glycosylated bioactive compounds in porcine milk...
TRANSCRIPT
Glycosylated bioactive compounds in porcine milk:Identification & quantification during lactation
and correlation with fecal metagenome
J. Salcedo1,S.A. Frese1, A. Mudd2, M. Chichlowsky3, B. Berg3, R.Dilger2, D.A, Mills1,
D. Barile1
Davis, 28th September 2016
1Food Science and Technology, UC Davis2Department of Animal Sciences, U. Illinois
3Mead Johnson Pediatric Nutrition Institute
Human milk importance
Human milk composition
Colostrum: 10-15 g/LMature milk: 5-10 g/L
3
4
5
67
8
Adapted from Petherick (2010) Nature
Oligosaccharides functionality
ten Bruggencate et al.(2014) Nutrition Reviews
Oligosaccharides composition
150 Different compounds
Animal sources
DSLNT
β1-4
β1-3
β1-3α2-3
α2-6
LNFP-V
α1-3
β1-3
β1-3
Neutral OS Fucosylated OS
Acidic OS
Neu5Ac Neu5Ac/Neu5Gc
Oligosaccharides in mammals
Human milk
Bovine milk
Highest diversity in linkages and structures Composition:
- Neutral with/without fucose OS 60-80%;- Acidic OS 10-20%
Concentration: 10-15 g/L in colostrum; 5-10 g/L mature milk
Lower number of structures (50-80)
Lower diversity of linkages compared to human milk (type I OS)
Composition: neutral OS 60-80%, acidic OS 20-35%, fucosylated OS 0-1.5%
Concentration: 1-1.5 g/L in colostrum; 0.1-0.3 g/L mature milk
The pig as a model
Cheap Little experience neededGnotobiotic animals
Intestinal and immune system not similar to human Neonatal pig has striking similarities in anatomy, physiology, immunology and metabolism with the human neonate.
Bassols et al. (2014) Proteomics Clinical Applications; Roura et al. (2016) Nutrition Research Reviews
Rat milk OS far in composition and concentration to human milk (Hallam et al. (2014) Obesity)
Aim of the study
Characterize and quantify the oligosaccharides in porcine milk over lactation and correlate with the microbiome
Study design
Part 1: Oligosaccharide characterization & quantification in sow milk
Part 2: Effect of milk oligosaccharides in shaping the gut microbiome of suckling piglets
Day 0 - 19
Characterization of the oligosaccharide content in porcine milk
Study 1
M. ChichlowskyA. T. MuddR. N. Dilger B. Berg
Study design: Porcine milk OS characterization
BirthDay 7 Day 186 Hours
Colostrum Mid lactation Late lactation
Seven multiparous Yorkshire sows
Ad libitum diet
(x7) (x7) (x7)
Analysis
-20°C
OS profile: nano LC-Chip QToF MS/MSOS quantification: HPAEC-PAD
Aqueous layer
4000rpm30min
4°C
SPE-PGC
Protein precipitation(EtOH, -20° C, 1h)
Sample preparation: OS identification
100 µL sample
100 µL H2O+
4000rpm,30min
4°C
Agilent nano-LC Chip QToF MS/MS
4000rpm,30min
4°C
Fat removal
Proteins removal
OS Clean up
Folch extraction(4 vol. CHCl3 : MetOH)
(2:1)
Martín-Ortiz et al.(2015) Journal of Chromatography A; Lee et al. (2016) J Agric Food Chem
( ) g p _
0 5 10 15 20 250 5 10 15 20 25
( ) g p _
( ) g p _
0 5 10 15 20 25
0 5 10 15 20 25
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
3.25
3.5
3.75
4
4.25
4.5
4.75
5
5.25
5.5
0 5 10 15 20 25 30 35 40
x 105
Retention Time (min) Retention Time (min) Retention Time (min) Retention Time (min)
Inte
nsity
(cps
)
Retention Time (min)
3-Hex2’-FL
3-Hex-1-HexNAc 3’-SL
Identification of OS present in porcine milk
Colostrum
0
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
+ Product Ion (19.387 min) (636.23472 -> **) Sow 3 day 0-2 MSMS.d
292.
1017
7
454.
1540
9
274.
0907
2
183.
0851
2
345.
1371
6
636.
2310
5
165.
0738
5
147.
0630
8
310.
1103
3
474.
1810
8
256.
0786
3
197.
0418
2
129.
0520
7
215.
0533
6
232.
0810
2
618.
2222
9
436.
1445
9
Counts vs. Mass-to-Charge (m/z)
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760
MS/MS Confirmation
Exact mass (expt) Exact mass
(calc)Retention time (min) Hex HexNAc Fuc Neu5Ac Neu5Gc
471.1616 471.1588 11.7 1 0 0 1 0488.1723 488.1742 14.37 2 0 1 0 0488.1737 488.1742 15.62 2 0 1 0 0504.1688 504.1691 16.45 3 0 0 0 0504.1690 504.1691 13.02 3 0 0 0 0504.1690 504.1691 13.38 3 0 0 0 0504.1692 504.1691 14.41 3 0 0 0 0504.1694 504.1691 12.4 3 0 0 0 0504.1696 504.1691 15.66 3 0 0 0 0529.1993 529.2007 13.73 1 1 1 0 0529.1994 529.2007 13.18 1 1 1 0 0545.1951 545.1956 15.7 2 1 0 0 0545.1953 545.1956 14.38 2 1 0 0 0545.1953 545.1956 13.96 2 1 0 0 0545.1953 545.1956 11.35 2 1 0 0 0545.1954 545.1956 12.05 2 1 0 0 0545.1954 545.1956 12.98 2 1 0 0 0633.2117 633.2116 23.37 2 0 0 1 0633.2118 633.2116 18.3 2 0 0 1 0666.2211 666.2219 2.42 4 0 0 0 0666.2212 666.2219 19.93 4 0 0 0 0666.2215 666.2219 12.72 4 0 0 0 0674.2368 674.2382 22.94 1 1 0 1 0674.2374 674.2382 16.77 1 1 0 1 0674.2377 674.2382 18.31 1 1 0 1 0691.2525 691.2535 12.97 2 1 1 0 0707.2477 707.2484 21.64 3 1 0 0 0707.2477 707.2484 15.23 3 1 0 0 0707.2478 707.2484 16 3 1 0 0 0707.2484 707.2484 17.14 3 1 0 0 0
Exact mass (expt)
Exact mass (calc)
Retention time (min) Hex HexNAc Fuc Neu5Ac Neu5Gc
748.2742 748.2750 15.28 2 2 0 0 0795.2641 795.2645 24.11 3 0 0 1 0795.2641 795.2645 25.19 3 0 0 1 0795.2644 795.2645 25.8 3 0 0 1 0836.2894 836.2910 14.96 2 1 0 1 0836.2899 836.2910 22.71 2 1 0 1 0836.2902 836.2910 13.79 2 1 0 1 0853.3067 853.3063 15.8 3 1 1 0 0869.3005 869.3012 27.54 4 1 0 0 0869.3012 869.3012 20.7 4 1 0 0 0869.3013 869.3012 19.69 4 1 0 0 0910.3274 910.3278 18.51 3 2 0 0 0910.3286 910.3278 19.39 3 2 0 0 0910.3288 910.3278 19.08 3 2 0 0 0998.3431 998.3438 24.73 3 1 0 1 0998.3448 998.3438 26.35 3 1 0 1 0998.3457 998.3438 28.69 3 1 0 1 01015.357 1015.3591 15.38 4 1 1 0 01031.352 1031.3541 22.8 5 1 0 0 01072.384 1072.3806 21.64 4 2 0 0 01113.409 1113.4072 20.12 3 3 0 0 01113.409 1113.4072 22.32 3 3 0 0 01152.379 1152.3803 17.83 7 0 0 0 01160.398 1160.3966 27.14 4 1 0 1 01160.398 1160.3966 27.54 4 1 0 1 01201.426 1201.4232 25.6 3 2 0 1 01363.478 1363.4760 28.93 4 2 0 1 01363.479 1363.4760 26.13 4 2 0 1 0
Unique compositions identified: 25Including isomers and anomers: 59
Neutral: 32 Fucosylated:7 Acidic: 20
Identification of OS present in porcine milk
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
0
1
0
2x 106
0
1
3
2
x 106
0.4
1.2
0.8
x 106
Retention time (min)
Inte
nsity
(cps
)
Day 0
Day 7
Day 18
3’-SL
Porcine milk OS characterization during lactation
Identification of OS present in porcine milk at day 0, day 7, day 18
0
15
30
45
Day 0 Day 7 Day 18
Abun
danc
e (%
)
Neutral OS
3 Hexose 3_1_0_0_0
0
5
10
15
20
Day 0 Day 7 Day 18
Abun
danc
e (%
)
Neutral fucosylated
2'-FL 2_0_2_0_0
0
10
20
30
Day 0 Day 7 Day 18
Abun
danc
e (%
)
Acidic OS
3'-SL 1_1_0_1_0
Variation of specific oligosaccharides during lactation
0.E+00
4.E+08
7.E+08
Day 0 Day 7 Day 18
Tota
l pea
k ar
ea O
S
Total OS
Significant increase in fucosylated OS with lactation
Sample preparation: OS quantification
Aqueous layerFolch extraction(4 vol. CHCl3 : MetOH)
(2:1)
4000rpm30min
4°C
Protein precipitation(EtOH, -20° C, 1h)
100 µL sample
100 µL H2O+
4000rpm,30min
4°C
HPAEC-PAD
4000rpm,30min
4°C
Eliminate fat
Eliminate proteins
Validated methodology for OS quantification
Kunz et al. (1998) Journal of Chromatography B; Lee et al. (2015) Journal of Dairy Science
OS quantification during lactationOligosaccharide Type
LNDFH Fucosylated
LDFT Fucosylated
2-FL Fucosylated
LNFP-I Fucosylated
LNT Neutral
LNnT Neutral
3 Hexose Neutral
2 Hexose-1 HexNAc Neutral
6’-SLN Acid
6’-SL Acid
3’-SL AcidColostrumDay 7Day 18
Porcine milk OS quantification during lactation
p < 0.05, HSD-Tukey Test
Most abundant OS: LNnH, 3’-SL Unique increase of 2’FL
Total amount of OS decrease with time: 225 mg/L in colostrum; ~100 mg/L later in lactation.
Neutral
Fucosylated
Acidic a
b
c
a
b
c
Agreement with oligosaccharide profile
Porcine milk OS characterization: conclusions
Porcine milk OS concentration decreases from 225 mg/L in colostrum to ~100 mg/L in mature milk. Lower than human milk and closer to bovine milk
Porcine milk OS diversity is similar to bovine milk, less complex than human milk
First time observing an increase in fucosylated OS
Although content is lower than bovine milk, OS are similar to human milk increase in fucosylation, type I OS (synthesis machinery in mammary gland)
Porcine milk is closer to human milk than bovine milkdespite its lower OS concentration
Effect of milk oligosaccharide shaping the gut microbiome in suckling piglets
Part 2
1 42
27 piglets
Analysis
Identification bacterial speciesMetagenomic sequencing
352114753
Nursing Weaning
Study design: Porcine milk OS and microbiome
D. A. MillsS. A. Frese
Sample preparation: microbiome identification
• Fecal samples collected and extracted by Zymo Research Fecal DNA kit
• Amplify portion of 16S ribosomal RNA gene, a ‘fingerprint’ for bacterial species
• Sequenced at UC Davis Genome Center
Sample preparation: metagenome analysis
Porcine milk OS and piglet microbiome
Colostrum and milk contain OS with sialic acid, N-AcetylHexosamineand fucose
We expect a shift in the piglet microbiome from bacteria able to consume complex oligosaccharides to bacteria consuming
starch and beta glucans
Nursing Weaning
Oat-based feedlacks sialic acid, N-AcetylHexosamine and fucose
Day 21
Nursing Weaning
METAGENOMICS
• Sialidades and Hexosaminidases found in Bacteroides
Correlating milk OS with piglet microbiome
• Fucose permeases found in Enterobacteriaceae
Frese et al. (2015) Microbiome
Conclusions
Porcine milk OS concentration decrease with time
Increased proportion of fucosylated OS during lactation
Ability to synthetize OS commonly present in human milk (Type I OS as LNT, 2´-FL)
Milk OS composition is reflected in the enzymatic potential of the gut metagenome (sialidase, fucosepermease)
Number of structures is lower than human milk
Ideal model for human nutrition
Neonatal pig has striking similarities in anatomy, physiology, immunology and metabolism with the human neonate.
Similarities with human milk OS composition
Salcedo et al. (2016) Journal of Dairy Science; Mudd et al. (2016) Frontiers in Nutrition
Acknowledgements
M. Chichlowsky
D. A. Mills
A. T. MuddR. N. Dilger B. Berg
S. A. Frese
D. Barile
Randall C. Robinson
Joshua C. Cohen
Tian Tian
Garn Bunyatratchata
Valerie Weinborn
Anisur Khan
Shalini Shankar
Mrittika Bhattacharya
Sercan Karav
Thank you for your attention!
Glycosylated bioactive compounds in porcine milk:Identification & quantification during lactation
and correlation with fecal metagenome
J. Salcedo1, S.A. Frese1, A.T. Mudd2, M. Chichlowsky3, B. Berg3, R.N. Dilger2, D.A, Mills1, D. Barile1
Davis, 28th September 2016
1Food Science and Technology, UC Davis2Department of Animal Sciences, U. Illinois
3Mead Johnson Pediatric Nutrition Institute
Late lactation
Three multiparous
Yorkshire sows
Colostrum Mid lactation
Day 7 Day 14Birth-1 Birth
Pre-colostrum
(x3) (x3) (x3) (x3)
Analysis
OS profile: nano LC-Chip QToF MS/MS
Porcine milk OS and piglet microbiome
6.102
46.36047.538
Day 0
26.411
23.411
50.178
Day 14
OS profile variation through lactation
Acidic OS decrease over lactation
Fucosylated OS increase over lactation