish. The hypothesis withstood testing in an independent study the following year. To conclude, (1)H NMR metabolomics proved suitable for identifying physiological responses not identified by more targeted assays used in parallel studies. Whether these changes are linked to adverse effects remains to be tested.

A metabolomics approach to elucidate effects of food deprivation in juvenile rainbow trout (Oncorhynchus mykiss).

The aim of this study was to evaluate whether NMR-based metabolomics is a suitable method to generate an integrated view on metabolic processes during food deprivation in salmonid fish. Juvenile rainbow trout (Oncorhynchus mykiss) weighing 43-115 g were either fed ad libitum or deprived of food for 28 days at 10°C to investigate catabolic effects on energy reserves and metabolite patterns. The NMR analysis of plasma, liver, and muscle extracts revealed significant fasting-induced changes in the metabolome. Altered plasma lipoprotein levels and tissue-specific patterns of fatty acid mobilization were the most prominent responses, emphasizing the role of lipids as the primary energy source during fasting. In plasma, very-low-density lipoprotein levels increased in food-deprived fish compared with fed fish, whereas levels of high-density lipoprotein decreased. Signs of muscle protein catabolism were also observed as changes in the alanine dynamics. The results further suggest that mechanisms to preserve liver glycogen are present in the food-deprived fish. This study thus demonstrates the utility of NMR-based metabolomics of tissue extracts and plasma to describe the integrated metabolic status of fish.

Contributions from metabolomics to fish research.

Metabolomics is a systems approach to studying the small, endogenous metabolites in an organ, biofluid or whole organism. It can be used as a screening tool for metabolite profiling, or to detect changes in the metabolome brought on by external or internal stressors. The purpose of this review is to summarize and evaluate the information obtained from the application of metabolomics in fish research and to discuss its future potential. It is already clear that metabolomics has contributed new knowledge about fish in areas such as basic physiology and development, disease, water pollution and aspects concerning fish as foodstuffs.