PC3

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0

0

100000

200000

300000

Inte

nsity

[µV]

AU

0,00

0,20

Minutes10,00 20,00 30,00 40,00

AU

0,00

0,10

Minutes0,00 10,00 20,00 30,00 40,00

AU

0,00

0,10

Minutes10,00 20,00 30,00 40,00

AU0,00

0,20

0,40

Minutes10,00 20,00 30,00 40,00

AU

0,00

0,10

Minutes10,00 20,00 30,00 40,00

β-Lg

α-Laβ-Lg

   

Abs

orba

nce

at 2

16nm

(arb

itrar

y un

its)

PC2

PCC

PC0

PC1

Retention Time (min)

Antihypertensive Whey Protein Hydrolysate Obtained from a Commercial Pepsin

Luísa O. L. Rosa1, Ana Iraidy S. Brigida2; José E.Silva-Santos3; Caroline Mellinger-Silva2*, Lourdes M. C. Cabral2.1Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil; 2EMBRAPA Agroindústria de

Alimentos, Rio de Janeiro, Brasil; 3Laboratório de Farmacologia Cardiovascular, Universidade Federal de Santa Catarina, Florianópolis, Brasil;

e-mail: caroline.mellinger@embrapa.br

INTRODUCTION RESULTS

CONCLUSION

MATERIALS AND METHODS

Whey is the milk’s aqueous portion which is separated from casein during the cheesemaking process, which contains great part of nutritional and functional properties of milk;

Whey proteins and its peptides have been constantly described in the literature for its bioactive properties, which includes the antihypertensive capacity. However, the costs of production of bioactive whey hydrolysates remains a barrier, mainly for small industries;

The aim of the present study was to generate antihypertensive whey peptides, through enzymatic hydrolysis, catalyzed by pure (PPA) and commercial (PPC) pepsins, comparing both obtainment processes.

Reversed-Phase High Performance Liquid Chromatography (RP-HPLC): Freeze-dried samples were ressuspended into ultrapure water (2mg mL-1). Analytical unit of HPLC with a C18 column was used. Two solvents were used: Solvent A – 0.1% TFA in ultrapure water (v/v); Solvent B – 0.1% TFA in CAN (v/v). Hydrolysates were eluted as follows: 0-2 min, 95% A; 2-15 min, 95-80% A; 15-20 min, 80-70% A; 20-25 min, 70-60% A; 25-28 min, 60-50% A; 28-32 min, 50-40% A; 32-34 min, 40-30 % A; 34-36 min, 30-20% A; 36-38 min, 2095% A; 3840 min, 95%A. Flow rate:1.0 mL min-1; Detection: 216 nm ; Running time: 40 min; Bovine α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) were used as standards. Protein Eletrophoresis: Proteins were analysed by SDSPAGE in a Bio-Rad vertical mini PROTEAN II xi cell (Hercules, CA, USA). Stacking and running gels were prepared by using 8% and 12% (w/v) acrylamide solutions, respectively. Voltage 100V; Running time: 2.5h. Antihypertensive Activity: Antihypertensive potential of the sample was evaluated into 6 rats’ denuded aortic rigs. Tension generation response to vasoconstrictor or vasodilator effect was evaluated through isometric force transductor coupled to a PowerLab® register and software LabChart v. 7.2 (both from AD instruments, São Paulo, Brasil). Phenylephrine was used as hypertensive control and acetylcholine as vasodilator control. Financial Support: FAPERJ, CAPES, CNPq. Special thanks to Alibra and Bela Vista Produtos. Enzimaticos

for the WPI and commercial pepsin supplies, respectively.

RP-HPLC

LSTD PCC PC0 PC1 PC2 zPC3 HSTD (kDa)

 103.0

 77.0

 

50.0

 

34.3

 

28.8

 

20.7

 

204.0

 123.0

 80.0

 

48.0

 

β-Lg

α-La

(kDa)

 

Lactoferrin

Serum albumin

Caseins

Caseínas

Protein Eletrophoresis

Figure 2: SDS-PAGE electrophoresis of whey pepsin hydrolysates using: A – PPA 0.4% (w/w); B – PPC 1.6% (w/w). LSTD - low molecular protein standard solution, PCC- control, whitouth the addition of enzyme, PC0 to PC3 – samples from 0h to 3h-of hydrolysis, HSTD - high molecular protein standard solution. β-Lg - beta-lactoglobulin, α-La - alpha-lactalbumin. Arrows indicate the migration direction.

Figure 3: Relaxation of phenylephrine-contracted rat aortic rings induced by: A – cumulative concentrations of PPA (0.4% w/w) whey hydrolysate (0.3, 1,3, 5, 10 mg mL-1 ); B – cumulative concentrations of PPC (1,6% w/w) whey hydrolysate (0.3, 1,3, 5, 10 mg mL-1 ).

Antihypertensive Activity

A B

A B

PPA 0.4%(w/w)

Enzymatic Hydrolysis (3h)

PPC 1.6%(w/w)

Whey Protein Isolate1.25% (w/v) pH2 37°C

RP-HPLC Eletrophoresis SDS-PAGE

Biological Analyses

Enzymatic Activity(PPA/PPC)

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0

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100000

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300000

Inte

nsity

[µV]

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0

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100000

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300000

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nsity

[µV]

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0

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400000

Inte

nsity

[µV]

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200000

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nsity

[µV]

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0

0

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[µV]

α-La β-Lg

α-La

Abs

orba

nce

at 2

16nm

(arb

itrar

y un

its)

PCC

PC0

PC1

PC2

PC3

Retention Time (min)

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0

0

100000

200000

300000

Inte

nsity

[µV]

Figure 1: Peptidic profiles of pepsin whey hydrolysates using: A - PPA 0.4%; (w/w); B – PPC 1.6% (w/w). (α-La) α-Lactalbumin; (β-Lg) β- -Lactoglobulin, PCC= control sample, without enzyme. PC0 to PC3 represents the hydrolysates, from 0 to 3 h-hydrolysis.

LSTD PCC PC0 PC1 PC2 PC3 HSTD

β-Lg

α-La

103,0

77,0

204,0

50,0

34,3

28,8

20,7

(kDa)

123,0

80,0

48,0

(kDa)

Lactoferrin

Serum Albumin

Caseins

Enzymatic Activity

Using hemoglobin as substrate, the pure pepsin (PPA) presented an enzymatic activity of 3.1 μU mL-1, 11-fold greater than the commercial one (PPC - 0.28 μU mL-1).

The results showed that an increment of 4 times in the commercial pepsin concentration was enough to generate a whey hydrolysate with high antihypertensive potential and lower cost of acquisition.