10/3/2016

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Milk glycoproteomics: Preserving, enhancing, and 

delivering bioactivity 

Daniela Barile 1,3

Jaime Salcedo 1, Annabelle Le Parc 2, Adam Sun 2, Sercan Karav 1, Joshua Cohen 1, Juliana DeMoura Bell 1

1 Department of Food Science and Technology, University of California, Davis

2 Prolacta Bioscience, 757 Baldwin Park Blvd, City of Industry, CA

3Foods for Health Institute, University of California, Davis, 

Milk as a model for functional ingredients

2

Infant

Adult

Bioactive components retained and amplified over 120 million years of evolution

Dr. Bruce GermanDirector FFHI

Dr. David MillsDairy Shields Chair

Dr. Carlito LebrillaDistinguished Professor

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Proteins

Peptides

Oligosaccharides

Glycolipids

Glycoproteins

How to get there: deconstructing milk bioactivity

Measure..measure..measure! Variety of monosaccharides

Numerous linkages and branching

Complex mixture

Issues of overlapping peaks

Intense sample purification required

O

O

CH2OH

OH

OH

OH

O

O

CH2OH

OH

OH

OH

Chiral CentersConnection sites

Retention time

ISOMERS

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Use  multiple  Analytical  techniques• High performance anion exchange chromatography

• Quantification with standards

• Fraction collection

• nano‐LC‐Q‐ToF MS/MS• Full profile with fragmentation

• Include isomers separation

• MALDI‐ToF MS• Rapid profiling

• Identification of contaminants

Donor milk

• Increasing awareness of donor human milk benefits

• Expansion of human milk banks and industries collecting and processing 

human milk 

• Need to study the effect of thermal treatment on bioactive milk glycosylated 

compounds

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Previous findings from the literatureYear Author (et al.) Target Compunds # of samples Heat treatment Results

1996 TroyanoMonosaccharides & aminosugars

(Bovine milk)4 UHT Slight increase

2008 E. Bertino Oligosaccharides 10 Holder Pasteurization No Significant Change 

2011 J. Ewaschuck

Lactose 12‐15 Holder Pasteurization No Significant Change

IgA 1‐36 Holder Pasteurization 0%‐48% Reduction

IgG 9 Holder Pasteurization 34% Reduction

Lactoferrin 1‐22 Holder Pasteurization 57%‐80% Reduction

Lysozome Activity 1‐36 Holder Pasteurization 0%‐60% Reduction

2013 C. Marx Oligosaccharides 5 Holder Pasteurization No Significant Change

2015 J. Ding Lactose 3 Retort Processing No Significant Change

2016 C. Peila

Lactose 10 Holder Pasteurization No Significant Change

IgA 1‐36 Holder Pasteurization 20%‐62% Reduction

IgG 1‐36 Holder Pasteurization 23%‐100% Reduction

Lactoferrin 1‐22 Holder Pasteurization 35%‐90% Reduction

…what about other thermal treatments?

Is there detectable/quantifiable difference in milk bioactive components due to various heat treatments and/or 

storage conditions?

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Study design

Heat Treatment #2high temp‐short time(HTST) 72 °C 15 sec

Heat Treatment #3extended shelf life (ESL) 127 °C 5 sec

Heat Treatment #4ultra‐high temperature

(UHT) 140 °C 6 sec

Heat Treatment #1Holder Pasterur.63 oC 30min

Filling under sterile conditions

Non‐treated milk (baseline control)

20L of pooled human milk from 5 donors

Abundance and number of structures for:

Human Milk OligosaccharidesProtein  N‐glycosylation

GlycolipidsNano LC Chip Q ToFBarile Lab

Sterile product outlet automatic filling control 

(MicroThermics)

UHT/HTST hybrid continuous pasteurizer

(MicroThermics)

Donor milk from Prolacta Bioscience Thawing & pooling

Investigate thermal effect on milk glycosylated components

triplicates

Mat & Methodsand Results

HMO

N‐GLYCANS

GLYCOLIPIDS

Dr. Adam Sun

Dr. Salcedo

Dr. LeParc

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Human Milk Oligosaccharides

D-Galactose

D-Glucose

N-acetyl-glucosamine

L-Fucose

Sialic acid

5 building blocks

Many possible linkages

TERMI

NAL

POSI

TI

ON

Human Milk Oligosaccharides Functions

Development of nervous system

Selective prebiotics

Decrease hypersensitivity 

reactionsGut barrier function

Antibacterial and antiviral

Immune system activation

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HMO isolation and analysis100 L milk + 100 L H2O

Fat removal(Centrifugation 4000rpm,30min,4°C)

Lower layer(lipids)

Folch extraction (CHCl3/MeOH 2:1) 

Pellet Upper layer(lipids)Supernatant

(Proteins, HMOs…)

Upper layer

(proteins, HMOs…)

Protein precipitation: EtOH (‐20C, 1h) + centrifugation

Pellet(proteins) Supernatant

(HMOs, salts….)

Reduction and solid phase extraction

Neutral Fucosylated

Neutral

Sialylated Fucosylated

Sialylated

HMO profile of the raw milk (control)

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49%

34%

10%

5%

OS Relative Abundance (%) by type

65 individual structures monitored for abundanceIn the Control and in the 4 heat‐treated samples

………………….

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Mat & Methods

and Results

HMO

N‐GLYCANS

GLYCOLIPIDS

Dr. Karav

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N‐glycans & health

Protein structure and function (antioxidant, antibacterial and antiviral activities, binding of iron, and immunomodulation)

Protein structure and function 

Selective prebioticactivity

Inhibition of pathogens and immunomodulation

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N‐glycan release: classical method

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Lipid Removal 

Lipids

Proteins, free oligosaccharides

pellet

Centrifuge30min 4 0C

Protein Precipitation

1V Milk + 4V cold ethanol

Enzymatic Release

Released N‐glycanspurification

Characterization bymass spectrometry

Ethanol precipitation+PGC

PNGase F

90 C

Sample heating for protein denaturation is required for 

PNGaseF

(it would mask the effect of thermal treatment)

N‐glycan release  optimization

Initial discovery  byDr. Daniel Garrido, (Dr. Mills’ Lab)

• Recombinant enzyme Endo BI‐1• Isolated from B. longum subsp. infantis• Active on native glycoproteins • No need for protein denaturation• Simulates N‐glycans accessibility in the gut of healthy breast‐fed infants

OptimizationBy Dr. Karav

(Mills & Barile Lab)

(Endo BI‐1)

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Modified protocol for N‐glycan Release

Lipid Removal 

Lipids

Raw Milk

Proteins, free oligosaccharides

Caseins

Centrifuge30min 4 0C

Protein Precipitation

HMO Removal

1V Milk + 4V cold ethanol

10 kDAmembrane separates proteins from HMOs

HMO

Proteins

Enzymatic Release: EndoBI‐1Glycan CollectionCharacterization byNano LC Chip QTOF

Ethanol precipitation+PGC

37 ᴼC

Glycan Diversity (Number/type of structures)  

Relative Abundance of all structures

N‐glycans analysis

Glycosidase

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N‐glycans abundance: comparing control, HT1, HT2

HT 1:     63 °C   30 min HT 2:      72 °C  15 sec

N‐glycans abundance:comparing control, HT3, HT4

HT 3: 127 °C    15 min HT 4: 140 °C   6 sec

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Mat & Methodsand Results

HMO

N‐GLYCANS

GLYCOLIPIDSDr. Salcedo

Glycolipids

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R

OH OH

NH

R

O

-O-Glucose -Galactose -Sia

Ceramide

Fatty acid(18-24 C)

Sphingoid base (16-20 C)

Oligosaccharide chain

Glycolipids: Glycosphingolipids containing at least one Sia moiety in the structure

> 100 compounds

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Glycolipids

Pathogens

inhibition

Decoy

Prevent infection

Prebioticactivity

bifidobacteria

Activate T cellsand Th1-Th2lymphocytes

Anti-inflammatoryeffects

Immuneresponse

Mielin stability

Nervous tissueregeneration

Neuronal development

Glycolipids and health

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Crude glycolipid extract

Clean up

C‐8 cartridges

N‐omega‐ CD3‐octadecanoyl GM3

(internal standard)

1.5 mL sample

Lipid extract Glycolipid extract

Lipid extract

+

MetOH:CHCl3:H2O(8:4:2)

+Glycolipid extraction

UPLC‐QQQ

Lebrilla’s lab

Glycolipid  extraction & purification

MetOH:H2O(2:1)

Glycolipid extract

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Total HMO abundance Glycolipids are stable Results

N‐glycans

Translation: Bovine Milk Oligosaccharides

Monitor oligosaccharides enrichment at the pilot scale from whey permeate 

Dr. JulianaDeMoura Bell

Josh Cohen(see Poster) 

10/3/2016

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What happens to oligosaccharides

during spray dry & storage?

As an emerging functional ingredient, 

there is a need to understand the

chemical reactivity of this ingredient 

within the dairy matrix

http://www.snexportgroup.com/

structural modifications

?

REDUCEDSOLUBILITY

?

Bioavailability?

Degradation products?

?

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http://www.gea.com/

PROBLEMATIC QUANTIFICATION

Good resolution& QUANTIFICATION

Maillard reaction

• Leads to the decrease in nutritional value of proteins and formation of brown compounds in milk

• Loss of essential amino acids

• Amadori Products can be measured by a number of chromatographic and enzymatic techniques (assess the reaction stage)

Reaction between reducing sugars and amino acids

Accelerated during heat treatment.

Continue to develop during long storage (RT) Louis-CamilleMaillard, 1916

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• Evaluate the formation of Amadori Products involving BMO

• Assess the nutritional impact of the reaction in infant formulaenriched with BMO

Prof. Vincenzo Fogliano

• Chair of Food Quality Design• Former President of the International

Maillard Reaction Society (IMARS) 

O

OH

O

NH2acrylamide

OO

glyoxal

O

O

methylglyoxal

O

H

HO

H

HO

H

OHOHH

H

OH

H2NH2N O

OH

lysine

aldosesugar

+amino

compoundN-subsituted glycosylamine + H2O

Amadori rearrangement

1-amino-1deoxy-2-ketose

- 3 H2O

Schiff base of HMFor furfural

- amino compound+ H2O

-2 H2O

reductones

+ -amino acidsStrecker degradation

-2H +2H

CO2

dehydroreductonesHMF or furfural

+ amino compound

aldimines

with or without amino compound

aldols and N-freepolymers

+ amino compoundaldimines orketimines

aldehyde

+ amino compound

aldimines

+

fissionproducts(acetol,

pyruvaldehyde,diacetyl etc)

O

2-methylbutanal

MelanoidinsBrown nitrogenous polymers and copolymersstyrene

O

HO

acetic acid

N

N

pyrazine

NH

pyrroline

Initial stage

Intermediate stage

Advanced stage

HMF

O

O

2,3-Butanedione

Maillard reaction: Hodge schema

Hodge; J.Agric Food Chem, 1953

BMO ?

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BMO Degradation and  Amadori Products formationBMO Degradation and  Amadori Products formation

Air forced circulating oven 

Exactive OrbitrapHRMS system 

Commercial Infant Formula

Same infant Formula Enriched with BMO

( UC‐Davis MPL)

BMO Model System

( Pure Standards + amino acids )

Oligosaccharidesaminoacids &

Amadori Products 

Thermal treatment Quantification

Whirlpool, 900W M420 Double Emission System)

Personal Compound Database and Library 

Many fucosylated oligosaccharides (albeit in low abundance)

2016

Exp. spectra comparison with spectral library produces a match score allowing rapid & confident compound identification

Dr. Otter 

10/3/2016

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Assembling more tools to achieve the full analytical characterization of bioactive glycosylated compounds in human milk and dairy products…

quoteaddicts.com

Disclosure: I am co-founder of a start-up company (Evolve BioSystems) that studies probiotics and prebiotics

The Peter J. Shields  Endowed Chair in Dairy Food ScienceThe Peter J. Shields  Endowed Chair in Dairy Food Science

10/3/2016

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Thank you for your attention

Speed Bump by Dave Coverly