Impact of enzymatic modification of whey proteins on their bioactive
properties
R.J. FitzGerald, A.B. Nongonierma and M.B. O’Keeffe
Department of Life Sciences and Food for Health Ireland (FHI), University of Limerick, Limerick, Ireland.
Bioactive peptides
•ACE inhibitory•Antioxidant
Satiatingpeptides
•Insulinotropic•DPP-IV inhibitors
Opioid peptides
Mineral binding
HypertensionObesity Diabetes Stress Bone health
Dyslipidemia
Hypocholesterolemic peptides
Metabolic Syndrome
Food Proteins
Fermentation In vitro hydrolysis Gastrointestinal digestion
Management and treatment of Type 2 diabetes
Lifestyle Diet Drugs Physical activity
Main issues:
1. Non-compliance to changes in lifestyle, exercise, drugs, etc.2. Side-effects of drugs3. Prohibitive cost of anti-diabetic drugs
Whey protein hydrolysates as a natural source of antidiabetic agents
Insulinotropic activity of a whey protein hydrolysate (WPH)
Gaudel et al. 2013, J. Nutr
Pancreatic BRIN BD11 beta cells
20 min in 16.7 mM Glucose buffer
Incubation with WPH*
Cell viability
Acute insulin secretion
*WPH: whey protein hydrolysate
Dose response insulinotropic effect of WPH in vitro
Krebs
WP 1m
g/ml
WPH 0.
01m
g/ml
WPH 0.
1mg/m
l
WPH 1m
g/ml
WPH 5m
g/ml
0
2
4
6
8
******
***
***
Insu
lin
(n
g/m
g p
rote
in)
In vitro insulinotropic response
***p<0.005 vs. WP
Antidiabetic whey protein hydrolysate
Ob/ob mouse
Better clearance of a glucose load in ob/ob mice with WPH
Glucose load 2 g kg-1 body weight
8 weeks administration of WPH*
(100 mg kg-1 body weight) by oral gavage
*p<0.05, **p<0.01, ***p<0.005 vs. control
0
10
20
30VehicleWPH
*****
Time (min)
Blo
od g
luco
se (
mm
ol/L
)
0
5
10
15
20
*
AU
C (
mm
ol/L
/min
)
ControlWPH
Control WPH
*WPH: whey protein hydrolysate; AUC: area under the curve Gaudel et al. 2013, J. Nutr
Mice group Glucose Insulin Ex vivo insulin mmolL-1 pmolL-1 fmolL-1islet -1
Control 16 ± 1a 2930 ± 164b 12 ± 2a
WPH 15 ± 1a 1500 ± 152a 29 ± 2b
In each column, figures with different superscript letters are significantly different (P< 0.05)
Pancreatic islets
20 min in 16.7mM glucose buffer
Acute insulin secretion
Blood Glucose & Insulin
Restoration of the insulin secretory function of pancreatic β-cells
Reduction of the plasma insulin concentration
Mechanism of action
Dipeptidyl peptidase IV (DPP-IV) inhibition?Gaudel et al. 2013, J. Nutr
DPP-IV inhibitory whey hydrolysates and peptides
DPP-IV inhibition is hydrolysate and peptide specific
Nongonierma & FitzGerald (2013a), Peptides
Compound DPP-IV IC50 (μM)* DPP-IV IC50 (mg.mL-1)*
IPI (diprotin A) 4.23 ± 0.08a 0.0015 ± 0.0001a
EK 3216.73 ± 2.12h 0.654 ± 0.001f
GL 2615.03 ± 612.80g,h 0.492 ± 0.115f
AL 882.13 ± 68.66f 0.178 ± 0.014e
VA 168.24 ± 7.96d 0.032 ± 0.001b
WV 65.69 ± 2.95b 0.020 ± 0.001b
FL 399.58 ± 10.81e 0.111 ± 0.003d
HL 143.19 ± 0.35c 0.038 ± 0.001c
SL 2517.08 ± 36.33g 0.549 ± 0.008g
LFH1 na 1.088 ± 0.106h,i
WPH1 na 1.430 ± 0.272h
WPH2 na 0.999 ± 0.077h
*Half maximal inhibitory concentrations (IC50)Na: not applicable; Figures with different superscript letter are significantly different P < 0.05
Predictive model for milk protein ranking
Nongonierma & FitzGerald (2014), Food Chem
Known DPP-IV inhibitors with an IC50 < 2000 µM
Algorithm
MW
nIC
PIi
correctedii
,
,50
1
Inhibitory potency index (PI)
Occurrence in whey proteins
Predictive model for milk protein ranking
Nongonierma & FitzGerald (2014), Food Chem
ProteinProtein
coverage (%)
Pcorrected (%)
# peptide sequences
PI (×106 (μM-1 g-1)
α-la 43.9 34.1 8 5.66
β-lg 34.0 25.9 8 3.27
LF 11.6 11.6 18 2.67
BSA 7.9 7.9 10 0.93
Whey proteins are a good source of DPP-IV inhibitory peptides
Food-drug interaction with Sitagliptin
Nongonierma & FitzGerald (2013b), Int Dairy J
Additive effect of whey hydrolysate with Sitagliptin
Compound
DPP-IV IC501 Apparent DPP-IV IC50
(0.006 ng.mL-1 Sitagliptin)1
IC50 reduction (%)2,3
(mg.mL-1) (mg.mL-1)
IPI 0.001454± 0.000218a 0.000701 ± 0.000013a 51.8*
WV 0.020 ± 0.001b 0.012 ± 0.001b 39.4*
VA 0.032 ± 0.001b 0.025 ± 0.001c 21.0*
WPH 1.33 ± 0.17c 1.149 ± 0.051d 13.6*1Values represent mean IC50 values ± confidence interval (P = 0.05) for triplicate determination (n=3). Within the same column, values with different superscript letters are significantly different (P < 0.05)
2
3 *P < 0.05 vs DPP-IV inhibition determined with diprotin A, Trp-Val, Val-Ala or WPH alone.
1001 aloneinhibitor tocompared ICapparent in Reduction 50
5050
IC
nsitagliptiwithICApparent
Antioxidative (AO) Peptides
Power et al., (2013), Amino Acids
Oxidative stress Antioxidant defence
Oxidative stress
Development of AO peptides from whey protein hydrolysates
Oxidants modify proteins, carbohydrates, lipids & nucleic acids
Oxidative stress metabolic syndrome (cardiovascular disease & diabetes)
ORAC activity of whey protein hydrolysates
O’Keeffe & FitzGerald (2014), Int. Dairy J.
ORAC: oxygen radical absorbance capacity;Alc: Alcalase; Neu: Neutrase; Cor: Corolase PP; Fla: Flavourzyme
5 kDa permeates 1 kDa permeates
Higher ORAC activities for hydrolysates & low molecular mass peptides
***: p<0.001 vs. WPC
HUVECs in culture
Glutathione increased on incubation with WPHs, differing effects of fractionation
HUVECs incubated with WPHs or media alone (vehicle) for 48 h
WPH: whey protein hydrolysate; HUVEC: human umbilical vein endothelial cells
Glutathione Catalase Microarray
O’Keeffe & FitzGerald (2014), Int. Dairy J.
5 kDa permeates 1 kDa permeates
*: p<0.05, **: p<0.01,***: p<0.001 vs. vehicle
HUVECs incubated with WPHs or media alone (vehicle) for 48 h
Glutathione Catalase Microarray
O’Keeffe & FitzGerald (2014), Int. Dairy J.
Catalase increased on incubation with WPHs
5 kDa permeates 1 kDa permeates
*: p<0.05, **: p<0.01, ***: p<0.001 vs. vehicle
Genes involved in AO system beneficially regulated
HUVECs incubated with WPHs** or media alone (vehicle) for 48 h
Glutathione Catalase Microarray
Gene Name Δ-Fold WPC Δ-Fold Alc Δ-Fold Neu
Glutathione peroxidase 3 (plasma) - +2.13 +1.78
NAD(P)H dehydrogenase, quinine 1 +1.62 +1.91 +1.78
NAD(P)H dehydrogenase, quinine 2 - +1.64 +2.04
Aldehyde dehydrogenase 3 family, member A1
- +1.76 +1.77
Aldehyde dehydrogenase 1 family, member B1
+1.81 - +1.84
Aldehyde dehydrogenase 1 family, member A3
- +1.85 -
Aldehyde dehydrogenase 3 family, member A2
- +1.94 -
O’Keeffe & FitzGerald (2014), Int. Dairy J.
Future perspectives:
Need for more detailed studies on mechanism(s) of action in vivo
Potential of in silico approaches for discovery of novel bioactive peptides
Enzymatic hydrolysis of whey proteins releases bioactive peptides
Extensive in vitro with limited in vivo evidence for whey hydrolysate bioactivity
Overall conclusions:
List of publications• Akhavan, T., Luhovyy, B. L., Brown, P. H., Cho, C. E., & Anderson, G. H. (2010). Effect of premeal consumption of whey protein and its hydrolysate on
food intake and postmeal glycemia and insulin responses in young adults. The American Journal of Clinical Nutrition, 91(4), 966-975.
• Cervato, G., Cazzola, R., & Cestaro, B. (1999). Studies on the antioxidant activity of milk caseins. International Journal of Food Sciences and Nutrition, 50, 291-296.
• Gaudel, C., Nongonierma, A. B., Maher, S., Flynn, S., Krause, M., Murray, B. A., Kelly, P. M., Baird, A. W., FitzGerald, R. J., & Newsholme, P. (2013). A whey protein hydrolysate promotes insulinotropic activity in a clonal pancreatic beta cell line and enhances glycemic function in ob/ob mice. The Journal of Nutrition, 143(7), 1109-1114.
• Geerts, B. F., van Dongen, M. G. J., Flameling, B., Moerland, M. M., de Kam, M. L., Cohen, A. F., Romijn, J. A., Gerhardt, C. C., Kloek, J., & Burggraaf, J. (2011). Hydrolyzed casein decreases postprandial glucose concentrations in T2DM patients irrespective of leucine content. Journal of Dietary Supplements, 8(3), 280-292.
• Hernandez-Ledesma, B., Davalos, A., Bartolome, B., & Amigo, L. (2005). Preparation of Antioxidant Enzymatic Hydrolysates from α-Lactalbumin and β-Lactoglobulin. Identification of Active Peptides by HPLC-MS/MS. Journal of Agricultural and Food Chemistry, 53(3), 588-593.
• Lacroix, I. M., & Li-Chan, E. C. Y. (2013). Inhibition of dipeptidyl peptidase (DPP)-IV and α-glucosidase activities by pepsin-treated whey proteins. Journal of Agricultural and Food Chemistry, 61(31), 7500–7506.
• Lacroix, I. M. E., & Li-Chan, E. C. Y. (2012). Dipeptidyl peptidase-IV inhibitory activity of dairy protein hydrolysates. International Dairy Journal, 25(2), 97-102.
• Lacroix, I. M. E., & Li-Chan, E. C. Y. (2014). Isolation and characterization of peptides with dipeptidyl peptidase-IV Inhibitory activity from pepsin-treated bovine whey proteins. Peptides, 54, 39–48.
• Liu, J.-R., Chen, M.-J., & Lin, C.-W. (2005). Antimutagenic and Antioxidant Properties of Milk−Kefir and Soymilk−Kefir. Journal of Agricultural and Food Chemistry, 53(7), 2467-2474.
• Manders, R. J., Hansen, D., Zorenc, A. H., Dendale, P., Kloek, J., Saris, W. H., & van Loon, L. J. (2014). Protein Co-Ingestion Strongly Increases Postprandial Insulin Secretion in Type 2 Diabetes Patients. Journal of medicinal food.
• Morifuji, M., Ishizaka, M., Baba, S., Fukuda, K., Matsumoto, H., Koga, J., Kanegae, M., & Higuchi, M. (2010). Comparison of different sources and degrees of hydrolysis of dietary protein: effect on plasma amino acids, dipeptides, and insulin responses in human subjects. Journal of Agricultural and Food Chemistry, 58(15), 8788-8797.
• Nongonierma, A. B., & FitzGerald, R. J. (2013a). Dipeptidyl peptidase IV inhibitory and antioxidative properties of milk-derived dipeptides and hydrolysates. Peptides, 39, 157-163.
• Nongonierma, A. B., & FitzGerald, R. J. (2013b). Dipeptidyl peptidase IV inhibitory properties of a whey protein hydrolysate: influence of fractionation, stability to simulated gastrointestinal digestion and food-drug interaction. International Dairy Journal, 32(1), 33–39.
• Nongonierma, A. B., & FitzGerald, R. J. (2014). An in silico model to predict the potential of dietary proteins as sources of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. Food Chemistry, 165, 489–498.
• O’Keeffe, M. B., & FitzGerald, R. J. (2014). Antioxidant effects of enzymatic hydrolysates of whey protein concentrate on cultured human endothelial cell. International Dairy Journal, 36(2), 128-135.
• Phelan, M., Aherne-Bruce, S. A., O'Sullivan, D., FitzGerald, R. J., & O'Brien, N. M. (2009). Potential bioactive effects of casein hydrolysates on human cultured cells. International Dairy Journal, 19(5), 279-285.
• Power, O., Jakeman, P., & FitzGerald, R. (2013). Antioxidative peptides: enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides. Amino acids, 44(3), 797-820.
• Silveira, S. T., Martínez-Maqueda, D., Recio, I., & Hernández-Ledesma, B. (2013). Dipeptidyl peptidase-IV inhibitory peptides generated by tryptic hydrolysis of a whey protein concentrate rich in β-lactoglobulin. Food Chemistry, 141(15), 1072–1077.
• Suetsuna, K., & Chen, J.-R. (2002). Studies on biologically active peptide derived from fish and shellfish- V Antioxidant activities fronm Undaria pinnatifida dipeptides derivatives. Journal of National Fisheries University, 51(1), 1-5.
• Suetsuna, K., Ukeda, H., & Ochi, H. (2000). Isolation and characterization of free radical scavenging activities peptides derived from casein. The Journal of Nutritional Biochemistry, 11(3), 128-131.
• Tulipano, G., Sibilia, V., Caroli, A. M., & Cocchi, D. (2011). Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors. Peptides, 32(4), 835-838.
• Uchida, M., Ohshiba, Y., & Mogami, O. (2011). Novel dipeptidyl peptidase-4–inhibiting peptide derived from β-lactoglobulin. Journal of Pharmacological Sciences, 117(1), 63-66
• Uenishi, H., Kabuki, T., Seto, Y., Serizawa, A., & Nakajima, H. (2012). Isolation and identification of casein-derived dipeptidyl-peptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL from gouda-type cheese and its effect on plasma glucose in rats. International Dairy Journal, 22(1), 24-30.
• WHO (2006). Definition and diagnosis of diabetes mellitus and intermediate hyperglycemia : report of a WHO/IDF consultation.
List of publications