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TRANSCRIPT
Conjuga(on of Par(ally Hydrolyzed Whey Protein to Reduce Protein Allergenicity
Yuansheng Gong, Lei Shelly Xu, and John. A. Lucey
Department of Food Science University of Wisconsin-‐Madison.
11/10/15
Background: 1. Milk protein allergy is es(mated to affect about
300,000 children, approximately 2.5 percent of children under the age of 3 in US.
2. Methods to reduce protein allergenicity – Heat treatment/protein denatura(on – Enzyme hydrolysis – Subs(tute with another protein – Structural modifica(on (e.g., Conjuga7on of protein with
polysaccharide .)
Previous work at UW-‐Madison: • Novel (patent pending) method to prepare whey protein
conjugates1,2,3,4, which has greatly enhanced func(onality. • Conjugates diges(on using infant diges(on model5. • Carbohydrate diges(bility of conjugates6. • Impact of molecular weight of polysaccharide on IgE
binding capacity of conjugates7.
1. Zhu et al., J. Agric. Food Chem., 2008 2. Zhu et al., J. Agric. Food Chem., 2010 3. Allelein et al., J. Chromatogr., 2012 4. Bund et al., J. Chromatogr., 2012 5. Bö]ger et al., Food Dig., 2012 6. Gong et al., ADSA 2014 7. Xu et al., ADSA 2015
Dextran (DX) Conjugate (covalently linked protein-‐polysaccharide ingredient)
Protein
Hypothesis Whey protein hydrolysis in combina(on with protein conjuga(on will reduce allergenicity by enzyma(cally cleaving epitopes or blocking them with polysaccharides.
β-‐LG
IgE
X
Hydrolysis
Conjuga(on DX
ObjecMve To inves(gate the impact of DH (degree of hydrolysis) and conjuga(on of whey protein on IgE binding capacity.
Material and Methods
• Trypsin and Chymotrypsin (from Sigma) • WPI, 10kDa DX. To avoid using a severe heat treatment to inac(vate the enzymes used for protein hydrolysis, we immobilized the enzymes.
Concentration of enzyme, optimum pH and temperature on the hydrolysis of whey protein catalyzed by trypsin and chymotrypsin 1,2,3,4.
1. CUSTÓDIO, et. al. Alim. Nutr., Araraquara 2005, v 16, n. 2, p. 105-109, 2. Galvao, et.al. Journal of Food Engineering 2009, (91) 109–117 3. Galvao, et. al. Applied Biochemistry and Biotechnology 2001, Vol. 91–93, 4. Pahud, et. al. Journal of Pediatric Gastroenterology and Nutri@on, 1985, 4: 408-‐413
Concentra(on mg/L pH Temperature ℃
Hydrolysis procedure chart
WPI (Davisco Food Interna(onal Inc. ) → → ( 40g/L in H2O) → Adjust pH to 9.0
Trypsin and Chymotrypsin at 0.2 g/L.
↓
↓
Take 1 ml at 0, 10, 30, 60,120,180 ,240 min.
↓
Cooling down and store at 4 °C. →
SDS-‐PAGE and Other analysis
Incubate at 50 °C
↓ →
CharacterizaMon of WPI Hydrolyzates
• SDS-‐PAGE electrophoresis • Protein profile by HPLC • Protein content by BCA • Molecular weight by SEC-‐MALLS • Degree of hydrolysis (DH) by OPA
M WPI 30 60 90 120 180 240 min
170
130
95
72
56
43
34
26
17
11
Mw,kDa
Results SDS-‐PAGE of hydrolyzed WPI , catalyzed by immobilized trypsin and chymotrypsin at pH 9.0 , 50 °C.
Increasing degree of hydrolysis
Size exclusion chromatogram of WPI hydrolyzates (WPIH) Co
ncen
tra(
on ( volts)
Pep(des
-0.2
0.0
0.2
0.4
0.6
20 30 40 50
LS
, A
UX
(v
olts
)
Volume (mL)
Chromatograms
WPIH-30minH-180minH-240min
BSA
β-‐Lg
α-‐La
-‐-‐-‐ WPI -‐-‐-‐ 30 min of hydrolysis -‐-‐-‐ 180 min of hydrolysis -‐-‐-‐ 240 min of hydrolysis
Pep(des
Table 1. Effect of temperature on DH of WPI hydrolyzates (WPIH) a]er 240 min incubaMon
Table 2. Effect of Mme on DH, and Mw of WPIH at 50C
Temperature 40C 45C 50C DH (%) 19.7 22.4 32.2
Time (min) 0 30 120 180 240
DH (%) -‐-‐ 21.0 24.4 27.7 32.2
Mw (kDa) 25.9 15.7 11.2 9.1 8.8
ConjugaMon procedure
• 10% WPI or hydrolyzates were mixed with 30% Dextran.
• Heated at 62°C and pH 6.5 in a water bath for 24 h.
• Conjugates were not purified from this mixture.
IgE-‐binding capacity: Comparison of WPI and three different WPIH–DX Conjugates
A-‐C Different superscript represent significant differences among different samples (P<0.05). * Significant difference from conjugates within samples aoer treatment (P<0.05).
0
0.2
0.4
0.6
0.8
1
1.2
WPI WPIH-‐1 WPIH-‐2 WPIH-‐3
IgE bind
ing capa
city / W
PI
WPI/WPIH
WPI/WPIH+DX Mixture
WPI/WPIH-‐DX conjugates
DH 18.7% DH 22.5% DH 27.1%
* C
*
C
*A
* B
1.6%
Conclusion: • The combinaMon of conjugaMon and parMal enzymaMc hydrolysis significantly reduced IgE binding capacity of whey protein.
Acknowledgement
This project was funded by the Na(onal Ins(tute of Food and Agriculture of the U.S. Department of Agriculture (Award number 2011-‐67017-‐20097).
Lucey’s Lab. Members. UW-‐Hospital Dr. Gern’s Lab.
