glycans from dairy coproducts Capturing the whey glycome Joshua L Cohen a Sercan Karav a Juliana MLN de Moura Bell a David A Mills ab Daniela Barile ab a Department ID: 530231
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Pilot scale isolation of bioactive glycans from dairy co-productsCapturing the whey glycome
Joshua L Cohena, Sercan Karava, Juliana MLN de Moura Bella, David A Millsa,b, Daniela Barilea,b aDepartment of Food Science and TechnologybFoods for Health InstituteUniversity of California, Davis. One Shields Ave, Davis CA 95616Slide2
Why we care about oligosaccharidesPotent bioactive compounds in milkLaundry list of biological functionalitiesWe don’t have a reliable source for human use or even to fully understand their complete suite of functions in clinical trialsSlide3
Bioactive glycans
Free
glycans
(oligosaccharides)
Conjugated
glycans
Glycolipids
GlycoproteinsSlide4
Sources of bioactive oligosaccharidesSlide5
Global whey production is ~200 million tonnes annually*
*As of 2013, Tetra Pak® Dairy Processing Handbook
Dairy co-products
Whey permeate
Cohen et al 2015, in
Agricultural Wastes: Types, Characteristics, and ManagementSlide6
Isolating milk oligosaccharides
Bovine milk oligosaccharides are typically low in concentration(0.2 – 1 g/L)It takes a lot of steps and equipment to isolate oligosaccharidesUC Davis Milk Processing LaboratorySlide7
Nanofiltration for BMO isolationSemi-permeable membraneShould retain oligosaccharides, allow everything else to pass throughComplicated process, as there are many membrane chemistries available with many propertiesRC, PES, SPES, PA, PS, SPS, CA, etc.
Range of MWCO values, but not absoluteSlide8
Our target oligosaccharides and lactose are very close in molecular weight
Lactose – 342 DaOligosaccharides – >600 Da
Concentrating whey permeate concentrates both oligosaccharides and lactose
Concentrated whey permeate
Whey permeate before processing
Lactose
Lactose
Oligosaccharide isolation using membrane filtration
BMO
BMO
Use of
β
-galactosidase (lactase) to cleave lactose into glucose and galactose
Slide9
Pressure
Nanofiltration
for BMO isolation
Membrane surface
Large molecules are retained by membrane
Smaller molecules permeate membrane, favoring monosaccharide elimination
Oligosaccharides
MonosaccharidesSlide10
Recovering BMO at pilot scaleNanofiltration
with diafiltrationInvestigate how operating parameters affect BMO and monosaccharide retentionUse nanofiltration and diafiltration to remove monosaccharides and salts by “washing”Use NF to concentrate BMOFermentation and nanofiltrationUse Saccharomyces cerevisiae to ferment glucose and galactoseMicrofiltration to remove yeast cells and colloidal material for clarificationUse NF to concentrate BMO
Enzymatic lactose hydrolysis
Prof. Juliana Bell
de Moura Bell et al 2016,
J Dairy
SciSlide11
How does
diafiltration improve purity?
HYDROSTATIC
PRESSURE
Semi-permeable membraneSlide12
Transmembrane pressure and pH affect glucose retention
Low glucose retention is favorableCohen et al (in final review)Slide13
Yields using membrane filtration and diafiltrationHigh BMO retention
High monosaccharide eliminationCohen et al (in final review)Slide14
Our glycan analysis platformnano-LC Q-TOF MS/MSFull profile with fragmentationNo absolute quantification
High performance anion exchange chromatography (HPAEC-PAD)Accurate quantification with standardsNo structural data without MSSample
Standard
Aldredge
et al, 2013.
GlycobiologySlide15
Oligosaccharide diversity
Hexose_HexNAc_Fucose_NeuAc_NeuGc>20 unique compositionsCohen et al (in final review)Slide16
We have greatly improved isolation of free bovine milk oligosaccharides, with high oligosaccharide purityHowever, there are glycans “locked up” in whey proteins
Next-generation prebioticsCAPTURING THE WHEY GLYCOMESlide17
Isolated from commensal bacteriumBifidobacterium longum subsp.
infantis ATCC 15697Endo BI-1Active on native glycoproteinsHeat stableGarrido et al, 2012. Molecular & Cellular ProteomicsRecombinant endo-β-N-acetylglucosaminidase
Deglycosylation after 95 °C for 5 min
Prof. David MillsSlide18
Protein
Molecular weight (kDa)Concentration in milk (g/kg)Type of glycosylationGlycomacropeptide71.5 g/L in wheyO-linkedLactoferrin
86
0.1
N-linked
Transferrin
76
0.01-0.03
N-linked
IgG
150
1.8
N-linked
IgA
385
0.4N-linkedIgM9000.2N-linkedOsteopontin600.018O-linkedɑ-lactalbumin14.21.2N-linked
Key whey glycoproteinsSlide19
Released Milk GlycansHuman Milk Oligosaccharides
Time (h)OD600nmB. infantisB. lactisB. infantisB. lactisKarav et al 2016 Applied and Environmental Microbiology
Sercan Karav
Next generation prebioticsSlide20
Who’s eating what?
Nano LC Chip Q-TOF analysis of N-glycans from bovine whey concentrateKarav et al 2016 Applied and Environmental Microbiology Slide21
Capturing the whey glycome
Ultrafiltration
BMO isolation
Enzymatic
deglycosylation
Released
glycansSlide22
We immobilize enzymes for several reasons:
Stabilize for pH and temperature Reuse of catalystHelps to separate enzyme from reaction mixtureAllows for multiple reaction configurations
http://www.abpischools.org.uk/
Wang and Uchiyama (2013)
Covalent binding
Adsorption
Entrapment
Work in progress: enzyme immobilizationSlide23
Barile Lab members
Prof. Juliana Bell
AcknowledgementsSlide24
Barile Lab members
Prof. Juliana Bell
Acknowledgements