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Hydrolysis on Reducing Capacity of Whey Protein Hydrolysate

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Abstract

The main objective of the present study was to optimize the operation conditions including pH, temperature and enzyme substrate ratio for the sequential action of pepsin and trypsin to hydrolyses whey protein isolate at the substrate concentration containing 8 per cent of protein content and investigate the effect of hydrolysis on reducing capacity of whey protein hydrolysate as compared to that of whey protein isolate. Results showed that the operation conditions including pH 1.5, temperature 37ºC and E/S ratio of 1:100 was optimum for pepsin with the degree of hydrolysis of 0.736 while trypsin had the degree of hydrolysis of 9.44 at pH 9, temperature 42ºC and E/S ratio of 1:100. The reducing capacity of whey protein hydrolysis was 2.20 for the digestion time of 3 1/2 hours for pepsin digestion followed by the action of trypsin for 31/2 hours. SDS PAGE analysis revealed that the cleavage of alpha lactalbumin resulted in the formation of lower molecular weight peptides could be contributed to the highest reducing capacity of whey protein hydrolysate as compared to that of whey protein isolate.

Keywords: - whey protein isolate; bioactive peptides; antioxidant; SDS-page

1. Introduction

Whey is soluble fraction of milk contain of various bioactive proteins, minerals and lactose that separated from casein throughout manufacturing of cheese or casein (Rock, 2010). Milk whey protein derived- component attribute the range of biological activity that influence digestion, metabolic response to absorbed nutrients, growth and development of specific organs and reduces the risk of chronic desease. Additionaly these protein peptides manifold, are manifested upon the proteolytic digestion which lead to bioactive peptides capable to strive specific biological activity such as antihypertensive, antimicrobial, antioxidant, immunomodulant or miniral (Yalcin, 2006; Hernandez-Leadsmen al., 2008; Madureira et al., 2010) . thes hydrolytic reaction must be strickly managed to produce hydroslysate with appropriate peptid profile for nutritional purposes. It s several aplicatication in specialty foods such as disinged to provide nutritional supportto individuals with particular physiological or nutritional needs not covered by conventional diets(Pacheco, Amaya-Farfan,Sgarbieri, 2002). Whey proteins complex of proteins including ᵦ- lactoglobulin(β-Lg ), α –lactalbunin (α-La), immunoglobulin ‘s(Igs), serum albumin and protease-peptone are soluble over a broad pH range (Multihull, 1992). Whey protein has processed in the three form ; whey protein concentrate(WPC) containing 35-85 percent protiens(Brans et al., 2004) ,whey protein isolate containing 90 percent and third is whey protein hydrolysate which processed from WPI or WPC WPH stands distinguished over WPI and WPC because of the peptides obtained with the specific physiological activity compared to the bioactivity of protein and its derived amino acids.

Whey protein contain high level of branch of amino acid leucine, isoleucine, valine that help in tissue growth and repairing identified as key role during the initiation of translation and sulphur contains methionine, cysteine amino acids that involve in inhancing immune function upon intracellular interconversion of glutathione (Marshal et al,2004). Whey protein hydrosylate has been contain broad range of antioxidant properties in an iron catalysed liposome emulsion (Pena-Ramos and Xing, 2001) or copper catalysed in liposome emulsion (Colbert & Decker, 1991), thats depending upon the used of proteases. Several studies has showed beta-casein (Kansci, Gemots, Meynier, Gaucheron, & Chobert, 2004; Rival, Boeriu, &Wichers, 2001) and αs1-casein (Suetsuna, Ukeda, & Ochi, 2000), antioxidantive peptides ,most found in bovine milk.beta lacto-globulin major protein antioxidative peptide (Hernandez-Ledesma, Davalos, Bartolome, & Amigo, 2005; Pena-Ramos, Xiong, &Arteaga, 2004). However Effective potential of alpha lactalbumin (α-LA; Swissprot accession number P00711), second aboundant protein in bovine milk has not mush (Hernandez-Ledesmaet al., 2005). s

2. Materials and Methods

2.1 Materials

Whey protein isolate is of one category of whey protein which can be obtained during cheese manufacturing. It is manufactured by two processes namely cross flow microfiltration and ion exchange. The former process yields a higher protein quality than the latter because use of chemicals like sodium hydroxide and hydrochloric acid may cause denaturation of proteins present in whey. Hence whey protein isolate obtained through cross flow microfiltration was used to carry out the study. Proteolytic enzymes including pepsin and trypsin and DPPH (1,1-diphenyl-2-picrylhydrazyl) from Sigma-Aldrich and Bovine Serum Albumin, Methanol, Ferric chloride, Potassium ferricyanide, Trichloro Acetic acid, Sodium dodecyl sulphate, Mercaptoethanol, Tris-HCl, Coomassie Blue dye, Bromophenol blue dye from Himedia, India were purchased. All other chemicals were of analytical grade.

2.2. Optimization of enzyme operation conditions:

Pepsin is a chemotrypsin enzyme digest up to 20% of ingested carbon bonds by cleaving preferentially after the N-terminal of aromatic amino acids such as phenylalanine, traptophan and tyrosine amino acid peptide linkage releasing peptides. It has been reported to be active in the pH ranging from1.5 to 2.5 and temperature from 37 to 40 similarly trypsin is a serine group enzyme it digests to N-terminals of lysine, arginine and c-terminal of carboxylic group of praline and produced peptides. It has been reported that to be active in the pH ranging 8.0 to 9.0 and temperature from 37 to 50 degree Celsius

Hence the optimization for pepsin at pH ranging 1.5 to 2.5and temperature ranging from 37 to 40 and enzyme substrate ratio 1:50,1:100,1:200 for the substrate concentration of 8 per cent on the basis of protein content in whey protein isolate was carried out based on degree of hydrolysis. Optimization of trypsin at pH 8.0 to 9.0 and temperature 37 to 50 at 1:50.1:100, 1:200 was also carried out based on degree of hydrolysis.

2.3 Degree of hydrolysis

Degree of hydrolysis is defined as directly propotional to peptides formation by using digestive enzyme and calculated as per the given formula using a pH- stat method (Adler-Nissen, 1986)

DH=(h ⁄ h tot)-100%,h=B×Nb×1/α×1/MP,B=base consumption(ml), Nb=concentration of base(1M NaOH),1/α=calibration factor for pH-stat(1/α=1.01 ),MP= mass of protein(g), and h= hydrolysis equivalents. For whey, htot=8.8 mmol/g protein.

2.4. Hydrolysis of whey protein

Whey protein hydrolysate was carried out by hydrolyzing the whey protein isolate first by pepsin at its optimized pH, temperature and enzyme substrate ratio for the substrate concentration(See section-2.2) of 8 percent on the basis of protein content in whey protein isolate. During the complete digestion of pepsin till degree of hydrolysis became constant, the required pH was maintained using 2M Hcl and the aliquots was taken out half an hour once by inactivating the hydrolysis using 2M NaOH bringing the pH to 6.6 for the analysis of the analysis of reducing power. Hence the pepsin digests that determined to have the highest reducing power was further taken for the subsequent 0 hr,30 minutes,1 hr,1.30 hr,2.30 hr digestion at optimized pH , temperature and enzyme substrate ratio till the degree of hydrolysis became constant. Aliquots of this subsequent digests were taken out by inactivating the hydrolysis using 2M Na2Co3 till the pH became 11 for the analysis of the subsequent digest having the highest reducing power.

2.5. Reducing power:

A method devised by Oyaizu (1986) for reducing power was followed with modifications. To 2.5 ml of the sample solution, 2.5ml of sodium phosphate buffer (0.2M, 6.6pH) was added, followed by the addition of 2.5ml of 1% potassium ferricyanide. The reaction mixture was then incubated at 50ºC in water bath for 20 minutes. It was cooled to 20ºC and then 2.5ml of 10% Trichloro Acetic acid was added to terminate the reaction. The mixture was centrifuged at 3000 rpm for 10 minutes. 0.1 ml of the supernatant was made up to 2.5 ml with distilled water and added with 1ml of 1% Ferric Chloride. The colour developed was read at 700 nm after 5 minutes reaction.

2.6. Protein content:

Whey protein hydrolysate was analysed for protein content according to Bradford method. 0.1 ml of the sample of whey protein hydrolysate and whey protein isolate was made up to 1ml using phosphate buffer saline (pH 7.4) was added with diluted dye solution (1:4) prepared by dissolving 100mg of Coomassie brilliant blue G250 in 50ml of 95% ethanol followed by the addition of addition of 100ml of conc. Phosphoric acid making up the solution to a final volume of 200ml with distilled water. Standard curve was made for bovine serum albumin in the concentration ranging from 0.1mg to 1mg/ml.

2.6. SDS-PAGE analysis:

SDS-PAGE was run with o.3gml stacking and 0.25 gml separating acryl amide gel to identify amino acid composition of whey protein hydrosylate in oder to molecular weight of fraction. The SDS-page sample was prepared by β-marcaptoethnol to prevent the cross linking of disulphide bond during the sample preparation. A 30 µg aliquot added to 1 mg/ml of protein loaded to each well.

2.7. Statistical analysis:

The collected data in triplicates were analyzed for the significant difference between means using SPSS version

3. Result and Discussion:

  1. Optimization of enzyme operation condition: Optimization of enzyme operation conditions including pH, temperature and enzyme substrate ratio at the constant substrate concentration containing 8 percent protein for enzymatic hydrolysis of whey protein isolate using individual and sequential action of pepsin and trypsin were determined based on the degree of hydrolysis as discussed below.

3.1.1 Operation conditions for individual pepsin

pH

Temperature (ºC)

 

Enzyme-Substrate Ratio

   
   

1:50

1:100

1:150

1:200

1.5

37

0.51±0.02

0.736±0.017

0.373±0.011

0.26±0.02

1.5

40

0.37±0.01

0.699±0.018

0.214±0.01

0.22±0.021

2.0

37

0.383±0.01

0.548±0.03

0.265±0.011

0.144±0.02

2.0

40

0.158±0.01

0.343±0.16

0.103±0.01

0.064±0.018

t value* =3.316

t value* =2.424

F* value=25.587

Sign=0.002

Sign.= 0.019

Sign.=0.000

3.1.2 Operation conditions individual for trypsin

pH

Temperature

(ËšC)

Enzyme substrate ratio

(w/w)

1:50

1:100

1:150

1:200

8

37

3.47±0.12

4.08±0.07

2.45±0.1

1.64±0.08

9

37

9.34±0.01

6.04±0.03

5.3±0.36

4.03±0.51

8

40

3.84±0.69

4.17±0.19

3.43±0.21

2.27±0.16

9

40

6.53±0.27

5.29±0.68

3.84±0.27

2.27±0.35

8

42

5.08±0.65

5.83±0.22

3.53±0.07

2.84±0.00

9

42

9.1±0.02

9.44±0.10

7.05±0.02

8.10±0.05

‘t’value*

-6.974

‘t’ value*

-6974

F value*=48.262

 

Sign=0.00

Sign=0.000

3.1.3 Operation conditions for sequential digestion using pepsin and trypsin

Enzyme

Ph

Temperature

(â-¦C)

E/S ratio(w/w)

1:50

1:100

1:150

1:200

F-value

Pepsin

1.5

37

0.51±0.02

0.736±0.017

0.373±0.011

0.26±0.02

25.587

Trypsin

9

42

9.10±0.02

9.44±0.10

7.05±0.02

8.10±0.05

59.147

Sig=0.000

3.2. Whey protein hydrolysate:

Whey protein isolate at the substrate concentration of 8 per cent on protein content basis was enzymatically hydrolysed using pepsin at pH 1.5, temperature of 37°C and E/S ratio of 1:100 for 3 hours of complete digestion followed by trypsin digestion by bringing the pH of pepsin digests to 9, temperature at 42°C the E/S ratio of 1:100 for 6 hours of complete digestion. After digestion, the digests was inactivated by bringing the digests to the pH of 10.10 using 2M Na2Co3. While digestion was going on, the required pH was maintained using 2M Hcl and 2M NaoH. For further analysis of reducing power and protein estimation, the aliquots of digests was taken out half an hour once till the complete digestion and stored at refrigerator conditions after inactivation of the enzyme in digest mixture. Similar to the present study the invitro digestion of WPI by pepsin at pH, temperature 1.5, 37°C and E/S ratio of 1:100 was carried out to study the immune enhancing ability of WPH (.Gauthier et al, 2006)

3.3 Determination of reducing of whey protein hydrosylate

3.3.1 Optimization of operation conditions for pepsin hydrolysis of whey protein isolate:

Digestion time (hours)

Degree of hydrolysis*(%)

Reducing power*

(Abs)

1/2

0e

0.137±0.03e

1

0.33±0.03d

0.155±0.01d

1.5

0.668±0.03c

0.158±0.02d

2

0.672±0.03c

0.291±0.02c

2.5

1.13±0.02b

0.311±0.01b

3

1.18±0.02a

0.324±0.01a

3.5

1.19±0.01a

0.331±0.01a

One way ANOVA*

F=7938.752

Sign=0.000

F=882.903

Sign. =0.000

3.3.2 Optimization of operation conditions for trypsin hydrolysis of whey protein isolate:

Digestion time

(hours)

Degree

of hydrolysis*

(%)

Reducing power*

(Abs)

1/2

0.433±0.006g

0.898 ±0.001g

1

0.945±0.005f

0.982 ±0.10f

1.3

1.405 ±0.005e

1.244 ±0.010e

2

1.632±0.005d

1.470± 0.016d

2.3

1.826±0.005c

1.555±0.052c

3

1.853 0.005b

2.068 0.022b

3.3

1.956 0.005a

2.134 0.110a

One-way ANOVA*=

F-value=51780.805

Sign=0.000

F-value=968.580

Sign=0.000

*Significant at 0.05 level

3.3.4 Sequential hydrolysis by pepsin and trpsin:

Digestion time

(hours)

Degree

of hydrolysis*

(%)

Reducing power*

(Abs)

1/2

0.00n

1.83± 0.18f

1

0.33±0.03m

1.94 ±0.007ce

1.30

0.668±0.03l

1.95 ±0.005de

2

0.672±0.03k

1.97 ±0.004d

2.30

1.13±0.02j

1.97± 0.002,de

3

1.18±0.02i

1.98 ±0.001,cde

3.30

1.19±0.01h

1.97 ±0.001,cde

4

0.433±0.03g

1.99 ±0.100cd

4.30

0.945±0.03f

1.99 ±0.007c

5

1.405 ±0.04e

1.99±0.075c

5.30

1.642±0.03d

2.05±0.017b

6

1.846±0.03c

2.20±0.046a

6.30

1.869±0.020b

1.94±0.046de

7

1.965±0.21a

1.99±0.005cd

One-way ANOVA*=

F-value=17940.21

Sing=0.000

F-value=30.175

Sing=0.000

*Significant at 0.05 level

3.4. PROTEIN CONTENT :

3.4.1 Hydrolysed by pepsin

Digestion time (hours)

Degree of hydrolysis *(%)

Protein Content (mg/ml)

0

0

2.202±0.01a

0.5

0

2.083±0.07b

1

0.333±0.01d

1.921±0.01c

1.5

0.668±0.01c

1.723±0.06d

2

0.672±0.01c

1.702±0.01de

2.5

1.13±0.01b

1.691±0.01de

3

1.179±0.01a

1.671±0.01de

3.5

1.189±0.01a

1.638±0.04e

One way ANOVA*

F=7938.752

Sig=0.000

F=96.865

Sig=0.000

*Significant at 0.05 level

S


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