biological activities of peptide concentrates obtained ... · com o objetivo de explorar...

Ana Raquel Rodrigues Santana

Biological activities of peptide concentrates obtained

from hydrolyzed eggshell membrane byproduct

Dissertation of Quality Control Master

Specialization in Food and Water

Performed under the supervision of:

Doctor Tânia Sofia Granja Tavares

and co-supervision of:

Prof. Doctor Isabel Maria Pinto Leite Viegas Oliveira Ferreira

July, 2015

Ana Raquel Rodrigues Santana

Atividades biológicas de concentrados peptídicos

obtidos através da hidrólise do subproduto

membrana da casca do ovo

Dissertação do Mestrado em Controlo de Qualidade

Especialização em Água e Alimentos

Desenvolvido sob a orientação de:

Doutora Tânia Sofia Granja Tavares

e co-orientação de:

Prof. Doutora Isabel Maria Pinto Leite Viegas Oliveira Ferreira

Julho, 2015

The full reproduction of this dissertation is authorized, only for

research purposes, by declaration of commitment of the interested

party.

É autorizada a reprodução integral desta dissertação apenas para

efeitos de investigação, mediante declaração do interessado, que tal se

conpromete.

Abstract

iv

Abstract

Over the years, hen’s eggs consumption has been increasing worldwide, including in

Portugal. This increase comprises both industrial and household, leading to an eggshell

accumulation that can be responsible for environmental and public health problems.

Eggshell treatment has become essential to minimize these effects, as well as to find

rentable applications.

Protein is the biggest constituent of eggshell membranes and, besides its high

nutritional value, plays a crucial role in some physiological functions due to the presence

of bioactive peptides. So, in order to explore a food application for the eggshell membrane,

extraction and solubilization methods were optimized. The first one was more efficient

with acetic acid 5 %(v/v) during 1 h; the second one through application of 3-

mercaptopropionic acid solubilization method (63 % of yield). Protein concentrate

obtained (100 % protein content) was subjected to an enzymatic hydrolysis with alcalase

from Bacillus licheniformis, viscozyme L and protease from Bacillus amyloliquefaciens.

Optimum conditions were determined by surface response, where reaction time and

enzyme/substrate ratio were used as parameters and degree of hydrolysis (DH),

angiotensin I converting enzyme-inhibitory (ACE) activity and antioxidant activity were

used as objective functions. The suggested model showed to be statistically suitable to

describe DH, ACE-inhibitory activity and antioxidant activity of hydrolysates. The

optimum conditions observed for each hydrolysis considering the studied biological

activities were: 6 h, 2.2 %(v/v) for alcalase; 6.6 h, 1.9 %(v/v) for viscozyme L; and 5.3 h,

2.9 %(v/v) for protease. The best results were presented by alcalase hydrolysates. For

optimum conditions alcalase hydrolysates presented ACE-inhibitory (IC50) of 34.5 ± 2.1 µg

mL-1, since in viscozyme L and protease hydrolysates verified an IC50 of 63.0 ± 4.2 µg mL-1

and 43.0 ± 8.5 µg mL-1, respectively. In what concerns to antioxidant activity, values of 4.2

± 0.2, 4.4 ± 0.1 and 3.8 ± 0.2 µmolTrolox equivalent mg-1hydrolyzed protein were observed for alcalase,

viscozyme L and protease hydrolysis, respectively in the case of ORAC method; and values

of 3.8 ± 0.0, 4.4 ± 0.0 and 5.2 ± 0.2 µmolTrolox equivalent mg-1hydrolyzed protein for alcalase,

viscozyme L and protease hydrolysis, respectively to ABTS method.

This work showed that eggshell membrane is an added-value byproduct, since it is

a precursor of biological activities and may have potential industrial application as a

functional ingredient.

Keywords: Eggshell membrane, bioactive peptides, hydrolysis, ACE-inhibitory activity,

antioxidant activity.

Resumo

v

Resumo

O aumento do consumo de ovos de galinha, a nível industrial e doméstico, que se

tem vindo a verificar por todo o mundo, incluindo em Portugal, levou a uma acumulação

das cascas de ovos, traduzindo-se num problema de saúde pública e ambiental. Desta

forma, torna-se essencial o tratamento deste subproduto, assim como a procura de

aplicações rentáveis para a sua utilização.

O constituinte principal das membranas das cascas do ovo são as proteínas que, para

além de possuírem um elevado valor nutritivo, desempenham um papel crucial em

determinadas funções fisiológicas devido à presença de péptidos bioativos. Sendo assim,

com o objetivo de explorar aplicações alimentares deste subproduto, procedeu-se à

otimização da sua extração e solubilização, tendo a primeira sido mais eficaz em ácido

acético a 5 %(v/v) durante 1 h e a segunda através da aplicação do método de solubilização

com o ácido 3-mercaptopropiónico (rendimento 63 %). O concentrado proteico obtido

(100 % proteína) foi submetido a hidrólise, catalisada por três enzimas distintas: a alcalase

do Bacillus licheniformis, a viscozyme L e a protease do Bacillus amyloliquefaciens. As

condições ótimas de hidrólise foram determinadas através do método de resposta de

superfície onde o tempo de reacção e a razão enzima/substrato foram usados como

parâmetros, e o grau de hidrólise (GH), a atividade inibidora da enzima conversora de

angiotensina (ECA) e a atividade antioxidante como funções objetivo. O modelo sugerido

mostrou-se estatisticamente apropriado para descrever o GH, e as actividades inibitória da

ECA e antioxidante exibida pelos hidrolisados. As condições ótimas encontradas para cada

hidrólise, tendo em consideração as atividades biológicas em estudo foram: 6 h, 2.2 %(v/v)

para a alcalase; 6.6 h, 1.9 %(v/v) para a viscozyme L; e 5.3 h, 2.9 %(v/v) para a protease.

Os melhores resultados foram obtidos com a alcalase. Os quais, para as condições ótimas

apresentaram uma inibição da ECA em 50 % (IC50) de 34.5 ± 2.1 µg mL-1, enquanto que

nos hidrolisados da viscozyme L e da protease valores de 63.0 ± 4.2 µg mL-1 e 43.0 ± 8.5 µg

mL-1, respetivamente. No que se refere à atividade antioxidante, observou-se uma

atividade de 4.2 ± 0.2, 4.4 ± 0.1 e 3.8 ± 0.2 molequivalentes trolox mg-1proteína hidrolisada para os

hidrolisados da alcalase, viscozyme L e protease, respetivamente através do método

ORAC, e para o método do ABTS valores de 3.8 ± 0.0, 4.4 ± 0.0 e 5.2 ± 0.2 molequivalentes

trolox mg-1proteína hidrolisada para os hidrolisados da alcalase, viscozyme L e protease.

Este trabalho permitiu concluir que a membrana da casca do ovo é um subproduto

de valor acrescentado, visto ser precursora de atividades biológicas, com potencial

aplicação industrial como ingrediente funcional.

Palavras-chave: Membrana da casca do ovo, péptidos bioativos, hidrólise, atividade

inibidora da ECA, atividade antioxidante.

Acknowledgements

vi

Acknowledgements

This dissertation only has been possible with the help of Faculty of Pharmacy of

University of Porto and of several people who I would like to express my gratitude.

I want to thank to Dr. Tânia Tavares, my supervisor, for everything that she teach

me, for her help, dedication and patience demonstrated during this year, that was

essential for my professional growth and certainly would be important to my future.

To Professor Dr. Isabel Ferreira, my co-supervisor, for her orientation and support

demonstrated during this work.

For all people in Bromatology and Hydrology Laboratory, but especially to

Armindo Melo, Rebeca Cruz, Zita Martins and Carina Pinho for all knowledge that they

shared with me, for their help and friendship.

To all my friends and master colleagues, for all support, understanding and happy

moments shared.

Finally and especially to my parents, because without them this wouldn’t have been

possible, for all their unconditional love, understanding, patience and for being there for

me when I must need.

Abbreviations

vii

Abbreviations

AAPH

ABTS

ACE

ADHD

ANOVA

AUC

BCA

BH4

CCD

CUPRAC

DH

DPPH

ECA

E/S

ET

FAPGG

F-C

FL

GH

HAT

HHL

HPLC

IC50

NADPH

NO

NOS

OC

OCX

OPA

OPN

ORAC

ROS

RP-HPLC

2,2'-azobis(2-amidino-propane) dihydrochloride

2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

Angiotensin converting enzyme

Dimetilaminohidrolase dimethylarginine

Analysis of variance

Area under the curve

Bicinchoninic acid Tetrahydrobiopterin

Central composite design Cupric ion reducing antioxidant capacity

Degree of hydrolysis

2,2 – diphenyl-1- picrylhydrazyl

Enzima conversora de angiotensina

Enzyme/Substrate

Electrons transference

Furanacryloyl-L-phenyl-alanylglycylglycine

Folin-Ciocalteu

Fluorescein

Grau de hidrólise

Hydrogen atom transfer

Hippuryl-L-histidyl-L-leucine

High performance liquid chromatography

Concentration to inhibit 50 % activity

Reduced nicotinamide adenine

Nitric oxide

Nitric oxide synthetase

Ovocleidin

Ovocalyxin

o-phthaldialdehyde

Osteopontin

Oxygen radical absorbance capacity

Reactive oxygen species

Reverse phase – high performance liquid chromatography

Abbreviations

viii

RSEE

RSM

SDS

SDS-PAGE

SEE

SOD

TNBS

TRAP

Trolox

Relative standard error of the estimate

Response surface methodology

Sodium dodecyl sulfate

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Standard error of the estimate

Superoxide dismutase

2, 4, 6 -trinitrobenzenesulphonic acid

Total radical absorption potential

(±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid

Contents

ix

Contents

Abstract ................................................................................................................................ iv

Resumo ................................................................................................................................. v

Acknowledgements ............................................................................................................... vi

Abbreviations ....................................................................................................................... vii

Contents ................................................................................................................................ ix

Figure contents ..................................................................................................................... xi

Table contents ...................................................................................................................... xii

Objectives ............................................................................................................................ xiii

Chapter 1 – Introduction ....................................................................................................... 1

1.1. Functional foods...................................................................................................... 2

1.2. Structure and formation of the egg ......................................................................... 3

1.2.1. Egg formation .................................................................................................. 3

1.2.2. Eggshell calcification ....................................................................................... 4

1.3. Egg’s composition ................................................................................................... 5

1.3.1. Edible part ....................................................................................................... 5

1.3.2. Non – edible part ............................................................................................. 6

1.4. Applications of eggshell and eggshell membranes ................................................. 7

1.4.1. Eggshell applications ....................................................................................... 7

1.4.1.1. Environment ................................................................................................ 7

1.4.1.2. Health ....................................................................................................... 8

1.4.1.3. Agriculture ................................................................................................ 8

1.4.1.4. Other applications .................................................................................... 8

1.4.2. Applications of eggshell membranes ............................................................... 9

1.4.2.1. Health ....................................................................................................... 9

1.4.2.2. Food industry ......................................................................................... 10

1.4.2.3. Cosmetic industry ................................................................................... 10

1.4.2.4. Other applications .................................................................................. 10

1.5. Bioactive peptides .................................................................................................. 11

1.5.1. Production of bioactive peptides ................................................................... 12

1.5.1.1. Enzymatic hydrolysis ................................................................................. 12

1.5.2. Biological activities ........................................................................................ 15

1.5.2.1. ACE-inhibitory activity ........................................................................... 15

Contents

x

1.5.2.2. Antioxidant activity ................................................................................ 17

1.6. Preparation and solubilization of eggshell membranes ........................................ 21

1.7. Peptide analysis ..................................................................................................... 23

Chapter 2 – Experimental ................................................................................................... 25

2.1. Sample preparation ............................................................................................... 26

2.1.1. Eggshell vs Eggshell membrane nutritional characterization ....................... 26

2.1.2. Eggshell membranes extraction . ................................................................... 26

2.1.3. Eggshell membranes solubilization ............................................................... 26

2.2. Enzymatic hydrolysis ............................................................................................ 27

2.2.1. Experimental design, modeling and optimization ........................................ 27

2.2.2. Performance of enzymatic hydrolysis ............................................................ 28

2.2.3. Determination of degree of hydrolysis (DH) ................................................. 29

2.2.4. Determination of ACE-inhibitory activity ..................................................... 30

2.2.5. Determination of antioxidant activity ........................................................... 31

2.2.5.1. ORAC assay ............................................................................................ 31

2.2.5.2. ABTS assay ............................................................................................. 32

2.3. Statistical Analysis ................................................................................................ 33

2.4. RP-HPLC ............................................................................................................... 33

Chapter 3 – Results and Discussion .................................................................................... 34

3.1. Eggshell vs Eggshell membranes nutritional characterization ............................ 35

3.2. Extraction and solubilization of eggshell membranes .......................................... 35

3.3. Experimental design, modeling and optimization ................................................ 38

3.4. Model validation ................................................................................................... 43

Chapter 4 – Conclusions ..................................................................................................... 47

Chapter 5 – Future prospects .............................................................................................. 49

Chapter 6 – References ........................................................................................................ 51

Figure contents

xi

Figure contents

Figure 1 – Schematic representation of egg (Hincke et al., 2012). ..................................... 3

Figure 2 – Hen’s oviduct scheme (Hincke et al., 2012). ..................................................... 4

Figure 3 – Reaction of TNBS with amino groups (Rutherfurd, 2010).............................. 14

Figure 4 – Scheme of ACE-inhibitors bioactive peptides mechanism (adapted from:

Huang et al., 2013). ............................................................................................................. 16

Figure 5 – Scheme of peptides with antioxidant power actions in hypertension (adapted

from: Huang et al., 2013). ................................................................................................... 18

Figure 6 – Reaction of ABTS●+ radical in the presence of the antioxidant compound

(Zulueta et al., 2009). .......................................................................................................... 19

Figure 7 – Reaction of the 2, 2'-azobis(2-amidino-propane) dihydrochlorideradical

(AAPH) during ORAC assay (Zulueta et al., 2009). ............................................................ 20

Figure 8 – Micrograph of outer surface of eggshell membrane treated with 3-

mercaptoproprionic acid and acetic acid (a) 0 h (b) 1 h (c) 3 h (d) 5 h (Yi et al., 2004). .... 22

Figure 9 – Most commonly techniques used to separate and identify peptides. .............. 23

Figure 10 – Graphic representations of patent solubilization method yield (%) obtained

for membranes (M) and eggshells (E) with different NaOH concentrations (5 % and 10 %)

and different temperatures (50 °C and 37 °C), before and after dialysis. ........................... 36

Figure 11 – Illustration of antioxidant capacity dependency on reaction time. Curves T1

to T5 represent the absorbance values obtained for increasing concentrations of Trolox. 44

Figure 12 – Variation of degree of hydrolysis, ACE-inhibitory activity and antioxidant

activity as a function of each term in the model – time and E/S ratio, obtained for three

enzymes – alcalase from Bacillus licheniformis, viscozyme L and protease from Bacillus

amyloliquefaciens................................................................................................................ 45

Figure 13 – Chromatogram of sample before and after hydrolysis and of <3 kDa and >3

kDa fractions. ....................................................................................................................... 46

Table contents

xii

Table contents

Table 1 – Chemical composition of edible egg part (Caballero, 2003). .............................. 5

Table 2 – Egg components and their biological activities (Kovacs-Nolan et al., 2005). .... 6

Table 3 – Biological activities of bioactive peptides from eggshell proteins. .................... 12

Table 4 – Advantages and disadvantages of two types of enzymatic hydrolysis (Korhonen

and Pihlanto, 2003, 2006). ................................................................................................. 13

Table 5 – Advantages and disadvantages of ABTS and ORAC assays (Thaipong et al.,

2006; Zulueta et al., 2009). ................................................................................................. 20

Table 6 – Trade, chemical names, activity and the optimum conditions of the studied

enzymes (Source from the supplier (Merck and SigmaAldrich)) and buffer solution used to

each enzyme. . ...................................................................................................................... 29

Table 7 – Protein results (%) obtained by Kjeldahl method and determination of ashes

(942.05 AOAC method) for membranes and eggshell. ....................................................... 35

Table 8 – Percentage of peptides formed during solubilization process for patented

method. . .............................................................................................................................. 37

Table 9 – Experimental design encompassing two processing parameters (reaction time

and E/S ratio) and results pertaining to three responses (degree of hydrolysis (DH),

antioxidant activity and ACE-inhibitory activity of total hydrolysates), obtained from three

enzymes, alcalase from Bacillus licheniformis, viscozyme L and protease from Bacillus

amyloliquefaciens. ............................................................................................................... 39

Table 10 – Best estimates of each term in the model – reaction time, T (linear and

quadratic), E/S ratio, R (linear and quadratic) and interaction thereof (linear), and

corresponding statistics – R2, SEE and RSEE, pertaining to three responses – degree of

hydrolysis, ACE-inhibitory activity and antioxidant activity for total hydrolysates obtained

for three enzymes – alcalase from Bacillus licheniformis, viscozyme L and protease from

Bacillus amyloliquefaciens. ................................................................................................. 42

Table 11 – Maximum ACE-inhibitory and antioxidant activities of total hydrolysates and

corresponding values of two processing parameters – time and E/S ratio, obtained for

three enzymes – alcalase from Bacillus licheniformis, viscozyme L and protease from

Bacillus amyloliquefaciens. ................................................................................................. 43

Table 12 – ACE-inhibitory and antioxidant activities of total fraction, <3 kDa fraction

and >3 kDa fraction at optimum conditions. ...................................................................... 44

Objectives

xiii

Objectives

The aim of the present work was to study the presence of bioactive peptides in

hydrolysates from eggshell membrane byproduct with a perspective to their reuse in the

future, reducing all environmental and health problems associated to their accumulation

as a waste. In order to achieve that objective, the proposed goals in this work were:

Extraction and solubilization of eggshell membrane.

Optimization and validation of enzymatic hydrolysis conditions of three

different enzymes via response surface methodology.

Determination of biological activities – inhibition of angiotensin converting

enzyme and antioxidant activities – study of combined effect of hydrolysis

parameters, namely, reaction time and enzyme/substrate ratio.

Chapter 1 – Introduction

1.1. Functional foods

1.2. Structure and formation of the egg

1.3. Egg’s composition

1.4. Applications of eggshell and eggshell membranes

1.5. Bioactive peptides

1.6. Preparation and solubilization of eggshell membranes

1.7. Peptides analysis

Chapter 1 - Introduction

2

1.1. Functional foods

Nowadays, consumers show a growing interest in choosing food with a benefic

effect on their health. In the last decades there was an increase in the development of

functional foods that can be natural or modified and has specific benefits for body

functions. The functional food concept was first created in Japan in 1980’s but in the

current days it spreads all over the world. The countries that are more interested are

mainly USA and Japan, however in Europe this kind of food has become popular in last

year’s, despite European’s being more critical and questioners about this. Portugal is one

of the European countries that have been embracing the functional foods to prevent

diseases, such as obesity, cerebral vascular accidents, hypertension, cancer and diabetes.

The reason for consumers interest about this food has arise from the desire of have a long

and healthy life, and to be independent until old age (Cornish, 2012; Moors, 2012; Ong et

al., 2014; Ozen et al., 2012, 2014).

This kind of food can be natural or processed food, such us cereals, coffee, tea,

wine, dairy products, chocolate, meat, fish, eggs and others, and their beneficial properties

come from physiological active components, such as proteins. Proteins are a source of

essential amino acids and energy for the organism, they also provide bioactive peptides

responsible for biological activities. Most of these peptides are inactive in the native

protein and only activated before their release, usually during gastrointestinal digestion or

in food processing. Bioactive peptides can be a good base for the new concept of nutrition;

however, there are some precautions that need to be taken into account to ensure its safety

(Choi et al., 2012; Korhonen and Pihlanto 2003, 2006, Ong et al., 2014).

Eggs are really important in human diet due to their nutritional value that is

almost perfect concerning protein, lipids, vitamins, minerals content and other

components. They also present important biological functions such as antioxidant,

antihypertensive, anti-inflammatory, anticancer, antimicrobial activities, among others

(Kovacs-Nolan et al., 2005).

Over the years, hen’s eggs consumption has increased all over the world, having

grown more than double since 1960. Currently, Asia is the biggest eggs producer, followed

by North of America and Europe (Caballero et al., 2003).

In Portugal, egg’s consumption has been growing since 1983 until 2014 and the last

studies shows that each portuguese eat 24 g egg/day, approximately (INE – Instituto

Nacional de Estatística, 2015). Beyond that, in USA and Europe about 30 % and 25 % of

the produced eggs, respectively, are used as higher-value egg product such as liquid, dried

or freeze eggs, boiled and pickled eggs. These products are used in bakeries, pastries and


3

other industries, and as their production method doesn’t change the nutrients content,

these products have the same properties. All this new products have increased the eggshell

byproduct that, if it not properly treated, it’s considered a pollutant; so it’s necessary

discover some useful applications for it (Bender, 1978; Caballero et al., 2003).

1.2. Structure and formation of the egg

The hen’s egg is a complex and highly differentiated reproductive cell and it consist

on a shell (9.5 %), albumen or egg white (63 %) and yolk (27.5 %) (Figure 1) (Caballero et

al., 2003; Kovacs-Nolan et al., 2005).

Figure 1– Schematic representation of egg (Hincke et al., 2012).

1.2.1. Egg formation

Before ovulation, the yolk goes to specific regions of the oviduct, where it’s going to

earn specific components of the egg. The oviduct is divided in 5 parts: infundibulum,

magnum, isthmus, uterus and vagina (Figure 2); it weighs about 77 g during egg’s

production and less of 6 g after egg is expelled. Before the vitelline membrane and egg

white have been deposited during the passage through the infundibulum and magnum,

respectively, the yolk and egg white are going to the white isthmus where the eggshell


4

membrane precursors are secreted about one hour, forming the two eggshells membranes

around egg white. When the egg in formation gets into red isthmus, mineralization starts

and organic aggregates are deposited in external shell membrane. Egg arrives at uterus

where eggshell membrane formation is finalized. The uterus fluid is constituted by 6 to 10

mM of ionized calcium and 70 mM of bicarbonate ions and promotes the calcium

carbonate precipitation in calcite on external shell membrane (Caballero et al., 2003;

Hincke et al., 2012; Nys et al., 2004).

Figure 2 – Hen’s oviduct scheme (Hincke et al., 2012).

1.2.2. Eggshell calcification

Eggshell calcification process develops into three phases and takes about 17 h. The first

phase is the beginning of mineralization; calcite crystals are nucleated on the external

membrane surface. The nucleation sites are the origins sites of mammillary cones and

allowed the formation of palisade layer. In the second phase there is a fast growth of

polycrystalline calcite to form palisade layer that occurs in the available free space and

that produce crystals that are perpendicular to the membrane surface with deposition of

calcium carbonate. Calcification ending is in the last phase with mineralization conclusion

in the uterine fluid that is supersaturated with calcium and bicarbonate ions (Hincke et

al., 2012).


5

1.3. Egg’s composition

1.3.1. Edible part

All egg’s components have important nutrients for the embryo development and

for the human diet. Table 1 represents the chemical composition of the edible egg part

(Froning, 1998).

Table 1 – Chemical composition of edible egg part (Caballero, 2003).

Whole egg (%) Egg white (%) Yolk (%)

Water 72.8 – 75.6 87.9 – 89.4 45.8 – 51.2

Proteins 12.8 – 13.4 9.7 – 10.6 15.7 – 16.6

Lipids 10.5 – 11.8 0.03 31.8 – 35.5

Carbohydrate 0.3 – 1.0 0.4 – 0.9 0.2 – 1.0

Ash 0.8 – 1.0 0.5 – 0.6 1.1

The amount of total proteins and lipids is not influenced by hen’s diet, however the

profile of lipids, vitamins and minerals can change with feeding (Froning, 1998).

Yolk is delimited by vitelline semi-permeable membrane, showing 0.025 mm of

thickness. Yolk material is deposited in concentric rings and in the middle of the yolk

there is the latebra – immature ovule. This is the most concentrate compound of the egg, it

has 48 % of water, it is rich in lipids and proteins and it has a small amount of

carbohydrates and ash (Belitz, 1999; Caballero et al., 2003).

Egg white or albumen is comprised by a thick layer that surrounds the vitelline

membrane and a thin layer which leads the join of the first one to the fiber constituents of

chalaza (allowed that the yolk keep centered in egg white). It has a turbid aspect, due to

the presence of CO2, it has 87 % of water and is rich in proteins, wherein the ovalbumin is

the main one followed by ovotransferrin and ovomucoid (Belitz, 1999; Caballero et al.,

2003).

Table 2 shows the relative amounts of different components present in yolk and

egg white and their biological activities.


6

Table 2 – Egg components and their biological activities (Kovacs-Nolan et al., 2005).

Egg’s part Components Biological activities

Yolk

Phosvitin

Antibacterial

Antioxidant

Enhancement of calcium solubility

Immunoglobulin Y Anti-adhesive

Antimicrobial

Cholesterol Cell membranes component

Albumen

Ovalbumin

Antibacterial

Antihypertensive

Immunomodelator

Ovotransferrin

Antibacterial

Antimicrobial

Immunomodelator

Ovomucoid

Serine protease inhibitor

Immunomodelator

Biospecific ligand

1.3.2. Non - edible part

The most important biological function of eggshell is to protect embryo from all

external environmental attacks and ensure all metabolic and nutritional needs (Hincke et

al., 2012; Nys et al.,2004). Eggshell has between 0.2-0.4 mm of thickness, consists,

mainly, of calcium carbonate ceramic in calcite form (98.2 %) and an organic matrix of

proteins, glycoproteins, phosphoproteins and soluble and insoluble proteoglycans that

represents about 2 % of calcified eggshell weight. To present date more than 500 proteins

were discovered in eggshell, more than in other egg compartments (Hincke et al., 2000,

2012). Eggshell is divided in three layers: mammillary, palisade and vertical crystal layer,

it also has two non-calcified membranes (King’ori, 2011). The internal membrane has ca.

0.02 mm thickness and has direct contact with the egg white; the external membrane has

0.05 mm thickness, their location is between internal membrane and calcified layer, it’s

linked to the eggshell by several cones on internal membrane surface, it has 6 layers of

mineralized fibers alternately in opposite directions (Huopalathi, 2007). The membranes

fibers are composed for 10 % of collagen (type I, V and X) and 70-75 % of other proteins

and glycoprotein rich in lysine residues (Hincke et al., 2012).

This egg constituent has also pores that allow the gas exchange by osmosis, being

the first barrier against bacterial invasion., cuticle which is an organic layer covers the

opening of the pores (Hincke et al., 2012; Ho et al., 2013; Nys et al., 2004).


7

1.4. Applications of eggshell and eggshell membranes

Every day tons of eggshell from agricultural stations, domestic use and food

industries are wasted. The biggest waste comes from food industries that produce higher-

value egg products, turning eggshell into a byproduct. In the USA the waste is about 30 %

while in Europe about 25 % where the annual production increased to 350,000 tons

(Soares et al., 2013).

The eggshell residues are a very serious problem. Usually they are disposed in

landfills without treatment, promoting unpleasant odors, flies and pathogenic agents that

have a negative effect in environment and human health (Cordeiro and Hincke, 2011; Ho

et al., 2013; Soares et al., 2013, 2012).

The European policies have been changing and thus the regulation (EC) Nº

1774/2002 of European Parliament and Council, says that the eggshell residues must be

recycled and reused and eggshell only could be put in soil after due treatment (Soares et

al., 2012). Thus, industries are more keen to invest in the development of processes in

order to use this residues (Cordeiro and Hincke, 2011; Galloway, 2013).

Eggshell byproduct presents high added value organic and inorganic compounds,

thus their reuse is a promising alternative for eggshell upgrade, leading to the elimination

as pollutant agent. Some efforts have been made in this way, however there are a lot to do

in this area (Intharapat et al., 2013; King’ori, 2011).

1.4.1. Eggshell applications

The eggshell consists mainly of calcium carbonate, about 98.2 %, and other

important constituents such as magnesium and phosphorus (King’ori, 2011). Because of

the high calcium content, several applications of the eggshell have been studied in

different areas, such as environmental, agricultural health, and constructions materials.

1.4.1.1. Environment

Ribeiro et al.(2012) showed that the eggshell is effective in adsorption capacity for

the treatment of wastewater of different industrial areas due to the pore composition.

Thus, water pollutants such as heavy metals and organic matter content can be removed

from wastewater in a cheaper way than with activated carbon technique. In the same line

of research, studies conducted by Park et al.(2007), show that heavy metal contaminated

soils can be treated if sprayed with eggshells.


8

1.4.1.2. Health

As mentioned before, the eggshells may be used for various purposes, in order to

minimize the impact on environment. One of them can be in healthcare, specifically in

nutrition and medicine.

An eggshell medium size has about of 750-800 mg of calcium; the eggshell powder

increases the mineral density of bones of people and animals with osteoporosis. In Japan

studies with eggshell enriched with vitamin D3 have been performed, and results showed

an significant increase of bone mineral density and in the blood calcium content (Cordeiro

and Hincke 2011; King’ori, 2011).

The eggshell and membranes have been used for new biomaterials synthesis, such

as bioactive materials, using the calcium phosphate in several clinical applications. An

example is the hydroxyapatite, which is a good substitute for the bones due to their

biocompatibility, osteoconductive properties and chemical similarities with mineralized

bone structure. Therefore, this structure has been widely used in bio-inert implants,

prostheses and bone fill (Cordeiro and Hincke, 2011; Elizondo-Villarreal et al., 2012; Ho et

al., 2013).

1.4.1.3. Agriculture

Eggshells are used for fertilizers production, which are cheaper and less pollutants

than the conventional ones, because of the material reused (Cordeiro and Hincke, 2011;

Ho et al., 2013). Since most plants prefer soil with a pH between 5.8 and 7.0, the calcium

level increase or neutralize the acidic pH of the soil promoting plants growth.

Other use within the agricultural area is as soil stabilizers that can replace the lime,

since they have the same chemical composition being cheaper process. This soil stabilizer

can be used, for example, in road construction (Cordeiro and Hincke, 2011; King’ori,

2011).

1.4.1.4. Other applications

The eggshell is a good source of calcium carbonate, which can be removed from the

eggshell and can be used as filler for polymeric materials agent (Intharapat et al., 2013).

Eggshells could also be used in the preparation of limestone cement. Due to its content of

carbonate one of its applications is as substitute for natural limestone and thereby assist in

the preparation of limestone cement (Andreola et al., 2010).


9

1.4.2. Applications of eggshell membranes

Eggshell membrane encompasses an inner and an outer membrane, composed by

bioactive compounds, such as glucosamine, chondroitin sulfate, hyaluronic acid, sulfur

proteins and collagen (Ruff et al., 2012). There are three types of collagen (type I, V and

X), it is a fibrous protein that plays a principal role in the connection and support tissues

such as skin, bones, tendons, muscles and cartilage. However, it also supports the internal

organs and is always present on teeth. The areas where collagen is widely used are:

cosmetics, biochemistry and pharmaceutical industries (Huang X et al., 2010; King’ori

2011; Ohto-Fujita et al., 2011).

1.4.2.1. Health

Collagen is very important for organism tissues when combined with elastin, since

their cover properties such as stiffness, strength and flexibility. It has been reported that a

supplement of the eggshell membrane, which has collagen, glucosamine, chondroitin and

hyaluronic acid, reduces significantly pain and stiffness, allowing the concomitant

improvement in tissue function.

In sports, collagen from the eggshell can also be used in order to quickly increase

muscle mass, leading to reduced recovery time, rebuild damaged muscle areas without

surgery and decrease cardiovascular diseases in athletes.

In cancer patients the collagen has an important role; the use of hydrolyzed

collagen in chronic arthritis can, also reverse it. Also has benefits in cardiovascular, liver,

prostate and lung disability in skin burns, in orthopedics, in dentistry, and is even used for

carrying out auxiliary pictures (King’ori, 2011).

Ruff et al. (2009) conducted a study in which they introduced the membrane of

natural eggshell as a therapy to joint diseases and connective tissues. It has been found

that the majority of patients were considerably helped by the membrane natural eggshell

supplement, where the pain decreased quickly and continuously. There are already several

commercially dietary supplements made 100 % from the eggshell membranes.


10

1.4.2.2. Food industry

When the collagen is hydrolyzed, produces a gelatin that could be used in flavored

foods as a thickening agent, as an emulsifier and gelling agent; however, both (collagen

and gelatin) have a low protein quality, since is constituted almost for glycine, proline and

lysine, being lysine the only essential amino acid (King’ori, 2011).

1.4.2.3. Cosmetic industry

Skin has collagen that starts to decrease with age and becomes thinner, weaker,

drier and less elastic leading the appearance of wrinkles. This deterioration is also caused

by the reduction of amino acids in skin structure. Thus, nowadays there are many

supplements on the market and creams with the goal of improving the appearance of skin,

having a number of studies that have proven their effectiveness (King’ori, 2011).

1.4.2.4. Other applications

Collagen is used in many appliances such as musical instruments, such as violin

and guitar. The eggshell membranes can be used to synthesize nanoparticles of different

sizes with possible applications in catalysis and medical imaging optics and also in the

paper industry, as pigment for coating paper printing (Cordeiro and Hincke, 2011;

King’ori, 2011).

As could be realized, eggshell and their membranes are of utmost importance at

many levels, because there is a wide range of applications that allow the reduction of

environmental impact. However most of the eggshell membranes applications are in

cosmetic and health areas; the food industry is still an unexplored area.


11

1.5. Bioactive peptides

Over the years, there has been a growing interest in the use of foods with bioactive

peptides as intervention agents against human chronic diseases and maintenance of

human well-being (Özen et al., 2014; Udenigwe and Aluko, 2012). The first bioactive

peptides to be discovered were the casein phosphopeptides by Mellander in 1950, when he

suggested that casein derived from phosphorylated peptides potentiated vitamin D in the

calcification of bones in stunted children (Korhonen and Pihlanto, 2003). The bioactive

peptides are by definition peptides having a biological activity at physiological level that

benefits the health of the body. Bioactive peptides have between 3 and 20 amino acids and

can be derived from animal or vegetable proteins. Their specific activity is determined by

the type and location of amino acid residues in the primary structure of proteins (Eckert et

al., 2013). Typically, the native protein sequence of these peptides is found in an inactive

form, being activated when released in the gastrointestinal tract by microbial activity or by

food processing. Once released, few peptides are absorbed in the intestinal tract and most

of them act directly in the intestinal tract or via receptors and cell signaling in the intestine

(Choi et al., 2012; Möller et al., 2008; Udenigwe and Aluko, 2012).

The bioactive peptides are very important constituents or ingredients of functional

food, being produced or added in the production process. In most cases, these peptides

have been shown to have higher bioactivity than the original proteins, and its

administration can affect in a positive way the cardiovascular, digestive, immune or

nervous systems (Hartmann and Meisel, 2007; Korhonen and Pihlanto, 2006; Udenigwe

and Aluko, 2012). Table 3 shows biological activities of bioactive peptides from eggshell

proteins.

Although bioactive peptides are very promising, it is important to note that

proteins and peptides with bioactive properties, may not present an effect on human

health, once they can be degraded during digestion and losing their primary structure or

they could not be absorbed and thus will not reach the bloodstream and tissues to

significant levels. There is also the problem that certain processing foods may influence

the bioactivity and may even be toxic, allergenic or even carcinogenic to humans.

Overcoming these problems, the bioactive peptides can be included as ingredients in

functional foods, dietary supplements and pharmaceuticals in order to improve human

health (Hartmann and Meisel, 2007; Korhonen and Pihlanto, 2006; Möller et al., 2008;

Udenigwe and Aluko, 2012).


12

Table 3 – Biological activities of bioactive peptides from eggshell proteins.

Proteins Sequence Biological

activity System References

Ovalbumin

Ovocinin (FRADHPPL)

Inhibition of ACE Cardiovascular Miguel and Aleixandre, 2006

Non-specific peptides

Immunomodelator Immune Iawaniak and Minkiewicz, 2007

AHK, VHH, VHHANEM

RADHPF

Antioxidant nervous and

cardiovascular

Eckert et al., 2013 and Huang W et al., 2010

Ovotransferrin

KVREGTTY Inhibition of ACE Cardiovascular Lee et al., 2006

OTAP-92 (F109 – 200)

Antimicrobial gastrointestinal

immune

Mine and Kovacs-Nolan, 2006

Lysozyme Non-specific

peptides Antimicrobial

gastrointestinal e immune

Mine and Kovacs-Nolan, 2006

1.5.1. Production of bioactive peptides

Most native proteins do not present functional properties. So, there is a need to

improve these functions by releasing bioactive peptides. There are different methods in

order to produce peptides: microbial fermentation through combination of

microorganisms, that will interact with each other, enzymatic hydrolysis, the most

common method, and also a combination of both methods (Eckert et al., 2013; Yamamoto

et al., 2003).

1.5.1.1. Enzymatic hydrolysis

There are many commercial enzymes that can be used in this process, such as

alcalase, protease, viscozyme L, thermolysin, pepsin and others. In this work alcalase,

viscozyme L and protease were used. Alcalase is an alkaline enzyme produced from

Bacillus licheniformis, it is a serine endopeptidase of microbial origin that is suitable to

hydrolysis of proteins (See et al., 2011); viscozyme L is a multi-enzyme complex of

carbohydrases with arabinase, cellulase, β-glucanase, hemicellulase and xylanase; and

protease, an enzyme with a microbial origin used to break down proteins by hydrolyzing

peptide bonds.


13

The hydrolysis can occur through two mechanisms: the mechanism one-by-one in

which it is necessary to unfold partially native proteins causing them the loose of stability

and exposing the peptide bonds, leading the enzyme to have access to peptide link; and

"zipper" mechanism wherein the native protein is rapidly converted into intermediates

products and occurs hydrolysis of the indirectly protein very slowly (Eckert et al., 2013).

There are two types of enzymatic hydrolysis: a conventional hydrolysis or a continuous

hydrolysis using ultrafiltration membranes, for which there are advantages and

disadvantages, which are presented on Table 4 (Choi et al., 2012; Korhonen and Pihlanto,

2003).

Table 4 -Advantages and disadvantages of two types of enzymatic hydrolysis (Korhonen and

Pihlanto, 2003, 2006).

Conventional hydrolysis Continuous hydrolysis

(ultrafiltration of membranes)

Advantages Disadvantages Advantages Disadvantages

- Product separation and catalyze recovery is done in a single operation.

- Expensive enzymes

- Less efficiency

- Greater efficiency due to membrane ultrafiltration reactors

- Increased productivity

- Expensive process

Enzymatic hydrolysis may modify and improve protein functional properties, such

as decrease viscosity, increase whipping ability and high solubility, without affecting its

nutritive value, but converting it into peptides with desired size, charged and surface

properties (Chen and Chi, 2012; Jamdar et al., 2010). However, certain hydrolysis

parameters such as incubation time, enzyme substrate ratio, enzyme concentration and

specificity, temperature, pH, ionic strength, presence of activators and inhibitors, degree

of hydrolysis (DH) of protein, and pre-treatment of protein before hydrolysis are very

important, since they could potentiate enzymatic hydrolysis and increase the interactions

between the enzyme and protein (Eckert et al., 2013; Udenigwe and Aluko, 2012).

Response surface methodology (RSM) is an important tool to optimize the process in an

effective way; it reduces the number of trials needed to evaluate multiple parameters being

a statistical tool to identify interactions. This methodology was already successfully used

to optimization of enzymatic reactions and it was, for this reason, used in this work

(Agarwal and Bosco, 2014; Contreras et al., 2011; Guan and Yao, 2008; See et al., 2011;

Tavares et al., 2011).


14

The bioactive peptides properties will depend on the degree to which the protein

has been hydrolyzed. So, it is necessary to determine the DH that is the percentage of total

number of peptide bonds that are cleaved during hydrolysis (Adler-Nissen, 1979; Chen

and Chi, 2012; Jamdar et al., 2010; Spellman et al., 2003).

DH can be determined by several methods, such as osmometry, pH stat,

determining the amount of nitrogen or free amino groups released during hydrolysis. The

determination of the free amino groups could be performed by formol titration or by

compounds which react specifically with amino groups such as 2, 4, 6 -

trinitrobenzenesulphonic acid (TNBS), o-phthaldialdehyde (OPA), ninhydrin or

fluorescemine. The most commonly used are OPA and TNBS method. The first one is

based on the specific reaction between OPA and primary amino groups to form isoindoles

that can be quantified spectrophotometrically at 340 nm or fluorometrically at 455 nm. A

disadvantage of this method is the low reactivity for proline and cysteine residues. The

TNBS method also reacts specifically with primary amino groups to form a chromophore

with a maximum absorbance at 420 nm (Figure 3). This reaction occurs with alkaline

conditions and is stopped when a lowering pH reagent is added. A caution to have with

this procedure is that TNBS is not completely selective for primary amino groups and also

reacts slowly with hydroxyl ions presents in the medium and this increase is stimulated by

light (Adler-Nissen, 1979; Jamdar et al., 2010; Nielsen et al., 2001; Panasiuk et al., 1998;

Spellman et al., 2003).

Figure 3 –Reaction of TNBS with amino groups (Rutherfurd, 2010).


15

1.5.2. Biological activities

1.5.2.1. ACE-inhibitory activity

Some bioactive peptides from functional foods are inhibitors of angiotensin

converting enzyme (ACE), i.e., are potential helpers in preventing very serious health

problems such as hypertension. According to the World Health Organization,

hypertension leads to death of about 7.1 million people a year. This disease is based on a

chronic medical condition in which blood pressure increases and there are many risk

factors such as high cholesterol, age, obesity, sleep apnea, diabetes, chronic kidney

disease, diet, etc.

Studies carried out show an inhibitory effect on ACE by some biopeptides and

thereby enable prevention of hypertension. The ovocinin I and II as well as the peptides

Phe-Arg-Ala-Asp-His-Pro-Phe-Leu (FRADHPFL), Arg-Ala-Asp-His-Pro-Phe (RADHPF),

Arg-Ala-Asp his-Phe-Leu (RADHFL), Glu-Ser-Ile-Ile-Asn-Phe (ESIINF) and Tyr-Gly-Arg-

Gly-Leu-Glu-Pro-Ile-Asn-Phe (YRGGLEPINF) are examples of bioactive peptides from

different types of peptide digests of ovalbumin protein with antihypertensive activity by

inhibiting ACE (Aleixandre et al., 2008; Miguel and Aleixandre, 2006).

Angiotensin converting enzyme is a dipeptidyl carboxypeptidase responsible for

vasoconstriction being important for blood pressure regulation. ACE converts the

decapeptide angiotensin I to octapeptide angiotensin II, a potent vasoconstrictor and

inactivates the vasodilator bradykinin. Moreover ACE inhibitors are involved in the

regulation of the rennin - angiotensin system preventing the formation of angiotensin II

and decreasing the metabolism of bradykinin involved in systemic dilatation of arteries

and veins leading to arterial blood pressure decrease. The inhibition of angiotensin II

formation decreases the secretion of aldosterone from the renal cortex, decreasing the

reabsorption of sodium and water with concomitant reduction of extracellular volume,

which reduces blood pressure (Figure 4). Peptides with an antihypertensive effect, i.e.,

with in vitro ACE-inhibitory activity, have been isolated from different food proteins,

replacing therefore synthetic drugs that can have secondary effects (Aleixandre et al.,

2008; Chen and Chi, 2012; Huang et al., 2013; Kitts and Weiler, 2003; Majumder and

Wu, 2010; Miguel and Aleixandre, 2006; Murray et al., 2004; Sentandreu and Toldrá,

2006; Tavares et al., 2011; Vermeirssen et al.,2002; Yamamoto et al., 2003).


16

Figure 4– Scheme of ACE-inhibitors bioactive peptides mechanism (Huang et al., 2013).

There are many methods to measure ACE activity and inhibition in vitro, such as

colorimetric, fluorimetric, radiochemical and liquid chromatographic procedures. The

most widely used are the spectrophotometric methods that use hippuryl-L-histidyl-L-

leucine (HHL) or furanacryloyl-L-phenyl-alanylglycylglycine (FAPGG) as substrate. In this

work ACE activity was quantified by a fluorimetric procedure, using the intramolecularly

quenched fluorescent tripeptide o-aminobenzoylglycyl-p-nitrophenylalanylproline (Abz-

Gly-Phe(NO2)-Pro) as substrate, developed by Sentandreu and Toldrá (2006). The

advantages of this method are that it is a rapid, simple and sensitive assay, allowing

continuous monitorization of ACE activity, involves only one-step reagent and a large

number of samples can be analyzed in a short time. These method allows the calculation of

the IC50 value, i.e., the concentration of inhibitor needed to inhibit ACE activity by 50

%(Quirós et al., 2009; Murray et al., 2004; Vermeirssen et al., 2002).

ACE Inhibitors

Angiotensin I

Angiotensin II

ACE

Bradykinin

Inactive

Angiotensin

Receptors

Vasoconstriction

Blood Pressure

Vasodilatation

Blood Pressure


17

1.5.2.2. Antioxidant activity

It is known that free radicals produced in the oxidation reactions promote various

diseases such as osteoarthritis, cancer, diabetes and cardiovascular disease, which can

cause damage to cells and tissues (Memarpoor-Yazdi et al.; 2012; Zambrowicz et al.,

2013). Therefore, and also due to the increased interest by consumers for health issues,

there was a need to identify natural antioxidants involved in public health prevention.

Antioxidants can help prevent and to control diabetes, obesity, hypertension,

atherosclerosis, cardiovascular and neurodegenerative diseases such as Alzheimer's and

Parkinson's disease (Lin et al., 2013).

Usually, the antioxidant peptides contain about 5-16 amino acid residues and its

antioxidant activities can be reported by several mechanisms. Antioxidant activity can be

determine by three different lines of defense: the first one consists in mechanisms such as,

ion chelation, induction of defense enzymes expression and inhibition of oxidative

enzymes that promote the appearance of reactive oxygen or nitrogen species; the second

one consists in elimination of reactive species before it causes an oxidative damage in

biomolecules; and the last one consists in clearing wastes, repair biological damage and

reconstitute the loss of function. Since there is a wide variety of radicals and pathways of

action there is not one method capable of representing antioxidant activity in a safe and

precise way, so normally more than one method is used (Magalhães et al., 2012, 2014).

While natural antioxidants are less effective than synthetic, they are safer (Chen

and Chi, 2012; Magalhães et al., 2014; Memarpoor-Yazdi et al., 2012). Studies suggest that

protein hydrolysates and biopeptides not only donate electrons to the free radicals, but

also form a protection on lipid membranes against oxidative processes. Several egg

bioactive peptides presenting antioxidant activity and beneficial to human health have

been studied (Moon et al., 2012). Examples of peptides derived from ovalbumin and

ovotransferrin with antioxidant capacity are: Ile-Ser-Gln-Val-Ala-His-Ala-His-Ala-Glu-

Asn-Ile-Ala-Glu-Gly-Arg (ISQAVHAHAEINEAGR), Ser-Val-Leu (LAS), Leu-Gln (Q) and

Ala-Pro (AP), Lys-Val-Arg (KVR), respectively (Zambrowicz et al., 2013).

In the case of hypertension, the antioxidant activity of the peptides will also be

significant, because there are studies that indicate oxidative stress as one of the causes for

endothelium availability of nitric oxide (NO) reduction. Nitric oxide, endogenously

synthesized from L-arginine, oxygen and nicotinamide adenine dinucleotide phosphate

(NADPH) by various NO enzymes; leads to smooth muscle relaxation resulting in

vasodilation, increasing blood flow and reducing blood pressure.

The increased production of reactive oxygen species (ROS) reduces the NO by

direct inactivation through high oxidation tetrahydrobiopterin (BH4), a cofactor enzyme


18

of NO and inhibition of dimetilaminohidrolase dimethylarginine (DDAH),

(dimetilarginines degrading enzyme, inhibitor of nitric oxide synthetase (NOS)).

Antioxidants reduce oxidative stress by eliminating free radicals such as ROS. Moreover,

improves endothelial function, reducing NADPH oxidase and increasing extracellular

superoxide dismutase (SOD) activity. The last one leads to a block of ROS production

which promotes the availability of endothelial and reduced blood pressure (Figure 5)

(Huang et al., 2013).

Figure 5 – Scheme of peptides with antioxidant power actions in hypertension (Huang et al., 2013).

The antioxidant activity of food products could be determined by many types of in

vitro assays because there is not a method that can represent all mechanisms of

antioxidant action. The different methods can be divided in two groups: assays based on

hydrogen atom transfer (HAT) which relies on competition between the antioxidant and

the substrate to form peroxyl radicals from the decomposition of azo-compounds, and

assays based on electrons transference (ET) that measure the capacity of an antioxidant to

Vasodilatation

Blood Pressure

NO

L-Arg + O2 + NADPH

NADPH oxidase Antioxidants

Superoxide SOD

ROS

BH4 Oxidation NO Inactivation DDAH Inhibition

Vasoconstriction

Blood Pressure

NOS

Antioxidants


19

reduce an oxidant. Examples of the first group are the Folin-Ciocalteu (F-C), the cupric ion

reducing antioxidant capacity (CUPRAC), radical scavenging capacity against 2,2 –

diphenyl-1-picrylhydrazyl radical (DPPH●) and 2,2 azinobis-3-ethylbenzothiazoline-6-

sulfonic acid radical cation (ABTS●+) and of the second are the oxygen radical absorbance

capacity (ORAC) and the total radical absorption potential (TRAP). The ABTS and ORAC

method are the most commonly used of each group. The first method consists on green

color disappearance register that indicated the scavenging of the ABTS●+ radical cation

promoted by the presence of antioxidants in the sample (Figure 6).

Figure 6 – Reaction of ABTS●+ radical in the presence of the antioxidant compound (Zulueta et al.,

2009).

The ORAC assay is a fluorimetric method that measures the ability of antioxidants

present in samples to prevent protein conformation loss due to oxidative damage caused

by peroxyl radicals. The less antioxidants the sample has more decrease in the

fluorescence protein is measure (Figure 7) (Dávalos et al., 2004; Magalhães et al., 2012;

Zulueta et al., 2009).

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20

Figure 7 – Reaction of the 2, 2'-azobis(2-amidino-propane) dihydrochlorideradical (AAPH) during

ORAC assay (Zulueta et al., 2009).

The advantages and disadvantages of these two assays are present in Table 5.

Table 5 – Advantages and disadvantages of ABTS and ORAC assays (Thaipong et al., 2006; Zulueta

et al., 2009).

ABTS assay ORAC assay

Advantages

Easy and inexpensive

Stable

Sensitive

Used for organic and aqueous

samples

Reproducible

Many absorbance maximum

Good solubility

Use of free radicals biologically

relevant

Standardized (allows value

comparison between lab)

Stable

Use of fluorescence – less

interference of color compost

present in samples

Disadvantages

Preparation of ABTS●+ radical

solution the day before

Not standardized (difficult value

comparison between lab)

Standard kinetic profile is

different from the antioxidants

presents in food samples.

Expensive

Variability between equipments

Require long times to quantify

results


21

1.6. Preparation and solubilization of eggshell

membranes

Calcium carbonate eggshell together with the membrane proteins do not have great

commercial value, so it is relevant to develop techniques that allow to separate the

eggshell membrane in order to increase the profitability of these products, giving them a

significant commercial value (Sonenklar, 1999; Wei et al., 2009).

There are already some industrial methods of eggshell membranes separations.

Such us, a machine developed by MacNeil (2001), which uses a multi-bladed knife to

scrape off the membrane’s surface that, with the aid of water, allow the separation of the

eggshell membrane. Another method described was the use of a food processor with

sterile water where eggshell were put and homogenized and then the product is placed in a

sterile container and allowed to stand for later be decanted and dried in vacuum (Ahlborn

et al., 2006). As well as a technique where eggshells are placed in a container with air

injected into the water flow, leading to membrane float and eggshell precipitation (yield of

96 %) (Yoo et al., 2009). Another techniques, use pulse energy in a liquid containing

eggshells (Oren, 2010); or steam heat vacuum and mechanical abrasion. This last method

is widely use because it allows the separation of several tons per day of these two products

(Adams, 2006). Besides those non-chemical separators, eggshell membranes can be

separated by cavitation in a tank with water and acetic acid (Vlad, 2009).

However, at laboratory-scale the most widely use method is the manual process,

where membranes are separated from the eggshell by hand, with the help of acid, washed

and dried at room temperature. Dried samples are reduced to fine powder and stored.

Nevertheless, industrially, this method is not profitable (Jia et al., 2012; Kaweewong et al.,

2013).

After the eggshell membrane separation, there are a few methods already

described, with the aim of membrane proteins solubilization. However, this process is

often difficult due to the fact that these proteins possess a large number of disulfide

crosslinks (Yi et al., 2003, 2004). The solution is the use of buffers and detergents,

however it has been found that extraction by this method are very difficult. The best result

was provide with the anionic detergent sodium dodecyl sulfate (SDS) (Ahlborn et al.,

2006). Kaweewong et al. (2013) used two lyses solutions, SDS and Triton X-100, which

were added to the powder of eggshell membranes. The lysate of the eggshell membranes

was centrifuged, and acetone was added to the supernatant in order to precipitate the

proteins. It was found that SDS was more effective than Triton X-100 (nonionic

detergent).


22

Considering a future utilization of the eggshell membranes proteins for

incorporation into food products, it is important to note that treatment with toxic agents

should be avoided. Thus, Strohbehn (2012), patented a method that allows the

solubilization without use of toxic agents, but a basic solution (NaOH). However, there is,

also the 3-mercaptopropionic acid method that allows the cleavage of disulfide bonds.

Despite of being toxic, when this agent is used at low concentrations it is eliminated

through a washing, after precipitation (Jia et al., 2012; Yi et al., 2003, 2004).

Figure 8 – Micrograph of outer surface of eggshell membrane treated with 3-mercaptoproprionic

acid and acetic acid (a) 0 h (b) 1 h (c) 3 h (d) 5 h (Yi et al., 2004).

Yi et al. (2004) studied what happens to the membrane during the process of

solubilization with 3-mercaptopropionic acid method and thus verified that the membrane

which initially had a thickness of 49 microns after 5 h of solubilization was 32 microns,

i.e., thickness had decreased, which suggests that dissolution starts at the surface

membrane and is always the surface that is dissolving due to the close contact with the 3-

mercaptopropionic acid. The outside surface of the membrane is a network composed of

microfibers and coalescing interlaced (Figure 8). During solubilization process fibers will

be destroyed. The inner surface of the membrane does not contain fibers. After a

treatment greater than 5 h the membrane is too weak to withdraw the suspension and so

gradually falls apart into small pieces eventually dissolve completely. Amino acid analysis

showed the loss of cysteine which indicates the modification of disulfide bonds with them

having been broken, which causes crosslinking.


23

1.7. Peptide analysis

In order to improve the bioactivity and to have a better efficacy of a peptide

concentrate obtained by hydrolysis of proteins, it can be processed according to their

physico-chemical and structural properties, such as size, hydrophobicity and charge. Thus,

the peptides with biological activities can be isolated and identified. The peptides may be

separated by several techniques such as chromatography, ultrafiltration, electrophoresis,

etc. (Figure 9) and those identification is usually performed by mass spectrometry.

Figure 9 – Most commonly techniques used to separate and identify peptides.

Chromatographic methods are based in separation of a mixture of various

components while they migrate with different rates in the stationary phase by means of

the mobile phase. HPLC (high performance liquid chromatography) is one of the many

chromatographic techniques and is the most important and universal type of analytical

procedure. It is used for both analytical and preparative separation of molecules and is a

widely used technique, because it allows the separation of complex mixtures giving

information on the different retention times of the separated compounds. The HPLC can

be of normal phase or reverse phase. Reverse phase (RP-HPLC) is the most used and has a

non-polar stationary phase and a polar mobile phase while normal phase is the oposite.

The detectors used in this technique can be of ultraviolet (UV), fluorescence, nuclear

magnetic resonance or mass spectrophotometry. Regarding separation techniques, the MS

is the most used since it is a qualitative and quantitative technique, has a high efficiency

for molecules identification, allows the analysis of complex samples without the need to

Liquid Chromatography

Capillary Electrophoresis

Gasosa Chromatography

SDS-PAGE

Supercritic fluid Chromatography

Ultrafiltration

Others


24

make a separation the sample and it is a fast and very sensitive technique (Udenigwe and

Aluko, 2012; Samanidou and Karageorgou, 2010; Ibañez and Cifuentes, 2001).

Electrophoresis is, also, a very used technique because it is a simple, rapid and

sensitive analytical tool that separates proteins and nucleic acids, allowing the

identification, based in the transport of charged molecules through a solvent by an

electrical field. In the case of proteins, SDS is an anionic agent that bonds to them and

charges them negatively. This bond is directly proportional to molecular weight.

In this work a RP-HPLC was used not to identify and quantify peptides but for a

qualitative analysis to compare samples after and before hydrolysis.

Chapter 2 – Experimental

2.1. Sample preparation

2.2. Enzymatic hydrolysis

2.3. HPLC

Experimental

26

2.1. Sample preparation

2.1.1. Eggshell vs Eggshell membrane nutritional

characterization

To determine the nutritive composition of eggshell membranes protein and ashes

were quantified. To quantify protein the Kjeldahl assay was performed based on 990.03

AOAC method and protein was expressed in percentage (%). Regarding ashes, the 942.05

AOAC method was performed.

2.1.2. Eggshell membranes extraction

The hen’s eggshells were picked from a pastry and washed with tap water in order

to remove egg white residues. To optimize the eggshell membranes extraction 6

experiments were performed. So, different concentrations of acetic acid (5 % to 10 %)

(VWR Chemicals, Fontenay-Sous-Bois, France), temperatures (room temperature and 40

°C with and without sonicator) and time (30, 60, 120 and 180 min) were tested.

Membranes were washed with water, dried at 50 °C, weighed in an analytical balance and

then triturated (7,000 rpm, 15 s) and stored at -20 °C (Baláž, 2014).

2.1.3. Eggshell membranes solubilization

For eggshell solubilization two methods were performed: 3-mercaptoproprionic

acid method described by Yi et al. (2003, 2004) and Jia et al., (2012) with some

modifications, and a method based in a patent (Strohbehn, 2012). Preliminary tests were

made in order to study the membranes solubilization without extracting them from

eggshell.

For the first one three preliminary tests were carried out; immersing 6 eggshells in

20, 40 and 100 mL of 1.25 M 3-mercaptopropionic acid (Merck, Darmstadt, Germany)

with 10 % acetic acid and allowed in a water bath at 90 °C overnight. In the case of

eggshell membrane (0.6 g) they were dispersed in 20 mL of 1.25 mol L-1 aqueous 3-

mercaptopropionic acid diluted in acetic acid 10 %, and then was held at 90 °C for ca. 12 h.

For both samples (eggshells and eggshell membranes), after cooling to room temperature,

the mixture was adjusted to pH 5 with 6 M NaOH. The white precipitate was collected by

suction filtration, washed with methanol or ethanol and dried at room temperature.

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Experimental

27

For the method described by Strohbehn (2012), membranes (0.6 g) and eggshells

(6) were submersed in 20 and 100 mL respectively of a basic solution (NaOH 5 % and 10

%). They were incubated overnight with different temperatures (37 °C and 50 °C), in a way

to optimize method. After cooling to 4–18 °C, the samples with eggshell were filter with

gaze, pH was adjusted between 6-8 with acetic acid, conductivity was measured for all

samples which were dialyzed through float-A-lyzer G2 dialysis device 0.500 – 1 kDa

(SPECTRUM, Rancho Dominguez, CA, USA) until ash were reduced to a conductivity

reading of 2-4 mS cm-1, samples were lyophilized and stored a -20 °C .

For both eggshell membrane solubilization methods protein was quantified by

three methods: Lowry, using Modified Lowry Protein Assay Kit (Thermo scientific,

Rockford, USA), Bradford with Bradford reagent (Bio-Rad, Hercules, CA, United States),

and bicinchoninic acid (BCA) based in the Pierce™ BCA Protein Assay Kit (Thermo

Scientific). Aiming the study of process steps yield, for the Strohbehn (2012) method

protein was determined before and after dialysis.

2.2. Enzymatic hydrolysis

2.2.1. Experimental design, modeling and optimization

Conditions for proteins of eggshell membrane hydrolysis were optimized using

RSM. Hydrolysis was developed with three different enzymes. For each one, experiments

were conducted with two independent variables, enzyme/substrate (E/S) ratio and

reaction time whereas response used in experimental designs was the DH, ACE-inhibitory

and antioxidant activities of the corresponding hydrolysates. In order to achieve that

propose the central composite design (CCD) consisted in a complete 22 factorial design,

with thirteen independent experiments (N=2k + 2k + n0). Of the 13 experiments, 4 were

accounted for by two levels (-1 and +1); another 4 were axial points (at a normalized

distance of ± √2); and the remaining 5 corresponded to center points (used as variance

estimators, at nil coordinate). This design permitted five distinct levels to be tested: 0.1,

0.5, 1.5, 2.5 and 2.9 %(v/v), for the E/S ratio; and 0, 1, 3.5, 6 and 7 h, for the reaction time.

The experiments were run in random order. The associated matrix of experimental design

and results is shown in Table 9.

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https://www.google.pt/search?biw=1280&bih=699&q=california&stick=H4sIAAAAAAAAAGOovnz8BQMDgwsHnxCXfq6-gXGBYa55ihIHiG2YZ16opZWdbKWfX5SemJdZlViSmZ-HwrHKSE1MKSxNLCpJLSo-zlxgXMpVUmPswFg25V6mzXa_oPkAu05mKWEAAAA&sa=X&ei=lNWKVayYLMbV7Aa8rIOYCA&sqi=2&ved=0CIsBEJsTKAIwEQ

https://www.google.pt/search?biw=1280&bih=699&q=estados+unidos&stick=H4sIAAAAAAAAAGOovnz8BQMDgysHnxCXfq6-gXGBYa55ihIniG2ZbF5uoKWVnWyln1-UnpiXWZVYkpmfh8KxykhNTCksTSwqSS0qvqMwMfotL9PZ_fn7bv-eYCW6QLP4EAD7MLGXYgAAAA&sa=X&ei=lNWKVayYLMbV7Aa8rIOYCA&sqi=2&ved=0CIwBEJsTKAMwEQ

28

The quadratic polynomial model proposed for each response variable, takes the form:

𝑌 = 𝛽0 + 𝛽1𝑅 + 𝛽2𝑇 + 𝛽1,1𝑅2 + 𝛽2,2𝑇2 + 𝛽1,2𝑅𝑇 + 𝜀

where: R denotes the E/S ratio and T the reaction time; β0 is the vertical intercept;

β1 and β2 are linear coefficients, β1,1 and β2,2 are quadratic coefficients, and , β1,2 is the

interaction coefficient; and ε denotes the experimental error.

Experimental results from the CCD were analyzed using response surface

regression. Assessment of the goodness of fit was made by analysis of variance (ANOVA).

Experimental design, data analysis and response surfaces were performed by Statgraphics

Centurion XVI.

2.2.2. Performance of enzymatic hydrolysis

Protein eggshell membrane substrate was submitted to hydrolysis brought about

by three enzymes (one experiment for each enzyme): alcalase from Bacillus licheniformis

(Merck), viscozyme L (Sigma-Aldrich, St. Louis, MO, USA) and protease from Bacillus

amyloliquefaciens (Sigma-Aldrich). For each enzyme the substrate (40 gprotein L-1) was

prepared by two different ways. For the first one, a suspension of protein isolate was

prepared by mixing protein powder in acetic acid 10 %, followed by stirring for 30 minutes

at 100 °C. The pH was adjusted according to the optimum conditions of each enzyme with

NaOH 10 M (Table 6) solutions were cooled at room temperature. For the second one,

protein powder was solved with specific pH buffer solution (Table 6). The E/S ratio for

each experiment was expressed on commercial enzyme extract volume basis.

The samples were incubated at the suitable temperature to each enzyme and were

taken by 0, 1, 3.5, 6 and 7 h (Table 6); quenching was by heating at 95 °C for 20 min. The

hydrolysates were centrifuged at 5,500 rpm for 30 min, and the supernatants are frozen at

-20 °C (and kept it until use). In order to validate the model the hydrolysis were repeated

in the optimum processing conditions. A hydrolysate portion was submitted to

ultrafiltration through a hydrophilic 3 kDa cut-off membrane (Merck) and the <3 kDa and

>3 kDa fraction obtained were freeze-dried and kept at -20 °C until used.

29

Table 6 – Trade, chemical names, activity and the optimum conditions of the studied enzymes

(Source from the supplier (Merck and SigmaAldrich)) and buffer solution used to each enzyme.

Trade Name Chemical Content Activity T (ºC) pH

Buffer

solutions

Alcalase from

bacillus

licheniformis

Protease ≥ 0.75 AU mL-1 55 6 - 8,5

Phosphate

buffer pH

7,58

Viscozyme L

Pectinase/

xylanase/glucanase/

cellulose

≥ 100 FBGU g-1 50 4,5 - 7

Acetate

buffer pH

4,6

Protease from

bacillus

amyloliquefaciens

Protease ≥ 0.8 U g-1 50 5 - 7

Phosphate

buffer pH

6,61

2.2.3. Determination of degree of hydrolysis (DH)

The DH was quantified by measuring the increase in free amino groups, using a

picrylsulfonic acid, TNBS (Sigma-Aldrich) solution, according to McKellar (1981). Each

sample (0.050 mL) was mixed with 0.5 mL of 1M potassium borate buffer (pH 9.2) and

0.2 mL of 0.015 %(w/v) of TNBS and incubated in the dark at 25°C for 30 min; then, 0,2

mL of 2 M Na2HPO4 (Sigma-Aldrich), containing 18 mM Na2SO3, was added to stop

reaction, and absorbance was measured spectrophotometrically at 420 nm. A standard

curve was prepared with L-Leucine covering the range (0 – 2.0 mM) and absorbance was

converted to µmolfree amino groups mL-1 using said curve. The total number of amino groups

was determined in a sample by complete hydrolysis using 6 M HCl at 105 °C during 24h

and the percent DH values were calculate using the following formula (Baek and

Cadwallader, 1995; Hsu, 2010):

%𝐷𝐻 = (𝐿𝑡– 𝐿0

𝐿𝑚𝑎𝑥 − 𝐿0) × 100

where Lt is the amount of liberated amino acid at time t, L0 is the amount of the amino

acid in the original substrate (blank) and Lmax is the maximum amount of the specific

amino acid in the substrate obtained after hydrolysis. All measurements were performed

in triplicate.

30

2.2.4. Determination of ACE-inhibitory activity

The ACE-inhibitory activity was measured using the fluorimetric assay by

Sentandreu and Toldrá (2006), with the modifications reported by Quirós et al. (2009).

Extraction and Preparation of ACE from rabbit lung acetone powder:

Rabbit lung acetone powder (1 g) (Sigma-Aldrich) was mixed in 10 mL of 150 mM Tris-

base buffer containing 5 %(v/v) glycerol at pH 8.3 using gentle magnetic stirring at 4 °C

overnight. The extract solution was then centrifuged at 14,000 rpm until the deposit be

minimal, in a centrifuge at 4 °C and the supernatant containing ACE activity was retained

and stored at -20 °C (~2,000 units ACE activity L-1) (Murray et al., 2004).

Working solutions: ACE stock solution was diluted 1/5 with 150 mM Tris-base

buffer, pH 8.3, containing 1 µM ZnCl20.1 mM. The fluorescent substrate 0.45 mM was

prepared dissolving the Abz-Gly-Phe(NO2)-Pro (Bachem, Torrance, USA), in 150 mM Tris-

base buffer (pH 8.3) containing 1.125 M NaCl.

Procedure: In this method, the microplate was prepared with a blank (80 µL of

ultra-pure water), a control (40 µL of ultra-pure water and 40µL of enzyme), blank

samples (40 µL of sample and 40 µL of water ultra-pure) and samples (40 µL of sample

and 40 µL of enzyme). The enzyme reaction was initiated by addition of 160 µL of 0.45

mM fluorescent substrate which was immediately mixed and incubated at 37 °C and the

fluorescence was measured at 0, 15 and 30 min. The microplate (96F untreated, NuncTM,

Denmark) was automatically shaken before each reading. The activity of each sample was

tested in triplicate. A Biotek, Synergy HT plate reader with 350 nm as excitation and 420

nm as emission filters was used. The software used was Gen 5 2.01.

Results: ACE-inhibitory activity was expressed as IC50 values - concentration of

inhibitor needed to inhibit 50 % ACE-inhibitory activity. A non–linear fit of the data

obtained was performed to calculate the IC50 values, using the GraphPad Prism 5, and data

were expressed as means ± SD (n=2).

31

2.2.5. Determination of antioxidant activity

As already discussed, there are many methods to measure the antioxidant activity.

However at this work were used two methods: ORAC and the modified ABTS method

presented by Ou et al. (2001) with the modifications reported by Dávalos et al. (2004),

and Ozgen et al. (2006), respectively. However ABTS method was only used to determine

antioxidant activity in optimum conditions.

2.2.5.1. ORAC assay

Working solutions: Initially, a stock solution of fluorescein (FL) sodium salt

(1.17 mM) (Sigma-Aldrich) was prepared and conserved at 4 °C for a month. Daily, AAPH

(46.6 mM) (Sigma-Aldrich) was prepared and FL was diluted (0.117 µM) from the stock

solution in 75 mM phosphate buffer (pH 7.4).

Standard Solutions: First, it was prepared a stock solution of Trolox ((±)-6-

hydroxy-2, 5, 7, 8-tetramethylchromane-2-carboxylic acid) (Sigma-Aldrich) 1 mM in 1 mL

of methanol and phosphate buffer 75 mM pH 7.4 (Vf = 50 mL). A T0 solution (0.1 mM)

was prepared in the same buffer used previously. The standard solutions were prepared

from T0 solution with concentrations between 10 and 70 µM in phosphate buffer 75 mM

pH 7.4.

Procedure: At the microplate 20 µL of standard solutions or hydrolysates

samples (between 1:500 and 1:4000) and 120 µL of FL solution in phosphate buffer 75

mM, pH 7.4, were added in each well. The mixture was pre-incubated for 10 min at 37 °C.

Finally, 60 µL of AAPH solution were added and the microplate was immediately place in

the reader. The reaction was carried out at 37 °C, during 80 min. and that fluorescence

was recorded every minute. A control was prepared with 20 µL of phosphate buffer

instead standards or samples. The reaction mixtures were prepared in duplicate and at

least three independent assays were performed for each experiment. The microplate

(Black 96F untreated, NuncTM, Denmark) was automatically shaken before each reading. A

Biotek, Synergy HT plate reader with 485-P excitation and 520-P emission filters was used

and the software used was Gen 5 2.01.

32

Results: Fluorescence measurements were normalized by the curve of the blank,

as proposed elsewhere (Hernández-Ledesma et al., 2005). From the normalized curves,

the area under the fluorescence decay curve (AUC) was calculated as:

𝐴𝑈𝐶 = 1 + ∑ 𝑓𝑖/𝑓0

𝑖=80

𝑖=1

where f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at

time i. The net AUC corresponding to a sample was calculated as follows:

𝑛𝑒𝑡 𝐴𝑈𝐶 = 𝐴𝑈𝐶𝑎𝑛𝑡𝑖𝑜𝑥𝑖𝑑𝑎𝑛𝑡 − 𝐴𝑈𝐶𝑏𝑙𝑎𝑛𝑘

The regression equation between net AUC and antioxidant concentration was

calculated. The slope of the equation was used to calculate the ORAC value, by using the

Trolox curve obtained for each assay. Final ORAC values were expressed as µmoltrolox

equivalent mg-1hydrolyzed protein.

2.2.5.2. ABTS assay

ABTS●+ radical working solution: Prepared through a chemical reaction by

mixing an ABTS (Sigma-Aldrich) stock solution 7 mM with potassium persulfate (Sigma-

Aldrich) 2.45 mM at equal volumes. This solution was left in the dark overnight at room

temperature. Then some dilutions of these solutions were prepared between 25 µM and

250 µM, in order to determine the dilution factor that would give an absorbance value of

0.700 ± 0.020 at 734 nm, after dilution in the microplate well (ABTS●+ concentration was

about 85 µM).

Standard Solutions: First, a stock solution of Trolox ((±)-6-hydroxy-2, 5, 7, 8-

tetramethylchromane-2-carboxylic acid) 1 mM in 1 mL of methanol and acetate buffer 50

mM pH 4.6 (Vf = 50 mL) was prepared. From this solution a T0 solution (0.1 mM) was

prepared in the same buffer used previously. The standard solutions were prepared from

T0 solution with concentrations between 10 and 70 µM in acetate buffer 50 mM pH 4.6.

Procedure: In the microplate, 100 µL of standard solutions or hydrolysates

samples (between 1:128 and 1:512) and 100 µL of ABTS●+ solution in acetate buffer 50

mM, pH 4.6, were added in each well. The kinetic profile of the ABTS●+ scavenging was

33

registered spectrophometrically at 734 nm, recording every minute for 300 minutes. A

control was prepared with 100 µL of acetate buffer instead of standards or samples, in

order to evaluate the absorbance of ABTS●+ in the absence of antioxidant species. The

reaction mixtures were prepared in duplicate and three independent assays were

performed for each experiment. The microplate (96-Well Flat Bottom, Corning USA) was

automatically shaken before each reading. A microplate reader (SPECTROstar Nano, BMG

LABTECH) was used controlled by SPECTROnano software.

Results: The antioxidant activity was calculated using the equation from Trolox

calibration curve. Final ABTS values were expressed as µmoltrolox equivalent mg-1hydrolyzed protein.

2.3. Statistical Analysis

All experimental results were analyzed by one-independent t-test, after normality

has been study, at a significance level p<0.05. The software used was SPSS version 23.0

(IBM Corporation, New York, EUA).

2.4. RP-HPLC

Reverse phase high performance liquid chromatography (RP-HPLC) was

performed in order to compare eggshell membrane protein before and after hydrolyses as

well as its < and >3 kDa fractions. For that purpose, an HPLC unit was used (Jasco, Tokyo,

Japan) equipped with Jasco PU-1580 HPLC pumps, a Jasco AS-2057 Plus auto injector,

an MD-910 Plus multi-wavelength detector, and with the Borwin PDA Controller Software

for data acquisition and control. The column was a reversed-phase Chrompack P300, C18

(8 µm of particle size, 300 mm of length and 4.6 mm of inner diameter). The HPLC was

carried out by gradient elution with a mixture of two solvents and a flow rate of 0.8 mL

min-1. Solvent A consisted in 0.37 % of trifluoroacetic acid (TFA) in water and solvent B

consisted in 0.27 % of TFA in acetonitrile. Elution was performed with a linear gradient:

0-5 min, 98 % of A and 2 % of B, 5-65 min, 55 % of A and 45 % of B and 65-80 min column

rise and re-equilibrium. Separations were carried out at room temperature. Absorbance of

the eluent was monitored at 214 nm. Samples were prepared in duplicate.

Chapter 3 – Results and Discussion

3.1. Eggshell vs eggshell membranes nutritional characterization

3.2. Extraction and solubilization of eggshell membranes

3.3. Experimental design, modeling and optimization

3.4. Model validation


35

3.1. Eggshell vs Eggshell membranes nutritional

characterization

Protein and ashes quantification were carried out in order to have an insight if

protein content in eggshell membranes could be nutritionally interesting. Results are

presented in Table 7.

Table 7 – Protein results (%) obtained by Kjeldahl method and determination of ashes (942.05

AOAC method) for membranes and eggshell.

Membranes Eggshell +

Membranes

Protein (%) 98.1 ± 0.7

4.8 ± 0.5

Ash (%) 3.8 ± 0.1 94.5 ± 0.0

Membrane’s protein content is close to 100 %, having only 3.8 % of ashes. This

result shows its nutritional importance. The eggshell consisted mainly of CaCO3 (about 95

%) and , unlike membranes, had a lower percentage of protein which was expected and

according with King’ori (2011) findings, as mentioned in Chapter 1, section 1.3.2.

3.2. Extraction and solubilization of eggshell membranes

Considering a future application in food industry, membrane extraction and

solubilization methods were studied in order to find the easiest, quickest, most efficient

and economical one. Concerning membrane extraction, it was observed that they could be

easily extracted when immersed in 5 % or 10 % of acetic acid at room temperature during 1

h.

In the case of solubilization, two methods were tested and the possibility for

membranes solubilization without removing them from eggshells was studied for each

one. However, for 3-mercaptopropionic acid method only the test with isolated

membranes was successful. For all experiments, protein content was quantified by three

different assays – BCA, Lowry and Bradford. The results obtained for BCA and Lowry tests

were similar, since they depend on the reduction of cupric ion to develop color and both

can quantify total protein (Lovrien and Matulis, 2005). In the case of Bradford assay, the

reagent used reacted more specifically with proteins larger than 13 kDa (Singh et al.,

2004); so, in this case, peptides can be determined using the protein quantified by this

method, through the following the formula:


36

% 𝑝𝑒𝑝𝑡𝑖𝑑𝑒𝑠 = 𝑇𝑜𝑡𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 − 𝐵𝑟𝑎𝑑𝑓𝑜𝑟𝑑 𝑝𝑟𝑜𝑡𝑒𝑖𝑛

𝑇𝑜𝑡𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛× 100

The protein obtained were 25.6 ± 0.4 mg mL-1, 25.9 ± 3.4 mg mL-1 and 23.7 ± 1.9

mg mL-1 by BCA, Lowry and Bradford assays, respectively. Comparing BCA and Lowry

results, as expected, they did not present statistically differences (p<0.05). So, BCA assay

was chosen to determine total protein throughout this work, since it is a good combination

of sensitivity and simplicity, the reagent is more stable under alkaline conditions, and has

one unique step procedure unlikely Lowry assay (Lovrien and Matulis, 2005). For the

studied solubilization method, the peptides percentage was 8.5 ± 6.7 %. Regardless the

washing solvent used, both ethanol and methanol can be used with a solubilization yield of

63 % (p <0.05).

In case of Strohbehn (2012) patented solubilization method, the process yield was

determined for each experiment before and after dialysis step (once it is a critical step)

(Figure 10) as well as the percentage of formed peptides (Table 8). As can be noticed, the

yield was higher before dialysis than after, however this step is very important in order to

eliminate the high content of NaOH salt from the samples, in order to obtain a protein

concentrate.

Figure 10 – Graphic representations of patent solubilization method yield (%) obtained for

membranes (M) and eggshells (E) with different NaOH concentrations (5 % and 10 %) and different

temperatures (50 °C and 37 °C), before and after dialysis.

0

10

20

30

40

50

60

70

80

90

100

Before dialysis After dialysis

M5% 50 °C M10% 50 °C E10% 37 °C M10% 37 °C E5% 50 °C E10% 50 °C


37

It was also observed that, before dialysis samples presented higher yield values

with higher concentrations of NaOH and temperature, either to membranes or to eggshell

with membranes (M10% 50 ºC – 42.6 ± 4.5 % and E10% 50 ºC – 78.7 ± 9.2 %). Although the yield

was remarkably lower after dialysis, the best results were observed for samples with less

NaOH and temperature. This result can be explained by the possibility of, during

solubilization, smaller peptides had been formed in samples with high NaOH content and

passed through dialysis membranes. That assumption was corroborated by the percentage

of peptides formed during process (Table 8), showing that samples with high amount of

NaOH presented a higher peptide percentage. Although the dialysis membrane used, had

a very low porosity (between 0.5 - 1 kDa), it may not be enough to retain small peptides.

So, in order to retain small peptides it may be necessary to lower the porosity of dialysis

membrane. Nevertheless, this step is very slow and the recovery of small amounts of

protein implies large solution volumes, which delays the passage of laboratory scale

method to the semi-industrial or industrial scale. Thus, acid method was considered as

being better than this one, since it is quicker and more efficient. The problem with this

method is the presence of a toxic agent (3-mercaptopropionic acid), that should be

avoided if the objective is the membranes incorporation into food products. Despite of

being toxic, as referred in Chapter 1 (section 1.6), this agent is used at low concentrations

and is eliminated through washing after precipitation (Jia et al., 2012; Yi et al., 2003,

2004).

Table 8 - Percentage of peptides formed during solubilization process for patented method.

Experiments % peptides

M 5% 50 °C 46.5 ± 6.1

M 10% 50 °C 59.2 ± 2.1

M 10% 37 °C 28.2 ± 8.9

E 5% 50 °C 25.7 ± 9.6

E 10% 50 °C 70.4 ± 4.7

E 10% 37 °C 28.0 ± 0.7

M – Membranes; E – Eggshell


38

3.3. Experimental design, modeling and optimization

Variables that mainly affect the enzymatic hydrolysis are E/S ratio (R) (v/v) and

reaction time (T) (h) (Contreras et al., 2011) so, in this work the influence of this two

factors was studied by quantifying DH, ACE-inhibitory activity and antioxidant activity of

eggshell membranes hydrolysates. In general, DH is an helpful parameter if the results

obtained in biological activities are a consequence of extensive hydrolysis, or due to some

intrinsic properties of the substrate(s) or enzyme specificity, since antihypertensive and

antioxidant activities depend considerably on DH of the protein substrates (Tavares et al.,

2011). The experiments were performed randomly with different parameters combinations

using statistically designed experiments. The values of the processing factors were selected

so as to cover a wide range of conditions – as shown in Table 9, but taking into

consideration the practical industrial constraints.

Besides going well beyond the goal of this study dealing only with optimization –

and implying a far larger number of experiments, the practical applicability of the results

to be generated in such a case would be narrow – because R and T are processing

parameters that can be easily manipulated a priori, unlike DH that would require

feedback control and monitoring before startup in an industrial setting.

Two different procedures were tested for each enzyme with the aim of determining

the best and most efficient way to prepare the substrate. Thus, protein powder obtained

from 3-mercaptopropionic acid solubilization method was either dissolved in acetic acid

10 % and in a specific buffer. For each substrate, the designed set of experiments were

applied and the degree of hydrolysis was determined and compared (Table 9). The results

show that there was not a statistically significant difference in DH (p<0.05). So, for this

reason the biological activities were quantified in hydrolysates obtained from substrates

prepared with buffer. Hydrolysates obtained from alcalase presented higher amount of

free amino groups, 38.4 ± 2.3 % (as measured by DH) than viscozyme L (18.4 ± 0.4 %)

and protease (8.5 ± 2.3 %), which showed the lowest values.


39

Table 9 - Experimental design encompassing two processing parameters (reaction time (T) and E/S ratio (R)) and results pertaining to three responses (degree of

hydrolysis (DH), antioxidant activity and ACE-inhibitory activity of total hydrolysates), obtained from three enzymes, alcalase from Bacillus licheniformis, viscozyme L and

protease from Bacillus amyloliquefaciens.

A – Eggshell membrane protein dissolved in acetic acid 10 %.

B – Eggshell membrane protein dissolved in specific buffer.

a Obtained by TNBS method (%).

b Obtained according to Sentandreu and Toldrá (2006), after modification by Quirós et al. (2009) (IC50, µg mL-1).

c Obtained by ORAC method (µmolTrolox equivalent mg-1hydrolysed protein).

Alcalase Viscozyme L Protease

Exp. T

(h) R

(%, v/v)

DH (A)a DH (B)a

ACE-inhibitory Activityb

Antioxidant Activityc

DH (A)a DH (B)a ACE-inhibitory

Activityb


DH (A)a DH (B)a ACE-inhibitory

Activityb


1 1 2.5 19.6 ± 5.9 18.8 ± 1.0 35.5 ± 2.8 2.7 ± 0.2 4.8 ± 0.2 5.2 ± 0.4 123.9 ± 11.1 3.4 ± 0.3 0.6 ± 0.4 4.4 ± 1.9 56.9 ± 8.0 2.7 ± 0.0

2 1 0.5 9.9 ± 0.1 11.3 ± 3.6 69.2 ± 11.0 2.1 ± 0.1 1.7 ± 0.8 5.3 ± 0.1 159.7 ± 4.2 2.0 ± 0.2 0.5 ± 0.1 3.3 ± 1.7 63.9 ± 8.0 2.3 ± 0.1

3 6 0.5 15.5 ± 2.3 21.9 ± 0.2 34.4 ± 1.4 4.1 ± 0.2 8.2 ± 0.7 7.1 ± 2.2 141.3 ± 6.1 4.2 ± 0.2 0.5 ± 0.0 4.7 ± 1.1 62.1 ± 1.2 3.2 ± 0.2

4 7 1.5 22.3 ± 1.5 27.1 ± 4.2 20.0 ± 1.1 4.9 ± 0.3 10.6 ± 1.2 12.3 ± 1.2 64.6 ± 6.4 4.6 ± 0.3 5.3 ± 0.1 5.1 ± 1.7 50.7 ± 3.3 3.6 ± 0.2

5 3.5 1.5 20.0 ± 0.6 22.8 ± 1.8 28.5 ± 0.8 4.1 ± 0.2 4.8 ± 0.5 9.9 ± 2.0 69.3 ± 0.2 4.7 ± 0.3 3.5 ± 0.5 5.8 ± 0.6 68.6 ± 11.3 2.8 ± 0.1

6 3.5 1.5 25.8 ± 2.4 23.5 ± 2.1 35.4 ± 4.9 3.4 ± 0.2 6.8 ± 1.2 10.9 ± 0.9 75.1 ± 0.2 4.1 ± 0.3 4.7 ± 0.9 5.7 ± 0.3 68.1 ± 5.1 3.5 ± 0.1

7 0.0 1.5 0.0 ± 0.0 0.0 ± 0.0 175.8 ± 0.4 1.8 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 155.8 ± 4.0 3.0 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 170.1 ± 21.6 2.2 ± 0.2

8 3.5 2.9 39.3 ± 7.9 31.3 ± 3.1 20.5 ± 0.6 4.3 ± 0.6 8.8 ± 1.5 11.5 ± 0.1 64.4 ± 7.2 4.0 ± 0.1 5.6 ± 0.8 8.5 ± 2.3 53.4 ± 6.9 3.7 ± 0.1

9 3.5 0.1 11.6 ± 1.6 17.3 ± 0.1 59.9 ± 6.4 3.5 ± 0.4 0.7 ± 0.0 5.3 ± 1.2 149.6 ± 10.5 2.8 ± 0.4 2.4 ± 0.6 3.7 ± 0.0 69.5 ± 1.3 2.7 ± 0.1

10 3.5 1.5 20.8 ± 0.5 24.5 ± 2.8 27.7 ± 1.7 4.3 ± 0.4 6.1 ± 0.1 10.3 ± 0.5 70.6 ± 1.1 4.0 ± 0.0 5.4 ± 0.1 5.4 ± 0.2 72.1 ± 11.3 3.3 ± 0.1

11 3.5 1.5 21.7 ± 2.3 25.7 ± 2.1 28.7 ± 3.5 4.1 ± 0.3 6.2 ± 2.5 10.4 ± 0.2 64.9 ± 5.7 4.1 ± 0.1 4.9 ± 0.1 5.6 ± 0.6 65.7 ± 3.3 3.3 ± 0.1

12 3.5 1.5 22.5 ± 1.7 23.3 ± 1.6 28.3 ± 4.0 4.7 ± 0.2 7.2 ±0.6 10.3 ± 0.5 72.4 ± 3.2 4.2 ± 0.2 5.6 ± 1.0 6.5 ± 0.5 63.5 ± 11.3 3.5 ± 0.2

13 6 2.5 36.7 ± 8.9 38.4 ± 2.3 23.4 ± 1.0 4.9 ± 0.3 8.1 ± 1.2 18.4 ± 0.4 42.4 ± 3.2 4.3 ± 0.2 8.2 ± 1.2 7.1 ± 1.1 52.3 ± 6.5 3.9 ± 0.1


40

Higher ACE-inhibitory activity was also observed for alcalase hydrolysates,

presenting a value of 20.0 ± 1.1 µg mL-1, followed by viscozyme L and protease

hydrolysates 42.4 ± 3.2 µg mL-1 and 52.2 ± 6.5 µg mL-1, respectively. The ACE-inhibitory

activity results appear to be notable compared with other research that exhibited an IC50

of 260.0 µg mL-1 in chicken collagen hydrolysates (Saiga et al., 2008), or in market

products, such as WE80BG (whey hydrolysates), EE90FX (egg white hydrolysates),

CE90STL (casein hydrolysates), SE50BT (soybean hydrolysates) and WGE80GPN (gluten

hydrolysates) that presented values of IC50 between 373 and 782 µg mL-1 (Murakami et

al., 2004); Calpis product (milk hydrolysates) with an IC50 of 266 µg mL-1 (Shou Tsai,

2008); and Biozate® (whey hydrolysates), soybean drink presented IC50 values of 450 and

80-360 µg mL-1, respectively (Nakamura et al., 1995). These values can be explained by

the solubilization treatment that membranes are subjected to before hydrolysis in order to

destroy the protein structure, as already discussed in Chapter 1 (section 1.6). The

membrane gets weak and dissolves completely, having the cysteine amino acid removed

(Yi et al., 2004). In this case, it is probable that the solubilization process has made a pre-

hydrolysis and could justify the high activity values observed for 0 h of hydrolysis in all

enzymes, alcalase, viscozyme L and protease – 175.8 ± 0.4 µg mL-1, 155.8 ± 4.0 µg mL-1

and 170.1 ± 21.6 µg mL-1, respectively (see Table 9), and consequently the greater value

obtained after enzymatic hydrolysis.

Although several studies have conveyed comparisons on the performance of

peptide mixtures versus captopril (i.e. the standard ACE-inhibition drug), note that the

core of this study was the optimization of crude enzyme hydrolysates, rather than

characterization of pure peptides – which were the only ones to deserve testing against a

pharmaceutical active principle. The type of peptides formed during hydrolysis could

explain ACE-inhibitory activity. Some studies showed that this enzyme prefers substrates

or inhibitors with hydrophobic (aromatic or branched side chains) amino acids at each of

three C-terminal positions, wherein Tyr, Phe, Trp and/or Pro are the amino acids more

present in C-terminal of ACE-inhibitors peptides (Hernández-Ledesma et al., 2011).


41

The three enzymes hydrolysates showed similar and remarkable ORAC values: 4.9

± 0.3, 4.7 ± 0.3 and 3.9 ± 0.1 µmoltrolox equivalent mg-1hydrolyzed protein by alcalase, viscozyme L and

protease hydrolysates, respectively. Some studies (Contreras et al., 2011; Hernández-

Ledesma et al., 2005; Huang et al., 2010) presented ORAC values of 1.7 µmoltrolox equivalent

mg-1hydrolyzed protein, from 0.7 to 1.1 µmoltrolox equivalent mg-1

hydrolyzed protein and from 0.7 to 3.0

µmoltrolox equivalent mg-1hydrolyzed protein, respectively, in egg white protein ovotransferrin and

whey proteins, as moderated antioxidant activities. Those results can be due to the

presence of peptides with branched amino acids, such as valine, leucine, isoleucine and

aromatic amino acids such as tyrosine, tryptophan and phenylalanine, since they have

indol and phenol groups capable of donating hydrogen’s (Ajibola et al., 2011; Saito et al.,

2003; Soladoye et al., 2015), leading to a higher antioxidant activity.

Regression analysis was performed to fit the response function and a final model

was obtained, the results are listed in Table 10, including a number of relevant statistics. A

good fitness of the model is showed in the same table, since the determination coefficient

(R2) was higher than 0.80 for all responses, and the relative standard error of estimate

(RSEE) presented variations between 3.0 % and 14.7 % (below 20 %), indicating that the

model is appropriate to describe the degree of hydrolysis, ACE-inhibitory activity and

antioxidant activity for three enzymes hydrolysates.

Curve analysis of response surfaces for experimental design allowed the prediction

of response function of the reaction time and E/S ratio in DH, ACE-inhibitory and

antioxidant activities (Figure 12). The convex response surface suggested well-defined

optimum variables (E/S ratio and reaction time) and in the case of ACE-inhibitory activity,

indicates that IC50 of the three enzymes hydrolysates decreased with the increase of E/S

ratio and reaction time, which means an increase of antihypertensive activity. Regarding

antioxidant activity, as can be seen, the activity increased with the increase of E/S ratio

and reaction time, with the exception of viscozyme hydrolysates case that starts to lightly

decrease after 3.5 h of hydrolysis.


42

Table 10 – Best estimates of each term in the model – reaction time, T (linear and quadratic), E/S ratio, R (linear and quadratic) and interaction thereof (linear), and

corresponding statistics – R2, SEE and RSEE, pertaining to three responses – degree of hydrolysis, ACE-inhibitory activity and antioxidant activity for total hydrolysates

obtained for three enzymes – alcalase from Bacillus licheniformis, viscozyme L and protease from Bacillus amyloliquefaciens.

Terms of the model


Degree of

hydrolysis1

ACE -inhibitory activity2

Antioxidant activity3

Degree of hydrolysis1

ACE-inhibitory activity2


Degree of hydrolysis1

ACE-inhibitory activity2


Constant 6.671 99.16 1.416 2.417 213.9 1.685 0.3051 55.61 1.6530

T 4.426*** -13.40*** 0.7262*** 1.784*** -26.54*** 0.4633*** 2.037*** 6.269 0.4348***

R 0.6955*** 37.33*** 0.2284*** 0.1523*** -70.79*** 1.516** -0.03147*** 7.639*** 0.4980***

T2 -0.4239** 0.7277** -0.05100** -0.2678*** 3.508*** -0.02860 -0.2380*** -0.9619*** -0.03940***

T x R 0.9000** 2.270** 0.04000 1.140*** -6.310*** -0.03000 0.1300 -0.2800 0.03000

R2 0.5431 5.612*** 0.006250 -0.5487 20.33*** -0.3788*** 0.2875 -3.633** -0.09625**

Statistics

R2a 0.972 0.975 0.961 0.967 0.985 0.888 0.923 0.905 0.982

SEEb 1.54 3.23 0.255 1.06 6.80 0.273 0.743 2.91 0.0951

RSEEc 6.47 9.42 6.89 11.8 7.06 7.00 14.7 4.68 2.97

1obtained by TNBS method (%).

2obtained according to Setandreu and Toldrá (2006), after modification by Quirós et al. (2009) (IC50, µg mL-1).

3obtained by ORAC method (µmoltrolox equivalent mg-1hydrolyzed protein).

Regression coefficient significantly different from zero: *p < 0.1, **p < 0.05, ***p < 0.01.

aR2 = coefficient of determination.

bSEE = standard error of the estimate.

cRSEE = relative standard error of the estimate – standard error of the estimate expressed as percent of mean value of response.


43

3.4. Model validation

In order to obtain the best active peptide concentrate, for each enzyme, optimum

processing conditions and corresponding prediction were achieved by multiple response

optimization tool of Statgraphics Centurion XVI. Antioxidant activity was maximized, in

contrast to ACE-inhibitory activity that was minimized – low IC50 values mean that a

small concentration of inhibitory substance is required to produce enzyme inhibition, so

the substance at stake presents a potent inhibitory activity. The optimum was determined

having into account the design point with highest predicted desirability (Table 11).

Table 11 – Maximum ACE-inhibitory and antioxidant activities of total hydrolysates and

corresponding values of two processing parameters – time (T) and E/S ratio (R), obtained for three enzymes –

alcalase from Bacillus licheniformis, viscozyme L and protease from Bacillus amyloliquefaciens.

Enzymes

Optimum processing conditions Predicted response

T

(h)

R

(%, v/v)

ACE-inhibitory activity

Antioxidant activity

Alcalase 6.04 2.21 20.0 ± 5.2 5.0 ± 0.4

Viscozyme L 6.61 1.90 51.4 ± 11.3 4.6 ± 0.5

Protease 5.32 2.90 49.1 ± 7.1 4.0 ± 0.2

In order to validate the model described, the optimum conditions were tested and

the same parameters were evaluated (Figure 12). Since molecular weight of ACE-

inhibitory peptides are usually below 3 kDa, hydrolysates were submitted to ultrafiltration

through a hydrophilic 3 kDa cut-off membrane and the biological activities determined for

the total hydrolysates (total fraction), <3 kDa fraction and >3 kDa fraction. The

antioxidant activity was determined by ABTS method too.

Antioxidant activity was performed by ABTS method, according to Magalhães et al.

(2012) a 5 h kinetic profile had to be carried out (Figure 11) with the aim to determine the

point of stability of the sample, since the standard (Trolox) used did not had the same

kinetic behavior that food samples (reacts fast with oxidizing species). So, unlike some

other related literature, it is important to pay attention to reaction time, since it has a big

influence in ABTS values, as can be seen in Figure 11. When the absorbance found at 30

min and 3.5 h are interpolated in Trolox calibration curve, the antioxidant activity values

determined will be different.


44

Figure 11– Illustration of antioxidant capacity dependency on reaction time. Curves T1 to T5

represent the absorbance values obtained for increasing concentrations of Trolox.

The results obtained for optimum conditions are presented in Table 12 and, as can

be seen, the activities obtained for total hydrolysate (total fraction) were similar to the

predicted response.

As expected, all hydrolysates presented better or similar activity values to <3 kDa

fractions corroborating with the idea mentioned above that peptides responsible for ACE-

inhibitory activity usually present a molecular weight below 3 kDa. Unlike <3 kDa

fractions, >3 kDa fractions showed low activities. In this case and thinking about a future

industrial application, these differences may not be significant enough to justify the cost of

this purification step and ultrafiltration could be dispensable. Nevertheless, to ascertain

this hypothesis an in vivo study should be performed. Antioxidant activity was different

for both methods, as expected and mentioned in Chapter 1, section 1.5.2.2. (Zulueta et al.,

2009), since each method studies a different way of action.

Table 12 – ACE-inhibitory and antioxidant activities of total fraction, <3 kDa fraction and >3 kDa

fraction at optimum conditions.

Enzymes

ACE – inhibitory

Activitya

Antioxidant activity

ORACb ABTSc

Total <3 kDa >3 kDa Total <3 kDa >3 kDa Total <3 kDa >3 kDa

Alcalase 34.5±2.1 28.5±0.7 86.5±2.1 4.2±0.2 4.1±0.2 3.0±0.1 3.0±0.2 3.8±0.0 1.9±0.1

Viscozyme L 63.0±4.2 45.5±2.1 103.5±13.4 4.4±0.0 3.8±0.1 1.9±0.3 4.8±0.5 4.4±0.0 3.1±0.3

Protease 43.0±8.5 40.5±9.2 118.5±0.7 3.8±0.2 3.4±0.2 1.3±0.1 4.2±0.4 5.2±0.2 1.7±0.1

a Obtained according to Sentandreu and Toldrá (2006), after modification by Quirós et al. (2009) (IC50, µg mL-1).

b Obtained by ORAC method (µmolTrolox equivalent mg-1hydrolyzed protein).

cObtained by ABTS method (µmolTrolox equivalent mg-1hydrolyzed protein).

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 1 2 3 4 5

T1

T2

T3

T5

T4

T1 T2 T3 T4 T5

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

[Trolox] (µM)[Trolox] (µM)

0.5 h3.5h 5h

Ab

so

rb

an

ce

Ab

so

rb

an

ce


45

Figure 12 -Variation of degree of hydrolysis, ACE-inhibitory activity and antioxidant activity as a function of each term in the model – time and E/S ratio, obtained for

three enzymes – alcalase from Bacillus licheniformis, viscozyme L and protease from Bacillus amyloliquefaciens.

0

40

80

120

160

200

240

R

T

0 2 4 6 8 01

23

30

40

50

60

70

80

R

T

0 2 4 6 80

12

3

0

1

2

3

4

5

6

R

T

0 2 4 6 8 01

23

0

20

40

60

80

100

120

R

T0

12

3

0 2 4 6 8

1,8

2,1

2,8

3,1

3,8

4,1

R

T


AC

E-i

nh

ibit

ory

ac

tiv

ity

An

tio

xid

an

t a

cti

vit

y

02 4 6 8 0

12

32

3

4

5

6

RT

01

23

0 2 4 6 8

R

T

R

T

R

T

T

R

0 2 4 6 8

T

01

23

R

0

10

20

30

40

50

De

gre

e o

f H

yd

ro

lysis

(A

)D

eg

re

e o

f H

yd

ro

lysis

(B

)


R

T

0 2 4 6 80

12

3

01

23

0 2 4 6 8

01

23

0 2 4 6 80

12

3

0 2 4 6 8 01

23

0 2 4 6 8

0

10

20

30

40

50

0

2

4

6

8

10

-5

-1

3

7

11

-5

-1

3

7

11

-3

2

7

12

17

22

27

0

40

80

120

160

200

240

R

T

0 2 4 6 8 01

23

30

40

50

60

70

80

R

T

0 2 4 6 80

12

3

0

1

2

3

4

5

6

R

T

0 2 4 6 8 01

23

0

20

40

60

80

100

120

R

T0

12

3

0 2 4 6 8

1,8

2,1

2,8

3,1

3,8

4,1

R

T


AC

E-i

nh

ibit

ory

ac

tiv

ity

An

tio

xid

an

t a

cti

vit

y

02 4 6 8 0

12

32

3

4

5

6

RT

01

23

0 2 4 6 8

Alcalase Viscozyme Protease

De

gr

ee

of

hy

dr

oly

sis

AC

E-i

nh

ibit

ory

ac

tiv

ity

An

tio

xid

an

t a

cti

vit

y


46

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Time (min)

Before hydrolysis

After hydrolysis

< 3 kDa fraction

> 3 kDa fraction

Less hydrophobic peptides

Hydrophobicpolypeptides

As mentioned above, all results point out to the fact that during this work, proteins

were hydrolyzed in the solubilization process. That led to better than expect activities after

subjected to enzymatic hydrolysis as well as low differences between total hydrolysate and

<3 kDa fraction. This hypothesis can be corroborated by chromatographic results showed

in Figure 13. According to Vieira et al. (2012) protein hydrolysates chromatographic

profiles obtained by RP-HPLC were divided into two major ranges: less hydrophobic

peptides and hydrophobic polypeptides eluted with acetonitrile between 2 – 27 % (5 – 40

min) and 27 – 42 % (40 – 60 min) respectively. Thus, all samples present less

hydrophobic peptides, since peptides concentrates were eluted from 5 -40 min as can be

seen on Figure 13. Although the presence of small peptides (less hydrophobic) can be

noticed in the sample before hydrolysis, these chromatograms illustrate the decrease of

polypeptides, with concomitant increase of the former after enzymatic hydrolysis. The

differences between total fraction (after hydrolysis) and <3 kDa fraction chromatograms

are not perceptible. This can justify the small differences obtained when the activities for

both extracts are compared and activity values showed for >3 kDa where is notorious the

absence of proteins. So, as theoretically expected, high peptides content might lead to a

bioactivities potentiation.

Figure 13 – Chromatogram of sample before and after hydrolysis and of <3 kDa and >3 kDa fractions.

Chapter 4 – Conclusions

Conclusion

48

The objective of this work was to investigate potential and relevant biological

activities presented by bioactive peptides of eggshell membrane hydrolysates that can be

used by the food industry. For that, firstly the extraction and solubilization methods were

studied and it was observed that the best results are: immersion of eggshell in acetic acid 5

%(v/v), during 1 h at room temperature to help in the manually separation of eggshell

membrane. For solubilization, 3-mercaptopropionic acid method demonstrated to be the

quicker, rentable (63 % of yield) and cheaper process. The hydrolysis performed in order

to study biological activities presented a degree of hydrolysis higher when hydrolysis was

performed with alcalase from Bacillus licheniformis. The optimum conditions to obtain

the best antihypertensive and antioxidant activity with the commercial enzymes, alcalase

from Bacillus licheniformis, viscozyme L and protease from Bacillus amyloliquefaciens

were: 6 h, 2.2 %(v/v); 6.6 h, 1.9 %(v/v); and 5.3 h, 2.9 %(v/v), respectively. The resulting

hydrolysates exhibited ACE-inhibitory activities characterized by an IC50 of 34.5 ± 2.1 µg

mL-1 (total fraction), 28.5 ± 0.7 µg mL-1 (<3 kDa fraction) for alcalase, 63.0 µg mL-1 (total

fraction) and 45.5 µg mL-1 (<3 kDa fraction), in the case of viscozyme; and for protease,

43.0 ± 8.5 µg mL-1 (total fraction) and 40.5 ± 9.2 µg mL-1. For antioxidant activities the

results obtained for ABTS and ORAC methods were different, since the mechanism of

action of both methods are different. For antioxidant ORAC method was observed values

of 4.2 ±0.2, 4.4 ± 0.1 and 3.8 ± 0.2 µmolTrolox equivalent mg-1hydrolyzed protein for alcalase, viscozyme L

and protease hydrolysis respectively, and for ABTS method the activity was 3.8 ± 0.0, 4.4

± 0.0 and 5.2 ± 0.2 µmolTrolox equivalent mg-1hydrolyzed protein for alcalase, viscozyme L and protease

hydrolysis, respectively. These results can be considered as high when compared with the

typical IC50 and antioxidant values of other food hydrolysates described in the literature.

In conclusion the eggshell membrane byproduct showed to have a great potential

and that its peptides could be consider a high added-value ingredient to be applied in

some functional food.

Chapter 5 – Future Prospects

Future Prospects

50

In this dissertation the presence of bioactive peptides responsible for physiological

functions was studied. It was of great importance, once the existing research on eggshell

membrane in food area is limited. However this was only the beginning of a big work

ahead, having many opportunities of futures researches.

More detailed studies are welcome, for a better understanding of antihypertensive

and antioxidant mechanisms; enzyme immobilization studies should also be carried out,

in order to optimize peptide release; and in the case of enzymes yield, other factors besides

substrate/enzyme ratio and reaction time should be optimized for each proteolytic system.

It is necessary submit hydrolysates obtained to a simulation of gastrointestinal digestion

in order to verify the bioavailability and stability of peptides. Try microencapsulation

would be a possible solution to increase efficiency in delivery of bioactive peptides in

foods, by protecting them against gastrointestinal digestion and releasing them only in the

colon. After that, in vivo studies (rats) should be pertained to ascertain if these peptides

concentrates present in vivo activity.

For a fundamental study, it is important identify and isolate peptides present in the

hydrolysates and understand which ones are the responsible for the detected activity and

follow all suggestions presented above of peptides submission to gastrointestinal digestion

simulation, and in vivo studies.

Finally, peptides should be tested in actual food systems for an industrial

application, being necessary precede to an evaluation of their rheological and sensory

behavior. To mask their bitter taste and avoid bioactive peptides degradation during

digestion, it could be apply an encapsulation of the compounds of interest. It also could be

done their incorporation in food, and check the stability of passage through the gastric

system and subsequent uptake of peptides in the colon and their bioavailability in the

bloodstream.

After study stability to gastrointestinal digestion and passage through the blood

barrier in vivo, the physiological function of bioactive peptides needs to be validated

through clinical trials with human volunteers.

Chapter 6 – References

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biological activities of peptide concentrates obtained ... · com o objetivo de explorar...

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