Embed Size (px)
Citation preview
Nanoencapsulation of Tea Catechins in Casein Micelles: Effects on Processing
and Biological Functionalities
by
Sanaz Haratifar
A Thesis
presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
in
Food Science
Guelph, Ontario, Canada
© Sanaz Haratifar, November, 2012
ABSTRACT
Nanoencapsulation Of Tea Catechins In Casein Micelles: Effects On Processing and
Biological Functionalities
Sanaz Haratifar Advisor:
University of Guelph, 2012 Professor M. Corredig
This thesis focuses on the interactions between milk proteins (caseins) and tea catechins and the
consequences of the interactions on the renneting properties and digestion of casein micelles as
well as on the biological functionality of the mixture, as measured using intestinal cell models.
The binding of epigallocatechin-gallate (EGCG) to casein micelles was quantified using HPLC
and fluorescence quenching, and it was shown that a substantial amount of EGCG can be
incorporated in the casein micelles. At concentrations < 2.5 mg/ml milk, most of the EGCG
added to milk was associated with the caseins. The formation of EGCG-casein micelles
complexes not only delayed the gelling point of milk, but they also affected the structure
formation of the gels.
EGCG is known to have antiproliferative activity on colon cancer cells.
Although nanoencapsulation of EGCG in casein micelles may have some advantages, such as
protecting this bioactive compound from degradation, it may also affect the bioavailability of
EGCG. To test this hypothesis, the effect of nanoencapsulation of EGCG in casein micelles on
the biological functionality of EGCG was tested by evaluating the cytotoxity and proliferation
behaviour of HT-29 colon cancer cells. It was demonstrated that nanoencapsulation did not
affect the bioefficacy of EGCG.
Similar experiments were also carried out on rat colonic cells, a normal line and its cancerous
tranformed line. For this study, nanoencapulated EGCG was subjected to in vitro digestion
before absorption. The results showed that EGCG-casein binding did not affect the digestion of
the milk proteins. In this case, the bioefficacy of EGCG was not diminished as well.
In addition, studies on normal cell lines demonstrated the specific effect of EGCG on cancer
cells, favoring normal cell survival whether EGCG was isolated or complexed with milk.
These experiments bring further evidence that milk can be employed as an appropriate platform
for the delivery of bioactive compounds.
iv
Acknowledgement
I would like to thank my advisor, Dr. Milena Corredig, for the opportunity to work on this
project. I appreciate all of the advice, time, and effort that Milena provided during my journey as
a PhD student . Without her direction and assistance this thesis would not have been possible.
I am also grateful to my advisory commitee: Dr. Gopinadhan Paliyath, Dr. Kelly Meckling and
Dr. Lisa Duizer for their advice during my study. Their comments and guidance, were always
comprehensive and constructive.
I am very grateful to Edita Verespej for all her technical support and would like to also thank
all my friends and lab mates, who I have had the opportunity to work with in the dairy lab and at
Canadian Research Institute for Food Safety (CRIFS).
I would like to show my gratitude to the Ontario Dairy Council and the Natural Sciences and
Engineering Council of Canada, through the Industrial Research Chair program, and the Canada
Research Chair Program for financial support of my study.
To my beloved family, especially my father, thank you for your support and encouragements
during all the ups and downs in my life.
Finally, to my sister, Farnaz, I am indebted in every possible way for all the love and
confidence you have in me. I can not imagine my life without you.
This thesis is dedicated to my mother and grandmother, my two angels in heaven.
v
Table of contents
CHAPTER 1 ............................................................................................................................................. 1
1.1. General Introduction ..........................................................................................................1
1.2. Research Hypotheses ..........................................................................................................4
CHAPTER 2 ............................................................................................................................................. 6
2.1. Polyphenols .........................................................................................................................6
2.1.1. Tea.................................................................................................................................8
2.1.2. Tea-bioavailability ....................................................................................................... 12
2.1.3. Nano encapsulation of Tea catechins ........................................................................... 15
2.2. Milk ................................................................................................................................... 17
2.2.1. Caseins ........................................................................................................................ 18
2.2.2. Whey Proteins.............................................................................................................. 21
2.2.3. Rennet induced gelation of milk ................................................................................... 21
2.3. Interactions between milk proteins and tea catechins ....................................................... 22
CHAPTER 3 ........................................................................................................................................... 28
INTERACTIONS BETWEEN TEA CATECHINS AND CASEIN MICELLES AND
THEIR IMPACT ON RENNETING FUNCTIONALITY .......................................................... 28
3.1 Abstract ............................................................................................................................. 28
3.2 Introduction ....................................................................................................................... 29
3.3 Materials and Method .................................................................................................. 31
3.3.1 Milk preparation and interaction studies ........................................................................ 31
3.3.2 Fluorescence spectroscopy ............................................................................................ 32
3.3.3 Quantification of polyphenol binding to casein micelles ................................................ 34
3.3.4 Rheological properties of rennet-induced gels ............................................................... 35
3.3.5 Release of casein macro- peptide (CMP) from the surface of the casein micelles .......... 35
3.3.6 Statistical analysis ......................................................................................................... 35
3.4 Results and Discussion ...................................................................................................... 36
3.4.1 Fluorescence spectroscopy studies ................................................................................ 36
3.4.2 Analysis of tea polyphenols by HPLC ........................................................................... 43
3.4.3 The effect of EGCG on rennet induced gelation of casein micelles ................................ 47
vi
3.5 Conclusions ........................................................................................................................ 55
CHAPTER 4 ........................................................................................................................................... 56
Anti-proliferative activity of nanoencapsulated tea catechin in casein micelles, using HT29
colon cancer cells ..................................................................................................................... 56
4.1. Abstract ............................................................................................................................ 56
4.2. Introduction ...................................................................................................................... 57
4.3. Materials and Methods .................................................................................................... 60
4.3.1 Milk samples preparation .............................................................................................. 60
4.3.2. Cell viability assay ....................................................................................................... 61
4.3.3. Cell proliferation assay ................................................................................................ 61
4.3.4. Statistical analysis ........................................................................................................ 63
4.4. Results and Discussion ..................................................................................................... 63
4.5. Conclusions ....................................................................................................................... 71
CHAPTER 5 ........................................................................................................................................... 72
THE EFFECT OF NANOENCAPSULATION OF TEA CATECHINS IN CASEIN
MICELLES ON THEIR BIOACCESSIBILITY STUDIED USING NORMAL AND
CANCEROUS RAT COLONIC CELLS ........................................................................................ 72
5.1 Abstract ............................................................................................................................. 72
5.2 Introduction ....................................................................................................................... 73
5.3 Materials and Methods ..................................................................................................... 76
5.3.1 Sample preparation ....................................................................................................... 76
5.3.2 In vitro gastro-duodenal digestion ................................................................................ 78
5.3.3 SDS-PAGE analysis...................................................................................................... 79
5.3.4 Cell proliferation assays ................................................................................................ 80
5.3.5 Statistical analysis ......................................................................................................... 81
5.4 Results and Discussion ...................................................................................................... 82
5.4.1 SDS-PAGE of milk and tea/milk complexes during the gastric phase ............................ 82
5.4.2 Proliferation of normal (4D) and tumorous (v-src) rat colonic cells exposed to EGCG
and EGCG -milk fractions before and after invitro digestion .................................................. 85
vii
5.5 Conclusion ......................................................................................................................... 92
CHAPTER 6 ........................................................................................................................................... 93
GENERAL CONCLUSIONS ............................................................................................................. 93
REFERENCES ...................................................................................................................................... 96
viii
Table of Figures
Figure 2.1 Chemical structure of flavonoids. ...............................................................................7
Figure 2.2 Chemical structure of flavan-3-ols. .............................................................................7
Figure 2.3. Structures of four catechins in tea infusions. ..............................................................9
Figure 3.2 Stern-Volmer plot for skim milk ( ) and caseins (♦) re-diluted in permeate. ............ 41
Figure 3.3 Ratio of tea catechin recovered in the colloidal phase (casein micelles) of skim milk,
as a function of the initial concentration of EGCG present in milk. . ......................................... 44
Figure 3.4 Scatchard plot for EGCG-milk caseins .................................................................... 46
Figure 3.5 Rennet coagulation process monitored by rheometer of the four samples………………….48
Figure 3.6 % CMP-release versus time after addition of rennet. ................................................ 52
Figure 3.7 % CMP-release versus time after addition of rennet. ................................................. 54
Figure 4.2 Amount of EGCG associated with the casein micelles in skim milk as a function of
the initial concentration of EGCG in milk……………………………………………………….65
Figure 4.3 % Viable cells after 24h exposure to isolated EGCG (solid) and EGCG encapsulated
in casein micelles. ................................................................................................................... 67
Figure 4.4 Proliferation of HT-29 cells exposed to isolated EGCG or EGCG in milk serum
(permeate) (A); milk whey (B) or encapsulated with the casein micelles in skim milk (C) ....... 70
Figure 5.2 SDS electrophoresis of milk digests . ....................................................................... 83
Figure 5.3 % Change of Tumor cell proliferation with increasing levels of isolated EGCG
(solid bars) and complexed EGCG-milk fractions before in vitro digestion ............................... 87
Figure 5.4 % Change of Tumor cell proliferation with increasing levels of EGCG in digests of
isolated EGCG (solid bars) and complexed EGCG-milk fractions. ............................................ 91
ix
LIST OF TABLES
Table 3.1. Gelling time and gel stiffness of different tea-milk samples.......................................50
1
CHAPTER 1
1.1. General Introduction
Interest for health promoting, natural food compounds has expanded in recent years due to the
increase in consumer awareness and the promotion of healthy eating and lifestyle.
However, challenges remain to ensure that such compounds not only remain in their active form
during processing and storage, but that their bioavailability is also maximized (Seymour et al.,
2008). Indeed, to be able to deliver the nutritional functionality, food products need to have not
only a nutritionally significant dose of bioactive molecules, but these molecules must also
remain fully functional for as long as necessary (Garti, 2008). Polyphenols are amongst the most
bioactive plant metabolites used in food and nutraceutical applications. Tea catechins, the focus
of this thesis, have been shown in numerous studies to have important preventive and therapeutic
roles for the most common human diseases such as cancer, cardiovascular and neurodegenerative
diseases (Manach et al., 2004; Sharma et al., 2009; Yang, 2009; Yang, 2012; Yuan et al., 2011).
However, higher amounts must be consumed to benefit from their bioactivity, as tea catechins
have been reported to have relatively poor absorption efficiency in vivo (Stevenson et al., 2007;
Papadopoulou et al., 2004; Green et al., 2007; Ferruzzi et al., 2010). The relations between
bioefficacy, concentration in food products, bioavailability and cell absorption are not straight
forward, and are the most challenging aspect in the development of food products with health
enhancing properties.
Market trends indicate that milk is an ideal vehicle for the delivery of bioactive compounds
targeting lifestyle diseases, as milk is a natural, multi-component, and nutrient-rich beverage that
is widely consumed around the world (Sharma et al., 2009). Many countries often consume tea
combined with milk (traditional consumption of black tea) and the number of new tea based
2
products containing dairy ingredients, such as ready-to-drink tea chai latte, has expanded in
recent years (Ferruzzi et al., 2005).
Dairy proteins are often used as ingredients in foods for their functionalities, for example,
gelling, foaming, emulsifying. However, in the context of delivery of bioactive compounds,
many structural and physicochemical properties of milk proteins point to their delivery functions
in nature. Milk caseins for example, have been designed by nature to deliver nutrients such as
calcium, phosphate and protein to the neonate (Livney, 2010). Studies have shown that casein
micelles bind to hydrophobic molecules such as curcumin and Vitamin D, which these
compounds penetrate into the core of the micelles. The encapsulation of Vitamin D provides
significant protection against UV light and in the case of curcumin the encapsulation does not
impair the cytotoxic effects of curcumin on HeLa cells compared to an equal dose of free
curcumin (Semo et al, 2007; Sahu et al., 2008; Livney et al., 2009).
Strong interactions between tea catechins and casein proteins have been reported in many studies
however, most studies have been so far conducted on isolated caseins and not with casein
micelles or skim milk (Arts et al., 2002; Jobstyl et al., 2004; Papadopoulou et al., 2005; Pascal et
al., 2007; Yan et al., 2009; Frazier et al., 2009; Yuksel et al., 2010). Interpretation of studies
using caseins in milk media has been difficult since, by the addition of milk, the product matrix
complexity increases, and obtaining accurate and reliable measurements for tea catechins
becomes more challenging. The effect of milk on the bioefficacy of polyphenols in tea is still a
matter of debate as some studies indicate that addition of milk has a negative impact on tea
catechins, for example, on the antioxidant properties, while some others indicate no such effect
(Langley-Evans, 2000; Arts et al., 2002; Kartosova et al., 2007; Sharma et al., 2009; Cilla et al.,
2009; Leenon et al., 2000; Keogh et al., 2007; Kyle, 2007; Green et al., 2007; Dubeau et al.,
3
2011; van der Burg-Koorevaar et al., 2011). It is important to state that there are differences in
the study designs and methodologies of the above mentioned studies which may contribute to the
controversy of the subject. Most studies have overlooked the strong interactions that take place
between the proteins and polyphenols, considering effective (i.e., bioavailable) only the material
recovered in the soluble phase.
This thesis focuses on the interactions between milk proteins (especially caseins) and tea
catechins, and their effect on processing functionality and bioavailability, as measured using
intestinal cell models. Although few studies have been reported on the effect of casein-tea
catechin interactions on the bioavailability of the catechin, rather limited experimental data is
available on the functional properties of caseins. Such studies are also needed to elucidate the
effects of polyphenols on protein functional properties that may influence the fate of polyphenol
application in food processing. Indeed, it is important to determine whether the functional
benefits of both compounds (tea and milk proteins) may work synergistically in a mixture, or
change the effect of the functionality of the components taken in isolation. Our work aims to
explore some of the effects of the addition of polyphenols to milk proteins and their relevance to
the design of improved dairy products.
In the first result chapter the interactions between tea catechins and casein micelles were
quantified using HPLC as well as fluorescence spectroscopy. Most binding studies carried out
so far have used isolated proteins, and this work advances our knowledge on the interactions
between EGCG and casein micelles in their native form. The use of two separate methods will
allow for stronger data interpretation. In this chapter, effect of tea catechin-milk casein
interactions on the rennet induced gelling of milk was evaluated.
4
The second part of the work (Chapters 3 and 4) focused on the effect of the milk casein and tea
catechin complexes on the biological functionality of tea polyphenols using cell culture models.
It was hypothesized that caseins, by binding to EGCG, will decrease the bioavailability of this
component. In Chapter 3 we report on the effect of the tea catechin-milk protein complexes on
the biological functionality of the tea catechin, by applying cell cytotoxity and proliferation
assays on HT-29 colon cancer cells. Different milk fractions were also used to better undersand
the role played by the various milk components in modulating bioactivivity. In Chapter 4, the
effect of the mixture of milk and EGCG on two parallel sets of cell lines was studied, to
determine the cytotoxicity and proliferation in normal versus cancer cell lines. Afterwards, an in
vitro digestion model mimicking the gastric stage and duodenal stage of human digestion was
employed to determine the significance of this association on the digestibility of the milk
proteins and on the bioaccessibility of the tea polyphenol epigalocatechin gallate (EGCG).
This thesis will be able to further our understanding of the interactions between EGCG and
casein micelles, and the impact of such interactions on the processing and biological
functionality of milk. Demonstration of the effective incorporation of bioactives into the milk
matrix is essential before using milk as a platform for delivery of polyphenols.
Hence, this research tested the following hypotheses:
1.2. Research Hypotheses
The main hypothesis of this study was that the interactions between EGCG and casein micelles
improve the bio-accessibility of EGCG without altering the functional properties of caseins as
well as their digestive breakdown.
Therefore, the main objectives studied in this research were:
5
1. Evaluate the interactions between EGCG and casein micelles, in their native form: milk.
2. Determine the effect of the EGCG-casein complexes on the gelation properties of caseins.
3. Determine if the complexes formed affect the biological functionality of tea polyphenols using
cell culture models.
4. Determine if the interactions between EGCG and milk affect the digestion of milk caseins and
if the complexes formed modify the bioactivity of tea polyphenols once digested.
6
CHAPTER 2
2.1. Polyphenols
Interest in polyphenols as health-promoting nutrients has expanded dramatically in recent years.
The main reasons for this interest are the recognition of their great importance in our diet, and
their likely role in the prevention of various diseases. Polyphenols are found in a variety of foods
such as: vegetables, fruits, wine, coffee and tea (Manach et al., 2004). Polyphenols are secondary
plants metabolites and are naturally involved in the defense mechanisms of the plant, for
example, against ultraviolet radiation or aggression by pathogens. Polyphenols may be classified
into different groups as a function of the number of phenol rings that they contain and of the
structural elements that bind these rings to one another. The main groups include phenolic acids,
flavonoids, stilbenes, and lignans. Polyphenols found in the daily diet mainly consist of
flavonoids, of which approximately 8000 molecular compounds have been reported. Flavonoids
share a common structure consisting of 2 aromatic rings that are bound together by 3 carbon
atoms that form an oxygenated heterocycle (Figure 2.1). They are usually divided into six main
subclasses; flavonols, flavones, isoflavones, flavanones, anthocyanidins, and flavan-3-ols
(catechins and proanthocyanidins) (Manach et al., 2004, Hakimmdun, 2006). The most common
group of flavonoids are flavan-3-ols (Figure 2.2) which in the human diet are considered
functional ingredients of beverages, fruits and vegetables, food grains, herbal remedies, dietary
supplements, and dairy products. Flavan-3-ols have been reported to exhibit several health
beneficial effects by acting as antioxidant, anticarcinogen, cardiopreventive, antimicrobial,
antiviral, and neuro-protective agents (Patel et al., 2005; Scalbert et al., 2005; Aron et al., 2008).
7
Figure 2.1 Chemical structure of flavonoids (Manach et al., 2004).
Figure 2.2 Chemical structure of flavan-3-ols (Aron et al., 2008).
8
2.1.1. Tea
One major dietary source of polyphenols is tea. Tea is one of the most concentrated sources of
polyphenols. These substances comprise up to 30% of its dry weight (Balentine et al., 1997;
Dubeua et al., 2010). Tea is one of the most widely popular beverages, consumed by over two-
thirds of the world's population due to its medicinal, refreshing and mild stimulant effects.
Drinking a cup of tea for pleasure or in times of stress is a part of daily life for millions of people
all over the world. The discovery and use of tea as a beverage has a long history. It may have
originated during the ―Shen-Nong‖ era of ancient China, around 5000 to 6000 years ago (Chen,
2002). Tea plant is a perennial evergreen plant with three varieties, Camellia sinensis, Camellia
assamica and Camellia cambodiensis (Karak et al., 2010; Voung et al., 2010).
There are many types of tea but three types of teas, green, black, and oolong are produced and
consumed abundantly in different regions of the world. Each of these types are obtained from the
same plant (C. sinensis) but are different because of their processing history and extent of
fermentation of the final product. Therefore, based on the tea manufacturing (fermentation)
process, teas from the genus Camellia can be divided into three categories: green tea
(unfermented), oolong tea (partially fermented), and black tea (fully fermented). Of the total
world tea production, 78% is black tea, 20% is green tea, and 2% is oolong tea (Sharma et al.,
2009; Wan et al., 2009).
Green tea has been regarded as a rich source of flavan 3-ols also known as catechins (Figure 2.3)
including (+)-catechin (C), (-)-epicatechin (EC), (-) -epigallocatechin (EGC), (-)-epicatechin
gallate (ECG), and (-)-epigallocatechin gallate (EGCG). The tea catechins present in Camellia
sinensis leaf are transformed to polymeric theaflavin and thearubigin by oxidation occurring
during fermentation of black tea.
9
Figure 2.3 Structures of four catechins in tea infusions. Epicatechin: R1=H, R2=H;
epigallocatechin: R1=OH, R2=H; epicatechin gallate: R1=H, R2=gallate (3,4,5-
trihydroxybenzoyl); epigallocatechin gallate: R1=OH, R2 = gallate (3,4,5-trihydroxybenzoyl).
(Ronner et al., 1998).
10
The distinctive reddish-black colour, reduced bitterness and astringency, and characteristic
flavour give black tea a marked distinction from green tea (Ferrara et al., 2001; Kim et al., 2011;
Sang et al., 2011). The catechins are colorless, astringent, water soluble compounds. They are
readily oxidizable, although their oxidation potentials vary. This property has been exploited
through their use as food antioxidants (Henning et al., 2003). More than 50% of the mass of the
catechins in green tea is composed of EGCG and it has been often suggested that EGCG is
responsible for the majority of the potential health benefits attributed to green tea consumption.
The potential health benefits attributed to green tea and EGCG include antioxidant effects,
cancer chemoprevention, improving cardiovascular health, enhancing weight loss, protecting the
skin from the damage caused by ionizing radiation, and others (Zaveri et al., 2006; Sharma et al.,
2009; Yang et al., 2009; Gonzalez et al., 2011; Citarasu et al., 2010).
Because of green tea’s alleged beneficial effects, green tea–based products have become the
fourth most commonly used dietary supplement in the United States. Exposure to green tea can
take place in several forms: consumed as dilute beverages or concentrated supplements or
applied topically (Chan et al., 2010). A recent search of the literature revealed more than 8000
citations that relate to the chemistry, bioactivity, production, and potential health benefits of
green tea (Nagle et al., 2006).
The possible preventive activities of green tea against cancer and cardiovascular diseases have
been studied extensively during the past three decades. Among the biological activities of tea, the
cancer-preventive activity has attracted the greatest attention. In particular, epigallocatechin-3-
gallate (EGCG), the main ingredient of green tea extract, has been shown to inhibit growth in a
number of tumor cell lines at different organ sites including the skin, oral cavity, esophagus,
stomach, intestine, lung, liver, pancreas, mammary gland, urinary bladder, and prostate.
11
The cancer-preventive processes of EGCG may include decreasing cell proliferation, increasing
apoptosis and suppression of angiogenesis. A large number of epidemiological studies have
examined the association between tea consumption and risk of cancers at various organ sites.
Some human cancer prevention trials with green tea polyphenol preparations have shown
promising results, more specifically causing reduced risk of upper gastrointestinal tract
cancers with diets characterized by a high intake of green tea. These studies have led to the
conclusion that EGCG is the most active compound to impart the cancer preventive effect of
green tea (Li et al., 2001; Chow et al., 2005; Lambert et al., 2007a; Yang 2009; Shpigelman et
al., 2010;Yuan et al., 2010; Yang et al., 2012).
In recent years the possible cancer preventive activity of EGCG has been extensively studied
using animal models and different cell line cultures from various organs. The inhibitory activity
of EGCG on cancer cells has been demonstrated during both the initiation and progression stages
of carcinogenesis (Vaidyanathan et al., 2003; Chen et al.,2004; Chen et al., 2007; Yang, 2009).
In vitro cell culture models are valuable tools for screening of chemo preventive agents and
provide preliminary data for in vivo studies. However, the specific cancer prevention
mechanisms related to EGCG, and in general to the molecules present in green tea, are still not
known. Since broad cancer-preventive effects have been observed in a variety of cell lines and
animal models, it is assumed that multiple molecular mechanisms, rather than a single receptor
or molecular target, may be involved. It has been seen that some of cancer preventive effects
may be by direct involvement of EGCG, such as binding to molecular targets while other effects
may be indirect, such as the inhibition of enzyme activities in certain cell pathways (Yang et al.,
2006; Yang et al., 2009; Shpigelman et al., 2010; Visioli et al., 2011).
12
2.1.2. Tea-bioavailability
To exert their biological properties, polyphenols must be bio-accessible and ultimately available
to some extent in the target tissue. Bioaccessibility is defined as the amount of ingested
compounds that are available for absorption in the gastrointestinal tract. Therefore, the
availability and luminal changes prior to absorption play a major role in ensuring bioavailability.
The amount of bioaccessible compounds can be equal to or less than the amount that is released
from the food matrix (Stahl et al., 2002; Aura, 2005; Saura-Calixto et al., 2007). Bioavailability
appears to differ greatly among the various polyphenols, and the most abundant ones in our diet
are not necessarily those that have the best bioavailability profile. Consequently, it is not only
important to know how much of a nutrient is present in a specific food or dietary supplement, but
also how much of it is bioavailable.
The bioavailability of dietary polyphenols is limited by a number of factors such as amount
consumed, physico-chemical properties of the molecule, enzyme and microbial-mediated
biotransformation and active reflux, instability under digestive conditions, intestinal absorption
and food matrix interactions (Stevenson et al., 2007; Papadopoulou et al., 2004; Green et al.,
2007; Ferruzzi et al., 2010).
Despite the epidemiological evidence that consumption of various teas is associated with positive
health benefits, the fate of tea catechins in the digestive tract is yet to be clearly identified. Some
studies have shown that, incubation of tea catechins at acid pH (as found in the stomach) had
little effect of the concentration of individual catechins. However, incubation at slightly alkaline
pH, similar to that found in the small intestine, resulted in a rapid decline in the concentrations of
EGC, EGCG and ECG but not as much to EC. However, the decline was not accompanied by a
13
reduction of a similar magnitude in the catechins antioxidant activity which is probably due to
the formation of different compounds of catechins (Roura et al., 2001; Cilla et al., 2009).
Intestinal factors are one of the most important factors that affect the bioavailability of
polyphenols. Following the ingestion of polyphenols, the absorption of some but not all
components occurs in the small intestine. The fraction of polyphenols that is not absorbed in the
small intestine reaches the colon, where it undergoes substantial structural modifications by the
colonic microflora which degrades the polyphenol fractions to simple phenolic acids. This
activity is of great importance for the biological action of polyphenols because specific active
metabolites are produced in such a way. During the course of absorption and prior to passage
into the bloodstream, polyphenols undergo many structural modifications due to conjugation
processes, which mainly include methylation, sulfation, and glucuronidation in the small
intestine and later in the liver which typically, enhances their likelihood of excretion in urine or
faeces. These modifications could affect the bioavailability of polyphenols and, consequently,
their biological activity. Therefore, it is clear that, during the course of absorption, polyphenols
are extensively modified. The metabolites present in blood, resulting from digestive and hepatic
activity, usually differ from the native compounds. Consequently, the compounds that reach cells
and tissues are often chemically, biologically, and in many instances functionally different from
the original dietary form. Therefore, due to the extensive biotransformation of polyphenols
during digestion, it is not adequate to predict their health effects by considering only their native
structures and the bioefficacy of their digests must also be assayed and observed (Manach et al.,
2005; Roura et al., 2008; Visioli et al., 2011).
Tea catechins, have shown relatively poor absorption efficiency in vivo, which among the
catechins, absorption of non-gallated forms (EC and EGC) has been more efficient than the
14
gallated ones (EGCG and ECG) from the same tea matrix (Aron et al., 2008; Henning et al.,
2008; Ferruzzi et al., 2010). Catechins, especially gallated ones, are expected to have the greatest
activity in the oral cavity and the colon, based on their low rates of absorption (Yang et al.,
2008). Catechin losses of approximately 80%, including almost total degradation of EGCG, have
been observed during simulated digestion of simple tea infusions (Green et al., 2007). As the
unabsorbed gallated EGCG goes through the colon, and even the absorbed EGCG is mostly
excreted into the intestine via the bile duct, intestinal environments are the optimal site for
studies observing cancer inhibition by tea polyphenols (Vaidyanathan et al., 2003; Yang et. al.,
2008; Visiolli et al., 2011). It may also be hypothesized that the intestine is one of the most
important sites of activity for polyphenols.
As bioavailability is an issue with polyphenolic compounds, the compounds or agents that are
not well absorbed systemically, will be in direct contact with the digestive tract and may be
important for the cancer-preventive activity at those sites. The intestine may actually be exposed
to high levels of polyphenols after ingestion of polyphenols. Therefore, in many studies
concerning the anti cancer effect of polyphenols, cell line cultures from the digestive tract such
as human colorectal adenocarcinoma cells HT-29 and CaCo-2 are used (Yang 1998; Hong et al.,
2002; Chen et al., 2003; Gonzalez et al., 2010). The proliferation inhibitory effects of tea
catechins extracts (mainly EGCG) against HT-29 or CaCo-2 growth have been noticeably strong
showing a potential source of unknown chemopreventive agents that could be incorporated into
future research on colon cancer (Gonzalez et al., 2010). The findings that tea catechins inhibit
growth in vitro may suggest a potential of this tea for the prevention of colorectal cancer in vivo.
15
2.1.3. Nano encapsulation of Tea catechins
It is questionable whether individuals can consume a large enough quantity of tea to obtain the
levels of catechins needed for health benefits. Therefore, utilisation of tea extracts in foods has
been considered as an alternative way to provide the health benefits from tea catechins (Voung et
al., 2011). Manufacturers have attempted to produce and package ready-to-drink green tea
infusions, some enriched with (-)-epigallocatechin gallate (EGCG) and/or other polyphenols, in
order to respond to the increasing market for functional foods that have potential health benefits.
Green tea extracts are also used as ingredients in many dietary supplements, such as vitamins and
weight reduction pills. However, the production of green tea beverages has been seen to be
problematic as the stability of EGCG and more other green tea catechins in solutions and drinks
is always a challenge (Kim et al., 2007; Bazinet et al., 2010; Yang et al., 2012). Food product
developers are therefore left with the challenge to preserve the stability, bioactivity and
bioavailability of these bioactive molecules, not only during processing and storage of foods, but
also during digestion. Indeed, to be able to deliver the nutritional functionality, food products
need to have not only a nutritionally significant dose of bioactive molecules, but these molecules
must also remain fully functional for as long as necessary (Garti, 2008). Encapsulation of these
bioactives may provide a solution to these challenges. EGCG seems to be an important candidate
for nanoencapsulation and enrichment in the diet (Shpigelman et al., 2010).
In nanoencapsulation technology, bioactives and drugs are loaded within the core of submicron
capsules or particles, which protect them from degradation (Reis et al., 2006). Interest in
nanocarriers as potential drug carriers in cancer therapy has expanded in the recent years. Milk
proteins are known to be natural carriers, as their structure facilitates the delivery of essential
micronutrients (calcium and phosphate), building blocks (amino acids), and immune system
16
components (immunoglobulins, and lactoferrin), from mother to the neonate. This unique
property of milk proteins has encouraged researchers to study possibilities of using these natural
nano carriers for the delivery of health-promoting and bioactive compounds such as polyphenols.
Milk proteins are natural, available, inexpensive raw materials with high nutritional value and
good sensory properties, and they have many structural properties and functionalities which
make them highly suitable as vehicles for delivering various bioactives (Livney 2010).
Researchers have suggested the potential of milk proteins to deliver nutraceuticals such as
vitamins D, curcumin, minerals (Fe2+
, Mg2+
), carotenoids (β–carotene), polyphenols (resveratrol,
caffeine), omega 3 oils and other health-promoting fatty acids, probiotics and other
microorganisms, cancer therapy drugs and even flavors or aromas. Studies have shown that both
whey proteins and caseins as well, usually in their isolated forms have been able to act as
delivery vehicles. For example whey proteins such as β-lactoglobulin are known to be carriers of
molecules such as vitamin D, omega-3 fatty acid DHA, or polyphenolic compounds like
resveratrol from grapes especially after β-lactoglobulin has been heated. α-lactalbumin can
apparently bind to retinol and palmitic acid, while bovine serum albumin is often proposed for
delivery tasks in the circulatory system for delivering chemotherapeutic drugs (5-fluorouracil).
Also, β-casein nanoparticles have been proposed to be used for the delivery of gastric cancer
treatment drugs (Livney, 2010; Rahimi Yazdi et al., 2012).
Recent developments have shown that curcumin can be delivered via caseins to tumour cells, and
that vitamin D can be protected from UV degradation by encapsulation in casein micelles,
showing the potential of these nanostructures in the delivery of neutraceuticals (Semu et al.,
2007; Sahu et al., 2008; Livney, 2010; Shpigelman et al., 2010).
17
Traditionally, in many countries, tea is consumed with the addition of milk to improve the
sensory properties and reduce the astringenty sensation caused by polyphenols.
Also, development of new products with tea ingredients formulated on dairy bases such as ready-
to-drink (RTD) tea beverages, chai latte beverages and other products containing dairy products
have expanded over the years (Ferruzzi et al., 2006). Milk is a natural, multi-component,
nutrient-rich beverage and is generally recognized as a source of beneficial substances for
growth and health in children and adults (Kalkwarf et al., 2003; Valeille et al., 2006). Also, for
oral delivery applications, biocompatibility of milk proteins is usually very good. Therefore,
milk is an ideal platform for delivery of other bioactive compounds and their additional benefits
to human health. Functional dairy beverages can satisfy the growing market need for health and
taste, but significant knowledge of the bioactive components and their interaction with dairy
components is required (Sharma 2005).
2.2. Milk
Milk is a fluid secreted by all mammals to supply to the neonate the nutrients required for its
growth. Milk is a colloidal suspension consisting of milk fat globules, milk casein proteins and
an aqueous phase consisting whey proteins, lactose, organic and inorganic salts dissolved in
water (Fox et al., 2000). In this polydispersed system, fat globules can be easily separated by
gravity or centrifugation. The typical composition of bovine milk is approximately 87.7% water,
4.9% lactose (carbohydrate), 3.4% fat, 3.3% protein, and 0.7% minerals. Bovine milk has pH of
approximately 6.7 (Fox et al., 1998). The protein fraction of milk is composed of caseins (αs1-,
αs2-, β-, and κ-casein) and whey proteins (mostly β-lactoglobulin, α-lactalbumin and bovine
serum albumin), accounting for ~80% and ~20%, respectively (Fox et al., 1998). The milk
18
serum, composed of lactose, small molecular weight components and salts, can be separated
from the proteins by filtering skim milk using an ultrafiltration membrane (Walstra et al., 2006).
2.2.1. Caseins
About 80% of the total proteins of bovine milk are caseins, defined as the proteins that
precipitate at pH 4.6 at 20 °C (Fox et al., 2000). There are four individual types of casein
molecules, αs1-, αs2-, β- and κ-casein, in approximate relative amounts of 4:1:3.5:1.5,
respectively. Caseins are small molecules about 20-25 kDa in molecular weight (Dalgleish,
1998). These protein assemblies are designed by nature to concentrate, stabilize and transport
essential nutrients, mainly calcium, phosphate and protein, for the neonate. These proteins are of
great importance in dairy technology, as their processing functionality (for example, their gel
forming abilities) are key to many dairy processes. The primary structure of caseins is
characterized by a high amount of proline, which impedes the formation of significant portions
of ordered structures. For this reason caseins are very flexible and are defined rheomorphic, as
they can adapt their conformation to changing environmental conditions (Holt et al., 1993).
There are four main caseins, αs1-, αs2-, β- and κ-casein, and they differ in some molecular aspects
such as, degree of phosphorylation, glycosylation of κ–casein, hydrophobicity and proline
content. All caseins are phosphorylated, most molecules of αs1-casein contain 8 or 9 PO4
residues, β-casein usually contains 5 PO4 residues, αs2-casein contains 10-13 PO4 residues and κ-
casein contains only 1 PO4 residue. The phosphate groups of the caseins are esterified as
monoesters of serine and most of the phosphoserine residues in the caseins occur in clusters. The
phosphate groups are very important from a nutritional view point but they also bind polyvalent
cations strongly. Therefore apart from κ-casein, all of the other caseins are calcium sensitive as
19
they precipitate in the presence of calcium ions (Fox et al., 2003). κ-Casein is the only
glycosylated casein which contains galactose, galactosamine and N-acetylneuraminic (sialic)
acid, that occurs either as trisaccharides or tetrasaccharides attached to theronine residues in the
C-terminal region. Caseins, especially β -casein, contain a high level of proline (cyclic amino
acid); in β -caseins 35 of 209 amino acid residues are prolines that are uniformly distributed
throughout the protein molecule, κ –casein contains 20 proline residues out of 169 amino acids,
αs1 and αs2-caseins contain 17 and 10 proline residues from 199 and 207 amino acids,
respectively. The caseins are relatively hydrophobic and, in particular, hydrophobic, polar and
charged residues on the caseins are not uniformly distributed throughout the sequences but occur
as hydrophobic or hydrophilic patches, giving the caseins strongly amphipathic structures.
αs1-casein has two hydrophobic and one hydrophilic region, that includes the phosphoserine
cluster, αs2-caseins has two hydrophobic and two phosphoseryl clusters, β–caseins has only one
hydrophobic and one phosphoseryl group and κ-casein has a hydrophobic N-terminal region but
no phosphoseryl cluster (Fox et al 2003; Farrell et al., 2006; Horne 2006; Dalgleish, 2011).
Nearly all caseins in milk (95%) are organized into colloidal particles known as micelles.
In bovine milk, the casein micelles are spherical particles of diameters ranging between 80-400
nm, with an average of about 200 nm and containing between 3-4 g of water per g protein
(Dalgleish, 2011). They contain 94% protein and 6% low molecular weight species (mainly
calcium and phosphate but also magnesium and citrate), which are collectively known as
colloidal calcium phosphate.
Many models for the structure of the casein micelles have been proposed and yet in regards to
the interactions involved in their assembly and maintaining the integrity of these particles,
dispute amongst researchers remains. However, there is a general agreement that caseins are held
20
together by an equilibrium between electrostatic and hydrophobic interactions (Horne, 2009;
Horne, 2006) as well as van der Waals forces (Dalgleish, 2011). The interior of the micelles is
mainly composed of αs1-, αs2-and β-caseins bound to calcium phosphate nanoclusters by their
phosphoserine domains. κ-casein exists mainly on the surface of the micelle with the hydrophilic
C-terminal part protruding into the solvent. Of particular relevance for milk coagulation, the
N-terminal of κ-casein (2/3 of the molecule, called para-κ-casein) is strongly hydrophobic,
whereas the C-terminus (called casein macropeptide, or CMP) is strongly hydrophilic and is
usually glycosilated.
κ-caseins are not evenly distributed on the surface, but are present in clusters that are easily
accessible for attack by chymosin (and related enzymes) (Fox et al., 2003; Holt et al.,1996; de
Kruif et al., 2003).
Milk proteins also contain tryptophan residues throughout their molecular structure where the
αs-caseins contain two tryptophan residues at positions 164 and 199 for αs1 and positions 109 and
193 for αs2, β- and κ-casein have just one tryptophan residue at position 143 and 76,
respectively. As described in the different models of casein micelles, it is widely accepted that
the αs- caseins and β-casein are mainly located in the interior hydrophobic core of the micelle,
whereas as κ- casein is mostly on the surface of the casein micelles (Dalgleish 2010). Therefore,
most of the tryptophan residues are located in the hydrophobic domains of the casein micelle.
Casein micelles are destabilized by the proteolytic activity of specific enzymes, by isoelectric
precipitation, by addition of an excess of calcium ions or ethanol which their destabilization is at
the basis of many dairy processes (de Kruif et al., 1996; de Kruif, 1999).
21
2.2.2. Whey Proteins
The other 20% of the total proteins in milk are whey proteins (WP) which are globular proteins
with high level of structural organization, causing susceptibility to thermal denaturation. The
compact and stable structure is given by intramolecular disulfide bonds, formed between the high
amount of cysteine residues. Unlike the caseins, which predominantly exist as associative
colloidal particles, the whey proteins are soluble in the milk serum (Fox et al., 2000).
The whey proteins include 50% β-lactoglobulin (β-LG), 20% α-lactalbumin (α-LA), 10% (BSA)
and 10% immunoglobulins (Ig). However, the first two are present in the greatest proportion and,
therefore, are of technological significance. At milk pH, β-lactoglobulin is a dimer of molecular
weight of 36.4 kDa while α-lactalbumin is a monomer of 14 kDa. Whey proteins are non-
phosphorylated and insensitive to calcium ions, are soluble at pH 4.6, soluble after rennet
coagulation of caseins and non sedimentable by centrifugation (Fox et al., 2003).
Following heat denaturation, β-lactoglobulin exposes its cysteine residues and forms complexes
with other cysteine containing proteins, such as α-lactalbumin, bovine serum albumin or
κ-casein (Livney et al., 2003).
2.2.3. Rennet induced gelation of milk
Milk proteins (caseins, whey proteins) are used in food products not only for their nutritional
value but for their functional properties as well. Major functionalities of milk proteins include
gelation (acid or/and rennet induced) emulsification, and foaming properties.
Rennet induced gelation (enzymatic coagulation) is the first step of the cheese-making process
and particularly relevant in dairy processing. This specific functionality of casein micelles occurs
without changes in pH and is induced by the enzymatic modifications of the surface of the casein
22
micelles. The process is composed of two stages, which partially overlap each other (Dalgleish
2010). During the first stage, the enzymatic action (by chymosin) destabilizes casein micelles,
while in the second stage the micelles aggregate, forming a coagulum.
During the first stage, the proteolytic enzyme chymosin cleaves κ-casein, which mostly resides
on the surface of the casein micelles. Chymosin is a proteolytic enzyme active on κ-casein,
specifically on the Phe105-Met106 peptide bond. While the hydrophobic para-κ-casein segment
(residues 1-105) remains associated with the micelle, the hydrophilic portion of κ-casein, the
casein macropeptide (CMP, residues 106-169) is released into solution (Dalgleish et al., 2012).
The polyelectrolyte layer of κ-casein stabilizes the micelles by generating steric repulsion, and
therefore, any process that removes the C-terminal region of κ-casein causes a decrease in the
colloidal stability of the micelles. When approximately more than 85% of the κ-casein is
hydrolyzed, the steric stabilization generated by the few remaining κ-casein hairs is insufficient
to keep the micelles apart, and they begin to aggregate and eventually gel. Therefore, gelling
occurs only after a nearly full release of CMP in solution. Once decreased steric repulsion allows
the micelles to approach one another closely, hydrophobic interactions cause bonding between
the particles, as does the amount of ionic calcium; increased calcium also allows aggregation at
progressively lower levels of CMP release (Horne, 1986; de Kruif, 1999; Horne 2006; Dalgleish
2012).
2.3. Interactions between milk proteins and tea catechins
The interaction between polyphenols and proteins is relevant to diverse fields of knowledge such
as medicine, toxicology, chemistry, food science, and agriculture. Hence, the reactions have been
23
studied throughout the years by numerous technical approaches (Goncalves et al., 2011). The
interactions between polyphenols and proteins are driven by hydrogen bonding between phenolic
hydroxyl and peptide carbonyl, and hydrophobic ―stacking‖ interactions between hydrophobic
amino acid residues and the aromatic rings of the phenols (Hofmann et al., 2006; Pascal et al.,
2008; Yan et al., 2008). In addition, binding is clearly affected by protein characteristics
including protein structure and amino acid composition, with proline-rich proteins having a
particularly high relative affinity for polyphenols, as well as polyphenol structure (eg.,
glycosylated or not) and molecular size (Poncet-legrand et al., 2006; Richard et al., 2006; Soares
et al., 2007; Frazier et al., 2009; Dubeau, 2010) .
It has been widely demonstrated that the astringency sensation is a consequence of the
interactions between proline-rich saliva proteins and polyphenols (O’Connell 2001; Charlton
2002; Hofmann et al., 2006). In a recent study, it has been shown that saliva proteins and
polyphenols interacts strongly and the astringency sensation is due to the free polyphenols rather
than the bound ones (Schwarz et al., 2008). In general, it has been found that catechins readily
interact with proteins rich in proline, with an open and flexible structure (Benick et al., 2002;
Papadopoulou et al., 2004; Maiti et al., 2006). Caseins contain high numbers of proline
residues evenly distributed throughout their amino acid sequences and have relatively open
structural features (like the salivary proline rich proteins), and are avid binders of polyphenols.
For this reason, isolated caseins have often been used as model proteins in various polyphenol-
protein studies (Jobstyl et al., 2004; Pascal et al., 2007; Yan et al., 2009). Catechins bind
strongest to the caseins (β-casein> αs-casein> κ-casein), followed by the whey proteins α-
lactalbumin, β -lactoglobulin and bovine serum albumin (Luck et al., 1994; Kartosova et al.,
2007). β-casein contains 209 amino acid residues, of which 35 prolines evenly distributed
24
through the sequence (Ribadeau et al., 1973). EGCG and β-casein interact by forming
hydrophobic interactions between the phenolic rings of the EGCG and prolines in β-casein,
wrapping the casein around the catechin (Jobstyl et al., 2006).
In agreement with recent studies on saliva proteins (Schwartz et al., 2008), sensory studies have
shown that caseins, especially β-caseins, due to their strong binding ability to tea catechins,
decrease the sensation of astringency caused by the tea polyphenols. It has also been suggested
that since binding reduces astringency, it may decrease the bioavailability of EGCG as well
(Jobstyl et al., 2006; Yan et al., 2009). Although, numerous studies have shown the strong
interactions of polyphenols and milk caseins in their isolated forms (most frequently β-casein),
few studies have been conducted to delineate the nature of association of polyphenols and casein
micelles in their natural matrix, milk.
Past studies present contradictory results regarding the effect of milk on the bio-availability of
tea catechins, whereas, the effect of the complexes formed between tea catechins and milk
proteins in terms of decreasing or increasing the bioefficacy of these bioactive molecules is still
controversial. Some studies have found a masking effect of the antioxidant capacity of catechins
in the presence of proteins, the extent of which depended on both polyphenol and protein types.
The tea polyphenol that showed the highest masking in combination with β-casein, was
epigallocatechin gallate (EGCG) (Arts et al., 2002; Kilmartin et al., 2003; Kartosova et al.,
2007). The masking effect was also seen in the blood among volunteers who drank tea with milk,
compared to those who drank tea without milk, which it has been suggested that the interactions
between milk proteins (especially caseins) and the tea catechins are responsible for the decline of
the antioxidant capacity of tea catechins (Langley-Evans, 2000). In contrast, other similar studies
showed no relevant effect of milk on the plasma antioxidant capacity of tea polyphenols (Leenon
25
et al., 2000; Keogh et al., 2007; Kyle, 2007). In addition, it has been shown that different
methods to determine the effect of milk on the antioxidant capacity of milk may lead to different
conclusions (Dubeau et al., 2011). Also, it has been shown by many studies that tea catechins,
more specifically EGCG, may exert their biological health benefits by not only acting as an
antioxidant but as well as a pro-oxidant. Therefore, determining the antioxidant capacity of tea
polyphenols may not be the most suitable marker or indicator of their bioacessability or bio-
availability.
It is important to state that the above discrepancies concerning the effect of milk on tea might be
due to the different study designs, different methodologies, and types of tea used. Most studies
have overlooked the strong interactions that take place between the proteins and polyphenols,
considering effective (i.e., bioavailable) only the material recovered in the soluble phase.
For example, one study claims that the addition of milk to fruit juices significantly decreases
their total polyphenol content compared to non-fortified fruit beverages, with flavan-3-ols
(catechins) being the most affected compounds. The decrease was more significantly seen in the
polyphenol content of fruit juice–milk digests after in vitro digestion (Cilla et al., 2009).
Sharma et al., 2008 also showed a significant decrease in the free phenolic content of black tea
after the addition of milk. In contrast, more recent studies have shown that the stability of
catechins in green tea post digestion is very poor, however green tea samples formulated with
50% bovine, soy, and rice milk, significantly increased total catechin recovery (Green et. al.,
2007; Ferruzzi, 2010).
Another factor that must be taken account is that during the course of gastric and intestinal
digestion, polyphenols undergo many structural and biological modifications that usually differs
them from the original compounds indigested. These modifications result in a complex
26
circulating profile of both free catechins, methylated, glucuronidated and sulfated catechin
metabolites which many of the metabolites are yet to be identified. Consequently, these
metabolites that reach cells and tissues, in many instances function differently from the original
dietary form. Therefore, due to the extensive biotransformation of polyphenols during digestion,
it is not adequate to predict their health effects by considering only their native structures and the
bioefficacy of their digests must also be assayed and observed. However, many studies test the
compound of little biological relevance when trying to identify the physiological mechanisms
involved in the health effects of polyphenols, rather than testing their active metabolites (Manach
et al., 2005; Roura et al., 2008; Visioli et al., 2011).
Considering the poor digestive stability of catechins from plain green tea infusions, the ability of
milk to potentially modulate the tea catechin profile must be further studied. The protein–
catechin interactions may provide a physical trapping of the reactive catechin species causing
more stability of the catechins which ultimately may impact the catechin bioavailability and
physiological profile in vivo.
Interpretation of such studies is difficult since by the addition of milk, the product matrix
complexity increases, and therefore accurate and reliable measurements of tea catechins becomes
more challenging (Ferruzzi et al., 2006).
To date most of the polyphenol-milk protein studies have been mainly focused on what impact
the complex may have on the bioavailibilty of polyphenols, whereas, the fate of milk proteins,
during digestion has not been observed. Therefore, it is of importance to understand the impact
of proteolytic activity on the polyphenol-milk complexes as several active peptides, which exert
various activities on the digestive, cardiovascular, immune and nervous systems, are produced
during the digestion of caseins (Korhonen et. al., 2006; Picariello et. al., 2010). Some studies
27
have shown, polyphenols may have an inhibitory effect on certain digestive proteases
(Tagliazucchi et. al., 2005; McDougall et al., 2008) while some other studies have seen a
stimulating effect on the rate of protein digestion (Siewright et al., 2006; McDougall et al., 2005;
Goncalves et. al., 2011). Although inhibition of proteases by polyphenol components can occur
in vitro, there is still not enough evidence to show that inhibition of protease activity occurs in
the digestive system in vivo (McDougall et al., 2008). However, this may still be a crucial factor
in casein digestion that must be observed when tea and milk complexes are consumed.
28
Chapter 3
Interactions between tea catechins and casein micelles and their impact on
renneting functionality
3.1 Abstract
Strong interactions have been reported between tea catechins and milk proteins in isolation, but
less is understood on the association of polyphenols with casein micelles, and more importantly,
the consequences of the interactions on processing and biological functionalities. It was
hypothesized that epigallocatechin-gallate (EGCG), the main catechin present in green tea forms
complexes with the casein micelles, and the association modifies the processing functionality of
casein micelles. In this study, the binding of EGCG to casein micelles was quantified using
HPLC and fluorescence quenching. It was demonstrated that the formation of catechin-casein
micelles complexes affected the rennet induced gelation of milk. Tea catechins affected both the
primary as well as the secondary stage of gelation. These experiments clearly identify the need
for a better understanding of the effect of tea polyphenols on the functionality of casein micelles,
before milk can be used as an appropriate platform for delivery of bioactive compounds.
Key words: Polyphenols, caseins, interactions, gelling, fluorescence
29
3.2 Introduction
Tea (Camellia sinensis) polyphenols have been reported to exhibit several beneficial health
effects by acting as antioxidant, anticarcinogen and cardiopreventive agents (Ferruzzi et al.,
2006). This is of particular interest as tea is the second most consumed beverage after water.
The most abundant group of polyphenols in tea are catechins. These compounds are water-
soluble, colorless compounds which contribute to astringency and bitterness in tea.
Major tea catechins include epicatechin, epigallocatechin, epigallocatechin-gallate, and
epicatechin-gallate in which epigallocatechin-gallate (EGCG) is the major catechin component
and also the most biologically active (Vaidyanathan et al., 2003; Henning et al., 2003). These
natural chemicals, especially EGCG, exhibit excellent radical scavenging and hence
antioxidative properties, and have been widely employed as food antioxidants (Graham et al.,
1992; O’Connell et al., 2001).
Traditionally, in many countries, tea is consumed with the addition of milk to improve the
sensory properties, i.e., to reduce the astringency sensation caused by polyphenols (Bennick
2002). Also, development of new products with tea ingredients formulated on dairy bases such as
ready-to-drink teas, chai latte and other beverages containing dairy products have expanded over
the years (Ferruzzi et al., 2005). Milk is a natural, multi-component, nutrient-rich beverage and is
generally recognized as a source of beneficial substances for growth and health in children and
adults (Kalkwarf et al., 2003; Valeille et al., 2006). For this reason, milk is an ideal platform for
delivery of other bioactive compounds, to obtain a new generation of dairy products providing
additional benefits to human health. Functional dairy beverages can satisfy the growing market
need for health and taste, but significant knowledge of the bioactive components and their
interaction with dairy components is required (Sharma 2005). It is important to determine
30
whether the addition of these compounds in milk may affect the processing functionality of the
casein micelles. Our work aims to explore some of the effects of the addition of polyphenols to
milk proteins and their relevance to the design of improved dairy products.
Strong interactions have been reported between tea catechins and purified milk proteins in model
solutions. Catechins bind strongest to the caseins, followed by whey proteins, namely α-
lactalbumin, β-lactoglobulin and bovine serum albumin (Luck et al., 1994; Kartosova et al.,
2007). Sensory studies have shown that caseins, especially β-caseins, due to their strong binding
ability to tea catechins, decrease the sensation of astringency caused by the tea polyphenols
(Lesschaeve et. al., 2005; Hofmann et. al., 2006; Soares et. al., 2007; Schwarz et. al., 2008).
It has also been suggested that since binding reduces the astringency, it may decrease the
bioavailability of EGCG (Jobstyl et al., 2006; Yan et al., 2009).
Catechins readily interact with proteins rich in proline, with an open and flexible structure
(Benick et al., 2002; Papadopoulou et al., 2004; Maiti et al., 2006). The astringency perceived
during in mouth processing of polyphenol rich foods is attributed to the free polyphenols, rather
than those bound to proline-rich saliva proteins (O’Connell, 2001, Charlton, 2002; Hofmann et
al., 2006; Schwarz et al., 2006). Caseins which constitute about 80% of the proteins of bovine
milk contain high numbers of proline residues evenly distributed throughout their amino
acid sequences and have relatively open structural features (like the salivary proline rich
proteins). For this reason, isolated caseins have often been used as model proteins in various
polyphenol-protein studies (Jobstyl et al., 2004; Pascal et al., 2007; Yan et al., 2009).
The effect of the interactions of polyphenols with casein micelles on the processing functionality
of milk proteins is not fully understood, apart from the fact that it has been recognized that the
31
presence of polyphenols enhances the heat stability of milk (O’Connell et al., 1999; O’Connell et
al., 2001).
The main objective of the proposed research is to further our understanding of the impact of the
milk protein/polyphenol interactions on the functional properties of milk proteins.
Although many studies have acknowledged these interactions, few studies have focused on the
association of polyphenols with casein micelles in milk. Indeed, most studies have been limited
to the interaction of isolated caseins (most frequently β-casein) with a range of polyphenols, but
not the casein micelles. To date, the possible effects of the interactions on the functional
properties of milk proteins have been studied to a lesser extent than the effects on the
bioavailability of the polyphenols. The effects of the complexes formed with the casein micelles
on the functional properties of caseins, in particular their rennet-induced aggregation, have yet to
be reported.
3.3 Materials and Method
3.3.1 Milk preparation and interaction studies
Fresh milk was collected from the Ponsonby Research Station of the University of Guelph
(Guelph, Ontario, Canada) and sodium azide (0.02% w/v) was immediately added to prevent
bacterial growth. Milk was skimmed at 6000 g for 20 min, at 20°C, in a Beckman–Coulter
centrifuge (Model J2-21, Beckman Coulter, Mississauga, ON, Canada) and filtered three times
through Whatman glass fibre filters (Fisher Sci.). Milk permeate was prepared from skim milk,
using a laboratory scale ultrafiltration cartridge (Millipore CDUF001LG; Fisher Scientific), with
a nominal cut-off of 10,000 Da and a nominal area of 1 ft2 (0.095 m
2).
32
A source of green tea polyphenol extract (containing minimum 90% EGCG) was chosen from
DSM Nutritional Products INC (Montreal, Canada). Different concentrations of EGCG was
obtained from a stock solution of EGCG in water (20 mg/ml water). When needed, dilutions
were carried out using permeate (milk serum), to reach high ratios of EGGC to caseins while
maintaining concentrations of EGCG below those critical for solubility.
Separation of the soluble proteins (whey) from the casein micelles was accomplished by
centrifugation (36,000 g), at 20°C, for 45 min (Beckman Coulter, Canada, Inc., Mississauga, On,
Canada), as previously described (Del Angel et al., 2006). Afterwards, the pellet containing the
micellar caseins was re-diluted in fresh ultrafiltration permeate (see above) for fluorescence
experiments.
3.3.2 Fluorescence spectroscopy
Fluorescence quenching has been widely studied as a source of information about biochemical
systems in which biochemical applications of quenching are due to the molecular interactions
that result in quenching (Rawel et al., 2006; Lakowicz, 2006). The binding of EGCG with milk
proteins was quantified by fluorescence spectrophotometry (Shimadzu RF-5301PC
spectrofluorometer, Shimadzu Corp., Tokyo, Japan). Milk was diluted 1:20 (v/v) in milk serum
(permeate) and the fluorescence spectra were collected at varying concentrations of EGCG. In
addition, similar experiments were also performed with casein micellar suspensions, prepared as
described above. The casein suspension was also diluted 1:20 (v/v) in milk permeate. Samples
were excited at wavelength of 280 nm and the emission spectra were recorded from 300 to 500
nm (with slit widths of 3 for both excitation and emission).
33
The Stern-Volmer equation (equation 3.1), was used to plot F0/F for the skim milk and casein
samples as a function of EGCG concentration added. F0 is the fluorescence intensity of control
skim milk or control caseins without the addition of EGCG, F is the fluorescence intensity after
fluorescence quenching (after addition of EGCG), [Q] is the concentration of the quencher
(EGCG) and Ksv is the Stern-Volmer quenching constant (Lakowicz, 2006).
F0/F = 1+ Ksv [Q] Eq. 3.1
Also, using a modified form of the Stern-Volmer equation (equation 3.2), the ratio of F0/ΔF was
plotted as a function of the concentration of EGCG where, the fa, which is the fraction of the
fluorescence that is accessible to quencher, and the Ka, the Stern-Volmer quenching constant of
the accessible fraction were calculated (Lakowicz, 2006).
F0/ ΔF = 1/ faKa[Q] + 1/ fa Eq. 3.2
In these experiments, the concentration of quencher was normalized by the amount of protein
present in the sample, specifically, 32 mg/mL for skim milk and 26 mg/mL for the casein
micelles suspensions.
34
3.3.3 Quantification of polyphenol binding to casein micelles
Different concentrations of EGCG were added to skim milk, and after approximately 20 min of
incubation at room temperature (22°C), the mixtures were centrifuged at 36000g for 45 min (see
above) to separate the colloidal phase (consisting of casein micelles) from the whey proteins and
soluble EGCG. After the separation, the amount of catechin present in the whey fraction (i.e.,
the centrifugal supernatant) was analyzed by reversed phase HPLC as previously published
(Ferruzzi et al., 2006). In brief, aliquots of whey were combined with 2% aqueous acetic acid
(1:1 v/v) and centrifuged at 14,000g for 5 min. Supernatants were collected and filtered through a
0.45 µm PTFE filter and immediately injected. A parallel set of tea catechin solutions containing
whey protein isolates (separated from skim milk as described above) were also prepared and
were used as standard curves to determine the effect of the presence of whey proteins in the
quantification of the EGCG. No differences were noted, when compared to samples containing
isolated EGCG fractions (without whey protein). Hence, the experiments were run using whey
protein fractions and EGCG fractions as standards.
To quantify the binding of EGCG in milk, Scatchard plot was used to determine the dissociation
constant (Kd) which is a useful measure to describe the strength of binding (or affinity) between
proteins and their ligands. Using the Scatchard equation (equation 3.3 ) the Kd between EGCG
and casein proteins was determined. Where , is the average number of ligand molecules bound
per protein molecule. Curved Scatchard plots can be decomposed into successive linear plots,
and the dissociation constant Kd can be derived from each of the slopes (Dickinson et. al., 2001).
/[L] = 1/Kd - /Kd Eq. 3.3
35
3.3.4 Rheological properties of rennet-induced gels
A fresh rennet solution was prepared from double strength rennet (Chymostar, Danisco,
Cranberry, NJ, USA) and immediately added to milk and tea-milk samples yielding a final rennet
concentration of 3.14x10-4
IMCU/ml. Immediately after rennet addition at 30°C, twenty-
milliliter samples were placed in concentric cylinders in a controlled-stress rheometer AR1000
(TA Instruments, New Castle, DE). The development of the gel structure was followed using
0.01 constant strain, 1.0 Hz frequency and temperature of 30°C. The time of cross-over between
G’ (storage modulus) and G’’ (loss modulus) was considered the gelation point.
3.3.5 Release of casein macro- peptide (CMP) from the surface of the casein micelles
Renneted milk and tea-milk samples were pipetted to several test tubes in a waterbath maintained
at 30°C. The rennet reaction was stopped at specific time intervals by addition of 3% perchloric
acid with subsequent vortexing. After overnight refrigerated storage, the supernatant was
collected after centrifugation at 4500g for 15 minutes, filtered through 0.45 μm filter, and
analyzed by reversed phase HPLC as previously published (López-Fandiño et al, 1993).
Intregration of the peaks was done by using ChromQuest software v.4.1 (ThermoFinnigan,
Burlington, ON, Canada) and compared to the maximum peak area for skim milk without tea
polyphenol added (control milk).
3.3.6 Statistical analysis
The analyses were repeated at least three times and evaluated by their means and SD.
36
3.4 Results and Discussion
3.4.1 Fluorescence spectroscopy studies
The fluorescence spectra and the values of maximum intensity as a function of EGCG
concentration are depicted in Figure 3.1 for samples of skim milk and samples of micellar
caseins. A control of EGCG solution measured under the same conditions yielded a very low
signal. In both skim milk and casein micelles suspensions (Figure 3.1 A and B, respectively),
with the increase of EGCG concentration, the maximum fluorescence intensity decreased until
reaching a plateau. This plateau value was statistically equivalent between skim milk and casein
micelles samples. As shown in the insets of Figure 3.1, the emission maximum for both skim
milk and casein micelle samples (approximately 335 nm) progressively shifted to longer
wavelengths with increasing EGCG concentrations, together with a decreasing in intensity.
Similar results were noted for micellar caseins as well as skim milk, suggesting that most of the
quenching effect of tryptophan can be attributed to the association of EGCG with the casein
proteins. Since the changes in emission maximum of tryptophan residues is closely related to the
polarity of the microenvironment around them, the observations suggest that a change in the
conformation of caseins took place as EGCG concentration was increased (Ghosh et al., 2008).
It is important to note that fluorescence measurements on purified caseins (α and β caseins),
showed a similar behavior with a shift to longer wavelengths but no peak broadening at the high
concentrations of polyphenols. Theses studies suggested that the upward shift of the protein
emission bands observed in the spectra of EGCG–casein is related to more exposure of
tryptophan residue and unfolding of protein structure, which was also shown in infrared results
that showed major secondary structural changes for caseins upon EGCG complexation (Hasni et
al., 2010). However, the binding of EGCG with whey protein isolates and β-lactoglobulin has
37
been previously studied whereas, the addition of EGCG to whey proteins did not cause a shift in
the wavelength, therefore indicating that the shift seen in Figure 3.1 is not related to the spectrum
of EGCG, but it is specific to the EGCG interactions with caseins (Shpigelman et al., 2010).
38
Figure 3.1 Fluorescence intensity and fluorescence spectra (inset) for (A) caseins and (B) skim
milk. Arrow indicates increasing concentrations of EGCG. Values are the average of three
independent experiments.
A
B
39
In the case of skim milk, the intrinsic fluorescence intensity is usually attributed to the caseins in
milk (αs1-, αs2-, β-, and casein) although α-lactalbumin and β-lactoblobulin (whey proteins), have
2 and 4 tryptophan residues, respectively (Brew et al., 1967). While the αs- caseins contain two
tryptophan residues at positions (164 and 199 for αs1) and positions (109 and 193 for αs2), β- and
κ-casein have just one Trp residue at position 143 and 76, respectively. As described in the
different models of casein micelles, it is widely accepted that the αs- caseins and β-casein are
mainly located in the interior hydrophobic core of the micelle, whereas as κ-casein is mostly on
the surface of the casein micelles (Dalgleish 2010). Therefore, most of the tryptophan residues
are located in the hydrophobic domains of the casein micelle.
The interactions between polyphenols and proteins are driven by hydrogen binding between
phenolic hydroxyl and peptide carbonyl, and hydrophobic ―stacking‖ interactions between
hydrophobic amino acid residues and the aromatic rings of the phenols (Hofmann et al., 2006;
Pascal et al., 2008; Yan et al., 2008). Furthermore, a study using purified caseins and tea
catechins suggests that the binding of the polyphenols to caseins seems to be more hydrophobic
than hydrophilic (Hasni et al., 2010).
To quantify the extent of the interactions, the fluorescence spectra of skim milk proteins and the
isolated casein micelles with EGCG were analyzed as shown in Figure 3.2. As casein proteins
are in the form of micelles and most of the tryptophan residues are located in the hydrophobic
domains of the micelle, each residue can be differently accessible to the quencher. The deviation
of linearity in the Stern Volmer plots shown in Figure 3.2 suggests that EGCG may not be able
to access all the sites within the casein micelles, as full quenching is not obtained with increasing
concentrations of EGCG in milk and in casein micelles as well, as that a plateu of quenching was
reached at approximately 0.04 mg EGCG per mg milk proteins. Whereas, recent studies with
40
curcumin have demonstrated that with this polyphenol molecule full quenching of the
tryptophans is achieved indicating the penetration of curcumin within the hydrophobic core of
the casein micelles (Rahimi Yazdi et al., 2012). It has been previously suggested that small
molecules have the ability to penetrate into the inner core of the casein micelles due to the
porosity of the micelles which contain water channels within their structure (Dalgleish, 2011).
41
Figure 3.2 Stern-Volmer plot for skim milk ( ) and caseins (♦) re-diluted in permeate.
Values are the average of three independent experiments.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
Fo
/F
EGCG concentration (mg/mg protein)
Milk
Caseins
42
The values of the Stern-Volmer quenching constant of the accessible fraction (Ka) and the
accessible fraction to the quencher (fa) were calculated using a modified Stern Volmer equation
(see Eq. 3.2). For skim milk the calculated fa and Ka are 0.80± 0.04 and 1.8 (± 0. 1) × 104 M
-1
respectively and for casein samples fa and Ka are 0.77± 0.06 and 1.3 (± 0. 2) × 104 M
-1,
respectively. While K values obtained for pure fractions of α-casein and β-casein with EGCG
were found to be 7.4 (±0.4) × 104 M
-1 for α-casein and 1.59 (±0.2) × 10
4 M
1 for β-casein , with
fa of 1.5 for both purified caseins (Hasni et al., 2011). The lower values obtained in our results
for milk and casein micelles compared to the purified α-casein and β-casein, show that while in
the molecular caseins there is access of EGCG, in the case of casein micelles, there is not full
access of EGCG to all the binding sites available in the molecular state.
The results clearly demonstrated that there were no significant differences in the binding values
of skim milk and caseins (P> 0.05), suggesting that the main sites of tryptophan quenching are
within the casein micelles. The slightly higher value of Ka for skim milk compared to the casein
micelles is probably caused by the presence of whey proteins.
43
3.4.2 Analysis of tea polyphenols by HPLC
To determine the binding of tea catechins to casein micelles in a wider range of concentration,
binding isotherms were obtained by analyzing the amount of EGCG recovered in the serum
phase of the mixtures after separation of the colloidal casein micellar phase by centrifugation.
Figure 3.3 illustrates the amount of catechins recovered in the colloidal phase as a function of
ligand per protein present in skim milk. It is important to note that whey proteins remained in
the serum phase, hence, any EGCG interacting with the whey was included in the fraction
recovered in the supernatant and subtracted from the total EGCG, allowing for an estimate of the
amount associated with the casein micelles. At low concentrations nearly all of the tea catechins
added to the milk (around 95±2%) were recovered with the casein micelles fraction (figure 3.3).
The high amounts of EGCG in the colloidal phase (pellet) of milk clearly indicated that
recoveries of the tea catechins in the serum phase of complex mixtures cannot be used as an
index of polyphenol bioavailability. As shown in Figure 3.3 the majority of the tea catechin was
recovered with the casein micelles up to a concentration of 0.06 mg/mg protein in milk, after
which the amount of unbound polyphenol started to increase. It is assumed that at this
concentration of EGCG per milk proteins, casein micelles are saturated with the tea catechin,
meaning there are no more binding sites on the caseins for interactions with EGCG. Therefore,
with increasing the concentration of EGCG, the recovery in the soluble phase (serum phase) was
also increased. Such binding isotherm for EGCG and casein micelles has never been reported
before.
44
Figure 3.3 % Tea catechin recovered in the colloidal phase (casein micelles) of skim milk, as a
function of the initial concentration of EGCG present in milk. Values are the average of 3
independent experiments (i.e., 3 milk batches) and bars represent the standard deviation.
0
10
20
30
40
50
60
70
80
90
100
0.00 0.01 0.02 0.03 0.06 0.13 0.26 0.51
% T
ea c
atec
hin
Initial EGCG concentration (mg/ mg protein)
45
Using the Scatchard equation (equation 3.3) the dissociation constant (Kd) between EGCG and
casein proteins was determined (figure 3.4). The ratio of bound/free EGCG continued to
decrease as a function of bound EGCG, and three dissociation constants were derived from the
slopes. The dissociation constants calculated were 2.7× 10-4
M , 6.6 ×10-4
M and 6.5×10
-3 M for
the first, second and third portion of the curve, respectively. These results indicated different
types of binding for EGCG with the casein micelles, decreasing in specificity and with a larger
value of the dissociation constant. Kd indicates the strength of binding between the ligand and
the protein in terms of how easy it is to separate the complex, therefore, the smallest Kd
calculated for the first type of binding (equivalent to low EGCG concentrations to EGCG
concentrations at the saturation point of the caseins, up to 0.06 mg EGCG per mg milk protein-
see figure 3.3), show the strongest binding compared to EGCG concentrations after saturation
point of the caseins. It is important to note that another study using small-angle X-ray scattering,
showed a similar concentration of EGCG for saturating casein micelles as indicated by our work
(Shukla et al., 2009).
The combination of the fluorescence and RP-HPLC techniques used in this study clearly
demonstrated that EGCG strongly interacts with the casein micelles, and a substantial amount of
EGCG is associated with the colloidal phase of the casein micelles. The RP-HPLC results
showed, that at approximately 0.06 mg EGCG per mg milk protein the casein micelles are
saturated however the fluorescence results indicated that at around 0.04 mg per mg milk proteins
the quenching reaches a plateau. These results indicate that up to approximately 0.04 mg per mg
milk proteins, EGCG is mainly penetrated within the hydrophobic core of the casein micelles
and thereafter, up to saturation of casein micelles (0.06 mg EGCG per mg milk protein ), is
mostly located at the surface of the casein micelles.
46
Bound/Total protein
0 2 4 6 8
(Bo
un
d/T
ota
l p
rote
in)/
Fre
e (M
-1)
0
2000
4000
6000
8000
10000
12000
14000
Figure 3.4 Scatchard plot for EGCG-milk caseins (see eq 3.3). The bound was calculated as the
amount of EGCG (M) that was within the casein micelle and free EGCG was calculated as the
amount of EGCG (M) that was quantified in the soluble whey proteins by RP-HPLC (see figure
3.3).
47
3.4.3 The effect of EGCG on rennet induced gelation of casein micelles
To determine if the presence of a complex with EGCG affects the processing functionality of the
casein micelles, their ability to form gels with rennet was tested. Rennet induced gelation was
selected as it is caused by a surface destabilization of the casein micelles via a specific hydrolysis
of the stabilizing layer of k-casein on the micelles. Rennet gelation is an enzymatic coagulation
particularly relevant in dairy processing, as it is the first step of the cheese-making process. It is
composed of two stages, which partially overlap each other. During the first stage, the enzymatic
action of the rennet (chymosin) destabilizes the casein micelles, while in the second stage the
k-casein depleted micelles aggregate, in the presence of calcium, and form a protein network
(Walstra, 1990).
Figure 3.5 shows the develelopment of the structure during rennet induced gelation for milk with
and without EGCG. In particular, the value of G’ (storage modulus), as a function of renneting
time for milk samples containing different amounts of tea catechins. Concentrations were
selected from the results of Figure 3.3, as low, medium and saturation concentrations. When the
tea catechin was added to skim milk at very low amounts (0.002 mg EGCG/mg protein, i.e.
much lower than the saturation of the casein micelles, see Figure 3.3) the gel time of the sample
slightly increased compared with the control skim milk sample (without the addition of tea
catechin). As the concentration of tea catechin in skim milk increased, there was a significant
increase in the gelling time. At concentrations higher than the saturation point, gelation was not
observed. The formation of catechin-casein micelles complexes not only delayed the gelling
point of milk, but they also affected the structure formation, as could be noted by the
development of the lower elastic modulus (G′) over time in the tea- milk mixtures compared to
48
control milk. Table 3.1 summarizes the difference in the gelation parameters as a function of
differnet concentration of EGCG added.
Time (min)
0 30 60 90 120 150 180 210 240
G' (
Pa)
0
20
40
60
80
Figure 3.5 Rennet coagulation process monitored by rheometer of the four samples: control
skim milk (●), skim milk with 0.002 mg EGCG/ mg milk protein (▲) skim milk with 0.02 mg
EGCG/ mg milk protein (■), skim milk with 0.08 mg EGCG/ mg milk protein (saturation point
of caseins).
49
Table 3.1. Gelling time and Gel stiffness of different tea milk samples
Gelling time
(min)
Gel stiffness (Pa)
After 80 min of
gelling
Milk (ctrl) 35 ± 5 65.9±7.2
0.002 mg EGCG/ mg milk
protein
41 ±2 36.4±2.6
0.02 mg EGCG/ mg milk protein 65±4 21.3±3
0.08 mg EGCG/ mg milk protein 170±7 0.17±0.04
Values are the means of three replicates.
The results shown in Table 3.1 clearly demonstrated that the presence of tea catechins strongly
affected the gelling behavior of the casein micelles. To further elucidate the origin of the impact
of these interactions, the release of casein macro peptide (CMP) from the surface of the casein
micelles was measured. This should allow determining if the actual enzymatic hydrolysis
occurred at the same rate in all the samples. Chymosin is a proteolytic enzyme active on
κ-casein, specifically on the Phe105-Met106 peptide bond. Following enzymatic cleavage,
para-κ-casein remains on the micelle and the soluble, hydrophilic moiety, casein macro peptide
(CMP) is released into the serum. Removal of CMP from κ-casein reduces steric and
electrostatic repulsion between micelles and increases the attractive forces between the micelles,
leading to aggregation (Anema et al., 1996). Once a sufficient amount (~ 85-90%) of CMP is
released, rapid aggregation of the micelles and a sharp increase in the values of the elastic
modulus are detected (Sandra et al., 2007; Gaygadzhiev et al., 2009).
Figure 3.6 depicts the effect of different concentrations of tea catechin (EGCG) on the release of
CMP from the surface of the casein micelles. The presence of EGCG led to a slower removal of
CMP as measured by the time taken to reach 90% release (as all samples contained the same
50
concentration of casein micelles, the maximum CMP released from the control skim milk was
taken as 100%). The control sample reached 90% of CMP released in about 33 min, while skim
milk with an EGCG concentration lower than the saturation point of the caseins reached the
same point after 60 min. Whereas, the EGCG concentration at saturation point of the caseins did
not reach the 90% of CMP during the time frame of the work.
The fact that even small amounts of EGCG inhibit aggregation and that there is a concentration
dependence, may suggest that the tea polyphenols are affecting the structure of the micelle and
the accessibility of the enzyme to substrate, as full inhibition occurs at concentrations close to the
saturation point of the caseins. Rennet containing the enzyme chymosin, specifically cleaves the
Phe105-Met106 bond of κ-casein. However, it has been shown that other residues near the cleaved
bond are also involved in the hydrolytic reaction of rennet on κ-casein.The peptide
corresponding to the residues 98-111 of κ -casein (His-Pro-His-Pro-His-Leu-Ser-Phe-Met-Ala-
Ile-Pro-Pro-Lys) was found to provide complete requirement for hydrolysis. Also, as proline is a
cyclic amino acid, it causes bends in the polypeptide chain of κ -casein therefore, facilitating
accessibility of the substrate to the shallow cleft of the enzyme (Chitpinityol et al., 1998). It is
proposed by the results found in this study that due to the fact that catechins have a strong
attraction to proline residues, EGCG is probably binding with the four proline residues on each
side of the cleaved bond therefore reducing accessibility of the enzyme to the substrate.
However another possibility of the delay in reaching critical CMP (~90%) for the tea-milk
samples may be due to the fact that catechins are know to have metal chelating properties
(Kumamoto et al.,2001; Green et al., 2007). It has been shown that once κ-casein is cut off from
the surface and a decrease is caused in steric repulsion, the casein micelles approach one another
closely. At this point hydrophobic interactions that cause bonding between the particles play an
51
important role, as does the amount of ionic calcium. Also, a partial loss of colloidal calcium may
reduce the charge interactions, causing the formation of a weaker, more flexible casein network
(Dalgleish, 2012).
52
Figure 3.6 % CMP-release versus time after addition of rennet. Control skim milk (■), 0.02 mg
EGCG/ mg milk protein (●) 0.08 mg EGCG/ mg milk protein (▲).
0
20
40
60
80
100
0 20 40 60 80 100 120
% C
MP
Rel
ease
Time (min)
53
To further test the mentioned hypotheses, another assembly of caseins was studied using sodium
caseinate. Sodium caseinate is made by the disruption of the casein micelle as the calcium
phosphate content is removed, therefore, caseins are no longer in the micellar form and now are
in monomeric forms. Sodium caseinate without the addition of EGCG was used as control as
well as another sample of sodium caseinate with the addition of EGCG at saturation point of the
casein micelles and the CMP release of these samples was analysed. As seen in Figure 3.7, the
results were similar as what was seen in the case of the CMP release of the skim milk and EGCG
samples (Figure 3.6), where the presence of EGCG led to a slower removal of CMP compared to
the control. These results not only confirmed that EGCG strongly interact with caseins but they
also inhibit the accessibility of the enzyme to the caseins, no matter what form the caseins may
be.
54
Figure 3.7 % CMP-release versus time after addition of rennet. Control sodium caseinate (■),
and 0.08 mg EGCG/ mg sodium caseinate (▲).
0
10
20
30
40
50
60
70
80
90
100
110
0 20 40 60 80 100 120 140
% C
MP
rel
ease
Time (min)
55
3.5 Conclusions
Numerous studies have shown that strong interactions take place between tea catechins and milk
proteins, which we also confirmed. However, most studies have been on isolated caseins and not
caseins in milk as well as the effect of polyphenols on functional properties of milk proteins is
not fully understood. In this study it was shown that the binding of EGCG to caseins in skim
milk reaches a saturation point where afterwards EGCG is then seen in whey proteins. As the
concentration of EGCG increases and saturation of caseins is reached, a more significant effect is
seen on the renneting properties of milk. It was shown with fluorescence quenching of
tryptophan that to a certain degree, EGCG is incorporated into the porous casein micelles and
thereafter may be on the surface of the micelles resulting in less accessibility of the enzyme to
the substrate (caseins) or may be due to chelating of calcium resulting in a weaker casein
network. This study clearly shows that demonstration of successful and effective incorporation
of bioactives into milk matrix is essential before using milk as a platform for delivery of
polyphenols.
56
CHAPTER 4
Anti-proliferative activity of nanoencapsulated tea catechin in casein micelles,
using HT29 colon cancer cells
4.1. Abstract
The present study tested the ability of casein micelles to deliver biologically active
concentrations of polyphenols to colon cancer cells (HT-29). Epigallocatechin-gallate (EGCG),
the major catechin found in green tea, was employed as model molecule, as it has been shown to
have antiproliferative activity on colon cancer cells. Casein micelles in milk may be an ideal
platform for the delivery of bioactive molecules such as EGCG, as casein proteins have been
shown to have strong interactions with polyphenols. In the present work, it was hypothesized that
the strong binding of caseins with EGCG causes a change in the bioavailability of EGCG.
The cytotoxity and proliferation behaviour of HT-29 colon cancer cells when exposed to EGCG
was compared to that of EGCG nanoencapsulated in casein micelles, in their native form, in
skim milk. EGCG-casein micelle complexes decreased the proliferation of HT-29 cells,
demonstrating that bioavailability was not reduced by the nanoencapsulation. As casein micelles
may act as protective carriers for EGCG in foods, it was concluded that nanoencapsulation of tea
catechins in casein micelles may not diminish their anticancer activity compared to free tea
catechins.
57
4.2. Introduction
Inrecent years, polyphenols have been recognized as important components in the diet, as their
consumption has increasingly been associated with the prevention of various chronic diseases
(Yang et al., 2009; Gonzalez et al., 2011; Yuan et al., 2011). The major sources of polyphenols
in the diet are vegetables, fruits, wine, coffee and tea (Manach et al., 2004). Tea (Camellia
sinensis), which is the second most consumed beverage in the world, is a rich source of
extractable polyphenols, more specifically of flavan-3-ols, commonly referred to as catechins.
The catechins in green tea represent up to 85% of the total polyphenols (Ferruzzi 2010). Recent
epidemiological and biological data have associated green tea consumption with a reduced risk
of cancer and diseases of the cardiovascular system. Major tea catechins include epicatechin,
epigallocatechin, epigallocatechin-gallate (EGCG), and epicatechin-gallate. EGCG is the major
catechin component in tea and it appears to be the most biologically active (Vaidyanathan et al.,
2003; Henning et al., 2004) (Figure 4.1). Human clinical intervention trials have demonstrated
positive links between green tea consumption and a reduced risk of cancer (Li et al., 2000;
Lambert et al., 2007a).
Scavenging of reactive oxygen and nitrogen species, chelating of redox active metals, inhibiting
cancer-related transcription factors, and inhibiting oxidative enzymes are amongst the reactions
involving catechins which may result in these beneficial health effects (Green et al., 2007).
Bioavailability is usually defined as the fraction of the bioactive constituents (or the metabolites)
that have been absorbed by the body and can exert their biological actions at specific target
tissue sites (Ferruzzi, 2010; Visioli et al., 2011). Bioavailability appears to differ greatly among
polyphenols, and for tea catechins it is generally considered to be poor (Ferruzzi, 2010).
58
Figure 4.1 Structure of (-) epi-gallocatechin gallate
59
Several factors may play a part in limiting the bioavailability of polyphenols, including
molecular structure, amount consumed, intestinal absorption and the degree of bioconversion in
the gut (Papadopoulou et al., 2004; Yang et al., 2008). In addition, the interactions between the
bioactive molecules and the food matrix are considered critical factors limiting or enhancing
bioavailability (Saura-Calixto et al., 2007).
Milk is known to be a multi-component and a nutritious source of compounds beneficial to
growth and health in children as well as adults. Studies have shown that both whey proteins and
caseins act as delivery vehicles for bioactive compunds (Livney 2010). Nanoencapsulation of
-lactoglobulin has been shown to protect EGCG against oxidative degradation (Shpigelman et
al., 2010). Bovine serum albumin has often been referred to as a carrier of chemotherapeutic
drugs in the circulatory system (Livney 2010). Recent studies have also demonstrated the
association of bioactive molecules such as resveratrol, vitamin D and curcumin (a polyphenol)
with casein micelles (Semu et al., 2007; Sahu et al., 2008; Rahimi Yazdi et al., 2012).
The encapsulation of vitamin D in casein micelles provided partial protection against UV-
induced degradation of vitamin D (Semu et al., 2007).
Polyphenols are known to have strong binding affinities with proteins rich in proline, and
proteins with an open and flexible structure such as caseins (Benick et al., 2002; Papadopoulou
et al., 2004). Studies have shown that the highest extent of binding of catechins with isolated
forms of caseins, was noted for β-casein, followed by αs-caseins and κ-casein (Luck et al., 1994;
Kartosova et al., 2007). In terms of bioaccessibility, researchers have shown that curcumin
encapsulated within casein micelles can be efficiently delivered to Hela tumour cell culture
(Sahu et al., 2008). However, the bioaccessibility and bioavailability of polyphenols in milk
mixtures is still a source of debate. Some studies have suggested that the presence of protein
60
masks the antioxidant capacity of tea catechins (Arts et al., 2002; Kartosova et al., 2007).
This masking effect was also reported in human clinical trials comparing tea drank with or
without milk (Langley-Evans 2000). In contrast, other reports showed no significant effect of
milk on the plasma antioxidant capacity of tea polyphenols (Leenon et al., 2000 Keogh et al.,
2007; Kyle 2007).
The discrepancies may derive from the differences designs and methodologies of the studies. It is
also important to note that stability of catechins during storage and digestion may be poor, but
when present in complex matrices such as bovine milk, soymilk or rice milk the stability may
increase (Green et al., 2007; Ferruzzi 2010).
The objective of this work was to test the effect of nanoencapsulation of EGCG in casein
micelles on its anticancer activity. Casein micelles can load a substantial amount of catechins
within their structure, acting as carriers for the catechin and therefore may affect their
bioavailability. The antiproliferative activities of EGCG on HT 29 colon cancer cells was tested
with free EGCG and EGCG in various milk matrices, namely, milk serum, milk whey and when
encapsulated in casein micelles.
4.3. Materials and Methods
4.3.1 Milk samples preparation
Commercial skim milk was purchased from local super market (Parmalat Canada Inc., Toronto,
Canada).
Separation of the soluble proteins (whey) from the casein micelles was accomplished by
centrifugation (36,000 ×g) at 20°C for for 45 min (Beckman Coulter, Canada, Inc, with rotor
type 70.1 Ti, Mississauga, On, Canada) as previously described (del Angel et al., 2006).
61
Milk permeate was prepared using a laboratory scale ultrafiltration cartridge (Millipore
CDUF001LG; Fisher Scientific), with a nominal cut-off of 10,000 Da and a nominal area of
1 ft2 (0.095 m
2).
A polyphenol extract from green tea (containing minimum 90% EGCG) was obtained from DSM
Nutritional Products Inc. (Montreal, Canada). Different concentrations of EGCG were obtained
from a stock solution of EGCG in water (20 mg/ml water). The amount of EGCG encapsulated
within the casein micelles was calculated using HPLC, as described in detail in chapter 3.
4.3.2. Cell viability assay
Human colorectal cancer cells (HT-29) were obtained from the American Type Culture
Collection (ATCC- HTB-38; Virginia, USA). HT-29 cells were maintained in Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2.2 g/l sodium
bicarbonate, 1% penicillin/ streptomycin and 1% L-glutamine (Sigma-Aldrich Canada Ltd.
Oakville, Ontario, Canada), and incubated at 37°C in 95% humidity and 5% CO2 is air (Forma
Series II Water-jacketed CO2 Incubator, model 3110 , Forma Scientific, California, USA).
Trypan blue dye exclusion is typically used to estimate cell viability. This large molecular
weight dye will be excluded unless the cell membrane has been damaged or its permeability
compromised in some way. Healthy cells exclude the dye while dead or dying cells take the dye
up. Therefore, this procedure is used to estimate the percentage of viable cells under specific cell
culture conditions (Ehrich et al., 2000).
HT-29 colon cancer cells were seeded and allowed to grow for 48 h, afterwhich different
concentrations of tea catechins, alone and in combination with milk, were added to the cells and
incubated for a further 24 h. Each sample was diluted in media at a ratio of 1:5.6 (sample:media)
62
(v/v). Control samples were also tested, either with media only (for free EGCG) or with milk,
with no EGCG (for tea-milk samples). After a continuous incubation of 24 h, trypsinized cells
were suspended in culture medium, counted using a Neubauer haemocytometer and viability was
measured using Trypan blue dye exclusion (see Ehrich et al., 2000 for details).
Cell viability was expressed as the ratio between the viable cells for each treatment (i.e. EGCG-
milk; EGCG free) and the number of viable cells present in the corresponding control.
Cell viability tests were used in combination with the sulforhodamine B cytotoxicity assay (see
below), to better determine changes in cell proliferation.
4.3.3. Cell proliferation assay
The anti-proliferation effect of EGCG on HT-29 cancer cells was determined using the
Sulforhodamine B (SRB) dye binding assay (see Shekhan et al., 1990 for details). SRB is a
colorimetric assay for the quantification of the total protein synthesis rate of cells in response to
different concentrations of tea catechin. SRB is an anionic dye that binds to synthesized proteins.
The fixed dye, measured photometrically after solubilization, correlates with protein synthesis
and with cell proliferation (Vichai et al., 2006).
HT-29 colon cancer cells were seeded on 96 well plates and allowed to adhere for 24 h.
Afterwards, different concentrations of tea catechin (EGCG) alone and in combination with milk,
were added to the cells and incubated for for an additional 24 h. Each sample was diluted in
media at a ratio of 1:5.6 (sample:media) (v/v). Control samples were also tested, either with
media only (for free EGCG) or with milk, with no EGCG (for tea-milk samples). After a
continuous incubation of 24 h, TCA-fixed cells were stained with SRB dye. At the end of the
staining period, SRB was removed and cultures were quickly rinsed with 1% acetic acid to
63
remove unbound dye. After drying, the bound dye was solubilized with 10 mM Tris base (pH
10.5).The adbsorbance of plates was measured using an automated 96-well plate reader (Multi
detetctor Microplate Reader, Biotek Synergy HT Model, Vermont , USA) at a wavelength of 570
nm. The results are reported as % proliferation, which is the OD of each fraction at 570 nm
divided by the OD of its control multiplied (at 570 nm)by 100 (Vichai et al., 2006).
4.3.4. Statistical analysis
The results presented are the average of at least three independent experiments. Each experiment
had at least 5 sub samples. The mean values, standard deviations, and statistical differences were
evaluated using analysis of variance (ANOVA). All statistics were performed by using R
software (R Project for Statistical Computing version 2.15.1).
4.4. Results and Discussion
Various studies have observed that tea polyphenols inhibit tumour formation and proliferation in
different cell lines, as well as in animal models (Yang et al., 2006; Yang et al.,2009).
The inhibitory activity can include decreasing cell proliferation, increasing apoptosis and/or
suppressing angiogenesis (Araújo et al., 2011). This study focused on the effect of the matrix on
the antiproliferative properties of EGCG on colon cancer cells. Figure 4.2 shows the EGCG
encapsulated in the native casein micelles as a function of the amount of EGCG added in skim
milk. At a concentration of 2.5 mg EGGC/ml of milk, most of the tea catechin is associated with
the colloidal phase of the micelles (see also Chapter 3). For the cell cytotoxicity and proliferation
64
assays, EGCG concentrations below saturation (0.2 and 0.5 mg/ml), at saturation (2.5 mg/ml)
and above saturation (5 mg/ml) were used.
The study of the effectiveness of EGCG on the inhibition of colo-rectal carcinogenesis is of great
interest (Gonzales et al., 2010). The majority of the studies related to the anti-proliferative
properties of polyphenols on colon cancer cells has so far been based on the effect of individual
polyphenols or crude polyphenol extracts (Hong et al., 2002; Chen et al., 2003; Na et al., 2006;
Bazinet et. al., 2010), and much less has been reported on polyphenols bound to proteins such as
in milk. However, gut epithelial cells are more likely to be exposed to complex food matrices,
and this, as previously discussed, may affect the bioavailability of polyphenols in the gut (Saura-
Calixto et al., 2007).
65
Figure 4.2 Amount of EGCG associated with the casein micelles in skim milk as a function of
the initial concentration of EGCG in milk. Results are the average of three independent
experiments and bars represent the standard deviation.
0
10
20
30
40
50
60
70
80
90
100
0.15 0.2 0.5 0.75 1 2 2.5 3.3 5
% E
nca
psu
late
d E
GC
G in
cas
ein
s
Initial concentration of EGCG in milk (mg/ml)
66
Figure 4.3 illustrates the difference in the viability of HT-29 cancer cells after 24h of incubation
with different concentrations of EGCG, either dispersed in water or in nanoencapsulated in the
casein micelles in skim milk. The final concentrations of EGCG in the wells are indicated in the
figure, and were 0.03, 0.075 mg/ml 0.15 mg/ml, 0.4 mg/ml and 0.75 mg/ml, corresponding 0.2,
0.5, 1, 2.5 and 5 mg/ml in the original milk. The viability of the cells exposed to unencapsulated
EGCG after 24 h of incubation (solid bars, Figure 4.3) decreased as a function of the amount of
EGCG present. This result was in full agreement with other studies using similar concentrations
of EGCG (Salucci et al., 2002; Bazinet et al., 2010). When the cells were exposed to the mixture
of EGCG-milk samples (Figure 4.3, empty bars), the same trend was observed, a decrease in
viability with increasing EGCG.
While at concentrations, < 0.15 mg/ml there were no differences in viability between
unencapsulated and encapsulated treatments, at higher concentrations, above saturation, viability
was significantly higher than for samples with EGCG in water. The lower decrease in viability
in the presence of milk is most probably due to differences in the composition of the media,
containing milk components.
67
EGCG concentration (mg/ml)
Ctrl 0.03 0.075 0.15 0.4 0.75
% V
iable
cel
ls
0
20
40
60
80
100
Figure 4.3 Viable cells after 24h exposure to isolated EGCG (solid) and EGCG encapsulated in
casein micelles. All samples were diluted in media 1:5.6 (sample: media). The control used for
isolated EGCG was media without EGCG and the control used for of EGCG-milk fractions was
milk without EGCG. Values are the average of at least 3 replicates, bars represent standard
deviations. Different letters within each concentration of isolated and encapsulated EGCG,
indicate statistical significant differences at p<0.05.
a a
a a
a
a
a
b
b
b
68
To better identify the effect of the medium, the matrix and the concentrations dependence of
EGCG encapsulated in casein micelles, the anti-proliferative effect of EGCG on HT-29 cancer
cells was studied. The results are summarized in Figure 4.4. When EGCG was non encapsulated
(solid bars Figure 4.4), even at very low concentrations (i.e. ≤ 0.02 mg/ml media corresponding
≤ 0.1 mg/ml water) it was able to significantly decrease the proliferation of the cancer cells.
At higher concentrations (i.e. ≥ 0.03 mg/ml media corresponding ≥ 0.2 mg/ml milk), the
decrease in proliferation reached a plateau.
The same trend in the proliferation of the HT-29 colon cancer cells was seen when they were
exposed to different concentrations of the EGCG-milk samples. Figure 4.4 compares the
proliferation of the cells incubated with EGCG dispersed in permeate, milk whey and skim milk.
In all cases, as the EGCG concentration increased, there was a decrease in the cancer cell
proliferation, eventually plateauing at high concentrations. Although at higher concentrations of
EGCG (i.e. ≥ 0.15 mg/ml media corresponding ≥ 1 mg EGCG /ml milk) no significant
differences were noted in the anti-proliferative activity of EGCG (p> 0.05), whether isolated or
present in a milk matrix, the bioefficacy of isolated EGCG seemed to be affected in the presence
of milk mixtures, as at low concentrations (<0.015 mg/ml) the extent of proliferation differed.
However, this could not be attributed to the encapsulation of EGCG in casein micelles (Figure
4.3 C), as the same effect was noted also for EGCG in milk serum (Figure 4.3 A) and in milk
whey (Figure 4.3 B). At the lower concentrations, one hypothesis is that the difference in
proliferation are due to a difference in the media composition, as milk serum, milk whey and
skim milk may contain growth factors not present in the water used to resuspend EGCG, nor in
the media used for cells.
69
At about 0.03 mg/ml (corresponding to 0.2 mg/ml milk), a significantly higher proliferation was
noted for the cells treated with EGCG in skim milk, compared to EGCG in whey, permeate or
control. Hence, the effect of the interactions between tea catechin and caseins may still be
apparent since the EGCG-milk fractions reach a plateau with some delay compared to the
EGCG-whey and EGCG- permeate fractions. When comparing the estimated IC50 values, 0.01
mg/ml, 0.016 mg/ml, 0.018 mg/ml and 0.02 mg/ml for isolated EGCG, EGCG-whey, EGCG-
permeate and EGCG-milk (caseins), respectively, it could be hypothesized that this decrease in
the apparent bioefficacy may be caused by the complexes formed between EGCG and casein
micelles. Therefore, the strong interactions between EGCG and milk caseins, may affect the
ability of EGCG to inhibit the proliferation of the cancer cells.
70
% P
roli
fera
tio
n
0
20
40
60
80
100 EGCG
EGCG in milk serum
% P
roli
fera
tio
n
0
20
40
60
80
100EGCGEGCG in milk whey
EGCG concentration (mg/ml)
ctrl 8e-3 0.015 0.03 0.08 0.15 0.4 0.8
% P
roli
fera
tio
n
0
20
40
60
80
100EGCG
EGCG in skim milk
Figure 4.4 Proliferation of HT-29 cells exposed to isolated EGCG (solid bars) or EGCG in milk
serum (permeate) (A); milk whey (B) or encapsulated with the casein micelles in skim milk (C).
Values are the average of at least three independent experiments. Bars represent standard
deviation. Different letters within each concentration of EGCG, indicate statistical significant
differences at p<0.05.
a a a a a a a a a a
b
b
a
a
a a a a a a a a a a
a
a
b
b
a a a a a a a
a
a
b
b
b
A
B
C
a
a a
71
4.5. Conclusions
EGCG is one of the most extensively investigated chemopreventive phytochemicals. By
modulate signal transduction pathways involved in cell proliferation, transformation,
inflammation, apoptosis, metastasis and invasion, EGCG could potentially block multiple stages
of carcinogenesis (Na et. al, 2006). However, in order to exert their biological health benefits in
vivo, polyphenols must be bioavailable. This study showed that in spite of the strong binding of
EGCG to the casein micelles, the bioefficacy of EGCG was not diminished, as cell proliferation
results showed that the EGCG-milk complexes were still able to significantly decrease the
proliferation of HT-29 cancer cells, similar to isolated EGCG. It is important to note though, that
the present studies were carried out on fresh EGCG. Mixed systems may ensure better stability
to tea catechins with storage and during digestion, ultimately improving their bioefficacy.
72
Chapter 5
The effect of nanoencapsulation of tea catechins in casein micelles on their
bioaccessibility studied using normal and cancerous rat colonic cells
5.1 Abstract
Numerous studies have demonstrated that tea catechins form complexes with milk proteins,
especially caseins. Much less work has been conducted to understand the metabolic conversions
of tea/milk complexes during gastro-duodenal digestion. The objective of this study was to
determine the significance of this association on the digestibility of the milk proteins and on the
bioaccessibility of the tea polyphenol epigalocatechin galate (EGCG). An in vitro digestion
model mimicking the gastric and duodenal phases of the human gastrointestinal tract was
employed to follow the fate of the milk proteins during digestion, and determine the bioefficacy
of EGCG free or encapsulated with the caseins. The samples, before and after digestion were
tested using two parallel colonic epithelial cell lines, a normal line (4D/WT) and its cancerous
transformed counterpart (D/v-src). EGCG digests showed a specific effect on proliferation of
cancer cells. In normal cells, neither free or encapsulated EGCG affected cell proliferation, at
concentrations <0.1 mg/ml. At higher concentrations, both free and encapsulated produced
similar decreases in proliferation. On the other hand, the bioefficacy on the cancer cell line
showed some differences at lower concentrations. The results demonstrated that regardless of the
extent of digestion of the nanoencapsulated EGCG, the bioefficacy of EGCG was not
diminished, confirming that casein micelles are an appropriate delivery system for polyphenols.
73
5.2 Introduction
Numerous studies have attributed many of the beneficial health effects associated with green tea,
to the catechins (Li et al., 2001; Lambert et al., 2007a, Yang 2008; Shpigelman et al., 2010).
In recent years the possible cancer preventive activity of epigallocatechin-3-gallate (EGCG) has
been extensively studied using animal models and different cell lines from various organs and
species. The inhibitory activity of EGCG has been demonstrated during both the initiation and
progression stages of carcinogenesis (Vaidyanathan et. al., 2003; Chen et. al., 2007; Yang 2009).
Since broad cancer-preventive effects in cell lines and animal models have been observed, it is
assumed that multiple molecular mechanisms, rather than a single receptor or molecular target,
may be involved (Jung et al., 2001; Hsu et al., 2003; Hwang et. al., 2007). For EGCG, binding to
molecular targets and inhibition of enzyme activities have been reported, as well as broader
spectrum antioxidant activities (Yang et. al., 2006; Yang et al., 2009; Li et al., 2010). Tea
catechins potently induce apoptotic cell death in tumor cells, and cancer cells are more
susceptible to the inhibitory and chemotherapeutic effects compared to normal and non-
transformed cells (Yamamoto et al., 2003; Friedman, et al., 2007; Yang 2009).
However, to exert their biological properties, polyphenols must be bio-accessible to the target
tissue. The bioavailability of dietary polyphenols is limited by a number of factors such as the
amount consumed, physiochemical properties of the molecule, enzyme and microbial-mediated
biotransformation and active reflux, instability under digestive conditions, intestinal absorption
and food matrix interactions (Papadopoulou et al., 2004; Green et al., 2007; Ferruzzi et.al.,
2010).
It has been shown that tea catechins are stable in the gastric lumen, under conditions of low pH
caused by the gastric juices of the stomach. However, there are many factors that influence the
74
extent and rate of absorption of ingested compounds by the small intestine (Spencer et al., 2003;
Visioli et al., 2011). Upon transfer from the stomach to the small intestine the pH rises to values
around neutral. It is well known that polyphenolic compounds such as catechins, oxidize in
neutral and alkaline pH environments. EGCG has been observed to rapidly oxidize, however, the
oxidation of EGCG resulted in the formation of dimerized products, which were observed to
possess greater superoxide radical scavenging activity than EGCG itself and had powerful iron
chelating properties (Spencer et al., 2003). However, most studies on the fate of polyphenols in
the digestive tract have been limited to specific types of polyphenols or polyphenols in
combination but not on the fate of polyphenols when incorporated in food matrices (Saura-
Calixto et. al., 2007).
Milk proteins are known to be natural carriers, as their structure facilitates the delivery of
essential micronutrients (calcium and phosphate), from mother to the neonate. Researchers have
suggested the potential of milk proteins, mostly in their isolated forms, to deliver nutrients such
as vitamins D, curcumin, minerals (Fe2+, Mg2+), carotenoids (β–carotene), polyphenols
(resveratrol, caffeine), cancer therapy drugs and even flavors or aromas (Semu et al, 2007; Sahu
et al., 2008; Livney 2010). A recent study has shown that casein micelles can take up a
substantial amount of EGCG (see Chapter 3), and that nanoencapsulation of EGCG with milk
proteins provides considerable protection to EGCG against oxidative degradation compared to
unprotected (non-encapsulated) EGCG (Shpigelman et al., 2010).
It has been previously demonstrated that the interactions taking place between the tea catechin
and the milk caseins in native casein micelles does not affect the antiproliferative activity of
EGCG using HT-29 colon cancer cells. However, studies on catechin-milk protein interactions
with respect to catechin availability after simulated digestion have not been reported.
75
Furthermore, the complexes formed between EGCG and caseins may affect the digestion of the
proteins.
The effects that the tea catechins may have on digestive enzymes, more specifically proteases,
must be considered as well. Recent studies have suggested an inhibitory effect of some
polyphenols on proteases (Siewright et al., 2006; McDougall et al., 2005; Goncalves et. al.,
2011). Although inhibition of proteases by polyphenol components can occur in vitro, there is
still not enough evidence to show that inhibition of protease activity occurs in the digestive
system in vivo (McDougall et al., 2008), and the results are still a source of debate (Tagliazucchi
et al., 2005; McDougall et al., 2008).
In this study, we aim to examine the effects of EGCG encapsulation in casein micelles on normal
and cancerous rat colonic cell lines, both before and after in vitro digestion.
76
5.3 Materials and Methods
5.3.1 Sample preparation
Commercial skim milk was purchased from a local supermarket (Parmalat Canada Inc., Toronto,
Canada). Green tea polyphenol extract (containing a minimum of 90% EGCG) was purchased
from DSM Nutritional Products INC (Montreal, Canada).
Different concentrations of EGCG were generated from a stock solution of EGCG in water (20
mg/ml water). The amount of EGCG encapsulated within the casein micelles was calculated
using HPLC, as described in detail in chapter 3). Figure 5.1 shows the percentage of
encapsulated EGCG at different concentrations of EGCG in milk. The EGCG concentration is
shown as mg EGCG per ml milk. These results showed that the casein micelles reach a
saturation point at around 2.5 mg EGCG per ml milk. Therefore, for cell cytotoxicity and
proliferation assays, concentrations of EGCG in milk at concentrations below saturation of
casein micelles, at saturation and above saturation were used.
77
Figure 5.1 Amount of EGCG associated with the casein micelles in skim milk as a function of
the initial concentration of EGCG in milk. Results are the average of three independent
experiments and bars represent the standard deviation.
0
10
20
30
40
50
60
70
80
90
100
0.15 0.2 0.5 0.75 1 2 2.5 3.3 5
% E
nca
psu
late
d E
GC
G i
n c
asei
ns
Initial concentration of EGCG in milk (mg/ml)
78
5.3.2 In vitro gastro-duodenal digestion
An in vitro digestion model mimicking the gastric and duodenal stage of human digestion was
applied on isolated EGCG, skim milk and EGCG-milk mixture (Malaki Nik et al., 2010).
Briefly, simulated gastric fluid (SGF) was added to an amber jar containing pre-incubated
samples at 37 ºC for 15 min. SGF was prepared by dissolving the desired amount of pepsin in
salt solution as previously reported in detail (Malaki Nik et al., 2010). The final concentration of
3.2 mg of pepsin per mL of mixture (samples plus SGF) and final pH of 2 were employed. The
mixture was then incubated at 37 ºC in a shakingwater bath (New Brunswick Scientific Co., Inc.,
NJ) at 250 rpm for 60 minutes.
Samples from the gastric phase were collected, and the reaction was stopped by raising the pH of
mixture to 6.5 using 1 M NaHCO3.
Immediately after the gastric phase, the mixture was treated with simulated bile fluids and
simulated duodenal fluids at a final pH of 6.5. The mixture was incubated at 37 ºC and 250 rpm
for 1 h, the final mixture contained 8 mM bile salts and 5 mg mL−1
pancreatin as previously
reported in detail (Malaki Nik et al., 2010).
To deactivate the trypsin enzyme activity, all samples were immediately cooled on ice and kept
frozen at -20◦C until further use. For the samples destined to cell culture studies, the samples
were diluted and stored in media containing 10% Fetal Bovine Serum (FBS) (1:6 digest: media
v/v) (Sigma-Aldrich Canada Ltd. Oakville, Ontario, Canada) and frozen until use.
79
5.3.3 SDS-PAGE analysis
To determine if the encapsulation affected the digestion behaviour of the casein micelles during
the gastric phase, Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
was conducted on milk controls as well as milk containing EGCG at a concentration above
saturation, 5 mg/ml (see Chapter 3). The samples were withdrawn from the digestion mixture at
5, 10, 20, and 30 min during in vitro simulated gastric digestion. SDS-PAGE under reducing
conditions was carried out using 15% resolving gels (Malaki Nik et al., 2011). Aliquots of
digested samples of milk and EGCG-milk were mixed with electrophoresis sample buffer,
containing 200 mM Tris–HCl, 2% SDS, 40% glycerol, 2% β-mercaptoethanol, and 1%
bromophenol blue. The solutions were then heated at 95 ◦C for 5min, centrifuged at 10,000 g for
5min using an Eppendorf centrifuge (Brinkmann Instruments, New York, USA). The resulting
subnatant was loaded (10µL) onto the gels and run at a constant voltage of 200V.After each run,
gels were immediately stained using Coomassie blue R-250 for 30min and destained with a
solution of acetic acid: water: methanol (1:4.5:4.5) for 2 h with two changes, and then destained
overnight with a destaining solution containing 22.5% methanol and 5% acetic acid. SHARP JX-
330 scanner (Amersham Biosciences, Quebec) was employed to scan the gels.
80
5.3.4 Cell proliferation assays
The cell lines used in this research were the immortalized non-tumorigenic cell line referred to as
FRC TEX CL 4D (4D/WT) and the derived line transformed FRC TEX CL D/v-src, established
by transfection of a v-src sequence into the 4D/WT parental line (Cha et al., 2005). The cell
lines were a generous gift from Dr. Susan E. Pories, New England Deaconess Hospital and
Harvard Medical School.
4D/WT and D/v-src cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal
bovine serum (FBS), hydrocortisone (0.02 g/ml), insulin (0.25 g/ml), transferrin (0.12 g/ml),
polybrene (1 g/ml) and penicillin-streptomycin (50 U/ml) (Sigma-Aldrich Canada Ltd.
Oakville, Ontario). in a humidified environment at 37°C in the presence of 10% CO2 in air.
Cell proliferation assays were conducted using the sulforhodamine B test (SRB). SRB is a
colorimetric assay for the quantification of the total protein synthesis rate of cells in response to
different concentrations of tea catechin. SRB is an anionic dye that binds to synthesized proteins.
The fixed dye, measured photometrically after solubilization, correlates with total protein
synthesis rate and therefore with cell proliferation (Vichai et al., 2006).
Cell proliferation assays were conducted using the sulforhodamine B dye-binding assay (SRB)
(Shekhan et al.,1990). The SRB assay was used to quantifythe total protein in cells, in response
to different concentrations of tea catechin. The fixed dye, measured photometrically after
solubilization, correlates with total protein and correlates well with cell proliferation (Vichai et
al., 2006).
4D/WT and D/v-src cells were seeded on 96 well plates and allowed to adhere for 24h,
afterwhich different concentrations of tea catechin alone and in combination with milk were
added to the cells and incubated for a further 24 h. Before digestion, all samples were diluted in
81
media 1:5.6 (sample: media). The digests of isolated EGCG and EGCG-milk were further diluted
to a final of 1:16 (sample: media) and delivered the the cells in 96-well plates. The control used
for EGCG digests was media without EGCG and the control used for of EGCG-milk digests was
digested milk.
After a continuous incubation of 24 h, TCA-fixed cells were stained with SRB dye. At the end
of the staining period, SRB was removed and cultures were quickly rinsed with 1% acetic acid to
remove unbound dye. After drying, the bound dye was solubilized with 10 mM Tris base (pH
10.5). The absorbance of plates was measured using an automated 96-well plate reader (Multi
detetctor Microplate Reader, Biotek Synergy HT Model, Vermont , USA) at a wavelength of 570
nm. The results are presented as percent control proliferation, which is calculated by dividing the
absorbance of each fraction at 570 nm by the absorbance of its control and multiplied by 100
(Vichai et al., 2006).
5.3.5 Statistical analysis
The results presented are the average of at least three independent experiments. Each experiment
had at least 5 sub samples. The mean values, standard deviations, and statistical differences were
evaluated using analysis of variance (ANOVA). All statistics were performed by using R
software (R Project for Statistical Computing version 2.15.1).
82
5.4 Results and Discussion
5.4.1 SDS-PAGE of milk and tea/milk complexes during the gastric phase
It is becoming clear that tea polyphenols, such as gallated catechins, have poor bioavailability
and/or stability in vivo (Ferruzzi 2010). Therefore, a large proportion of ingested polyphenols
from tea is not taken up into the circulation and passes through the gastrointestinal tract (GIT)
unabsorbed. Therefore the potential health benefits of catechins will be critically dependent on
their metabolic handling and delivery within the GIT.
The SDS-PAGE results of milk samples and milk-EGCG samples withdrawn during the gastric
phase are shown in Figure 5.1. The electrophoretic analysis of milk during simulated gastric
digestion demonstrated an extensive proteolysis of the caseins after 5 min of incubation. Only a
faint band was recovered for undigested β-lactoglobulin.
However, the objective of the study was to observe the effect of EGCG-casein complexes on
casein digestion which the SDS-PAGE results of milk proteins, whether complexed with EGCG
or alone (figure 5.2) show no differences in the digestion patterns of the milk proteins.
These results indicate that the complex formation between EGCG and milk caseins does not
make the caseins less susceptible to proteolysis. Although, it has been suggested by some studies
that polyphenols may inhibit protease activity of some gastric enzymes, such a situation was not
seen in our results (Siewright et al., 2006; McDougall et al., 2005; Goncalves et. al., 2011).
83
Figure 5.2 SDS electrophoresis of milk digests at 5, 10, 20 and30 minutes of gastric phase (a,b,c
and d) and EGCG-milk digests at 5, 10, 20 and 30 minutes of gastric phase (e,f,g and h) .
84
Our results are in accordance with the results obtained by van der Burg-Koorevaar et. al., 2011
when black tea –milk digestates were examined by SDS-PAGE.
Caseins, having a flexible structure, are sensitive to proteolysis; purified bovine caseins are
rapidly cleaved by digestive proteases during in vitro digestion in various models (Dupont et. al.,
2010). The extensive proteolysis of the caseins after 5 minutes in the skim milk samples may be
due to an excessive amount of enzyme, such as pepsin, used during the gastric process. It has
been shown that heat treatments of milk may cause protein denaturation which alters the
separation of proteins in SDS-PAGE therefore this may be the reason for the extensive
proteolysis of the caseins in our study, as the skim milk utilized was pasteurized (Dupont et. al.,
2010). Studies have noted that the rate of hydrolysis differs between caseins and whey proteins
in bovine milk, and that β-lactoglobulin is relatively highly resistant to digestion in bovine milk
samples (Inglingstad et. al., 2010 ; Dupont et. al., 2010 ; Picariello et. al., 2010).
85
5.4.2 Proliferation of normal (4D) and tumorous (v-src) rat colonic cells exposed to EGCG
and EGCG -milk fractions before and after invitro digestion
As green tea catechins are very unstable in neutral and alkaline solutions and are known to have
poor absorption and therefore low bioavailability, using milk as a natural nano carrier may be
beneficial. However, the impact of milk formulation on the bioaccessibility and bioavailability of
EGCG must be considered. By studying the affect of EGCG, both in isolation and complexed
with milk, on cell proliferation, we can get a better understanding of the bioaccessability of
EGCG.
For this assay, the EGCG was either dissolved in water or in skim milk, hence associated with
the casein micelles (see Chapter 3). The concentrations used for EGCG whether in water
(isolated form) or in milk (encapsulated with caseins) were derived from figure 5.1; lower
concentrations of EGCG before saturation of casein micelles (i.e. 0.2 mg/ml, 0.5 mg/ml, 1
mg/ml), a concentration of EGCG at saturation of casein micelles (2.5 mg/ml) and a
concentration of EGCG above saturation (5 mg/ml).
However, in order to expose the cell line to the EGCG and EGCG-milk fractions, further dilution
with media took place on the samples before digestion. Each fraction was diluted in media with
a ratio of 1: 5.6 (fraction: media (v/v)). Therefore, the final concentrations of EGCG in media
that were delivereded to cells correspond to concentrations of EGCG before saturation of casein
micelles (i.e. ≤0.19 mg/ml media), EGCG at saturation (i.e. 0.38 mg/ml media) and after
saturation of casein micelles (0.75 mg/ ml media). Also, it is important to note that the control
for isolated EGCG fractions was media, however, for EGCG-milk fractions, the control was
milk.
Figure 5.3 A and B show the results for normal (4D) and tumorigenic (v-src) rat colonic cells
when exposed to increasing concentrations of isolated EGCG and encapsulated EGCG-milk
86
before applying invitro digestion. The results showed in terms of cell proliferation of tumor cells,
a significant difference was seen at lower concentrations of EGCG (P < 0.05), as a lower effect
on inhibiting the proliferation of tumorigenic (v-src) cells was seen meaning that the bioefficacy
of EGCG at lower concentrations was affected by skim milk. However, at higher concentrations
of EGCG (≥ 0.09 mg/ml) similar effects were seen whether EGCG was isolated or bound to
milk. These results indicate that the strong binding of EGCG and milk casein not affect the bio
efficacy of the tea catechin, nor does it possibly affect the mechanism(s) responsible for the
inhibitory activity on tumor cells.
In the case of normal cells exposed to increasing concentrations of isolated EGCG and
encapsulated EGCG-milk before applying in vitro digestion (Figure 5.3 b), although a significant
difference was not seen between EGCG-milk fractions and fractions with isolated EGCG
(P> 0.05), the higher concentrations of EGCG were toxic for the normal cells (≥ 0.4 mg/ml).
However, the anti proliferative effect of EGCG on normal cells was much less than the effect on
tumor cells.The half maximal (50%) inhibitory concentration (IC50) of isolated EGCG on tumor
cells was 0.012 mg/ml and for EGCG-Mik was 0.02 mg/ml. For both isolated EGCG and
EGCG-milk fractions, the IC50 value was 0.19 mg/ml for normal rat colonic cells.
87
EGCG concentration (mg/ml)
ctr 6e-3 0.01 0.02 0.05 0.09 0.2 0.4 0.8
% P
roli
fera
tion
0
20
40
60
80
100
120
140
Tumor-EGCG
Tumor-EGCG+Milk
EGCG concentration (mg/ml)
ctr 6e-3 0.01 0.02 0.05 0.09 0.2 0.4 0.8
% P
roli
fera
tio
n
0
50
100
150
200Normal-EGCG
Normal-EGCG+Milk
Figure 5.3 % Change of Tumor cell proliferation with increasing levels of isolated EGCG
(solid bars) and complexed EGCG-milk fractions before in vitro digestion (A). % Change of
Normal cell proliferation with increasing levels of isolated EGCG (solid bars) and complexed
EGCG-milk fractions before in vitro digestion. The control used for isolated EGCG was media
without EGCG and the control used for EGCG-milk fractions was milk without EGCG. Different
letters within each concentration of EGCG, indicate statistical significant differences at p<0.05.
a a a a
a a
a a
a
b b
a
a a a a
a a
a a a a
a a
a a a a
b
a
a
b
88
However, these results indicate the bioaccessibility of EGCG when the caseins are still intact, as
they are fractions used before in vitro digestion. To more closely mimic the physiological case,
the digests of the fractions were also delivered to the normal and tumor cell lines. Many studies
have shown that polyphenols undergo biotransformation during their digestion and the products
may not necessarily have the same biological activities (Spencer et al., 2003; Auro 2005; Visioli
et al, 2011). Tea beverages are commonly consumed along with milk, so the effects of the tea-
milk complexes on the bioavailibilty of the digests should also be examined.
The concentrations used for EGCG digestion, whether in water (isolated form) or in milk
(encapsulated with caseins), were derived from figure 5.1 as explained above. However, in order
to expose the cell lines to the EGCG digests and EGCG-milk digests, further dilution with media
took place, with each digest being diluted in media with a ratio of 1: 16 (digest: media (v/v)).
Therefore, the final concentrations of EGCG in media that were delivered to cells corresponded
to concentrations of EGCG before saturation of casein micelles (i.e. ≤0.15 mg/ml media), EGCG
at saturation (i.e. 0.15 mg/ml media) and after saturation of casein micelles (0.3 mg/ ml media).
Figure 5.4 A and B shows the anti-proliferative activity of EGCG digests and EGCG-milk
digests on normal (4D) and tumorigenic (v-src) rat colonic cells. The inhibitory effect of EGCG
did not differ between cell lines (p>0.05), whether EGCG was bound to milk or in isolated form.
However, at higher concentrations of EGCG in normal cells (≥ 0.15 mg/ml), EGCG was seen to
be toxic to the normal (4D) cells as well.
In most studies, the effect of polyphenols on inhibiting the proliferation of cancer cells is
observed, whereas, fewer studies have studied the effects on normal cell lines. However, some
studies that have examined the cytotoxicity of polyphenols against both cancer and normal cell
89
lines, have reported the selective cytotoxicity against the cancer cell line (Chen et. al., 2003;
Yamamoto et. al., 2003; Hakimuddin et al., 2006).
Although the main factors that inhibit tumor cell proliferation but do not affect normal cell
proliferation are not clearly clarified, some possible explanations for this phenomenon have been
proposed. One explanation may be that as cancer cells have a rapid growth rate, usually caused
by the over expression of a few oncogenes, and if EGCG blocks the activity of some of these
oncogenes, it will result in the inhibition of the cell division. Conversely, growth and survival of
normal cells depends on the expression of many pathways, some of which may be inhibited by
EGCG, but others may not be inhibited. Therefore, in this case cell survival would not be
significantly affected (Weinstein et. al., 2008). Another study has hypothesized that some cells
that are more commonly in contact with plant-derived polyphenols, such as cells found in the
epidermis, oral mucosa, and digestive tract, and have naturally developed mechanism to tolerate
higher concentrations of polyphenols. However, cells from other organs that do not posses the
same mechanism(s), or cancer cells that may have lost their protective mechanisms, show
sensitivity to high concentrations of polyphenols (Yamamoto et. al., 2003).
Other proposed cancer prevention mechanisms include the antioxidant and pro-oxidant actions of
green tea catechins such as EGCG. Green tea catechins have been shown to act as antioxidants
that scavenge free radicals to protect normal cells. However, it has been demonstrated that
EGCG, mainly at higher concentrations, can cause the generation of reactive oxygen species
(ROS) intermediates, inducing oxidant stress and activating stress signalling cascades resulting
in tumor cell apoptosis (Chen et. al., 2003; Ebling et al., 2005; Na et. al., 2006).
However, the pro-oxidant activity of green tea catechins at higher concentrations, which may
ultimately cause apoptosis, has been shown in both transformed and nontransformed human
90
bronchial cells as well as some other normal cell lines (Hsu et al., 2002; Yamamoto et. al., 2003;
Yang et al., 2006). Some case reports have shown that when excessive amounts of tea extracts
were consumed, liver toxicity was reported and they concluded that it may be due to a pro-
oxidant mechanism induced by tea catechins (Bonkovsky et. al., 2006; Lambert et.al., 2007b).
This may be the reason toxicity at higher concentrations of EGCG was seen for the normal (4D)
rat colonial cells for both forms of EGCG; isolated and complexed with milk (Figure 5.4 B).
However, Chow et al. (2003) concluded that it is safe to take green tea polyphenol products in
amounts equivalent to the EGCG content of 8–16 cups of green tea, once a day. There results
showed that following high EGCG concentration consumption (800 mg EGCG once a day),
systemic availability of free EGCG after chronic green tea polyphenol administration was
increased by more than 60% .
91
EGCG concentration (mg/ml)
ctrl 2e-3 5e-3 0.01 0.02 0.04 0.08 0.15 0.3
% P
roli
fera
tion
0
20
40
60
80
100
120
EGCG concentration (mg/ml)
ctrl 2e-3 5e-3 0.01 0.02 0.04 0.08 0.15 0.3
% P
roli
fera
tio
n
0
20
40
60
80
100
120
Figure 5.4 % Change of Tumor cell proliferation with increasing levels of EGCG in digests of
isolated EGCG (solid bars) and complexed EGCG-milk fractions (A). % Change of Normal cell
proliferation with increasing levels of EGCG in digests of isolated EGCG and complexed
EGCG-milk fractions (B).The control used for isolated EGCG was media without EGCG and the
control used for EGCG-milk fractions was digested milk without EGCG.Within each
concentration at each of the cell lines, no statistical significant differences was seen (p˃0.05).
A
B
92
5.5 Conclusion
In spite of the complex formation of casein micelles and EGCG, the caseins are still susceptible
to proteolysis during in vitro gastric digestion. As casein micelles are recognized for their
valuable functionality in the delivery of bioactives, the cell proliferation results demonstrate that
EGCG-milk complexes before digestion have similar bioefficacy to isolated EGCG. However, in
tumor cells, at lower concentrations of EGCG, there was a lower extent of inhibition of cancer
cell proliferation by the complexes compared to the free EGCG (≤ 0.4 mg/ml). However, after
gastro-duodenal digestion, there were similarities between complexed and free EGCG. These
results indicate that the strong binding of EGCG and milk casein does not affect the bio efficacy
of the tea polyphenols. In addition there were clear differences in the bioefficacy of EGCG for
normal or cancer cells, showing that the mechanism(s) applied by EGCG for the selectivity of
inhibiting the proliferation of tumor cells rather than normal cells was still maintained even when
complexed with milk.
Overall, these findings support previous data, that casein micelles can be employed as an
appropriate platform for delivery of bioactive compounds. In addition to establishing the
inhibitory effects of EGCG-milk complexes on rat cancer cells, we have shown that they are
protective towards normal cells. Such components may be useful in adjuvant cancer therapeutics.
Developing effective prevention/treatment strategies using natural food sources may help solve
some of the other problems encountered with conventional chemotherapeutic treatments
(Hakimuddin et al. 2006).
93
Chapter 6
General Conclusions
Numerous reports are available on the affinity of tea catechins for caseins; however, in most
studies isolated caseins were employed and not casein micelles (Arts et al., 2002; Jobstyl et al.,
2004; Yan et al., 2009; Frazier et al., 2009; Yuksel et al., 2010). Former studies have suggested
the potential use of casein micelles as nanocarriers of hydrophobic compounds such as vitamin
D2 and curcumin (Livney et al., 2009; Sahu et al., 2008). The incorporation of the bioactive
molecules in casein micelles may be beneficial, not only because a higher concentration of these
molecules could be carried in the food product, but also because they may be protected against
adverse chemical reactions and/or interactions with other components in the food. However,
possible changes in the bioefficacy of bioactives when encapsulated in casein micelles needs to
be studied.
The results from this study clearly demonstrated that tea catechins, and specifically EGCG was
incorporated in casein micelles, and when <2.5 mg/ml were added to milk, most of the EGCG
interacted with the casein proteins, as it was recovered in the colloidal phase of milk, after
centrifugation. Furthermore, it was demonstrated that the tea catechin-caseins interactions
affected the processing behaviour of the casein micelles. Rennet gelation of casein micelles was
selected as it is caused by a surface destabilization of the casein micelles via an enzyme, and no
changes in environmental conditions occur during gelation, making data interpretation less
challenging. The results showed the association of the EGCG with the casein proteins in the
micelles hindered to some extent the renneting reaction, and more importantly, it affected the
94
formation of a gel. Indeed, at high enough concentrations (above saturation point) gelation was
hindered.
To evaluate differences in the bioaccessibility of EGCG, the effect of the tea catechin-milk
protein complexes on cell cytotoxity and proliferation of HT-29 colon cancer cells were tested.
The results demonstrated that the incorporation of EGCG in casein micelles did not hinder the
wanted decrease of proliferation of HT-29 cells. The differences in the extent of the decrease
between free EGCG and encapsulated were attributed to the difference in the matrix, as EGCG
suspended in milk serum and milk whey showed similar behaviors compared to EGCG
incorporated with the micelles.
However, to be able to indicate a similar bioefficacy for nanoencapsulated EGCG, a more
relevant testing model should be used, where the samples are tested not only before digestion,
but also after in vitro digestion. Although the relevance of the in vitro digestion to in vivo is still
under debate, due to the heterogeneities occurring during in vivo digestion, it is possible to
assume that the gastrointestinal cells will be exposed to a wide population of nanoencapsulated
EGCG, ranging from non digested to fully digested. Hence, the present study also observed the
effect of EGCG-casein micelles complexes before and after in vitro digestion. In the study, both
normal and cancerous cells were used.
It was demonstrated that the interactions between EGCG and milk caseins did not cause the
proteins to be less susceptible to gastric proteolysis. The disappearance of casein micelles during
pepsin digestion was similar with or without EGCG. The results for both normal (4D) and tumor
(v-src) rat colonic cells showed that the EGCG-milk digests, when compared to isolated EGCG
digests, did not show a significant difference in cell proliferation (P> 0.05). These results
indicated that the strong binding of EGCG and milk casein does not affect the bioefficacy of the
95
tea catechin, nor does it possibly affect the mechanism (s) applied to inhibit tumor cell growth. In
addition there were clear differences in the bioefficacy of EGCG for normal or cancer cells,
showing a selectivity of EGCG in inhibiting the proliferation of tumor cells rather than normal
cells whether in isolated form, or as complexed with milk.
The present research project contributed to better understanding of two aspects of the protein-
polyphenol interactions; 1. The impact of EGCG-casein complexes on the caseins by studying
protein functionality (rennet gelation) and protein digestion (in vitro). 2. The impact of EGCG-
casein complexes on the bioefficacy of EGCG by applying assays on normal and tumor cell lines
before and after in vitro digestion.
Further research is needed to explain the practical aspect of application of casein micelles for
delivery of bioactive compounds.
96
References
Anema S.G., Klostermeyer H., (1996). Zeta-Potentials of casein micelles from reconstituted
skim milk heated at 120 degrees C. International Dairy Journal 6, 673-687.
Araújo J.R., Gonçalves P.,Martel F.,(Chemopreventive effect of dietary polyphenols in
colorectal cancer cell lines. (2011). Nutrition Research, 31 (2) , 77-87.
Aron P.M., Kennedy J. A. (2008). Review: Flavan-3-ols: Nature, occurrence and biological
activity. Mol. Nutr. Food Res., 52, 79 – 104.
Arts M. J. T., Haenen R. M. M., Wilms L., Sasja A. J., Heijnen C. G., et al., (2002). Interactions
between Flavonoids and Proteins: Effect on the Total Antioxidant Capacity. J. Agric. Food
Chem., 50 (5), 1184-1187.
Auro A.M., (2005). In vitro digestion models for dietary phenolic compounds. Espoo. VTT
Publications 575, 107, 57.
Balentine D. A., Wiseman S.A., Bouwens L.C., (1997). The Chemistry of Tea Flavonoids.
Critical Reviews in Food Science and Nutrition, 37 (8), 693-704.
Bazinet L., Araya-Farias M., Doyen A., Trudel D., Têtu B., (2010). Effect of process unit
operations and long-term storage on catechin contents in EGCG-enriched tea drink. Food
Research International 43, 1692–1701.
Bennick.A., (2002). Interaction of plant polyphenols with salivary proteins. Crit. Rev. Oral Biol.
Med. 13(2), 184-196.
Bonkovsky H. L., (2006) Hepatotoxicity associated with supplements containing Chinese green
tea (Camellia sinensis). Ann. Intern. Med. 144, 68–71.
Brew K., Vanaman T.C. , Hill R.L. (1967). Comparison of amino acid sequence of bovine α-
lactalbumin and hen’s egg white lysozyme. J. Biol. Chem. 242, 3747-3749.
Cha M. C., Lin A. and Meckling K. A., (2005). Low dose docosahexaenoic acid protects normal
colonic epithelial cells from araC toxicity. BMC Pharmacology, 5(7).
Chan P. C., Ramot Y., Malarkey D.E., Blackshear P. et., al., (2010). Fourteen-Week Toxicity
Study of Green Tea Extract in Rats and Mice. Toxicologic Pathology, 38, 1070-1084.
Charlton A.J., Baxter N. J., Lokman Khan M., Moir A. J. G., Haslam E., Davies A. P., and
Williamson M. P., (2002). Polyphenol/Peptide Binding and Precipitation. J. Agric. Food Chem.,
50 (6), 1593-1601.
Chen M.I., (2002). Tea and Health—An Overview. In Tea: Bioactivity and Therapeutic
Potential. edited by Yong-Su Zhen
97
Chen C., G. Shen, Hebbar V., Hu R., Owuor E. D. and Kong A.N., (2003). Epigallocatechin-3-
gallate-induced stress signals in HT-29 human colon adenocarcinoma cells. Carcinogenesis 24
(8), 1369-1378.
Chen D., Daniel K.G.,. Kuhn D.J., Kazi A., Bhuiyan M., Li L., Wang Z., et al., (2004). Green tea
and tea polyphenols in cancer prevention.Frontiers in Bioscience 9, 2618-2631.
Chen L., Zhang H.Y., (2007). Review: Cancer Preventive Mechanisms of the Green Tea
Polyphenol Epigallocatechin-3-gallate. Molecules 12, 946-957.
Chow H. H., Cai Y., Hakim I. A., Crowell J. A., Shahi F., Brooks C. A., et al. (2003).
Pharmacokinetics and safety of green tea polyphenols after multiple-dose administration of
epigallocatechin gallate and polyphenon E in healthy individuals. Clinical Cancer Research 9,
3312–3319.
Chow H-H. S., Hakim I.A, Vining D.R., Crowell J.A., Ranger-Moore J., et. al., (2005). Effects
of Dosing Condition on the Oral Bioavailability of Green TeaCatechins after Single-Dose
Administration of Polyphenon E in Healthy Individuals. Clin Cancer Res.,11(12).
Chitpinityol S., Crabbe M.J.,(1998). Review: Chymosin and aspartic proteinases. Food
Chemistry, 61 (4), 395-418.
Cilla A., González-Sarrías A., Tomás-Barberán F. A., Espín J.C, Barberá R., (2009). J. Agric.
Food Chem.114, 813–820.
Citarasu, T. (2010). Herbal biomedicines: a new opportunity for aquaculture industry. Aquacult.
Int. 18, 403-414.
Dalgleish, D.G. (1998). Casein micelles as colloids: Surface structures and stabilities.J.Dairy
Sci. 81, 3013-3018.
Dalgleish D.G. (2011). On the structural models of bovine casein micelles - review and possible
improvements. Soft Matter.7:7 (6), 2265-2272.
Dalgleish D.G., Corredig M., (2012). The Structure of the Casein Micelle of Milk and Its
Changes During Processing. Annu. Rev. Food Sci. Technol. 3, 449–67.
De Kruif C. G., Zhulina E. B., (1996). κ-casein as a polyelectrolyte brush on the surface of
casein micelles. Colloids and Surfaces A. 117, 151–159.
De Kruif C.G. (1998). Supra-aggregates of casein micelles as a prelude to coagulation. J.Dairy
Sci. 81:3019-3028. De Kruif, C. G., Holt C. (2003). Casein micelle structure, functions and
interactions. In PF Fox and PLH McSweeney (eds). Advnced Dairy Chemistry. London : Kluwer
Academic/Plenum. 233-276.
98
del Angel C.R., Dalgleish D.G., (2006). Structures and some properties of soluble protein
complexes formed by the heating of reconstituted skim milk powder. Food Res. Int. 39, 472-479.
Dickinson E., Miller R., Relkin P.,Vermersh J., (2001). Binding properties of vanillin to whey
proteins: Effect on protein conformational stability and foaming properties. Food Colloids
Fundamentals of Formulation. Royal Society Chemistry. 282-292
Dubeau S., Samson G., Tajmir-Riahi H.A., (2011). Dual effect of milk on the antioxidant
capacity of green, Darjeeling, and English breakfast teas. Food Chemistry 122, 539–545.
Dupont D., Mandalari G., Molle D., Jardin J., Rolet-Repecaud O., Duboz G., Leonil J., Mills
C.N., and Mackie A.R., (2010). Food processing increases casein resistance to simulated infant
digestion. Mol. Nutr. Food Res.,54, 1677–1689.
Ehrich M., Sharova L., (2000). In Vitro Methods for Detecting Cytotoxicity. Current Protocols in
Toxicology. 3:2.6.1–2.6.27.
Elbling L., Weiss R., Teufelhofer O., Uhl M., Knasmueller S., Schulte-Hermann R., Berger R.,
Micksc M., (2005). Green tea extract and (–)-epigallocatechin-3-gallate, the major tea catechin,
exert oxidant but lack antioxidant activities. The FASEB Journal express article 10.1096/fj.04-
2915fje.
Farrell H.M Jr, Malin E.L., Brown E.M., Qi P.X., (2006) Casein micelle structure: What can be
learned from milk synthesis and structural biology? Current Opinion in Colloid & Interface
Science 11, 135–147.
Ferrara L., Montesano D., Senatore A.,(2001).The distribution of minerals and flavonoids in the
tea plant (Camellia sinensis) . Il Farmaco 56, Issues 5-7, 397-401.
Ferruzzi M. G., Green R. J., (2006). Analysis of catechins from milk–tea beverages by enzyme
assisted extraction followed by high performance liquid chromatography. J. Agric. Food Chem.
99, 484–491.
Ferruzzi M. G., (2010). The influence of beverage composition on delivery of phenolic
compounds from coffee and tea. Physiology & Behavior 100, 33–41.
Fox P.F., Mc Sweeny P.L.H., (1998). Dairy chemistry and biochemistry. 1st. Ed, Springer, New
York.
Fox P.F., Guinee T.P., Cogan T.M., McSweeney P.L.H., (2000). Fundamentals of cheese
science. Gaithersburg, MD: Aspen Pub.
Fox P.F., McSweeney P.L.H., (2003). Milk proteins: general and historical aspects. In Advanced
dairy chemistry. Vol. 1: Proteins. 3rd ed. New York, USA: Kluwer Academic/Plenum Publisher.
99
Frazier R. A., Deaville E.R., Green R.J., Stringano E., Willoughby I.,Plant J., Mueller-Harvey I.,
(2009). Interactions of tea tannins and condensed tannins with proteins. J. Pharm. Biomed. Anal.,
doi:10.1016/j.jpba.2009.05.035
Friedman M., Mackey B., Kim H.J., Lee I.S., Lee K.R., Lee S.U., Kozukue E., KozukuE N.,
(2007). Structure−Activity Relationships of Tea Compounds against Cancer Cells. J. Agric. Food
Chem.,55,243−253.
Garti N.( 2008). Delivery and controlled release of bioactives in foods and nutraceuticals. CRC
Press, Woodhead: Boca Raton, Fla.: Cambridge. 1-7.
Gaygadzhiev Z., Alexander M., Corredig M., (2009). Sodium caseinate-stabilized fat globules
inhibition of the rennet-induced gelation of casein micelles studied by Diffusing Wave
Spectroscopy. Food Hydrocolloids 23 (4), 1134-1138.
Ghosh K.S, Sahoo B.K, Dasgupta S., (2008). Spectrophotometric studies on the interaction
between epigallocatechin gallate and lysozyme. Chemical Physics Letters 452, 193–197.
Goncalves R, Mateus N., Pianet I., Laguerre M., de Freitas V., (2011). Mechanisms of Tannin-
Induced Trypsin Inhibition: A Molecular Approach. Langmuir, 27, 13122–13129.
Gonzalez de Mejia E., Song S.Y., Heck C.A., Ramirez-Mares M.V., (2010).Yerba mate tea (Ilex
paraguariensis): Phenolics, antioxidant capacity and in vitro inhibition of colon cancer cell
proliferation. J. Functl. Foods, 2, 23 – 34.
Graham H. N., (1992). Green Tea Composition, Consumption, and Polyphenol Chemistry.
Preventive Medicine 21, 334-350.
Green R. J., Murphy A.S., Schulz B., Watkins B.A., Ferruzzi M. G., (2007). Common tea
formulations modulate in vitro digestive recovery of green tea catechins. Mol. Nutr. Food Res.,
51, 1152 – 1162.
Hakimuddin F., Paliyath G., Meckling K., (2006). Treatment of Mcf-7 Breast Cancer Cells with
a Red Grape Wine Polyphenol Fraction Results in Disruption of Calcium Homeostasis and Cell
Cycle Arrest Causing Selective Cytotoxicity. J. Agric. Food Chem., 54 (20), 7912–7923.
Hasni I., Bourassa P., Hamdani S., Samson G., Carpentier R., Tajmir-Riah H.A., (2011).
Interaction of milk α- and β -caseins with tea polyphenols. Food Chemistry. 126 (2), 630–639
Henning S. M., Fajardo-Lira C., Lee H. W., Youssefian A., Go V. L. W., and Heber D., (2003).
Catechin Content of 18 Teas and a Green Tea Extract Supplement Correlates With the
Antioxidant Capacity. Nutrition And Cancer, 45(2), 226–235.
100
Hofmann T., Glabasnia A., Schwarz B., Wisman K. N., Gangwer K. A. and Hagerman A.E.,
(2006). Protein Binding and Astringent Taste of a Polymeric Procyanidin, 1,2,3,4,6-Penta-O-
galloyl-β-D-glucopyranose, Castalagin and Grandinin. J Agric Food Chem. 54(25), 9503–9509.
Holt C., Sawyer L.,(1993). Caseins as rheomorphic proteins - interpretation of primary and
secondary structures of the αs1-caseins, β-caseins and κ-caseins. J. Chem. Soc. Faraday Trans.
89, 2683-2692.
Holt C., Horne D.S., (1996). The hairy casein micelle: evolution of the concept and its
implications for dairy technology. Neth. Milk Dairy J. 50, 85-111.
Hong J., Lu H., Meng X., Ryu J.H, Hara Y., Yang C.S., (2002). Stability, Cellular Uptake,
Biotransformation, and Efflux of Tea Polyphenol Epigallocatechin-3-Gallate in HT-29 Human
Colon Adenocarcinoma Cells. Cancer Research 62, 7241–7246.
Horne D.S., (2006).Casein micelle structure: models and muddles. Current Opinion in Colloid
and Interface Science.11:11 (2-3), 148-153.
Horne D.S., (2009). Casein micelle structure and stability. in Milk Proteins-From Expression to
Food. ed. Thompson, A., Boland, M. and Singh, H., Academic Press, New York. pp.442-473.
Hsu S, Bollag W.B., Lewis J., Huang Q., Singh B., Sharawy M., Yamamoto T., and Schuster
G.S., (2003) Tea polyphenols induce differentiation and proliferation in epidermal
keratinocytes.J Pharmacol Exp Ther 306, 29–34.
Hwang J.T., Ha J., Park I.J., Lee S.K., Baik H.W, Woon H., Min Kim Y., Park O.J., ( 2007).
Apoptotic effect of EGCG in HT-29 colon cancer cells via AMPK signal pathway. Cancer
Letters 247, 115–121.
Inglingstad R.A., Devold T.G., Eriksen E.K., Holm H., Jacobsen M., Liland K.H., Rukke E.O.,
Vegarud G.E., (2010). Comparison of the digestion of caseins and wheyproteins in equine,
bovine, caprine and human milks by human gastrointestinal enzymes. Dairy Sci. Technol., 90,
549–563.
Jobstyl E., O'Connell J., Fairclough J. P. A., and Williamson M. P., (2004). Molecular Model for
Astringency Produced by Polyphenol/Protein Interactions. Biomacromolecules, 5 (3), 942-949.
Jobstyl E., Howse J. R., Fairclough J. P. A., and Williamson M.P., (2006). Noncovalent Cross-
Linking of Casein by Epigallocatechin Gallate Characterized by Single Molecule Force
Microscopy. J. Agric. Food Chem., 54 (12), 4077-4081.
Jung Y.D., Kim M.S., Shin B.A. , Chay K.O., Ahn B.W., Liu W., Bucana C.D., et al.,(2001).
EGCG, a major component of green tea, inhibits tumour growth by inhibiting VEGF induction in
human colon carcinoma cell. British Journal of Cancer 84(6), 844–850.
101
Kalkwarf HJ., Khoury JC., and Lanphear B.P., (2003). Milk intake during childhood and
adolescence, adult bone density, and osteoporotic features in US women. The American journal
of clinical nutrition. 77, 257-265.
Karak T., Bhagat R.M., (2010). Review:Trace elements in tea leaves, made tea and tea infusion.
Food Research International 43, 2234–2252.
Kartsova L. A. and Alekseeva A. V., (2008). Effect of Milk Caseins on the Concentration of
Polyphenolic Compounds in Tea. Journal of Analytical Chemistry, 63(11), 1107–1111.
Keogh J.B., Mcinerney J., and Clifton P.M., (2007). The Effect of Milk Protein on the
Bioavailability of Cocoa Polyphenols . J. Food Science. 72 ( 3).
Kilmartin P. A., Hsu C. F., (2003). Characterisation of polyphenols in green, oolong,and black
teas, and in coffee, using cyclic voltammetry. Food Chemistry, 82, 501–512.
Kim M., Murakami A., Kawabata K., Ohigashi H., (2005). Epigallocatechin-3-gallate promotes
pro-matrix metalloproteinase-7 production via activation of the JNK1/2 pathway in HT-29
human colorectal cancer cells. Carcinogenesis 26, 1553-1562.
Kim Y., Goodner K.L., Park J.D., Choi J., Talcott S.T., (2011) Changes in antioxidant
phytochemicals and volatile composition of Camellia sinensis by oxidation during tea
fermentation. Food Chem. 129, 1331–1342.
Korhonen H., Pihlanto A., (2006). Bioactive peptides: Production and functionality.
International Dairy Journal 16, 945–960.
Kyle J. A., Morrice P. C, Mcneill G., and Duthie G. G., (2007). Effects of Infusion Time and
Addition of Milk on Content and Absorption of Polyphenols from Black Tea. J. Agric. Food
Chem. 55, 4889−4894.
Kumamoto M., Sonda T., Nagayama K., Tabata M., (2001). Effects of pH and metal ions on
antioxidative activities of catechins. Biosciences Biotechnology Biochemistry 65 (1), 126–132.
Lakowicz J.R., (2010). Quenching Of Fluorescence. Principles of Fluorescence Spectroscopy
Third Edition. 227-331.
Lambert J.D., Lee M.J., Diamond L., Ju J., Hong J., Bose M., Newmark H.,Yang C. S., (2007a).
Dose-dependent levels of epigallocatechin-3-gallate in human colon cancer cells and mouse
plasma and tissues. Drug metabolism and disposition. 34 (1), 8-11.
Lambert J.D., Sang S., Yang C.S., (2007b). Possible Controversy over Dietary Polyphenols:
Benefits vs Risks. Chem. Res. Toxicol., 20, 583 585.
Langley-Evans S.C., (2000).Consumption of black tea elicits an increase in plasma antioxidant
potential in humans. International Journal of Food Sciences and Nutrition (2000) 51, 309–
315.
102
Leenan R., Roodenburg A., Tijburg L., Wiseman S., (2000). A single dose of tea with or without
milk increases plasma antioxidant activity in humans. European Journal of Clinical Nutrition, 54,
87–92.
Lesschaeve I., Noble A.C., (2005). Polyphenols: factors influencing their sensory properties and
their effects on food and beverage preferences. Am J Clin Nutr.,81.
Li L., Hang Chan T., (2001). Enantioselective Synthesis of Epigallocatechin-3-gallate (EGCG),
the Active Polyphenol Component from Green Tea. Organic Letters. Vol. 3 (5), 739 -741.
Li G.H., ChenY.k., Hou Z., Xiao H., Jin H., Lu G., Lee M.J., Liu B., Guan F., et. al., (2010).
Pro-oxidative activities and dose–response relationship of (2)-epigallocatechin-3-gallate in the
inhibition of lung cancer cell growth: a comparative study in vivo and in vitro. Carcinogenesis 31
(5), 902–910.
Livney Y. D., Verespej E. and Dalgleish D. G., (2003) .Steric effects governing disulfide
bond interchange during thermal aggregation in solutions of beta-lactoglobulin B and
alpha-lactalbumin.J.Agric.Food Chem.51,8098-8106.
Livney Y.D., Dalgleish D.G. (2009). Casein micelles for nanoencapsulation of hydrophobic
compounds. U.S. Patent US2009311329 (A1).
Livney Y. D. (2010). Milk proteins as vehicles for bioactives. Current Opinion in Colloid &
Interface Science 15, 73–83.
López-Fandiñoa1 R., Olanoa1 A., San Joséa C. and Ramos M., (1993). Application of reversed-
phase HPLC to the study of proteolysis in UHT milk. Journal of Dairy Research, 60: 111-116
Luck G., Liao H., Murray N. J., Grimmer H. R., Warminski E., Williamson M. P., Lilley T. H.
and Haslams E., (1994). Polyphenols, astringency and proline-rich proteins. Phytochemistry,
37(2), 357-371.
Maiti T.K,. Sundar Ghosh K, and Dasgupta S., (2006). Interaction of Epigallocatechin-3-Gallate
With Human Serum Albumin: Fluorescence, Fourier Transform Infrared,Circular Dichroism, and
Docking Studies. Proteins: Structure, Function, and Bioinformatics 64, 355–362.
Malaki Nik A., Wright A. J., Corredig M., (2010). Surface adsorption alters the susceptibility of
whey proteins to pepsin-digestion. Journal of Colloid and Interface Science 344, 372–381.
Malaki Nik A., Wright A. J., Corredig M., (2011). Impact of interfacial composition on emulsion
digestion and rate of lipid hydrolysis using different in vitro digestion models. Colloids and
Surfaces B: Biointerfaces 83, 321–330
103
Manach C., Scalbert A., Morand C., Rémésy C., and Jimenez L., (2004). Polyphenols: food
sources and bioavailability. Am J Clin Nutr. 79, 727–747.
Manach C., Scalbert A., Williamson G., Morand C., Rémésy C., (2005). Bioavailability and
bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin
Nutr2005;81(suppl):230S–42S.
McDougall G.J., Stewart D., (2005). The inhibitory effects of berry polyphenols on digestive
enzymes. BioFactors 23, 189–195.
McDougall G.J., Kulkarni N.N., Stewart D., (2008). Current developments on the inhibitory
effects of berry polyphenols on digestive enzymes. BioFactors 33,1–8.
McLoughlin P., Roengvoraphoj M., Gissel C., Hescheler J., Certa U., Sachinidis A., (2004).
Transcriptional responses to epigallocatechin-3 gallate in HT 29 colon carcinoma spheroids.
Genes to Cells. 9, 661–669.
Na H.k., Surh Y.J., (2006).Review: Intracellular signaling network as a prime chemopreventive
target of (–)-epigallocatechin gallate. Mol. Nutr. Food Res., 50, 152 – 159.
Nagle D.G., Ferreira D., Zhou Y.D., ( 2006).Epigallocatechin-3-gallate (EGCG): Chemical and
biomedical perspectives. Phytochemistry 67,1849-1855.
O’connel J.E., Fox P.D., Tan-Kintia R.and Fox P.F., (1998). Effects of tea, coffee and cocoa
extracts on the colloidal stability of milk and concentrated milk. Int. Dairy Journal.689-693.
O’Connell J.E., Fox P.F., (2001). Significance and applications of phenolic compounds in the
production and quality of milkand dairy products: a review. International Dairy Journal 11,103–
120.
Papadopoulou A. and Frazier R. A., (2004). Characterization of protein–polyphenol interactions.
Trends in Food Science & Technology 15,186–190.
Pascal C., Poncet-Legrand C., Cabane B., Vernhet A., (2008). Aggregation of a Proline-Rich
Protein Induced by Epigallocatechin Gallate and Condensed Tannins: Effect of Protein
Glycosylation. J. Agric. Food Chem., 56 (15), 6724-6732.
Picariello G., Ferranti P., Fierro O., Mamone G., Cairaa S., et. al., (2010). Peptides surviving the
simulated gastrointestinal digestion of milk proteins: Biological and toxicological implications.
Journal of Chromatography B, 878, 295–308.
Poncet-Legrand C., Edelmann A., Putaux J.L., Cartalade D., Sarni-Manchadoa P., Vernhet A.,
(2006). Poly(L-proline) interactions with flavan-3-ols units: Influence of the molecular structure
and the polyphenol/protein ratio. Food Hydrocolloids 20, 687–697.
104
Rahimi Yazdi S., Corredig M., (2012). Heating of milk alters the binding of curcumin to casein
micelles. A fluorescence spectroscopy study. Food Chemistry 132, 1143–1149.
Rawel H.M., Frey S. K., Meidtner K., Kroll J., Schweigert F.J., (2006). Determining the binding
affinities of phenolic compounds to proteins by quenching of the intrinsic tryptophan
fluorescence. Mol. Nutr. Food Res., 50, 705 – 713.
Reis C.P., Neufeld R.J., Ribeiro, A.J. and Veiga F., (2006). Nanoencapsulation I. Methods for
preparation of drug-loaded polymeric nanoparticles. Nanomed. Nanotech. Biol. Med..2, 8-21.
Ribadeau B, Mercier J.C.,Grosclau F., (1973). Amino-acid composition and sequence
of bovine αs1-caseins and β-caseins. Neth. Milk Dairy J. 27, 304-312.
Richard T., Lefeuvre D., Descendit A., Quideau S., Monti J.P., (2006). Recognition characters in
peptide–polyphenol complex formation. Biochimica et Biophysica Acta 1760, 951–958.
Roura E., Lacueva C. A., Estruch R., Lourdes Mata Bilbao M., Izquierdo-Pulido M. and.
Lamuela-Raventos R. M., (2008). The effects of milk as a food matrix for polyphenols on the
excretion profile of cocoa epicatechin metabolites in healthy human subjects. British Journal of
Nutrition, 100, 846–85.
Sahu A., Kasoju N., and Bora U., (2008). Fluorescence Study of the Curcumin Casein Micelle
Complexation and Its Application as a Drug Nanocarrier to Cancer Cells. Biomacromolecules 9,
2905–2912.
Salucci M., Stivala L. A., Maiani G., Bugianesi R. and Vannini V., (2002). Flavonoids uptake
and their effect on cell cycle of human adenocarcinoma cells. British J.Cancer 86, 1645-1651.
Sandra S., Alexander M., Dalgleish D. G., (2007). The rennet coagulation mechanism of skim
milk as observed by transmission diffusing wave spectroscopy. Journal of Colloid and Interface
Science. 308, 364–373.
Sang S., Lambert J.D., Ho C.T., Yang C.S., (2011).The chemistry and biotransformation of tea
constituents. Pharmacol Res., 64(2), 87-99.
Saura-Calixto F., Serrano J., Gon I., (2007). Intake and bioaccessibility of total polyphenols in a
whole diet. Food Chemistry. 101, 492–501.
Scalbert A., Manach C., Morand C., Remesy C., (2005). Dietary Polyphenols and the Prevention
of Diseases. Critical Reviews in Food Science and Nutrition, 45, 287–306.
Schwarz B. and Hofmann T., (2008). I s there a direct relationship between oral astringency and
human salivary protein binding. Eur Food Res Technol 227, 1693–1698.
Semo E., Kesselman E., Danino D., Livney Y.D., (2007). Casein micelle as a natural nano-
capsular vehicle for nutraceuticals. Food Hydrocolloids 21, 936-942.
105
Sharma R., (2005). Market trends and opportunities for functional dairy beverages. Australian
Journal of Dairy Technology. Melbourne: 60 (2).
Sharma V.and Rao L.J.M., (2009). A Thought on the Biological Activities of Black Tea. Critical
Reviews in Food Science and Nutrition, 49:, 379–404.
Skehan P., Storeng R., Scudiero D., Monks A. , McMahon J., Vistica, D., et al.,( 1990). New
Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. Articles. 82, 13, 1107-1112.
Shpigelman A., Israeli G., Livney Y. D., (2010). Thermally-induced proteinepolyphenol co–
assemblies: beta lactoglobulin-based nanocomplexes as protective nanovehicles for EGCG. Food
Hydrocolloids 24, 735-743.
Shukla A., Narayanan T. Zanchi D., (2009). Structure of casein micelles and their complexation
with tannins. Soft Matter 5, 2884–2888.
Soares S., Mateus N., de Freitas V., (2007). Interaction of Different Polyphenols with Bovine
Serum Albumin (BSA) and Human Salivary -Amylase (HSA) by Fluorescence Quenching .J.
Agric. Food Chem., 16, 6726-6735.
Spencer J.P.,(2003). Metabolism of Tea Flavonoids in the Gastrointestinal Tract. American
Society for Nutritional Sciences. 3255- 3261.
Stahl W., van den Berg H., Arthur J., Bast A., Dainty J., Faulks R.M., et al. (2002).
Bioavailability and metabolism. Molecular Aspects of Medicine. 23, 39-100.
Stevenson D. E., Hurst R. D., (2007). Review: Polyphenolic phytochemicals – just antioxidants
or much more? Cell. Mol. Life Sci., 64, 2900 – 2916.
Tagliazucchi D., Verzelloni E., Conte A.,(2005). Effect of Some Phenolic Compounds and
Beverages on Pepsin Activity during Simulated Gastric Digestion. J. Agric. Food Chem., 53,
8706−8713.
Vaidyanathan J.B.and Walle T., (2003). Cellular Uptake and Efflux of the Tea Flavonoid
Epicatechin-3-gallate in the Human Intestinal Cell Line Caco-2. Journal Pharmacology and
Experimental Therapeutics 307, 745–752.
Valeille K., Ferezou J., Parquet M., Amsler G., and Gripois D., Quignard-Boulanque A., and
Martin J.C., (2006). The natural concentration of the conjugated linoleic acid, cis-9,trans-11 in
milk fat has antiatherogenic effects in hyperlipidemic hamsters. Journal of Nutrition, The. 136,
1305-1310.
van der Burg-Koorevaar M. C. D., Miret S., and Duchateau G.,(2011). Effect of Milk and
Brewing Method on Black Tea Catechin Bioaccessibility. J. Agric. Food Chem., 59, 7752–7758.
106
Vichai V.,Kirtikara K., (2006).Sulforhodamine B colorimetric assay for cytotoxicity screening.
Nature protocols 1 (3), 1112.
Visioli F., De La Lastra C.A., Andres-Lacueva C., Aviram M., Calhau C. et al., (2011).
Polyphenols and Human Health:A Prospectus. Critical Reviews in Food Science and Nutrition,
51.
Vuong U.V. Stathopoulos C.E., Nguyen M.H., Golding J.B., Roach P.D., (2011).Isolation of
Green Tea Catechins and Their Utilization in the Food Industry. Food Reviews International,
27,227–247.
Walstra P., Wouters J.T.M., Geurts T. J., (2006). Dairy science and technology. Taylor &
Francis, New York.
Weinstein I. B., Joe A., (2008). Oncogene addiction. Cancer Res. 68, 3077–3080; discussion
3080
Willig J.A.,(2009). Thesis: The effect of anthocyanin acylation on the inhibition of ht-29 colon
cancer cell proliferation. Graduate School of The Ohio State University.
Yamamoto T., Hsu S., Lewis J., Wataha J., Dickinson D., Singh B., Bollag W. B. , Lockwood P.,
Osaki T., and Schuster G., (2003). Green Tea Polyphenol Causes Differential Oxidative
Environments in Tumor versus Normal Epithelial Cells. J. Pharmacology and Experimental
Therapeutics. 307 (1).
Yan Y., Hu J., and Yao P., (2009). Effects of Casein, Ovalbumin, and Dextran on the
Astringency of Tea Polyphenols Determined by Quartz Crystal Microbalance with Dissipation.
Langmuir 25, 397- 402.
Yang G., Liao J., Kim K., Yurkow E.J., Yang C.H.,(1998). Inhibition of growth and induction of
apoptosis in human cancer cell lines by tea polyphenols. Carcinogenesis. 19 (4 ), 611–616.
Yang C. S., Sang S., Lambert J.D., Hou Z., Ju J., Lu G., (2006). Possible mechanisms of the
cancer‑preventive activities of green tea. Mol. Nutr. Food Res.50, 170–175.
Yang C. S., Sang S., Lambert J.D., Lee M.J., (2008). Review: Bioavailability issues in studying
the health effects of plant polyphenolic compounds. Mol. Nutr. Food Res., 52, 139 –151.
Yang C. S., Wang X., Lu G. and Picinich S. C., (2009). Cancer prevention by tea: animal studies,
molecular mechanisms and human relevance. Nature reviews:cancer 9, 429-439.
Yang C. S., Pan E., (2012). Review: The effects of green tea polyphenols on drug metabolism.
Expert Opin. Drug Metab. Toxicol. 8(6), 677-689.
Yuan J.M., Sunb C., Butlerc L.M., (2011). Review: Tea and cancer prevention: Epidemiological
studies. Pharmacological Research 64,123– 135.
107
Yuksel, Z., Avci, E., & Erdem, Y. K., (2010). Characterization of binding interactions between
green tea flavanoids and milk proteins. Food Chemistry, 121(2), 450-456.
Zaveri N.T., (2006). Green tea and its polyphenolic catechins: Medicinal uses in cancer and
noncancer applications. Life Sciences 78, 2073–2080.
