Milk proteins as precursors of bioactive peptides.pdf

ACTA Acta Sci. Pol., Technol. Aliment. 8(1) 2009, 71-90

MILK PROTEINS AS PRECURSORS OF BIOACTIVE

PEPTIDES

Marta Dziuba, Bartłomiej Dziuba, Anna Iwaniak

University of Warmia and Mazury in Olsztyn

Abstract. Milk proteins, a source of bioactive peptides, are the subject of numerous re-

search studies aiming to, among others, evaluate their properties as precursors of biologi-

cally active peptides. Physiologically active peptides released from their precursors may

interact with selected receptors and affect the overall condition and health of humans. By

relying on the BIOPEP database of proteins and bioactive peptides, developed by the De-

partment of Food Biochemistry at the University of Warmia and Mazury in Olsztyn

(www.uwm.edu.pl/biochemia), the profiles of potential activity of milk proteins were de-

termined and the function of those proteins as bioactive peptide precursors was evaluated

based on a quantitative criterion, i.e. the occurrence frequency of bioactive fragments (A).

The study revealed that milk proteins are mainly a source of peptides with the following

types of activity: antihypertensive (A max = 0.225), immunomodulating (0.024), smooth

muscle contracting (0.011), antioxidative (0.029), dipeptidyl peptidase IV inhibitors

(0.148), opioid (0.073), opioid antagonistic (0.053), bonding and transporting metals and

metal ions (0.024), antibacterial and antiviral (0.024), and antithrombotic (0.029). The en-

zymes capable of releasing bioactive peptides from precursor proteins were determined

for every type of activity. The results of the experiment indicate that milk proteins such as

lactoferrin, α-lactalbumin, -casein and -casein hydrolysed by trypsin can be a relatively

abundant source of biologically active peptides.

Key words: bioactive peptides, milk proteins, in silico proteolysis

INTRODUCTION

Milk proteins and other food proteins are analysed mainly as a source of amino acids

indispensable for proper bodily functions. Other evaluation criteria are also taken into

account, including the consumed protein's effect on body weight, the type and content

of antinutritional compounds occurring together with proteins and their allergenic prop-

erties [Bush and Hefle 1996, Friedman 1996, Fukudome and Yoshikawa 1992]. Accord-

ing to the present level of knowledge, in addition to its primary function, every protein

may be a precursor of biologically active (bioactive) peptides. This hypothesis postu-

Corresponding author – Adres do korespondencji: Dr inż. Bartłomiej Dziuba, Department of

Industrial and Food Microbiology of University of Warmia and Mazury in Olsztyn, Cieszyński

Square 1, 10-957 Olsztyn, Poland, e-mail: niklema@uwm.edu.pl

M. Dziuba ...

lates that every protein may be a reserve source of peptides controlling the life processes

of organisms [Karelin et al. 1998]. A new, additional criterion for evaluating proteins as

a potential source of biologically active peptides has been proposed [Dziuba et al. 1999 a].

Biologically active peptides in the protein sequence are defined as fragments that re-

main inactive in precursor protein sequences, but when released, for example by prote-

olytic enzymes, they may interact with selected receptors and regulate the body's

physiological functions. The effect exerted by such peptides may be positive or negative

[Schlimme and Meisel 1995, Meisel and Bockelmann 1999].

Milk proteins are the best researched precursors of biologically active peptides

[Meisel 1998, Pihlanto-Leppl 2001, Dziuba et al. 1999 b, 2002, Gobbetti et al. 2002,

Kilara and Panyam 2003, Schanbacher et al. 1997]. Casein and whey proteins are rich in

motifs exhibiting antihypertensive, opioid, antibacterial and immunomodulating activ-

ity. Proteases naturally occurring in food products, such as milk plasmin, hydrolyse

proteins and release bioactive fragments during processing or storage. Many types of

bacteria applied in the production of fermented food and occurring naturally in the gas-

trointestinal tract are capable of producing biologically active peptides. Cheese contains

phosphopeptides which are further proteolysed in the process of cheese ripening, lead-

ing to the formation of various ACE inhibitors [Saito et al. 2000]. In a study of indus-

trial cultures of milk fermenting bacteria, Pihlanto-Leppl et al. [1998] concluded that

the investigated bacteria do not form ACE inhibitor peptides from casein or whey pro-

teins and that they are released during continued proteolysis. The results of a study of

lactic acid bacteria ( Lactobacillus subsp.) which are present in fermented dairy prod-

ucts, but which are not found in starter cultures, as well as in the human digestive tract

indicate that their proteolytic ability is comparable to that of Lactococcus lactis . Pro-

teinases found in the cell walls of Lactococcus lactis (PI and PIII) catalyse the first

stage of casein hydrolysis. Proteinase PI is β-casein-specific, and proteinase PIII is α s -

-casein and β-casein-specific. The above findings have been validated by many research

teams [Juillard et al. 1995]. The short sections of both hydrolysable forms of casein

create fragments containing up to 10 amino acid residues. Casein-derived peptides make

up a populous group, and those fragments correspond to bioactive peptide sequences.

Fragments of β-casein 60-68 and 190-193 correspond to sections of β-casomorphin-11

and immunopeptide, respectively [Korhonen and Pihlanto-Leppl 2001]. Several

casokinins have also been obtained as a result of the effect that serine proteinase from

Lactobacillus helveticus CP790 has on β-casein, while milk fermentation with the in-

volvement of starter cultures containing Lactobacillus helveticus CP790 and Saccharo-

myces cerevisiae led to the formation of β-casokinin. An antihypertensive fragment of

β-casein (residues: 169-175, KVLPVPE) was isolated from a casein hydrolysate with

the application of extracellular proteinase from Lactobacillus helveticus. This peptide

exhibited weak ACE inhibitor activity (IC 50 > 1000 μmol/l). A corresponding hexapep-

tide with KVLPVP sequence, obtained after splitting off the C-terminal glutamine resi-

due (E), displayed much stronger antihypertensive activity (IC 50 = 5 μmol/l) [Meisel

and Bockelmann 1999]. ACE inhibitor peptides were also obtained from milk fer-

mented by Lactobacillus delbruecki subsp. bulgaricus SS1 and Lactococcus lactis

subsp. cremoris FT4 bacteria [Gobbetti et al. 2000].

In addition to analytical methods, many research laboratories resort to computer-

aided techniques for evaluating food components, including proteins. The process of

modeling the physical and chemical properties of proteins [Lackner 1999], predicting

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Milk proteins as precursors of bioactive peptides

their secondary structure [Bairoch and Apweiler 2000] or searching for a homology

between proteins to identify their functions [Kriventseva et al. 2001, Bray et al. 2000]

requires analyses supported by databases of protein sequences or sequence motifs [Ben-

nett et al. 2004, Colinge and Masselot 2004]. A complementary part of such research is

the strategy of examining proteins and bioactive peptides.

The objective of this study was to evaluate milk proteins as bioactive peptide pre-

cursors based on a profile of potential biological activity, the occurrence frequency of

bioactive fragments in the protein sequence and the possibility of bioactive peptide

release by proteolytic enzymes.

MATERIALS AND METHODS

The evaluation of milk proteins as bioactive peptide precursors and their in silico

proteolytic release was carried out based on the BIOPEP database of proteins and bioac-

tive peptides, developed by the Department of Food Biochemistry (www.uwm.edu.pl/

biochemia). A total of 23 types of activity were analysed: antiamnestic, antithrombotic,

antihypertensive, immunomodulating, chemotactic, contracting, toxic, embryotoxic,

antioxidative, dipeptidyl peptidase IV inhibiting, opioid and opioid antagonistic, stimu-

lating red blood cell formation, hemolytic, binding and transporting metals and metal

ions, bacterial permease ligand, anorectic, activating ubiquitin-mediated proteolysis,

regulating ion flow, neuropeptide inhibiting, regulating gastric mucosa activity, antibac-

terial, antiviral, regulating phosphoinositol function. Peptides with the investigated

types of activity were selected in view of the frequency of their occurrence and other

health and technological properties. The experiment involved 16 protein amino acid

sequences from the BIOPEP database.

Functions of the BIOPEP application

The following analytical functions are available in the “Analysis” window of the

BIOPEP application: developing a list of proteins or bioactive peptides with a given

type of activity based on the “List of proteins” or “List of peptides with given activity”

option; determining the type, number and location of active protein fragments – identi-

fying the peptide profile (“Profiles of protein's biological activity”); computing parame-

ters A, B and Y to determine the value of a given protein as a source of bioactive pep-

tides (“A, B, Y Calculation”); performing in silico proteolysis with the use of the “En-

zyme action” option. The value of proteins as bioactive peptide precursors was evalu-

ated based on the occurrence frequency of bioactive fragments in the protein chain (A)

defined as:

A =

where:

a – number of fragments with given activity in the protein chain,

N – number of amino acid residues in the polypeptide chain of a protein mole-

cule.

Acta Scientiarum Polonorum, Technologia Alimentaria 8(1) 2009

M. Dziuba ...

In silico proteolysis of milk proteins

The in silico proteolysis of milk proteins was carried out with the use of a single en-

zyme (24 enzymes). The BIOPEP database contains information on the following 24

proteolytic enzymes: chymotrypsin A, trypsin, pepsin, proteinase K, pancreatic elastase,

propyl oligopeptidase, V-8 protease (glutamyl endopeptidase), thermolysin, plasmin,

cathepsin G, clostripain, chymase, papain, ficain, leukocyte elastase, chymotrypsin C,

metridin, thrombin, bromelain, pancreatic elastase II, glutamyl endopeptidase II, oli-

gopeptidase B, calpain and glycyl endopeptidase.

Verification of results by mass spectrometry

The molecular mass and amino acid sequences of peptides released by proteolytic

enzymes were studied by matrix-assisted laser desorption/ionization mass spectrometry

with the use of an Ettan MALDI-ToF Pro (Amersham Biosciences) mass spectrometer.

For the purpose of determining the molecular mass of the released peptides and their

identification, trypsin hydrolysates of selected proteins were analysed in reflectron

mode by PMF (peptide mass fingerprinting) analysis. The positive ions analysis func-

tion and 20 kV accelerating voltage were used. Samples were prepared by the dried-

droplet method. Proteins were hydrolysed in an ammonium bicarbonate solution (pH of

8.5) with the use of trypsin for a proteomic analysis (Sigma) at a 1:50 enzyme to sub-

strate ratio (w/w). Hydrolysis was performed for 24 h at a temperature of 37°C. Samples

were subjected to an MS analysis.

RESULTS AND DISCUSSION

The development of the BIOPEP database and its built-in software options support a

comprehensive analysis of proteins and bioactive peptides to determine whether they

can be derived from protein precursors. The experiment relied on in silico studies to

evaluate proteins as precursors of bioactive peptides as well as on a computer-aided

simulation of the proteolysis process. The obtained results have to be verified by ana-

lytical methods such as two-dimensional electrophoresis, high performance liquid

chromatography (HPLC) and mass spectrometry.

Tables 1-6 present the biological activity profiles of the main milk protein se-

quences, including the values of parameter A for all types of activity noted in the ana-

lysed proteins. The predominant milk protein fragments exhibit antihypertensive and

dipeptidyl peptidase IV inhibiting activity. The obtained values of parameter A for those

types of activity were relatively the highest, reaching: as regards antihypertensive activ-

ity – from 0.047 for lactoferrin and serum albumin to 0.225 for β-casein, and as regards

dipeptidyl peptidase IV inhibiting activity – from 0.024 for α-lactalbumin to 0.148 for

β-casein. Fragments with other types of activity are also found in milk proteins (Tables

1-6). None of the published sources account for the fact that casein contains fragments

corresponding to peptides with dipeptidyl peptidase IV inhibiting activity, i.e. a prote-

olytic enzyme involved in digestion processes [Pereira and Ciclitira 2004]. Many pep-

tides with the above types of activity were obtained by enzymatic hydrolysis of casein.

Coste et al. [1992] relied on this method to produce fragments of β-casein (residues

193-209) with immunomodulating activity.

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Milk proteins as precursors of bioactive peptides

Table 1. Profile of potential biological activity of cow‟s ( Bos taurus ) α s1 -casein (genetic variant

A) with the values of discriminants A – BIOPEP

Protein sequence (186 amino acid residues, ID 1086)

RPKHPIKHQGLPQPFPQVFGKEKVNELSKDIGSESTEDQAMEDIKEMEAESISSSEEIVPNSVEQKHIQ

KEDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKQGIHAQQKEPMIGVNQELAYFYPE

LFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPLW

Sequence

Location in protein chain

Antihypertensive activity (A = 0.134)

[87-88], [106-107]

FGK

[19-21]

[77-78]

[18-19]

[185-186]

AYFYP

[130-134]

YKVPQL

[91-96]

AYFYPE

[130-135]

[15-16]

DAYPSGAW

[144-151]

LAYFYP

[129-134]

TTMPLW

[181-186]

PLW

[184-186]

[80-81]

[78-79], [81-82]

[136-137]

[132-133], [140-141]

LAY

[129-131]

[130-131], [145-146]

[133-134], [146-147]

Immunomodulating activity (A = 0.011)

EAE

[48-50]

LGY

[79-81]

Opioid activity (A = 0.027)

PLG

[155-157]

TTMPLW

[181-186]

YLGYLE

[78-83]

[78-79], [81-82]

Opioid antagonist activity (A = 0.011)

RYLGYLE

[77-83]

RYLGYL

[77-82]

Acta Scientiarum Polonorum, Technologia Alimentaria 8(1) 2009

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