Edited by: Pietro Celi, DSM Nutritional Products, USA
Reviewed by: Barry Bradford, Kansas State University, USA; Ricardo F. M. Teixeira, University of Lisbon, Portugal
†Austin T. Mudd and Jaime Salcedo are equally contributing first authors.
Specialty section: This article was submitted to Animal Nutrition and Metabolism, a section of the journal Frontiers in Nutrition
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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.
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.
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.
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 (
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.
The gastrointestinal tract is known to directly influence physiology of the entire organism. Relative to the rest of the body, the highest number of immune cells and a diverse microbiome are present in the gut. In the developing infant, gut maturation is influenced by oligosaccharides (OSs) present in the mother’s milk and OS composition of human milk changes throughout lactation, which likely has specific physiological implications in the developing infant. Piglets are considered a suitable model for human infant nutrition due to similarities in the development of intestinal physiology and nutrient requirements (
Oligosaccharides are increasingly recognized as bioactive components of milk, and are believed to confer benefits throughout neonatal development (
Sialic acid (SA)-containing structures are essential in the first stages of life for optimal development due to the biological processes in which they are involved: inhibition of pathogen binding, brain development, and immune system maturation (
While the functions of these molecules are clear, sensitive evaluation of the changes in OS profiles and concentrations throughout lactation is lacking. Due to their structural diversity in both the composing monosaccharides and their linkages, the identification and quantification of OS has proven difficult. Recent technological developments elevated mass spectrometry as one of the most valuable and widely used tools for characterization of OS in mammalian milks and other biological samples (
Seven Yorkshire sows from the University of Illinois Imported Swine Research Laboratory (ISRL) were bred to Yorkshire boars and housed in standard gestation and farrowing crates throughout the study. Sows were provided custom gestation and lactation diets as described below. Two replicates of sows from consecutive farrowing groups were used with 3–4 sows in each replicate. Sows were allowed
Ingredient, g/kg | Gestation | Lactation |
---|---|---|
Ground corn | 785.0 | 652.0 |
Molassed dried sugar beet pulp | 70.0 | 65.0 |
Soy protein isolate |
60.0 | 118.0 |
Cornstarch | 39.7 | 101 |
Dicalcium phosphate | 20.0 | 20.0 |
Corn oil | 10.0 | 30.0 |
Limestone | 7.5 | 6.5 |
Vitamin and mineral premix |
3.0 | 3.0 |
Choline chloride |
3.0 | 2.4 |
Bacitracin |
0.0 | 2.1 |
1.5 | 0.0 | |
0.3 | 0.0 | |
Choline | 1,887 | 1,591 |
Folate | 2.80 | 2.60 |
Dry matter, % | 90.6 | 88.8 |
Organic matter | 95.6 | 95.6 |
Crude protein | 13.7 | 18.3 |
Crude fat | 2.69 | 2.58 |
Amino acids | ||
Lys | 0.64 | 0.99 |
Met | 0.42 | 0.25 |
Cys | 0.23 | 0.25 |
Arg | 0.75 | 1.11 |
Ile | 0.54 | 0.77 |
Leu | 1.42 | 1.57 |
Val | 0.70 | 0.87 |
Phe | 0.66 | 0.91 |
Thr | 0.46 | 0.66 |
Trp | 0.15 | 0.21 |
Milk samples were collected from each sow at three time-points throughout lactation. Colostrum was collected within 6 h of farrowing. To ensure representative samples of mature and weaning milk, samples were collected over 3-day collection periods, i.e., days 6–8 and days 17–19. Approximately 250 mL of milk were collected per sow at each time period. To facilitate milk let-down, piglets were removed from the sow for 1 h and placed in an adjacent, empty farrowing crate with access to supplemental heat and water. An intramuscular injection of oxytocin (2 mL, OxoJect, Henry Schein Animal Health, Dublin, OH, USA) was administered after 1 h without piglets, and milk was manually expressed into 50 mL conical tubes and stored at −20°C. Prior to analysis, milk samples were thawed, combined to create a homogeneous milk sample for each sow, and divided into aliquots; only one freeze–thaw process occurred prior to nutrient quantification procedures.
Acetonitrile (ACN), formic acid (FA), trifluoroacetic acid (TFA), sodium hydroxide (NaOH), sodium chloride (NaCl), and sulfuric acid (H2SO4) were obtained from Thermo Fisher Scientific (Waltham, MA, USA); sodium acetate (NaAc) was purchased from Sigma-Aldrich (St Louis, MO, USA). All solvents were MS grade. OS standards for lacto-N-difucohexaose I (LNDFH-I), 2′-fucosyllactose (2′-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), N-acetylgalactosaminyllactose, α1-3,β-4-
Oligosaccharides were isolated and purified according to a previously published method (
Nano-LC Chip–QTOF-MS/MS analysis was performed with an Agilent 6520 accurate-mass Quadrupole-Time-of-Flight (Q-TOF) LC/MS with a microfluidic nano-electrospray chip containing an enrichment and an analytical column packed with porous graphitized carbon (Agilent Technologies, Santa Clara, CA, USA). Nano-LC QTOF MS/MS parameters were as described previously (
Composition of eluted OS is listed as a set of the five individual monomers composing the OS using the following identification nomenclature: Hex_HexNAc_Fuc_Neu5Ac_Neu5Gc. Abbreviations for the components are as follows: Hex, hexose (glucose or galactose); HexNAc, N-acetylhexososamine; Fuc, fucose; Neu5Ac, N-acetylneuramic acid; Neu5Gc, N-glycolylneuramic acid. The number of each individual monomer present within an identified or quantified OS is represented using the aforementioned nomenclature. It should be noted that the analysis by Nano-LC Chip–QTOF MS separated multiple isomers for each OS; however, here we indicate the relative percentage of composition 3_1_0_0_0 (3 Hex, 1 HexNAc) as the sum of LNT and LNnT. Similarly, the relative percentage of composition 4_2_0_0_0 (4 Hex, 2 HexNAc) was the sum of LNH and LNnH due to close eluting times of the isomer pairs (i.e., co-elution).
Quantification of eight neutral (LNDFH, LDFT, 2′-FL, LNFP-I, LNT, LNnT, N-acetylgalactosaminyllactose, and 3-Hex) and 3 acidic (6′-SLN, 6′-SL, and 3′-SL) OSs was carried out using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD ICS-5000; Thermo Fisher Scientific, Waltham, MA, USA). Diluted OS solutions were filtered through a 0.22 μm membrane and injected using a 25 μL loop. Chromatographic separation was carried out on a CarboPac PA200 analytical column (3 mm × 250 mm; Dionex, Sunnyvale, CA, USA) and a CarboPac PA200 guard column (3 mm × 50 mm; Dionex, Sunnyvale, CA, USA) with 0.5 mL/min elution and a non-isocratic gradient: 0–10 min 50% B, 10–50 min 45% B – 10% C. The column was equilibrated for 5 min with 10% B followed by 10 min with 50% B. Solvent A was deionized water, solvent B 200 mM NaOH, and solvent C was 100 mM NaAc in 100 mM NaOH. Quantification was assessed by external calibration using a mixture of all OS standards ranging from 0.0001 to 0.03 g/L.
To evaluate total SA content, an acid hydrolysis was performed: 200 μL of sample was mixed with 800 μL of 0.05 M H2SO4, heated (60 min at 80°C), cooled to room temperature and centrifuged (13,000 ×
A one-way repeated measures ANOVA was performed using the MIXED procedure of SAS 9.3 (SAS Institute, Cary, NC, USA) to evaluate differences in the relative proportion of OS and individual concentrations of OS and SA in porcine milk throughout lactation. Day of lactation served as the repeated measure, and an HSD-Tukey adjustment was applied during the
Sixty OSs (including isomers and anomers) were identified and confirmed by Nano-LC Chip–Q-TOF MS/MS in this study (Table
# | Composition1 | Formula | Exact mass(exact) | Retention |
Type2 | Relative abundance |
±SEM | Presence in human milk4 | |||
---|---|---|---|---|---|---|---|---|---|---|---|
0 | 7 | 18 | |||||||||
1 | 2_0_1_0_0 | C18 H32 O15 | 488.1738 | 12.11 | NF | 2.37b | 1.87b | 0.270 | ✓ | ||
2 | 2_0_1_0_0 | C18 H32 O15 | 488.1723 | 14.37 | NF | 0.40 | 0.36 | 0.41 | 0.055 | 0.5507 | ✗ |
3 | 2_0_1_0_0 | C18 H32 O15 | 488.1737 | 15.62 | NF | 0.71 | 0.71 | 0.74 | 0.106 | 0.9579 | ✗ |
4 | 3_0_0_0_0 | C18 H32 O16 | 504.1694 | 12.40 | N | 4.31a | 1.43b | 0.559 | ✗ | ||
5 | 3_0_0_0_0 | C18 H32 O16 | 504.1690 | 13.02 | N | 3.55a | 1.91b | 2.03b | 0.366 | ✗ | |
6 | 3_0_0_0_0 | C18 H32 O16 | 504.1690 | 13.38 | N | 6.54a | 2.74b | 2.90b | 0.671 | ✗ | |
7 | 3_0_0_0_0 | C18 H32 O16 | 504.1692 | 14.41 | N | 2.48a | 2.06b | 2.05b | 0.247 | ✗ | |
8 | 3_0_0_0_0 | C18 H32 O16 | 504.1696 | 15.66 | N | 0.58a | 0.80a | 1.54b | 0.212 | ✗ | |
9 | 3_0_0_0_0 | C18 H32 O16 | 504.1688 | 16.45 | N | 0.30a | 0.27a | 0.51b | 0.082 | ✗ | |
10 | 1_0_0_1_0 | C18 H32 O15 | 470.1508 | 15.78 | A | 0.131 | 0.5029 | ✓ | |||
11 | 2_0_0_1_0 | C23 H39 N O19 | 633.2118 | 18.30 | A | 1.65 | 1.09 | 1.60 | 0.273 | 0.3404 | ✓ |
12 | 2_0_0_1_0 | C23 H39 N O19 | 633.2117 | 23.37 | A | 9.73 | 5.98 | 4.76 | 1.610 | 0.0816 | ✓ |
13 | 1_1_0_1_0 | C25 H42 N2 O19 | 674.2374 | 16.77 | A | 0.25a | 0.025 | ✗ | |||
14 | 1_1_0_1_0 | C25 H42 N2 O19 | 674.2377 | 18.31 | A | 1.16a | 0.105 | ✓ | |||
15 | 1_1_0_1_0 | C25 H42 N2 O19 | 674.2368 | 22.94 | A | 0.29a | 0.028 | ✓ | |||
16 | 3_1_0_0_0 | C26 H45 N O21 | 707.2477 | 15.23 | N | 1.07a | 0.69b | 0.50b | 0.087 | ✓ | |
17 | 3_1_0_0_0 | C26 H45 N O21 | 707.2478 | 16.00 | N | 0.99 | 0.88 | 0.307 | 0.5746 | ✓ | |
18 | 3_1_0_0_0 | C26 H45 N O21 | 707.2484 | 17.14 | N | 6.89 | 4.90 | 5.68 | 1.404 | 0.6366 | ✗ |
19 | 3_1_0_0_0 | C26 H45 N O21 | 707.2477 | 21.64 | N | 0.90a | 0.97a | 0.56b | 0.102 | ✗ | |
20 | 2_2_0_0_0 | C28 H48 N2 O21 | 748.2742 | 15.28 | N | 0.65a | 0.33b | 0.26b | 0.082 | ✗ | |
21 | 3_0_0_1_0 | C29 H49 N O24 | 795.2641 | 24.11 | A | 0.03 | 0.29 | 0.45 | 0.162 | 0.2025 | ✗ |
22 | 3_0_0_1_0 | C29 H49 N O24 | 795.2641 | 25.19 | A | 0.77ab | 1.06b | 0.182 | ✗ | ||
23 | 3_0_0_1_0 | C29 H49 N O24 | 795.2644 | 25.80 | A | 0.39a | 0.63ab | 0.87b | 0.099 | ✗ | |
24 | 4_1_0_0_0 | C32 H55 N O26 | 869.3013 | 19.69 | N | 5.36 | 7.37 | 7.11 | 0.672 | 0.1088 | ✗ |
25 | 4_1_0_0_0 | C32 H55 N O26 | 869.3012 | 20.70 | N | 5.43 | 7.69 | 7.44 | 1.076 | 0.2922 | ✗ |
26 | 4_1_0_0_0 | C32 H55 N O26 | 869.3005 | 27.54 | N | 1.16 | 2.14 | 1.93 | 0.384 | 0.1309 | ✗ |
27 | 3_2_0_0_0 | C34 H58 N2 O26 | 910.3274 | 18.51 | N | 0.65 | 0.249 | 0.4526 | ✗ | ||
28 | 3_2_0_0_0 | C34 H58 N2 O26 | 910.3288 | 19.08 | N | 1.05 | 1.06 | 0.361 | 0.5082 | ✗ | |
29 | 3_2_0_0_0 | C34 H58 N2 O26 | 910.3286 | 19.39 | N | 1.58 | 0.680 | 0.8237 | ✗ | ||
30 | 3_1_0_1_0 | C37 H62 N2 O29 | 998.3431 | 24.73 | A | 0.36 | 0.30 | 0.129 | 0.2926 | ✓ | |
31 | 3_1_0_1_0 | C37 H62 N2 O29 | 998.3457 | 28.69 | A | 1.73a | 0.31b | 0.131 | ✓ | ||
32 | 3_1_0_1_0 | C37 H62 N2 O29 | 998.3448 | 26.35 | A | 0.10 | 0.11 | 0.07 | 0.025 | 0.3424 | ✓ |
33 | 4_1_1_0_0 | C38 H65 N O30 | 1015.3573 | 15.38 | NF | 0.10a | 0.024 | ✗ | |||
34 | 4_2_0_0_0 | C40 H68 N2 O31 | 1072.3836 | 21.64 | N | 18.71a | 30.75ab | 29.66b | 3.038 | ✗ | |
35 | 4_1_0_1_0 | C43 H72 N2 O34 | 1160.3979 | 27.14 | A | 1.12b | 0.70b | 0.163 | ✗ | ||
36 | 4_1_0_1_0 | C43 H72 N2 O34 | 1160.3982 | 27.54 | A | 0.78a | 2.04b | 1.15a | 0.340 | ✗ | |
37 | 4_2_0_1_0 | C51 H85 N3 O39 | 1363.4791 | 26.13 | A | 7.03 | 6.19 | 1.324 | 0.0704 | ✓ | |
38 | 4_2_0_1_0 | C51 H85 N3 O39 | 1363.4783 | 28.93 | A | 3.12a | 0.286 | ✓ | |||
39 | 1_0_0_1_0 | C17 H29 N O14 | 471.1616 | 11.70 | A | 0.14ab | 0.21b | 0.043 | ✓ | ||
40 | 1_1_1_0_0 | C20 H35 N O15 | 529.1994 | 13.18 | NF | 0.15a | 0.13a | 0.06b | 0.025 | ✗ | |
41 | 1_1_1_0_0 | C20 H35 N O15 | 529.1993 | 13.73 | NF | 0.12a | 0.05b | 0.022 | ✗ | ||
42 | 2_1_0_0_0 | C20 H35 N O16 | 545.1953 | 11.35 | N | 1.02ab | 1.94b | 0.335 | ✓ | ||
43 | 2_1_0_0_0 | C20 H35 N O16 | 545.1954 | 12.05 | N | 0.60a | 1.48ab | 2.55b | 0.590 | ✓ | |
44 | 2_1_0_0_0 | C20 H35 N O16 | 545.1954 | 12.98 | N | 3.31b | 4.66b | 0.482 | ✓ | ||
45 | 2_1_0_0_0 | C20 H35 N O16 | 545.1953 | 13.96 | N | 0.77ab | 1.88b | 0.320 | ✓ | ||
46 | 2_1_0_0_0 | C20 H35 N O16 | 545.1953 | 14.38 | N | 0.30a | 0.77b | 0.72ab | 0.226 | ✓ | |
47 | 2_1_0_0_0 | C20 H35 N O16 | 545.1951 | 15.70 | N | 0.21 | 0.34 | 0.099 | 0.2928 | ✓ | |
48 | 4_0_0_0_0 | C24 H42 O21 | 666.2211 | 2.42 | N | 0.28a | 0.33ab | 0.75b | 0.109 | ✗ | |
49 | 4_0_0_0_0 | C24 H42 O21 | 666.2215 | 12.72 | N | 0.16 | 0.18 | 0.10 | 0.044 | 0.338 | ✗ |
50 | 4_0_0_0_0 | C24 H42 O21 | 666.2212 | 19.93 | N | 0.16 | 0.11 | 0.09 | 0.023 | 0.065 | ✗ |
51 | 2_1_1_0_0 | C26 H45 N O20 | 691.2525 | 12.97 | NF | 0.18b | 0.10ab | 0.034 | ✗ | ||
52 | 2_1_0_1_0 | C31 H52 N2 O24 | 836.2902 | 13.79 | A | 0.108 | 0.4052 | ✓ | |||
53 | 2_1_0_1_0 | C31 H52 N2 O24 | 836.2894 | 14.96 | A | 1.02 | 0.409 | 0.2439 | ✓ | ||
54 | 2_1_0_1_0 | C31 H52 N2 O24 | 836.2899 | 22.71 | A | 0.21 | 0.078 | 0.5575 | ✓ | ||
55 | 3_1_1_0_0 | C32 H55 N O25 | 853.3067 | 15.80 | NF | 0.35a | 0.06b | 0.07b | 0.054 | ✓ | |
56 | 5_1_0_0_0 | C38 H65 N O31 | 1031.3523 | 22.80 | N | 0.15 | 0.07 | 0.031 | 0.0571 | ✗ | |
57 | 3_3_0_0_0 | C42 H71 N3 O31 | 1113.4093 | 22.32 | N | 1.15a | 0.130 | ✗ | |||
58 | 3_3_0_0_0 | C42 H71 N3 O31 | 1113.4092 | 20.12 | N | 3.13a | 1.18b | 0.67b | 0.392 | ✗ | |
59 | 7_0_0_0_0 | C42 H72 O36 | 1152.3789 | 17.83 | N | 0.02 | 0.007 | 0.4861 | ✗ | ||
60 | 3_2_0_1_0 | C45 H75 N3 O34 | 1201.4255 | 25.60 | A | 0.18 | 0.17 | 0.061 | 0.4814 | ✗ |
Mature milk contained mainly neutral non-fucosylated OS, 4 Hex-2 HexNAc, and 3-Hex comprising more than 35% of the total OS. Neutral OSs were the most abundant at each lactation stage (69–81%), followed by acidic-sialylated OS (16–29%) and neutral-fucosylated OS (2–4%) (Figure
Eight neutral OS (LNDFH-I, LDFT, 2′-FL, LNFP-I, LNT, LNnH, 2Hex-1HexNAc, and 3 Hex) and three acidic (6′-SLN, 6′-SL, and 3′-SL) were quantified using reference standards. Six OSs were present in all the samples analyzed across lactation (LDFH-I, 2′-FL, LNFP-I, LNnH, 3 Hex, 3′-SL; Figure
Total quantified OS concentrations in porcine milk decreased (
The concentrations of total, free, and bound SA were determined using an enzymatic reaction with fluorescence detection (Figure
Oligosaccharides are a class of molecules that have gained considerable attention because of the biological processes in which they impact the first stages of life (
Sixty OSs (including isomers and anomers) were identified in porcine milk during the first 18 days of lactation by Nano-LC Chip–Q-TOF MS, with 24 structures matching those identified in human milk (
Quantification of OSs is generally limited to a few structures due to the paucity of commercial standards, which greatly impedes quantification of the diverse set of compounds that can be identified by Nano-LC Chip–Q-TOF MS. In the present study, eight neutral and three acidic OSs, which are found in human and/or bovine milk, were accurately quantified. The general trends in quantified OS match with observations of the global profiling by Nano-LC Chip–QToF MS, where sialylated OS decreased, and fucosylated OS increased, in abundance during lactation. The higher abundance of fucosylated versus sialylated OS structures places porcine milk OS (albeit in lower concentrations) as structurally closer to OS profiles found in human milk, rather than bovine milk. Additionally, the conspicuous absence of Neu5Gc-containing OS and the simultaneous presence of typical human milk OS, such as LNDFH-I, places porcine milk far closer to human milk than previously thought. Although porcine milk appears to closely resemble human milk composition, as stated above, there are some characteristics that tend to align more closely with bovine milk, namely individual OS concentrations in porcine milk tended to be lower than those found in human milk (
Sialic acid is present in mammals’ biological fluids and tissues, contributing to the formation of complex structures, such as glycolipids or glycoproteins. Only 5% of the total SA typically exists in the free or unbound form (
At the first stages of life, enzymes involved in SA synthesis and incorporation into other structures are not mature or active. In quantitative terms, this developmental insufficiency may increase the dietary SA requirement to maintain biological processes and ensure optimal development. As infant enzyme systems supporting SA production mature throughout the lactation period, their dietary requirements for free SA decrease, hence the biological basis for milk SA to decrease throughout lactation (
The present work expands on previous work in analysis of porcine OS by characterizing additional novel OS present in porcine milk, while also quantifying changes in SA across lactation. Moreover, this study suggests 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. Much of the similarities are based on the increased proportion of fucosylated OS during lactation and the ability of sows to synthetize OS commonly present in human milk. These findings support the use of pigs as an ideal model for studies on human nutrition, not only because of the striking similarities in intestinal, immune system, and brain development but also due to their similarities in milk OS composition.
RD, CG, SJ, LA, AM, BB, and MC were involved in project conceptualization. CG, SJ, LA, and AM were involved in daily project operations and data collection. JS and DB were responsible for analytical procedures. All authors were involved in data analysis, interpretation, and have reviewed and approved this manuscript.
BB and MC are employees of Mead Johnson Pediatric Nutrition Institute. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to thank the University of Illinois Imported Swine Research Laboratory staff for their contributions to the preparation and follow-through of the animal phase of this study. We would also like to thank Diana Flanigan from Mead Johnson Nutrition Analytical Sciences for her role in sample preparation, storage, and handling.
This work was partially supported by the United States Department of Agriculture National Institute of Food and Agriculture, Hatch project 1009051. The USDA funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This project was also partially funded by Mead Johnson Pediatric Nutrition Institute.