The Thoroughbred is among the most valuable horse breeds, and its husbandry is a major industry. Mare's milk plays a major role in the health of neonatal foals. Although the main components of mare's milk are broadly characterized, free oligosaccharides (OS), which possess various bioactivities in many mammalian milks, have not been fully profiled in Thoroughbreds. The aim of this study was to identify and quantify OS in Thoroughbred mare's milk during the first week of lactation, when foals typically consume mare's milk exclusively. A total of 48 OS structures (including isomers and anomers), corresponding to 20 unique compositions, were identified by nano LC-Chip QToF-MS and confirmed by tandem mass spectrometry. Neutral OS were the most abundant glycans (58.3%), followed by acidic OS containing Neu5Ac (33.3%), a minor presence of fucosylated OS structures (6.25%) and one structure containing NeuGc (2.1%). Comparison with other well-characterized mammalian milks revealed that mare's milk shared 8 OS structures with human, bovine, pig and goat milk (i.e., 2 sialyllactose isomers, 3 hexose, LNH, LNT, and OS with the composition 3 Hex-1 Neu5Ac). Additionally, there were seven unique OS not previously found in other mammal milks. During the first 7 days of lactation, the percentage of neutral and fucosylated OS increased, whereas acidic OS decreased and the total OS concentration ranged from 217.8 mg·L^-1 to 79.8 mg·L^-1.

Peptidomics and glycomics are recently established disciplines enabling researchers to characterize functional characteristics of foods at a molecular level. Milk-derived bioactive peptides and oligosaccharides have garnered both scientific and commercial interest because they possess unique functional properties, such as anti-hypertensive, immunomodulatory and prebiotic activities; therefore, the objective of this work was to employ peptidomic and glycomic tools to identify and measure relative and absolute quantities of peptides and oligosaccharides in widely consumed dairy products. Specifically, we identified up to 2117 unique peptides in 10 commercial dairy products, which together represent the most comprehensive peptidomic profiling of dairy milk in the literature to date. The quantity of peptides, measured by ion-exchange chromatography, varied between 60 and 130 mg/L among the same set of dairy products, which the majority originated from caseins, and the remaining from whey proteins. A recently published bioactive peptide database was used to identify 66 unique bioactive peptides in the dataset. In addition, 24 unique oligosaccharide compositions were identified in all the samples by nano LC Chip QTOF. Neutral oligosaccharides were the most abundant class in all samples (66-91.3%), followed by acidic (8.6-33.7%), and fucosylated oligosaccharides (0-4.6%). Variation of total oligosaccharide concentration ranged from a high of 65.78 to a low of 24.82 mg/L. Importantly, characterizing bioactive peptides and oligosaccharides in a wider number of dairy products may lead to innovations that go beyond the traditional vision of dairy components used for nutritional purposes but that will rather focus on improving human health.

Background

Oligosaccharides (OS) are indigestible carbohydrates naturally found in milk. The composition of porcine colostrum OS may influence the growth and the health of the neonate and consuming optimal concentrations of OS may reduce piglet susceptibility to illness. In this manner, targeted supplementation of animal feed with OS is being explored as a health management tool in the livestock industry. The variation in OS composition between different breeds of pig and its association with the litter performance is currently unknown. The aim of this study was to characterize the colostrum OS composition from sows of different breed and parity and correlate this data with sow maternal traits.

Methods

Eighty-three colostrum samples from parities 1 to 8 were gathered from 3 different breeds of sow: 44 Large White sows, 27 Landrace sows and 12 Duroc sows. Samples were taken between the birth of the first and the last piglet from sows that were not pharmacologically induced to farrow. OS were purified from the samples and analysed by MALDI-ToF mass spectrometry (21 OS compositions detected). The farrowing season and the maternal data were recorded for each sow, including the number of live piglets and the litter body weight at birth, at day (d) 3 and at weaning.

Results

Five OS compositions, including isomers of the bifidogenic Sialyllactose, Lacto-N-Tetraose and Lacto-N-Hexaose series, were detected in all the samples. Twelve other OS were identified in at least 50% of samples, and their abundances were affected by breed (P < 0.05; 6 of 12), marginally affected by season (P < 0.10; 3 of 12) and never by parity number. The abundances of each OS component were standardized by Z-score scaling (μ = 0 and SD = 1), transformed by principal component analysis, and four similarity clusters were generated. Cluster membership was associated with litter weight gain within 3 days (P = 0.063) and at weaning (P < 0.05), but not with piglet mortality within 3 days.

Conclusions

OS composition of colostrum may partially explain the variability in maternal performance within and between different breeds of sow. The obtained OS data can provide useful information for the development of novel prebiotic food supplements for suckling and weaning pigs.

Changes of the abundance of caprine milk oligosaccharides (CMO) at different lactation stages have been evaluated by hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-Q MS) and nanoflow liquid chromatography-quadrupole-time-of-flight mass spectrometry (nano-LC-Chip-QTOF MS). Eight major oligosaccharides (OS) were quantified at different lactation stages by HILIC-Q MS, while the use of nano-LC-Chip-QToF MS allowed expanding the study to forty-nine different OS by monitoring neutral non- and fucosylated species, as well as acidic species containing not only N-acetyl-neuraminic acid or N-glycolyl-neuraminic acid residues but also the combination of both sialic acids. Overall, the most abundant OS decreased with lactation time, whereas different trends were observed for minor OS. 6'-Sialyl-lactose was the most abundant acidic OS while galactosyl-lactose isomers were identified as the most abundant neutral OS. This is the first time that a comprehensive study regarding the changes of the abundance of CMO, both neutral and acidic, at different lactation stages is carried out.

Human milk oligosaccharides play a vital role in the development of the gut microbiome in the human infant. Although oligosaccharides derived from bovine milk (BMO) differ in content and profile with those derived from human milk (HMO), several oligosaccharide structures are shared between the species. BMO are commercial alternatives to HMO, but their fate in the digestive tract of healthy adult consumers is unknown. Healthy human subjects consumed two BMO doses over 11-day periods each and provided fecal samples. Metatranscriptomics of fecal samples were conducted to determine microbial and host gene expression in response to the supplement. Fecal samples were also analyzed by mass spectrometry to determine levels of undigested BMO. No changes were observed in microbial gene expression across all participants. Repeated sampling enabled subject-specific analyses: four of six participants had minor, yet statistically significant, changes in microbial gene expression. No significant change was observed in the gene expression of host cells exfoliated in stool. Levels of BMO excreted in feces after supplementation were not significantly different from baseline and were not correlated with dosage or expressed microbial enzyme levels. Collectively, these data suggest that BMO are fully fermented in the human gastrointestinal tract upstream of the distal colon. Additionally, the unaltered host transcriptome provides further evidence for the safety of BMO as a dietary supplement or food ingredient. Further research is needed to investigate potential health benefits of this completely fermentable prebiotic that naturally occurs in cow's milk.

An ongoing study at the University of Nebraska-Lincoln (which included 14 batches of gilts; n = 90 gilts/batch) demonstrated that energy restriction during the developmental period of a gilt increases longevity and may also have beneficial effects on progeny health and growth, particularly, parity 1 progeny. Therefore, we hypothesized that energy restriction during gilt development may affect milk nutrient profile, milk oligosaccharides (OS), and postnatal progeny biomarkers. During the development period, batch 14 gilts (n = 128, 8 gilts/pen) were fed 3 dietary treatments including the following: 1) Control diet formulated to NRC (2012) specifications (CTL); 2) Restricted (20% energy restriction via addition of 40% soy hulls; RESTR); and 3) CTL diet plus addition of crystalline amino acids equivalent to the SID Lys:ME of the RESTR diet (CTL+). All diets were fed ad libitum and applied in a 3-phase feeding regimen during gilt development (days 123 to 230 of age). Average daily feed intake was used to estimate daily metabolizable energy intake (Mcal/d) during each phase (Phase 1: 10.13, 6.97, 9.95; Phase 2: 11.25, 8.05, 10.94; and Phase 3: 9.47, 7.95,11.07) for CTL, RESTR, and CTL+, respectively. After 230 d of age, gilts were bred and fed a common diet. Milk samples were collected from batch 14 gilts (n = 7 per treatment) on days 0 and 14 postfarrowing for compositional analysis of N, CP, dry matter (DM), GE, insulin, and OS. Piglet blood samples (n = 6 piglets/gilt) were obtained on days 1 and 15 postfarrowing for quantification of glucagon-like peptide-2 (GLP-2) and insulin. No effects of developmental diet were observed for milk N, CP, DM, or GE; however, N, CP, DM, and insulin were increased (P < 0.05) on day 1 compared with day 14. A total of 61 different milk OS were identified. Milk OS profile was significantly different for neutral and acidic OS (P < 0.05) on day 0, but there were no significant differences on day 14. For piglet GLP-2, a treatment by day interaction was observed (P < 0.009); specifically, on day 1 GLP concentrations were greater (P < 0.001) in CTL+ compared with RESTR (6.73 vs. 1.21 ng/mL). For serum insulin, a treatment by day interaction was observed (P < 0.01); specifically, insulin in RESTR progeny was greater (P < 0.03) than CTL on day 1. In conclusion, nutritional management of the developing gilt may affect milk nutrient composition, milk OS profile, and piglet serum biomarkers.

Donor milk is the best option when mother's own milk is unavailable. Heat treatments are applied to ensure donor milk safety. The effects of heat treatments on milk gangliosides-bioactive compounds with beneficial antibacterial, anti-inflammatory, and prebiotic roles-have not been studied. The most abundant gangliosides in non-homogenized human milk were characterized and quantified by liquid chromatography-mass spectrometry (LC-MS)/MS before and after pasteurization treatments mimicking industrial conditions (63 °C/30 min, 72 °C/15 s, 127 °C/5 s, and 140 °C/6 s). Ganglioside stability over a 3-month period was assessed following the storage at 4 and 23 °C. Independent of the heat treatment applied, gangliosides were stable after 3 months of storage at 4 or 23 °C, with only minor variations in individual ganglioside structures. These findings will help to define the ideal processing and storage conditions for donor milk to maximize the preservation of the structure of bioactive compounds to enhance the health of fragile newborns. Moreover, these results highlight the need for, and provide a basis for, a standardized language enabling biological and food companies, regulatory agencies, and other food stakeholders to both annotate and compute the ways in which production, processing, and storage conditions alter or maintain the nutritive, bioactive, and organoleptic properties of ingredients and foods, as well as the qualitative effects these foods and ingredients may have on conferring phenotype in the consuming organism.

Background

Milk oligosaccharides (OSs) are bioactive components known to influence neonatal development. These compounds have specific physiological functions acting as prebiotics, immune system modulators, and enhancing intestine and brain development.

Objectives

The pig is a commonly used model for studying human nutrition, and there is interest in quantifying OS composition of porcine milk across lactation compared with human milk. In this study, we hypothesized that OS and sialic acid (SA) composition of porcine milk would be influenced by stage of lactation.

Methods

Up to 250 mL of milk were collected from seven sows at each of three time points: day 0 (colostrum), days 7-9 (mature), and days 17-19 (weaning). Colostrum was collected within 6 h of farrowing and 3-day intervals were used for mature and weaning milk to ensure representative sampling. Milk samples were analyzed for OS profiles by Nano-LC Chip-QTOF MS, OS concentrations via HPAEC-PAD, and SA (total and free) was assessed by enzymatic reaction fluorescence detection.

Results

Sixty unique OSs were identified in porcine milk. Neutral OSs were the most abundant at each lactation stage (69-81%), followed by acidic-sialylated OSs (16-29%) and neutral-fucosylated OSs (2-4%). As lactation progressed, acidic OSs decreased (P = 0.003), whereas neutral-fucosylated (P < 0.001) and neutral OSs (P = 0.003) increased throughout lactation. Six OSs were present in all samples analyzed across lactation [lacto-N-difucohexaose I (LNDFH-I), 2'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-neohexaose (LNnH), α1-3,β-4-d-galactotriose (3-Hex), 3'-sialyllactose (3'-SL)], while LDFT was present only in colostrum samples. Analysis of individual OS concentrations indicated differences (P = 0.023) between days 0 and 7. Conversely, between days 7 and 18, OS concentrations remained stable with only LNnH (P < 0.001) and LNDFH-I (P = 0.002) decreasing over this period. Analysis of free SA indicated a decrease (P < 0.001) as lactation progressed, while bound (P < 0.001) and total (P < 0.001) SA increased across lactation.

Conclusion

Concentrations of OS differ between colostrum and mature milk in the pig, and SA concentrations shift from free to bound forms as lactation progresses. Our results suggest that although porcine milk OS concentration and the number of structures is lower than human milk, the OS profile appears to be closer to human milk rather than to bovine milk, based on previously published profiles.