Protein Purification ‐
Separation of Egg Components
Proteins of Egg White
Ovotransferrin • A major avian egg white protein (12-15% of total egg
white protein)
• A monomeric glycoprotein consists of a 686-residue
single polypeptide chain. Molecular mass: ~ 78 kDa
• pI: 6.73 for apo-form (iron-free), 5.78 for holo-form
• Iron binding protein (reversible, two iron atoms/molecule,
as Fe3+).
• Metal-free (apo) form is easily destroyed by physical and
chemical treatments, while holo-form (iron bound) is a
salmon-pink colored
• Iron-complex is stable to proteolytic hydrolysis and
thermal denaturation.
Important Characteristics for Ovotransferrin Separation
• The release of Fe3+ from ferric transferrin requires the
presence of a simple anion such as pyrophosphate,
sulfate, and chloride
• Ovotransferrin is the most heat liable protein in egg white
– Forms aggregation by heating at 60 C
– Iron-binding increases the chemical and heat
resistance of ovotransferrin
• pH is important for iron-binding
• Bicarbonate is essential for iron binding
– Anion is needed for binding and release of iron
Ovotransferrin Separation Methods
•
Precipitation of ovalbumin by shaking
•
Ammonium sulfate precipitation
•
Ion exchange chromatography
– CM‐Cellulose cation exchange
– DEAE‐cellulose anion exchange
– Q‐Sepharose Fast Flow column
– Duolite C464 and C476) chromatography for industrial‐scale production •
Immobilized metal affinity chromatography (a copper‐loaded Sepharose 6B column) from undiluted, blended egg white •
A bi‐functional dye‐ligand chromatography – DEAE Affi‐Gel Blue, as the first step to fractionate egg white proteins. – Fast flow liquid chromatography for purification
•
A two‐step chromatographic procedure – gel permeation on a Superose‐6 Prep Grade column
– anion‐exchange chromatography on a Q‐Superose Fast Flow
Molecular formulas of cellulose-based
ion exchangers
Ion – exchange Chromatography
New Separation Goals
• Should fit for use in human foods
– Selection of solvent or chemicals are very important
• Scale‐up capability
• Simple and economical
– Minimize sample volumes
• Maintain antimicrobial capabilities
– Iron‐free form has stronger antimicrobial capabilities
Binding of Iron to Ovotransferrin
• Requires 1 molecule of HCO3‐ per atom of Fe3+ • pH is important • Presence of anion is required
Egg white after diluting with water
After adding FeCl3 to saturate ovotransferrin in egg white
Precipitation of egg white proteins using ethanol ‐ 43% Effect of pH on iron binding capacity of ovotransferrin Ovotransferrin conc. (mg/ml)
a
a
a
a
12.0
10.0
8.0
6.0
b
4.0
b
2.0
0.0
4
5
6
7
pH
8
9
A bsorbance at 468 nm
Iron saturation of ovotransferrin in egg white solution
0.6
c
c
0.5
b
0.4
0.3
0.2
a
a
0.1
0.0
0.25
0.5
1
2
3
20 mM FeCl3.6H 2O (ml)/100ml egg white*
*2 x diluted egg white solution, pH 9.0
Effect of pH on the yield of ovotransferrin from 2x‐
diluted egg white solution pH 7 8 9 First extraction Conc. Vol. 9.40ab 61.3a 8.94b 61.7a 9.69a 62.3a Second extraction Conc. Vol. 2.48b 14.0ab 2.82b 13.7b 6.62a 14.3a Vol. x Conc.
total
Yield (%)
610.7ab 87.18
581.4b 83.07
671.5a 95.88 The solution obtained from the first extraction with 43% ethanol. Conc: mg/ml; vol: ml
*The yields were calculated based on theoretical value (7.0 mg/ml) of ovotransferrin in 2x‐
diluted egg white solution. The yields of each supernatant obtained by adding 43% ethanol to egg white solution
Extraction Concentration (mg/ml) Volume
(ml) Vol. x Conc. (mg)
Yield*
(%) First 11.99 43.95 527.30 75.33
Second 7.94 22.32 170.56 24.37
First Extraction: 43% ethanol extraction and then 59% ethanol precipitation. Second extraction: 64% ethanol was used to precipitate ovotransferrin. *The yields were calculated based on theoretical value (700 mg) of ovotransferrin in 100 ml of 2x‐diluted egg white solution.
Effect of ethanol concentration on the extraction of holo‐OTF from egg white
43% ethanol
Ovotransferrin
Ovoalbumin
1 2 3 4 5 6 7 8 9
Lane 1: 2x‐diluted egg white
Lanes 2~5: supernatants of 20%, 33%, 43% and 50% of ethanol extraction
lanes 6~9: precipitates of 20%, 33%, 43%, and 50% of ethanol extraction. Precipitation of extracted holo-OTF using ethanol
59% ethanol ppt
59% ethanol supernatant
Ovotransferrin
Ovoalbumin
1 2 3 4 5 6 7 8 9
Lane 1: diluted egg white,
Lanes 2 ~ 5: precipitates by 53%, 56%, 59% and 62% ethanol
extraction
Lanes 6~9: supernatant by 53%, 56%, 59%, and 62% ethanol
extraction
Why should lactoferrin be activated?
http://www.activinlf.com/activinlf/ActivatedLactoferrinTech/alt_whatis.htm
Activation of Ovotransferrin
• Removal of iron from holo‐ovotransferrin
– Bicarbonate is essential
– A simple anion such as pyrophosphate, sulfate, and chloride is required in vitro
– The anion‐induced Fe3+ release is closely related to the opening of a domain in either lobe
– pH: citrate facilitate the release of iron
• Removal of iron from ovotransferrin solution
– Chelex
– AG ®1‐X2 resin (chloride form) Antibacterial activity against E. coli
¥
: Free ovotransferrin + 50 mM NaHCO3
¥ O : Ovotransferrin immobilized on
Spharose 4B + 50 mM NaHCO3
¥ 5 mg/ml of ovotransferrin
¥ Higher bacteriostatic activity of matrixbound ovotransferrin might be ascribed to
higher stability of matrix-bound protein
Antimicriobial Agents and Chemotheraphy 1982. 21(5): 840-841
Iron Removal from Holo‐Ovotransferrin
Residual iron (ppm)
4.0
a
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
ab
b
a
a
c
c
a
b
b
0.1
0.2
0.3
0.6
0.9
AG1-X 2 resin (g)/ 100 ml solution *
- Residual irons were determined by Ferrozine test
- OTF concentration of the solution was around 6.5 mg/ml
first
Second
The influence of citrate on the residual iron in ovotransferrin solution
Treatment Protein (mg/ml)
Ovotransferrin (mg/ml) Residual iron (ppm) Without
dialysis 8.59 8.00 0.52
Dialysis 8.57 8.42 0.53
Residual iron: The amount of iron remaining in the solution after iron removal. Protocol for Ovotransferrin Purification
Dilution of egg white with 1 volume distilled water Add NaHCO3, NaCl, FeCl3 and adjust pH to 9.0
Stir for 30 min, add ethanol (43%, final), and centrifuge at 3,220 x g for 20 min
Supernatant (1st) Precipitate
Re‐extract with 43% ethanol Centrifugation at 3,220 x g, 20min
Supernatant (2nd)
Purification method‐continued
Add ethanol (59%, final) and centrifuge
Dissolve precipitant with distilled water pH adjustment to 4.7 with 50 mM citric acid
Add 0.6 g of AG ®1‐X2 resin to 100 ml holo‐ovotransferrin solution
Stir for 1 hr and filter through Whatman #1 paper
Apo‐ovotransferrin
Freeze dry The yield of final ovotransferrin using the developed method Sample Conc. (mg/ml) Vol. (ml) Vol. x Conc. (mg) Yield (%)
Egg white 7.00 100 700 100 Final solution 6.36 108 693
99.03
The theoretical concentration of ovotransferrin in 2x‐diluted egg white solution used was 7 mg/ml. Final solution: Apo‐ovotransferrin solution produced after iron removal. Purity of ovotransferrin
200 kD
97 kD
66 kD
OTF
45 kD
ovalbumin
1 2 3 4 5 6
Lane 1: marker
3: 2x diluted egg white
4: Supernatant after ethanol extraction
5: Supernatant after precipitation of holo-OTF
6: Precipitant
Summary
• Dilution of egg white and iron saturation of apo‐
ovotransferrin to holo‐form using FeCl3 • Extraction of holo‐ovotransferrin by precipitating egg white proteins with 43% ethanol
• Precipitation of extracted ovotransferrin with 59% ethanol
• Irons removal from holo‐OTF using AG1‐X2 resin
• Freeze drying of ovotransferrin
Advantages of the Developed Protocol
•
•
•
•
Can be scaled‐up easily Simple process
Compatible to use in human foods
Materials can be recycled Significance of using Ovotransferrin as an Antimicrobial Agent
• Prevent cross contamination
• Extend self-life of food products by inhibiting growth of foodborne
pathogen during processing or storage
• Developing an economical, large-scale preparation method of
ovotransferrin from egg white will increase the value and use of
egg
• High possibility of becoming a GRAS material like lactoferrin
• A natural antibacterial agent
• No addition of any hazard material during separation
Lysozyme
• Lysozyme constitutes approximately 3.5% of hen egg white • Egg white lysozyme consists of 129 amino acid residues with a MW of 14.4 kDa • Binds to ovomucin, transferrin or ovalbumin in egg white
• The thermal stability of lysozyme is partly due to its four disulfide bonds
• Highly stable in acidic solution and heating at 100 C for 1‐2 minutes
• In nature, found mainly as a monomer but also exist as a reversible dimer
• The dimeric form of lysozyme exhibits therapeutic, antiviral and anti‐inflammatory properties
Lysozyme
• Demonstrates antimicrobial activity against a limited spectrum of bacteria and fungi • Its enzyme activity can be enhanced by certain substances including •
•
•
•
EDTA
Butylparaben
Tripolyphosphate other naturally occurring antimicrobial agents
Methods of lysozyme isolation – Direct crystallisation and precipitation
(1) Crystallisation with 5% sodium chloride (2) Lysozyme recovery usually ranges from 60‐80% (3) Extremely time consuming and the residual egg white is too salty for further food application
(4) Ultrafiltration and diafiltration to desalt the remaining egg white material after lysozyme separation can reduce the salt content and the resultant egg white retains its foaming ability and foam stability Methods of lysozyme isolation – direct membrane filtration
• Only small amounts of lysozyme (20%) are able to pass through ultrafiltration membranes. • A 2‐step ultrafiltration process combining the high flux of the 50 kDa MWCO membrane with the high selectivity of the 25 kDa MWCO membrane is useful for the efficient purification of chicken egg white lysozyme
Methods of lysozyme isolation ‐ ion‐
exchange chromatography
• A cation exchange resin can be stirred with egg white until the bulk of the enzyme has been adsorbed. • Residual egg white is decanted or filtered off and used for food applications. • Amberlite, CM‐cellulose, CM‐Sephadex and Duolite have been successfully applied as cation exchangers employed a co‐
polymer of methacrylic acid and divinylbenzene (Duolite C‐
464) with a rigid macroporous structure for the recovery of lysozyme. • Freeze‐drying and spray‐drying can be successfully used to dehydrate liquid lysozyme preparations without loss of enzyme activity.
Egg white (mucin-free egg white, 2x dil)
ethanol (30%) , pH 5.8
Centrifugation 4000 rpm, 30 min
Supernatant
Precipitate Adjustment pH to 7.4)
adjust to 40% ethanol
Centrifugation
Cation exchange chromatography
Conalbumen
Washing
( Phosphate + Nacl)
Elution
(Phosphate+NaCl)
Lysozyme
Ion Exchange Chromatography (con’d)
Methods of lysozyme isolation ‐ Affinity chromatography
• Use glucochitin (deaminated chitin) or chitosan (deacylated chitin)
• Not adapted for large‐scale isolation of lysozyme
‐ high cost of affinity supports or ligands, ‐ poor flow rates of undiluted egg white, ‐ expensive and time‐consuming elution procedures ‐ non‐specific adsorption of other egg white proteins . • A dye affinity membrane system produced lysozyme with a purity of 88% and a recovery of 92%. Why lysozyme has been used as an antimicrobial agent in various foods
• Heat stable • Active in a broad range of temperatures (from 1 C to nearly 100 C) • Withstands boiling for 1‐2 min • Stable in freeze‐drying and thermal drying • Not inactivated by solvents,
• Maintains its activity when redissolved in water
• Has optimum activity at pH 5.3 to 6.4 (i.e. typical for low‐
acidic food) • The presence of other proteins in food, however, can reduce its stability by the formation of sulfide bridges
Avidin
• Avidin is a trace component (0.05%) of egg white
• A tetrameric, strongly basic glycoprotein protein
• Composed of subunits of identical amino acid composition and sequence (15.6 kDa and 128 amino acids each) • Combines with biotin to form a stable complex, which is incapable of absorption by the intestinal tracts of animals. • Avidin binds with 4 biotin molecules.
• The binding between biotin and avidin is so strong that separation requires heating the complex at 120 C for 15 minutes. • The high affinity constant of avidin for biotin has been widely used in molecular biology, affinity chromatography, molecular recognition and labelling, Enzyme Linked ImmunoSorbent Assay (ELISA), histochemistry and cytochemistry
Affinity Chromatography
•
•
Other proteins can be separated by this method based on their
affinity for specific groups or compounds.
Examples:
Antigen Antibody
Antibody
Antigen
Substrate
Enzyme
Concavalin A
Glycoprotein
Hormone
Binding Protein/Receptor
Lipid and Protein Components of Egg Yolk
Lipids (31%)
Neutral Lipids (65%)
Phospholipids (30%)
‐ PC (83%)
‐ PE (14%)
‐ Sphingomyelin (2.5%)
‐ Phosphatidylinositol (0.5%)
Cholesterol (5%)
Carotenoids (carotenes)
Xantophylls (lutein, zeaxanthin) Proteins (17%)
Lipovitellins (α‐ and β‐): 69%
‐ α‐ Lipovitellins: 58% ‐ β‐ Lipovitelins: 11%
Livetins: 12%
‐ α‐ livetin (serum albumin): 4%
‐ β‐ livetin (glycoprotein): 5%
‐ γ‐ livetin (γ‐globulin): 2%
Phosvitin: 7% apo Low‐density lipoproteins: 12%
PROTEINS AND LIPOPROTEINS IN YOLK –
Yolk can be separated to granules and plasma by high‐
speed centrifugation Plasma
• Make up of 78% of yolk.
• Composed of 49% moisture. • On a dry basis, plasma consists of 77‐81% lipid, 2.2% ash, 18% protein.
• Proteins in plasma are composed of livetin and LDL‐proteins
• Livetins (α‐, β‐, γ‐) are fat‐free globular proteins that represents 10.6% of the total yolk solids.
• LDL proteins contains 89% lipid which consists of 74% neutral lipid, 26% PL (75% PC, 18% PE, 8‐9% sphingomyelin and lysophospholipids) Granules
• Make up of 22% of yolk.
• Composed of 44% moisture. 34% lipids, 60% protein, 6% ash on a moisture‐free basis.
• Phospholipids (85% PC, 15% PE) makes up 37% of total lipid. • Proteins in granules are composed of 70% α‐ and β‐
lipovitellines, 16% phosvitin, and 12% low‐density lipoprotein.
• Phosvitin binds ferric iron • Lipovitellines contain about 20% lipids, which include 40% neutral lipids and 60% PL.
• LDL contains 84% lipid which consist of 3.7% cholesterol, 31% phospholipid, and 65% neutral lipid.
Separation of IgY
IgY
• The molecular mass of IgY is ~ 180 kD (light chain ~25 kilo Dalton [kD] each: heavy chain ~ 65‐68 [kD] each). • Yolk antibodies are highly resistant to acid and heat
– IgY lost little of antibody activity by incubation for 10 min at pH 7.2 at 60 °C or for 10 min at 40 °C above pH 4.0. – Antibody activity were significantly diminished by incubation above 65 °C or below pH 4.0. – 30‐50% (w/v) sucrose reduced the heat denaturation of IgY at 75‐
80 °C – The addition of high levels of sucrose, maltose, glycerol or glycine displayed effective additional protection against thermal denaturation of IgY.
IgY
• The major serum antibody in chicken is IgG, but IgG is also transported to egg in a manner similar to the placental transfer of IgG in mammals. • The protection of relatively immuno‐incompetent newly hatched chicks from pathogens is through the transmission of antibodies from the mother via the egg. • In the egg, chicken IgG is found mainly in egg yolk, and the concentration in egg white is very low. Methods studied for purification of IgY
ƒ Ammonium sulfate precipitation (Schwarzkopf and Thiele 1996)
ƒ Dextran sulfate precipitation (Jensenius 1981)
ƒ Polyethylene glycol (PEG) precipitation (Polson and Wechmar 1980)
ƒ Caprylic acid extraction (McLaren et al. 1994)
ƒ Propanol-aceton extraction (Bade and Stegemann 1984)
ƒ Affinity chromatography (Chen et al. 2002)
ƒ Ion exchange chromatography (Akita and Nakai 1992)
ƒ Ultrafiltration (Kim and Nakai 1996, 1998)
ƒ Gel filteration (Akita and Nakai 1992)
Other Studies
ƒ 40 ~ 70% loss of IgY by ultrafilteration (Akita and Li-chan1998)
ƒUse of 0.1% carageenan exhibited a 14% loss of IgY from the
doubly diluted yolk in natural pH (Chang, 2000)
ƒ Akita and Nakai (1992)
- pH 4.6-5.2 removed lipids and incubation at cold room
for 2 ~6 hrs made WSF clear
- 60% ammonium sulfate precipitation resulted in 89% IgY
recovery with 30% purity
- The best purification scheme was combination of salt
precipitation, ultrafilteration, and gel filteration,
resulting in over 100 mg of pure IgY production per egg
ƒ Ammonium sulfate precipitation (25 ~45%) provided high
purity and 58% recovery of IgY (Svendsen, 1995)
Final Purification steps for IgY
ƒ
Gel filtration: Devi et al. (2002)
ƒ
Ammonium sulfate fractionation: Svendsen et al. (1995)
ƒ
Ion exchange chromatography
‐ Cation exchange (CM‐Cellulose): Fitchtali et al. (1992, 1993) ‐ Anion exchange (DEAE‐Sephacel): Akita and Nakai (1992)
ƒ
Affinity chromatography ‐ Fe3+ affinity: Greene and Holt (1997)
‐ Immunoaffinity chromatography: Chen et al. (2002)
Separation of water soluble proteins ƒ Dilution of egg yolk
‐ 10 times cold phosphate buffer, 20 mM ‐ Adjustment of pH to 5.0 helps lipid removal and
lower ionic strength helps the aggregation of lipoproteins
‐ Refrigeration of egg yolk soln. before centrifugation ƒ Centrifugation ‐ at 2,800 x g, 40 min, 4oC ‐ water soluble protein extraction ‐ lipid precipitation
Removal of lipoproteins and Concentration ƒ
After centrifugation, 0.02% carageenan or 0.01%
charcoal is added to WSF following filteration with
Whatman No. 1 paper
ƒ
ƒ
Concentration of prepared WSF through ultrafiltration
Ultrafiltration
- Using Labscale TM TFF System with Pellicon XL
Biomax 50 membrane filter
- Cut-off size : 50 kDa
Final step of purification
•
Cation exchange chromatography or Ammonium
sulfate precipitation for production of high purity
IgY
• Dialysis : after ammonium sulfate precipitation
removal of remaining salt by dialysis
• ELISA : measuring activity of IgY
•
Electrophoresis : optical estimation of IgY purity
Effects of pH adjustment on diluted
egg yolk solution
Treatment
Lipid (%)
Turbidity*
Protein (%)
Control
1.0
2.97
0.41
pH to 5.0
0.08
0.33
0.51
* Turbidity: Abs. at 600 nm
Effect of temperature on extraction of WSF* by Centrifugation
Treatment
Supernatant
vol. (%)**
Turbidity
(A600)
IgY
(mg/ml)
Total IgY ***
(yield x IgY)
Control
77.03a
0.065a
1.11a
86.73a
Refrigeration
78.23a
0.040b
1.12a
88.78a
Freezing
72.03b
0.014c
1.24a
90.05a
* means water soluble proteins obtained by centrifugation
after storage at different temp of diluted egg yolk soln.
** indicates the volume obtained after centrifugation
*** After centrifugation and diafilteration. The content of IgY was
measured through the ELISA method
Addition of Carrageenan to WSF % carrageenan
pH condition
0.4
0.3
0.3
Ig Y (m g /m l)
Ig Y ( m g /m l)
0.4
0.2
0.2
0.1
0.1
0.0
0
Control
0.01
0.02
0.05
0.1
0.2
Charcoal
pH 4
pH 5
pH 6
pH 7
Control : without carageenan
The best conditions selected for carageenan to WSF
- 0.02% concentration and pH 9.0
pH 8
pH 9
Charcoal treatment to WSF isolated egg yolk
3.0
2
Ig Y (m g /m l)
2.5
IgY (mg/ml)
pH Condition
2.0
1.5
1.0
Charcoal %
1.5
1
0.5
0.5
0
0.0
4.0
5.0
6.0
7.0
8.0
9.0
0.005
Best conditions for Charcoal to WSP
- pH 4.0 and 0.01% concentration
0.01
0.05
0.1
Conditions of Ammonium sulfate ppt and Cation exchange chromatography
40% Ammonium sulfate
Cation exchange chromatography
IgY (mg/ml)
7.0
6.0
I g Y (m g / m l )
6.0
5.0
4.0
5.0
3.0
4.0
2.0
3.0
1.0
2.0
0.0
4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 C*
pH
C* : total IgY amount before loading
Washing : 5 Vol. of 20 mM Citrate phosphate
buffer(CB), pH 4.8
Elution : 2 Vol. of 200 mM CB, pH 6.4
4.0
5.0
6.0
7.0
8.0
9.0
pH
Using 40% ammonium sulfate
At pH 9.0 : the best high IgY recovery
Comparison of purity on SDS page gel
Ammonium sulfate precipitaton
M A
1 2
3
4 5
200k
Cation ion exchange chromatography
1
2 3 4
5
6 7 8
9
IgY
45k
M : Marker
A : Chicken IgG
1 : Centrifugation
2 : Charcoal + Diafilteration
3 : Ultrafilteration
4 : Ammounium sulfate 1st ppt
5 : Ammounium sulfate 2nd ppt
1 : Loading, at pH 4.8 after Ultrafilteration
2~8 : Washing, 20mM CB buffer, pH 5.4
9 : Elution, 200 mM CB buffer, pH 6.4
Recovery of IgY
Purification step
Yield (%)
Water soluble fraction *
100
Charcoal and Diafilteration
103.47 ± 0.03
Ultrafiltration
80.02 ± 13.72
Ammonium sulfate (1st)
74.82 ± 12.57
Ammonium sulfate (2nd)
69.07 ± 7.58
Water soluble fraction of egg yolk was obtained from centrifugation
Conclusion
ƒ Developed more simple, efficient, rapid purification method for large‐scale IgY production from egg yolk.
ƒ Addition of 0.01%charcoal to water soluble fraction, pH 4.0, resulted in concentrating IgY and reduced attachment of IgY to ultrafiltration membrane.
ƒ IgY with 70% recovery and high purity was obtained by 40% ammonium sulfate precipitation at pH 9.0 and 0.01% charcoal addition.
Final preferred method for IgY
purification
Liquid egg yolk
10x dilution with phosphate buffer
pH adjustment to 5.0 with 1 N-HCl
Centrifugation (2,800 x g, 40 min, 4oC)
0.01% charcoal at pH 4.0 and
filteration with Whatman No. 1 paper
Ultrafiltration
40% Ammonium sulfate precipitation at pH 9.0
Dialysis
Phosvitin
• Phosvitin is a principal phosphoprotein present in egg yolk (approximately 16% of egg yolk proteins) • Contains ~10% phosphorus. Thus, phosvitin has an excellent metal (iron and calcium) binding capacity. • The calcium binding properties of phosvitin is influenced by pH. • The calcium binding capacities of native phosvitin were 20 mol Ca++/mol of phosvitin at pH 3.6 and 148 mol Ca++/mol phosvitin at pH 7.0 . • Phosvitin was capable of inhibiting lipid oxidation in phosphotidylcholine liposomes, muscle homogenates, and ground pork. Amino Acid Sequence of Egg Yolk Phosvitin
Phosvitin is a principal phosphoprotein present in egg yolk with
molecular mass of ~35 kD. Phosvitin contains ~10%
phosphorus.
O-
1
51
101
151
201
⏐
O = P – O⏐
O
⏐
AEFGTEPDAKTSSSSSSASSTATSSSSSSASSPNRKKPMDEEENDQVKQA
RNKDASSSSRSSKSSNSSKRSSSKSSNSSKRSSSSSSSSSSSSRSSSSSS
SSSSNSKSSSSSSKSSSSSSRSRSSSKSSSSSSSSSSSSSSKSSSSRSSS
SSSKSSSHHSHSHHSGHLNGSSSSSSSSRSVSHHSHEHHSGHLEDDSSSS
SSSSVLSKIWGRHEIYQ
123 phophoserines
↓
Metal Chelating Capability
Phosphorous Source
50
100
150
200
217
Use of Phosphopeptides
• Calcium supplement
– Soluble calcium phosphate formation
– Inhibiting the formation of insoluble calcium phosphates – CPP is currently sold as a calcium‐supplementing agent and mineral absorption enhancing agent in Japan and Sweden • Iron supplement
– Soluble iron phosphate formation
• Antioxidant
– Iron chelating effect
Separation strategies of phosvitin
•
Phosvitin is present in yolk granules where electron‐dense lipovitellin micells are attached to thread‐like phosvitin complexes (Chang et al., 1977; Radomski and Cook, 1964). •
The yolk granules are insoluble in hypotonic solution but can be disrupted at 1.71M NaCl solution (Chang et al., 1977). •
Phosvitin can be isolated from egg yolk by precipitating with a magnesium sulfate (Mecham and Olcott, 1949), and using chromatography (Connelly and Tarbosky, 1961)
•
Extracting phosvitin from lipid‐free egg yolk powder prepared with ethanol and hexane combination (1:3, Lesso and Nakai, 1995) •
Extracting phosvitin from liquid egg yolk with NaCl and then precipitating it with MgSO4 (Castellani et al., 2003). Separation of Lipids, Phospholipids and Proteins from Egg Yolk
Composition of egg yolk
Sample preparation
Moisture
Lipids
Protein
Ash
Whole yolk
48.7
33.2
16.6
1.5
Yolk after water
69.2
20.8
8.9
1.0
10.0
0.8
86.1
3.1
extraction (pH 5.0)
Yolk after lipid extraction
Selection of Solvents for Lipid Extraction from Yolk
1. Low toxicity: Essential for human consumption
2. Extractability: Lipid and phospholipids
3. Easy to handle
4. Price
5. Recycling
Characteristics of Solvents
• Neutral lipids are soluble in hexane and acetone
• Leithin: soluble in ethanol (1:12 vol), insoluble in acetone
• Cephalins: slightly soluble in ethanol, insoluble in acetone
• Cholesterol: soluble in ethanol (1.3 g/100 ml), soluble in hot ethanol (28 g/100 ml). • Sphinomyelins are freely soluble in hot alcohol.
Ethanol Extraction of Neutral Lipid and Phospholipids from Egg Yolk
• Ethanol at room temperature first, then 60 C, and finally at 79 C ethanol (84%, final concentration). • 95% of lipid extraction from liquid egg yolk. • Further fractionation using the cold temperature crystallization and then filtration.
• Yielded > 90% pure lecithin (Sim, 1994).
Extract of Egg Yolk with Acetone and Ethanol
TG
Std
TG
Chol
PE
PC
Actn
9 vol.
Actn
9 vol.
EtOH
9 vol.
EtOH
4 vol.
PE
PC
Ethanol and Hexane Extraction of Neutral Lipid and Phospholipids
• Water insoluble fraction of diluted egg yolk (67‐69% moisture content) • Mixing with 4 volumes of 100% ethanol yielded 87% and 9 volume produced 93% ethanol (final concentration)
• One ethanol extraction (5 vol.) produced mostly PC and PE, and little TG.
• Repeated (3‐5 times) extraction with ethanol or higher ethanol volumes extracted more neutral lipids
• Decrease in ethanol concentration (down to 71%) reduced the extractability. Extraction of Lipids from Egg Yolk
90% ethanol
81% ethanol
72% ethanol
63% ethanol
Further purification of phospholipids and neutral lipids
•
•
•
•
•
•
Hexane extraction (4 volumes, 3 times): Mostly neutral lipids and some phospholipids
Proteins were collected as the final component of water‐insoluble fraction of egg yolk. The ethanol and hexane extract of water‐insoluble fraction of egg yolk were dried using a rotary evaporator. The dried lipids were redissolved in 85% hot ethanol and then placed in a water bath to warm up. Lipid layers were separated using a separatory funnel. The lipid separated from the top was phospholipids ( > 95% purity), middle layer was neutral lipid ( > 95% purity), and bottom layers were phospholipids and neutral lipids mix.
Flow chart for large-scale isolation of lipids and protein from egg yolk
Yolk precipitant (69% moisture)
Extraction of phospholipids with 4 vol. of 100% ethanol
(87% ethanol, final concentration)
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Centrifugation at 2,800 x g, 15 min at 4oC Supernatant (Mainly PL) Drying with rotary evaporator (mostly phospholipids) Precipitant
(Neutral lipids and protein)
Hexane extraction (4 vol., 3x extraction)
(Protein residues are separated)
Drying lipid extracts
redissolving in ethanol Separation of PL and NL
Challenges
• The purity of the bottom layers were not very high and need further purification. • Removal of cholesterol from the phospholipids is necessary
• Removal of color: pigments such as xantophyll
(lutein, zeaxanthine) and carotene.
• Further fraction of phospholipids to PC, PE, sphingomyelin and phosphatidylinositol.