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:

Acknowledgements

Alice NongoniermaMartina O’KeeffeCeline GaudelPhilip NewsholmeOrla Power

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