Porcine Milk Oligosaccharides and Sialic Acid Concentrations Vary Throughout Lactation

Abstract

BackgroundMilk 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.ObjectivesThe 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.MethodsUp 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.ResultsSixty 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.ConclusionConcentrations 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.

Milk Glycans and Their Interaction with the Infant-Gut Microbiota

“…Milk from a number of animals, including cow (Albrecht et al 2014; Barile et al 2010; Tao et al 2008, 2009), sheep (Albrecht et al 2014, Martinez-Ferez et al 2006), goat (Albrecht et al 2014, Chaturvedi & Sharma 1988, Martin-Ortiz et al 2016, Martinez-Ferez et al 2006, Thum et al 2015), pig (Albrecht et al 2014, Mudd et al 2016, Salcedo et al 2016, Tao et al 2010), horse (Albrecht et al 2014, Nakamura et al 2001, Urashima et al 1991), and camel (Albrecht et al 2014, Alhaj et al 2013, Fukuda et al 2010), contains oligosaccharides similar to those found in human milk. There is a growing interest in examining the structures and concentrations of these animal milk oligosaccharides to identify additional sources of compounds with similar bioactivity to HMOs.…”

“…Relative abundances of acidic, acidic fucosylated, neutral fucosylated, and neutral nonfucosylated oligosaccharides in human, bovine, and porcine milk, as demonstrated by selected glycomics studies (Mudd et al 2016, Ninonuevo et al 2006, Tao et al 2009). …”

“…There is a growing interest in examining the structures and concentrations of these animal milk oligosaccharides to identify additional sources of compounds with similar bioactivity to HMOs. Comprehensive glycomic studies of bovine and porcine milks have shown a diverse collection of structures, with the relative proportion of acidic oligosaccharides being higher than that of human milk (Mudd et al 2016, Tao et al 2009). Although the type of animal used for industrial milk production can vary by country, bovine milk is generally produced in large quantities and is a potential source for industrial-scale oligosaccharide recovery.…”

An improved method for the purification of milk oligosaccharides by graphitised carbon-solid phase extraction

“…When purifying milk OS by PGC SPE, the solid phase is normally washed with water after sample loading (Mudd et al, 2016; Sundekilde et al, 2012; Tao et al, 2008), and water was therefore used as a control to the effectiveness of washing with low concentrations of ACN and TFA. TFA was included in all washing solutions to improve the recovery of acidic OS, as acidic pH increases the affinity of acidic OS for graphitised carbon (Pabst & Altmann, 2008).…”

“…Graphitised carbon solid phase extraction (SPE) is a well-established glycan purification technique that is commonly used in many glycomics workflows, including characterisation of protein N-glycomes (Aldredge, An, Tang, Waddell, & Lebrilla, 2012; Hua et al, 2013; Stumpo & Reinhold, 2010), biomarker discovery (Alley, Madera, Mechref, & Novotny, 2010; Balog et al, 2012; Vieira et al, 2012), and profiling of free milk OS (Marx et al, 2014; Mudd et al, 2016; Tao et al, 2008). Although previous publications have characterised the excellent retention characteristics of graphitized carbon in the context of glycan desalting and preconcentration (Packer, Lawson, Jardine, & Redmond, 1998; Redmond & Packer, 1999; Ward, 2009), the efficiency of the technique in separating milk OS and lactose has not been well-characterised, despite its widespread use for this purpose.…”