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