sh anshan li - universiteit gent
TRANSCRIPT
IImpact
Master
Mast
Facul
Ac
of Lipid
Protein
Promo
r’s disser
re
er of Scie
M
ty of Bio
cademic
d Oxida
ns in Oi
Sh
otor: Prof. Tutor: Mon
rtation su
equiremen
ence in N
ain subje
oscience
year 20
ation on
il-in-Wa
hanshan
dr. ir. Brunnica Oban
bmitted i
nts for the
Nutrition a
ect: Huma
e Engine
013 – 20
n Digest
ater Em
Li
no De Meudo Chaves
n partial f
e degree
and Rural
an Nutriti
eering
014
tibility
mulsions
lenaer s
fulfillmen
of
l Develop
on
of Dair
s
t of the
pment
ry
Promotor: Prof. dr. ir. Bruno De Meulenaer
Email: [email protected]
Tutor: Monica Obando Chaves
Email: [email protected]
Author: Shanshan Li
Email: [email protected]
I
Abstract
Background: Dairy products are usually used for PUFAs fortification. Unfortunately,
due to the high susceptibility of PUFAs to oxidation and the natural presence of
photosensitizer in dairy products, protein deterioration is a big problem during
processing and storage of this type of food.
Objective: This study aims at evaluating the impact of lipid oxidation on protein
digestibility
Study Design: Modification on whey protein and sodium caseinate were assessed
after being incubated with oils containing different amounts of polyunsaturated fatty
acids (fish, sunflower and soybean oil) under autoxidizing (70 °C) and
photo-oxidizing conditions. Finally a validation was carried out in PUFAs enriched
milk subjected to light oxidation.
Methods: Accumulation of malondialdehyde (MDA) and hexanal were monitored
during incubation by high-performance liquid chromatography (HPLC) and gas
chromatography-mass spectrometry (GC-MS), respectively. Protein molecular
changes were shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) under reducing condition. Protein digestibility was determined after in
vitro digestion and nitrogen determination in the pellet obtained after precipitation of
non-digested proteins in 15% trichloroacetic acid (TCA).
Results: In samples containing either whey protein or sodium caseinate, the highest
level of MDA was obtained in fish oil emulsions under both conditions and the
highest hexanal was observed in sunflower oil emulsions upon auto-oxidation and in
soybean oil emulsions upon photo-oxidation. Protein aggregates were visible in all
samples after long time of thermal or light treatment and more pronounced in fish oil
emulsions. Digestibility of whey protein and casein was always decreasing incubated
at 70 °C while light exposure increased whey protein and casein digestibility in
II
protein solutions and soybean oil emulsions but decreased them in fish oil emulsions
(p < 0.05). No significant changes (p < 0.05) were detected in milk validation.
Conclusion: The digestibility of dairy proteins increased after low extent of
modification while decreased if highly oxidized.
Key words: Dairy proteins Long-chain Polyunsaturated Fatty Acids Auto-oxidation Photo-oxidation Protein digestibility
III
Acknowledgements
I wish to express my sincere gratitude to my supervisor Prof. dr. ir. Bruno De
Meulenaer for taking me into the fascinating world of food chemistry, for approving
my thesis topic that has equipped me in so many ways and for providing me with
necessary facilities.
I owe my greatest gratitude to my tutor Monica Obando Chaves for helping me
through the hard laboratory work and for reviewing of the manuscripts carefully and
repeatedly as well as giving her valuable comments. And she has never tired of
answering my questions towards the work.
To Ir. Anne-Marie De Winter and Marian MAREEN: I am deeply grateful to each
of you for your unwavering support throughout my stay in Belgium, most especially
for being there for me at all times, providing for me what you know best including
motherly assistance and care.
IV
TABLE OF CONTENTS
Abstract ...................................................................................................................................... I
Acknowledgements ................................................................................................................. III
List of abbreviations ................................................................................................................ VI
1 Introduction ...................................................................................................................... 1
2 Literature review ............................................................................................................. 3
2.1 Dairy proteins .............................................................................................................. 3
2.1.1 Dairy protein composition ................................................................................. 3
2.1.2 Influence of heat treatment on dairy proteins .................................................... 3
2.1.3 Influence of photo-oxidation on dairy proteins ................................................. 4
2.2 Lipid oxidation ............................................................................................................. 4
2.2.1 Lipid oxidation in oil-in-water emulsions ......................................................... 5
2.2.2 Measurement of lipid oxidation in food systems .............................................. 6
2.3 Protein modification upon lipid oxidation ................................................................... 8
2.3.1 Lipid-protein interaction in food systems ......................................................... 9
2.3.2 Impact of oxidation on dairy protein digestibility ........................................... 10
3 Materials and methods .................................................................................................. 10
3.1 Materials and chemicals ............................................................................................. 10
3.2 Lipid oxidation measurements .................................................................................... 11
3.2.1 Peroxide value .................................................................................................. 11
3.2.2 P-anisidine value .............................................................................................. 11
3.3 Sample preparation ..................................................................................................... 11
3.3.1 Emulsions for photo-oxidation ......................................................................... 11
3.3.2 Incubation of samples under photo-oxidation condition ................................. 12
3.3.3 Preparation and storage of samples for auto-oxidation ................................... 12
3.3.4 Milk validation ................................................................................................ 12
3.4 In vitro digestion (static model) ................................................................................. 13
3.5 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ............ 14
3.6 Protein digestibility determination ............................................................................. 14
V
3.7 Malondialdehyde (MDA) determination ................................................................... 14
3.8 Hexanal determination ............................................................................................... 15
3.9 Amino acid analysis ................................................................................................... 16
3.10 Determination of tryptophan, N-formylkynurenine (NFK) and lipid-protein adducts
......................................................................................................................................... 16
3.11 Statistical analysis .................................................................................................... 17
4 Results ............................................................................................................................. 17
4.1 Auto-oxidation ........................................................................................................... 17
4.1.1 Malondialdehyde (MDA) ................................................................................ 17
4.1.2 Hexanal ........................................................................................................... 18
4.1.3 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) . 19
4.1.4 Digestibility ..................................................................................................... 23
4.2 Photo-oxidation .......................................................................................................... 24
4.2.1 Malondialdehyde (MDA) ................................................................................ 24
4.2.2 Hexanal ........................................................................................................... 24
4.2.3 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) . 25
4.2.4 Digestibility ..................................................................................................... 29
4.3 Validation with milk ................................................................................................... 30
4.3.1 MDA in milk ................................................................................................... 30
4.3.2 Hexanal in milk ............................................................................................... 31
4.3.3 Amino acid composition .................................................................................. 31
4.3.4 N-formylkynurenine (NFK) and lipid-protein adducts ................................... 33
4.3.5 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of
milk .......................................................................................................................... 34
4.3.6 Digestibility of milk ..................................................................................... 36
5 Discussion ....................................................................................................................... 36
6 Conclusion ...................................................................................................................... 42
Reference ................................................................................................................................ 44
VI
List of abbreviations
PUFAs long-chain polyunsaturated fatty acids
DHA docosahexaenoic acid
EPA eicosapentaenoic acid
TCA trichloroacetic acid
MDA malondialdehyde
SPME-GC/FID solid phase microextraction-gas chromatography/flame ionization
detection
HNE 4-hydroxynonenal
TBA 2-thiobarbituric acid
HPLC high-performance liquid chromatography
DNPH 2,4-dinitrophenylhydrazine
BSA bovine serum albumin
PBS Phosphate-buffered saline
TRIS tris (hydroxymethyl) aminomethane
SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
TEP 1,1,3,3-tetraethoxypropane
HS-SPME headspace solid-phase microextraction
GC-MS gas chromatography-mass spectrometry
NFK N-formylkynurenine
BHA Butylated hydroxyanisol
OPA o-phthaldialdehyde
FMOC 9-fluorenyl-methyl-chloroformate
ALA α-lactalbumin
BLG β-lactoglobulin
αs1-CN αs1-casein
αs2-CN αs2-casein
β-CN β-casein
1
1 Introduction
Nowadays an increasing interest has aroused in the fortification of foods with
omega-3 long-chain polyunsaturated fatty acids (PUFAs), for especially
docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5),
because several investigations have shown that the consumption of them in the typical
Western diet is inadequate [1-3]. Due to their various known health benefits [4, 5],
efforts have been made to incorporate marine oils, rich in ω-3 PUFAs, into various
food products [6].
Dairy products, as a group of healthy and frequently consumed food, are suitable
for PUFAs fortification. Unfortunately, due to the high susceptibility of these PUFAs
towards oxidative deterioration and the presence of riboflavin, the photosensitizer in
dairy products [7], specific precautions upon processing and storage should be taken
to ensure the quality and safety of food products enriched with ω-3 PUFAs.
Successful incorporation of ω-3 fatty acids into dairy foods would most likely be
in the form of lipid dispersions which are referred to as oil-in-water emulsions [8], e.g.
milk. Whey protein and casein are the two main components except water in dairy
products, and owing to both hydrophobic and hydrophilic regions of protein, they
usually act as emulsifiers in foods. Due to strong interaction between food
components in emulsions, the oxidation reactions of lipids can be easily transferred to
proteins, which are supposed to have an impact on the functional properties, enzyme
activity and nutritional value of food protein.
Proteins, as macromolecular nutrients which provide essential amino acids as
well as energy for human, must be broken down into amino acids or small peptides
within the digestive tract and pass through the small intestine wall that can be used by
human body. So the digestive process is essential to ensure the nutritional value of
protein component in the diet. Furthermore, protein digestibility is also related to
human health because non-digested or partially digested proteins in the small intestine
can be further hydrolyzed by colonic flora in the large intestine [9, 10], which could
2
be a cause of colon cancer.
A considerable amount of studies have been conducted to study lipid oxidation in
food systems including food emulsions [11-16]. In protein-stabilized emulsion
systems, lipid oxidation can proceed rapidly mainly owing to the large interfacial area
of the oil and aqueous phase that facilitates interaction between lipids and
water-soluble pro-oxidants, like transition metals [17]. Furthermore, some researchers
revealed that interfacial proteins can impact lipid oxidation due to their ability to
scavenge free radicals [18] and can react with lipid oxidation products [19]. However,
studies about dairy products deterioration mainly focused on lipid oxidation and its
impact on sensorial properties and safety of food. Recently, although some
researchers shift their attention towards the interaction between whey proteins and
light-induced lipid oxidation [20, 21], there is still too little information known about
the impact of lipid oxidation on protein modification. To our knowledge, the
digestibility of dairy proteins upon oxidation has not been investigated.
Thus the objective of the present study was to evaluate the impact of lipid
oxidation on protein digestibility, when emulsions containing whey protein or sodium
caseinate and oils with different levels of PUFAs subjected to both photo-oxidation
and auto-oxidation. It was hypothesized that oxidation would reduce the protein
digestibility and change the products profile, because of changes in accessibility to the
cleaving sites of proteases. Secondary lipid oxidation products were monitored during
incubation and protein digestibility was determined after in vitro digestion and
nitrogen determination in the pellet obtained after precipitation of non-digested
proteins in 15% trichloroacetic acid (TCA). Finally a validation was carried out in
PUFAs enriched milk subjected to light oxidation.
3
2 Literature review
2.1 Dairy proteins
2.1.1 Dairy protein composition
Dairy protein consists of two major categories: casein and whey protein. Casein
is account for approximately 82% and whey protein makes up of about 18% of
protein component in cow’s milk [22].
The casein family is made up of several subunits: αs1-casein (αs1-CN), αs2-casein
(αs2-CN), β-casein (β-CN) and κ-casein (κ-CN), and these subunits are held together
by calcium phosphate bridges inside the coil structure. The κ-casein, which helps to
stabilize the micelle in solution, is located near the outside surface of the casein
micelle [23]. The amino acid composition and oxidative stability of each subunit are
different.
The whey protein family is rather more complex, which consists of
approximately 50% β-lactoglobulin (BLG), 20% α-lactalbumin (ALA) and many
minor proteins [24]. Like other major milk components, each whey protein has its
own characteristic composition and variations. There is no phosphorus, by definition,
in whey protein, but it contains a large amount of sulfur-containing amino acids.
Disulfide bonds formed by these amino acids within the protein can cause the chain to
form a compact spherical shape.
2.1.2 Influence of heat treatment on dairy proteins
Casein is relatively stable to heat treatment and they can be heated to boiling
without adverse effects [25]. Along with some calcium phosphate, β-CN will migrate
in and out of the micelle upon changes in temperature, but this does not affect the
nutritional properties of the protein and minerals.
4
Whey protein is more sensitive to thermal treatment than casein. Higher heat
treatments may cause denaturation of BLG, the whey protein ALA, however, is very
heat stable. Denaturation can cause changes in the physical structure of proteins, but
generally would not affect the amino acid composition and thus the nutritional
properties. Severe heat treatments such as ultra-high pasteurization may cause some
damage to heat sensitive amino acids and slightly decrease the nutritional content of
the milk [26].
High temperature treatments can also cause interactions between casein and
whey protein that affect the functional but not the nutritional properties. For example,
at high temperatures, BLG can form a layer over the casein micelle that prevents curd
formation in cheese.
2.1.3 Influence of photo-oxidation on dairy proteins
Dairy products are particularly prone to photo-oxidation since they are rich in the
photo-sensitizer riboflavin. Under illumination, excited triplet riboflavin will be
produced and it can not only react directly with food components, but also interact
with triplet oxygen resulting in singlet oxygen which will readily attack the major
food components [27]. Proteins are known as major targets of singlet oxygen and it is
reported that upon photo-oxidation, dairy proteins can be severely modified [28].
However, the oxidative reactivity of protein depends strongly on the amino acid side
chain composition, because only tryptophan, histidine, tyrosine, methionine and
cysteine are susceptible to singlet oxygen [29, 30].
Casein is more sensitive to photo-oxidation than whey protein. The vulnerable
amino acids mentioned before will be oxidized to protein carbonyls and dityrosine
resulting in the aggregation of α-CN and β-CN [31].
2.2 Lipid oxidation
The oxidation of lipids in dairy products has long been recognized as a leading
5
cause of quality deterioration affecting both the sensory and nutritional properties of
foods [32-34] and it is often the decisive factor in determining on the reduction of
food products shelf-life. Different pathways for lipid oxidation have been proposed:
the radical mechanism or auto-oxidation, the singlet oxygen-mediated mechanism or
photo-oxidation and also the enzymatic oxidation, which is catalyzed by
lipoxygenases. A complex chain of reactions is involved when primary oxidation
products and a series of secondary oxidation products, small fragments with three to
nine carbons in length are produced.
Peroxides as the first compounds formed during oxidation process, especially
hydroperoxides which have little to no flavor or odor, are called primary oxidation
products. Because they are relatively stable, the peroxide value is usually used to
assess lipid oxidation status in food samples. Yet once exposed to extended oxidation
conditions, hydroperoxides usually suffer further oxidation and give rise to secondary
oxidation products, including aldehydes, ketones, epoxides, hydroxy compounds,
oligomers and polymers. Most of these secondary oxidation products are shown to
contribute to the undesirable sensorial and nutritional degradation of food quality
[35].
Among the wide variety of secondary oxidation products, both volatile and
non-volatile compounds can be found, hexanal or malondialdehyde (MDA) as main
representatives, respectively [36]. Hexanal, which originates from the oxidation of
linoleic acid and has a low odor threshold, is a known indicator thanks to be a major
product of lipid oxidation and to increase with storage [37]. It has an odor described
as “grassy” which contributes to off-flavors. MDA, based on its electrophilicity, has
high reactivity towards nucleophiles, such as basic amino acid residues (i.e. lysine and
histidine) of proteins.
2.2.1 Lipid oxidation in oil-in-water emulsions
Oil-in-water emulsions such as milk, infant formula, cream, and some desserts
6
are one of the most common forms of lipids in foods. The mechanism for lipid
oxidation in oil-in-water emulsions differs from bulk lipids mainly in two aspects,
which make lipid oxidation occur rapidly in oil-in-water emulsions. Firstly, the
existing of an aqueous phase in emulsion contains water-soluble components like
prooxidants and antioxidants. Secondly, the large surface area between oil and water
phase facilitates the interaction between the lipids and water-soluble prooxidants.
Many factors play key roles in influencing the rate of lipid oxidation in
oil-in-water emulsions: fatty acid composition, pH and ionic composition, oxygen
concentration, type and concentration of antioxidants and prooxidants. Moreover,
lipid droplet characteristics (e.g. particle size, concentration and physical state) and
emulsion droplet interfacial properties (e.g. thickness, charge, rheology, and
permeability) can have significant impacts on lipid oxidation progress.
In protein-stabilized emulsions, only a fraction of the proteins actually act as
emulsifier and absorb to the emulsion droplets, with the rest remaining in the aqueous
phase. The antioxidant activity of these continuous phase proteins in
protein-stabilized emulsions was confirmed by Habibollah et al. [38]. In their
experiments, protein was removed from the continuous phase of the emulsions
through repeated centrifugation and resuspension of the emulsion droplets (washed
emulsion). Based on hydroperoxide and headspace propanal formation, they found
that unwashed emulsions (with protein in aqueous phase) were more oxidative stable
when compared with washed emulsions.
2.2.2 Measurement of lipid oxidation in food systems
Evaluating lipid oxidation status is a challenging task due to a number of facts
such as the different compounds which are formed depending on the time, the extent
of oxidation and the mechanism involved. Therefore, choosing just one or two
parameters to evaluate the extent of lipid oxidation is a rather coarse approach and it
should be promoted to combine methods involving assessment of large set of
7
compounds [39].
Lipid peroxidation of milk has been studied using numerous indicators of lipid
oxidation, such as the measurement of conjugated dienes, peroxide value and O2
intake [40, 41]. The presence of volatile oxidation products is an established primary
indicator of lipid oxidation in some food systems. Hexanal, 2-(E)-hexenal are major
volatile aldehydes generated from lipid peroxidation which were usually quantified
using solid phase microextraction-gas chromatography/flame ionization detection
(SPME-GC/FID) [37]. These methods, however, can lack specificity due to
interference with non-lipid components in the food matrix, or have variable reactivity
that depends on assay conditions.
Other important reactive aldehydes that originate from lipid peroxidation of
PUFAs are dialdehydes, including MDA, 4-hydroxynonenal (HNE), glyoxal, and
acrolein. MDA is in many instances the most abundant individual aldehyde generated
during secondary lipid oxidation and also probably the most commonly used as
oxidation marker.
MDA is known to be mutagenic to humans because it can form adducts with
proteins and DNA [42]. The most common method to determine MDA in foods is the
spectrophotometric measurement of the pink-colored adduct of MDA with
2-thiobarbituric acid (TBA), which gives a maximum absorbance at 532-535 nm. All
of the spectrophotometric methods have been criticized because of their unspecificity
toward MDA [43]. It is well accepted that components present in food matrices, such
as browning reaction products and protein and sugar degradation products, participate
in the formation of the TBA color complex [44]. Application of high-performance
liquid chromatography (HPLC) analytical techniques has offered better specificity
and sensitivity towards MDA determination in foods and biological systems based on
the analysis of the MDA-TBA complex [45]. Furthermore, there are available HPLC
methods based on the analysis of MDA derivatives with hydrazine compounds, such
as 2,4-dinitrophenylhydrazine (DNPH) [46], or on the direct measurement of MDA
[47].
8
2.3 Protein modification upon lipid oxidation
It is well established that reactive products of lipid peroxidation, such as MDA,
HNE and acrolein can cause protein oxidation by means of reactions with
nucleophilic amino acid residuals in protein molecules [42, 48], leading to changes of
the primary structure of protein, and simultaneously the protein three-dimensional
structure. These changes can have an impact on the functional properties and
nutritional value of protein, including loss of enzyme activity and loss of essential
amino acids [49-51].
Protein oxidation can occur via either free-radical reactions or non-covalent
complex formation. In the former pathway, peroxyl radicals (ROO·) formed during
lipid oxidation abstract hydrogen atoms from protein molecules (PH) (Reaction 1)
leading to the formation of protein radicals (P·), which in turn create a protein net
(P-P) due to the cross-linking (Reaction 2). When strong attractions, like electrostatic
or hydrophobic interaction, between lipid oxidation products and the nitrogen or
sulfur centers of reactive amino acid residues of the protein exist, the reaction via the
latter pathway can happen (Reaction 3) [49, 52].
ROO· + PH P· + ROOH Reaction 1
P· + P· P-P Reaction 2
ROOH + PH [ROOH---HP] RO· + P· + H2O Reaction 3
The lipid hydroperoxides and the secondary lipid oxidation products can also
physically interact with protein by hydrophobic association or hydrogen bonds,
forming various types of complexes through covalent bonds [53]. One or more
secondary lipid oxidation products, such as HNE and MDA, can react with protein
molecules present at the interface of the oil droplets in the oil-in-water emulsion [54].
The reaction between secondary lipid oxidation products and certain nucleophilic
amino acid residues probably occur at the oil-water interface with the initial sites of
modification being located in the hydrophobic regions of the protein molecules. Due
9
to their structures, tryptophan, histidine, proline, lysine, cysteine, methionine and
tyrosine are the most sensitive residuals to oxidation where the hydrogen atom is
abstracted either form OH-, S- or N- containing groups [49, 52].
Oxidation of proteins by lipid oxidation products can furthermore lead to the
cleavage of the peptide bonds and peptide fragments can be formed when the
oxidative cleavage occurs in the main chain of the peptide [55].
2.3.1 Lipid-protein interaction in food systems
Due to the interaction between proteins and lipids in the food system, relatively
stable protein-lipid complexes can be formed rapidly which have a specific
fluorescence at the excitation wavelength around 350 nm [21].
Lipid oxidation products can interact with protein amino acid residues leading to
changes in the primary structure of the protein, which induced changes in the
secondary and tertiary structure of proteins [56]. The primary products of lipid
oxidation are found to cause oxidative changes in the tryptophan and cysteine residues
of β-lactoglobulin (BLG) [57].The reaction of amino acid residues with secondary
lipid oxidation products leads to the formation of carbonyl groups in the proteins and
lipid-protein adducts with fluorescence [21]. Primary lipid oxidation products (e.g.
hydroperoxides) are shown to cause less damage to amino acid residues than
secondary products of lipid oxidation such as aldehydes [58].
In addition, Schiff bases and Michael adducts can be produced via condensation
reaction of carbon atom of the aldehyde with the nucleophilic amino acid residues.
Subsequently the formed adducts can further react with another protein molecular to
produce protein-protein cross-linked derivatives. For example, the aldehyde group of
an HNE-protein adduct of one protein can interactive with the ε-NH2 group of a lysine
residue in another protein [59].
10
2.3.2 Impact of oxidation on dairy protein digestibility
The proteolysis of different model proteins modified by reaction with the lipid
oxidation products has been studied by several researchers. By incubating bovine
serum albumin (BSA) with lipid peroxidation product 4,5(E)-epoxy-2(E)-heptenal for
different periods of time with eight concentrations of the epoxyalkenal and, then,
treated for 24 h with chymotrypsin, pancreatin, pronase, or trypsin, Rosario Zamora
and Francisco J. Hidalgo found that the digestibility of BSA always decreased
compared with that of native BSA, and this inhibition of the proteolysis was related to
the concentration of the epoxyalkenal and the reaction time [60]. On the contrary,
Lars Wiking and his partner [61] confirmed that addition of MDA to skim milk
increased the plasmin hydrolysis of β-CN, evidenced by more detected γ-caseins (a
proteolysis product of β-CN) with increasing concentration of MDA and addition of a
plasmin inhibitor blocked the formation of γ-casein.
3 Materials and methods
3.1 Materials and chemicals
Whey protein (Lacprodan® DI-9224) was obtained from Acatris Food Belgium
(Londerzeel, Belgium). Sodium caseinate (Microdan® 30) was kindly provided by
Arla Foods (Wageningen, Netherlands) and skim milk powder was from
Frieslandcampina (Lochem, Netherlands). Sunflower oil and soybean oil were
purchased from a local store. Fully refined fish oil was kindly provided by Desmet
Ballestra (Zaventem, Belgium). Phosphate-buffered saline (PBS) (pH 6.8) consisted
of 0.135 M NaCl, 1.5 mM KH2PO4, 8 mM NaH2PO4⋅12H2O and 2.7 mM KCl. These
reagents were supplied by Chem-Lab (Zedelgem, Belgium). Acetic acid,
mercaptoethanol, HPLC grade acetonitrile and HPLC grade methanol were obtained
from VWR (Leuven, Belgium). TCA, tris (hydroxymethyl) aminomethane (TRIS) and
sodium dodecyl sulphate (SDS) were obtained from Acros Organics (Geel, Belgium).
Precast gel, precision plus protein standard and Biosafe Coomassie for sodium
11
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were from
Bio-Rad (Nazareth, Belgium). Solvents were of analytical grade except for hexane
which was of technical grade. All other chemicals and standards were of analytical
grade and obtained from Sigma-Aldrich (Bornem, Belgium).
3.2 Lipid oxidation measurements
The extent of oil oxidation was expressed by peroxide value and p-anisidine
value.
3.2.1 Peroxide value
According to AOAC official method [62], 10 mL solvent consisting of glacial
acetic acid and chloroform (3/2, v/v) was added to 1.0 g oil, followed by mixing with
0.2 mL of 1.4 g/mL potassium iodide solution. Sodium thiosulphate solution of 0.01
M was used for titration.
3.2.2 P-anisidine value
The p-anisidine value was evaluated following the AOCS official method. In
brief, 0.5 g oil was dissolved in isooctane and diluted to 25 mL. The absorbency of
the solution with and without p-anisidine reagent was measured at 350 nm.
3.3 Sample preparation
3.3.1 Emulsions for photo-oxidation
Oil-in-water emulsions were prepared following the method described by
Mestdagh et al. [20]. Emulsions containing 10 mg/mL oil and 6 mg/mL protein (whey
protein or sodium caseinate) in the presence of 2µg/mL riboflavin were well mixed in
10 mM PBS (pH 6.8) by an Ultraturax (Janke and Kunkel, IKA-Werk, Staufeb,
Germany) at 9200 rpm for 2 min. To get stable emulsions, the samples were
12
microfluidized by a Microfluidiser 110S (Microfluidics Corporation, Newton,
Massachusetts, USA) having its heat exchange coil immersed in a water bath at 60 °C.
Microfluidization was performed at a compressed air pressure of 2 bar, corresponding
to a liquid pressure of about 280 bar. Protein and riboflavin solutions without oil were
used as control samples. A total of 150 mL of emulsion/solution was filled into Duran
bottles (250 mL). In order to suppress microbial growth during the storage experiment,
the emulsions/solutions were pasteurized for 3 min at 70 °C in a water bath.
3.3.2 Incubation of samples under photo-oxidation condition
The samples of whey protein or sodium caseinate containing fish oil or soybean
oil together with the control group were stored under homogenous fluorescent tube
illumination (2200 lux) at 4 °C on an orbital shaker (Edmund Bühler, Hechingen,
Germany) for 0, 15 and 30 days, respectively. The reference samples, not subjected to
illumination, were kept in the dark by covering with aluminum foil.
3.3.3 Preparation and storage of samples for auto-oxidation
Emulsions containing 10 mg/mL oil and 6 mg/mL protein (whey protein or
sodium caseinate) were well mixed in 10 mM PBS (pH 6.8) by an ultraturax for 2 min
at 9200 rpm. Fish oil, soybean oil and sunflower oil were used in this step, and protein
solutions without oil were prepared as the control group. Falcon tubes (50 mL) were
filled with 15 mL of each sample and heated at 70 °C for 0, 24, 48, 96 and 144 h,
respectively. For each time point, five tubes per sample were prepared, three of which
would be used for digestion.
3.3.4 Milk validation
Skim milk powder (12.5% protein, w/w) and oil (1.5% and 3.5% of fish oil, w/w)
were dispersed in distilled water. Recombined milk were heated at 60 °C and
pre-homogenized by an Ultraturax for 2 min at 9200 rpm and homogenized by a
13
Microfluidiser 110S (Microfluidics Corporation, Newton, Massachusetts, USA) at
280 bar having its heat exchange coil immersed in a water bath at 60 °C. Conditions
of pasteurization and incubation were the same above indicated for photo-oxidation
experiment. Duran bottles (250 mL) filled with 100 mL milk were heated at 70 °C for
5 min to suppress microbial growth. The samples were stored in triplicate under
homogenous fluorescent tube illumination with constant shaking for 0, 15 and 21 days
respectively at 4 °C. The reference samples, not subjected to illumination, were stored
in the dark by covering with aluminum foil. Milk without oil was used as control
samples.
3.4 In vitro digestion (static model)
To simulate the human gastrointestinal digestion process, digestion of proteins
was performed by a sequential exposure to proteases of the digestive tract (pepsin,
and a mixture of trypsin and a-chymotrypsin) under conditions of pH and temperature
mimicking gastric and intestinal fluids [63]. Emulsions (containing 90 mg protein)
after light or thermal treatment were transferred to 50 mL falcon tubes and the pH
value was adjusted to 2.0 with 8 M HCl. Proteins were first digested by gastric pepsin
(P6867 Sigma-Aldrich) to an enzyme/substrate ratio of 1/250 (w/w) at 37 °C for 2 h
with constant shaking. To simulate the duodenal phase digestion in human body, the
pH value of the emulsions was changed to 6.5 with 8 M NaOH, and at the same time,
240 mg lipase (100-400 units/mg L3126 Sigma-Aldrich), 600 mg bile salts (B8631
Sigma-Aldrich) and 15 µL of 1 M CaCl2 were added to every 15 mL samples before
being hydrolyzed by a mixture of trypsin (T0303 Sigma-Aldrich) and chymotrypsin
(C4129 Sigma-Aldrich) (ratio 1/250) at 37 °C for 2.5 h. The digestion was terminated
by addition of 8 M HCl to get the final pH of 5.0.
14
3.5 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE)
Protein aggregation or fragmentation in digested and non-digested samples was
examined by SDS-PAGE under reducing conditions. SDS-PAGE was performed
following the method described by Laemmli [64] using 15% polyacrylamide gels.
Protein concentration was adjusted to 1 mg/mL prior to being diluted 1:1 (v/v) with
Laemmli buffer (Bio-Rad, Nazareth, Belgium) containing 0.05% mercaptoethanol.
After being heated at 95 °C for 5 min and centrifuged at 10 000 rpm for 5 min, 20 μL
of each sample was loaded in the gel along with 10 μL molecular weight marker as
the first lane. Electrophoresis was performed at a constant voltage of 160 V. The gels
were stained with Biosafe Coomassie (Bio-Rad), and scanned using Gel DocTM EZ
Imager (Bio-Rad). A precision plus protein standard (Bio-Rad) was used as reference
to determine the molecular weight of the proteins of interest.
3.6 Protein digestibility determination
Proteins in samples were precipitated by TCA 15% (final concentration) in 50
mL falcon tubes for 10 min under cold temperature (on ice). After centrifugation at
13000 g for 10 min at 4 °C, the pellet on the bottom was used to determine nitrogen
content. The procedure was conducted using Kjeldahl method and the digestibility of
proteins was calculated using the following formula: Digestibility (%) = (1 - Nitrogen
(in digested samples)/Nitrogen (in non-digested samples)) * 100.
3.7 Malondialdehyde (MDA) determination
The method of Papastergiadis, Mubiru, Van Langenhove and De Meulenaer [45],
was followed to determine MDA. TCA was added to the samples until final
concentration of 15% to precipitate the protein and centrifugation at 13000 g was
done to get a clear supernatant. Top layer was discarded and 1 mL of supernatant was
15
mixed with 3 mL of TBA reagent (40 mM dissolved in 2 M acetate buffer at pH 2.0)
in a test tube and heated in a boiling water bath for 40 min. The reaction mixture was
cooled prior to the addition of 1 mL of methanol, and 20 μL of the sample was
injected into a Varian C18 HPLC column (5 μm, 150 × 4.6 mm), held at 30 °C. The
mobile phase consisting of 50 mM KH2PO4 buffer solution, methanol, and acetonitrile
(72:17:11, v/v/v, pH 5.3) was pumped isocratically at 1 mL/min. Fluorometric
detector excitation and emission wavelengths were set at 525 and 560 nm,
respectively. For quantification, standard solutions of MDA in 7.5% TCA were
prepared from 1,1,3,3-tetraethoxypropane (TEP) and calibration curves were prepared
at a concentration ranging from 0.3 to 10 μM.
3.8 Hexanal determination
Hexanal formation in emulsions was evaluated using headspace solid-phase
microextraction (HS-SPME) combined with gas chromatography-mass spectrometry
(GC-MS). A total of 1 mL emulsion was placed in a glass vial (size 10 mL, 22.5 mm ×
46 mm) and mixed with 2 mL of 0.02 M Na2HPO4, 0.02 M KH2PO4 pH 2.0 buffer.
Butylated hydroxyanisol (BHA) dissolved in methanol was added in the vial at a final
concentration of 2.8 M and a known amount of hexanal-d12 was incorporated in the
sample. The vial was sealed with a PTFE septum cup and was subjected to HS-SPME
extraction. The SPME fiber (75 μm Carboxen/PDMS, Supelco, Bellefonte, PA, USA)
was inserted into the headspace of the vial and left there for 30 min at 75 °C. Volatile
compounds were desorbed by inserting the fiber into the injection port of an Agilent
7890A chromatograph (Agilent Technologies, Palo Alto, CA) operated in splitless
mode for 10 min at 240 °C. Helium was used as carrier gas with a constant flow rate
of 1.3 mL/min. The compounds were separated on a DB-624 column measuring (60
m × 0.25 mm x 1.4 μm). The oven temperature program began with 5 min at 50 °C
for 5 min, increase to 4 °C/min to 140 °C, and then 30 °C/min increase to 240 °C for
10 minutes. An Agilent 5975C inert XL mass spectrometry detector was used and
detection was carried out on the total ion current obtained by electron impact at 70 eV.
16
The selected ion was 56 for hexanal.
3.9 Amino acid analysis
Amino acid analysis of the protein samples was carried out after acid hydrolysis
[65]. The samples in a test tube with screw cap were hydrolyzed with 6 M HCl for 24
h at 110 °C and then neutralized carefully with 10 M NaOH to bring the pH to 2.2.
The HPLC system employed consisted of an Agilent 1100 model (Agilent
Technologies, Diegem, Belgium). The chromatographic column was ZORBAX
Eclipse AAA Rapid Resolution column 4.6 × 150 mm, 3.5 micron (Agilent
Technologies) operated at 40 °C at a flow rate of 2 mL min-1. The chromatographic
separation was achieved by injecting 0.5 mL sample and using a gradient elution of
mobile phase A (40 mM NaH2PO4 with 0.2 g L-1 NaN3 pH 7.8) and mobile phase B
(acetonitrile/methanol/water in a 45/45/10 ratio) and allowed separation of the amino
acids in 28 min (Agilent application note). Primary amino acids were derivatized with
0.5 mL of 75mM o-phthaldialdehyde (OPA) and detected at excitation and emission
wavelengths 340/450 nm. The secondary amino acid proline was derivatized with 0.5
mL 10 mM 9-fluorenyl-methyl-chloroformate (FMOC) and detected at excitation and
emission wavelength of 266/305 nm. Internal standards norvaline and sarcosine were
used for the quantification.
3.10 Determination of tryptophan, N-formylkynurenine (NFK) and
lipid-protein adducts
The loss of tryptophan, formation of NFK and lipid-protein adducts were
detected by fluorescence spectroscopy of intact proteins with a Spectramax Gemini
XPS fluorometer (Molecular Devices, Brussels, Belgium) [28]. After incubation, 50
μL sample was diluted in 250 μL 10 mM PBS (pH 6.8) and these parameters were
measured using excitation and emission wavelengths of 280 and 330 nm, 330 and 440
nm, 350 and 440 nm, respectively.
17
3.11 Statistical analysis
All calculations were carried out using the SPSS software package (version 19.0).
Statistical comparisons between groups were made using one-way ANOVA with
significant level of p < 0.05. When the differences were detected, multiple
comparisons were performed by Tukey’s test.
4 Results
4.1 Auto-oxidation
4.1.1 Malondialdehyde (MDA)
During incubation at 70 °C, the quantity of MDA produced in each sample was
measured to evaluate the degree of lipid oxidation (Table 1). MDA was below the
limit of detection of the method in the control samples and in the soybean oil
emulsions upon incubation (data not shown). The level of MDA remained stable at
around 0.1 µg/mL over the 144 h in sunflower oil emulsions containing either whey
protein or sodium caseinate. In addition, emulsions containing fish oil always had the
highest amount of MDA.
Fish oil emulsion containing whey protein experienced a surge from the
beginning to 24 h of incubation, with the value from 0.2 to 3.0 µg/mL, followed by a
gentle drop to the value around 0.3 µg/mL after 144 h of incubation. Similar trend but
lower amount of MDA than in fish oil emulsion containing whey protein was found in
fish oil emulsion containing sodium caseinate, which demonstrated a sharp increase
during the first 24 h followed by an abrupt decline in the next 24 h. After that, the
level fluctuated around 0.2 μg/mL.
18
Table 1. MDA in protein emulsions (soybean, sunflower or fish oil) after auto-oxidation under 70 °C1
Samples MDA (µg/mL sample)
0h 24h 48h 96h 144h
Whey protein
Sunflower oil emulsion 0.1±0.0a -- ND 0.1±0.0a 0.1±0.0a
Fish oil emulsion 0.2±0.1b 3.0±0.5a 2.9±1.1a 1.3±0.4ab 0.3±0.1b
Sodium caseinate
Sunflower oil emulsion ND 0.1±0.1a ND 0.2±0.0a 0.1±0.0a
Fish oil emulsion 0.1±0.1b 1.9±0.2a 0.4±0.0b 0.1±0.1b 0.3±0.1b
1Values represent mean values ± SD of two independent determinations ND: not detected Different letters within the same row indicate statistically significant differences (p < 0.05)
4.1.2 Hexanal
The production of hexanal was monitored every 48 h in all samples as shown in
Table 2. Hexanal was below the limits of detection of the method in all fresh samples
and in the two control groups upon incubation (data not shown). After 48 h, hexanal
started to be detected in fish oil emulsions containing whey protein and in sunflower
and fish oil emulsions containing sodium caseinate. Moreover, in samples containing
either whey protein or sodium caseinate, sunflower oil emulsions shown to produce
highest hexanal than all other samples and the peak levels were reached at 96 h (0.4
µg/mL in emulsions containing whey protein and 3.8 µg/mL in emulsions containing
sodium caseinate). The same trend was displayed with soybean oil emulsions.
However, fish oil emulsions containing sodium caseinate reached the peak at 48 h of
incubation. Furthermore, hexanal levels were lower in soybean or fish oil emulsions
than in sunflower oil emulsions.
19
Table 2. Hexanal in protein emulsions (soybean, sunflower or fish oil) after auto-oxidation under
70 °C1
Samples Hexanal (µg/mL sample)
24h 48h 96h 144h
Whey protein
Soybean oil emulsion ND 0.1±0.0a 0.1±0.1a ND
Sunflower oil emulsion -- 0.2±0.0b 0.4±0.1a ND
Fish oil emulsion 0.1±0.0a 0.1±0.1a 0.1±0.0a ND
Sodium caseinate
Soybean oil emulsion ND ND 0.1±0.0b 0.5±0.0a
Sunflower oil emulsion 0.1±0.0c 0.6±0.2bc 3.8±0.4a 1.3±0.2b
Fish oil emulsion 0.1±0.0a ND ND ND
1Values represent mean values ± SD of two independent determinations ND: not detected Different letters within the same row indicate statistically significant differences (p < 0.05)
4.1.3 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE)
SDS-PAGE of digested and non-digested samples was performed under reducing
condition in order to observe modifications in whey protein (Fig.1) and casein (Fig.2)
patterns induced by auto-oxidation. The broad-range molecular weight marker was
used to identify the protein bands.
As shown in Figure 1, in all non-digested fresh samples the subunits BLG and
α-lactalbumin (ALA) of whey protein were observed clearly at 14 and 18 kDa,
respectively. Degradation of ALA was considerably fast and in all samples the bands
corresponding to ALA almost disappeared after 48 h of heat-treatment. While the loss
of BLG was slightly slower than ALA and it could still be seen in the control sample
after 144 h of incubation. The rate of basic bands smearing out and accumulation of
20
aggregates and fragments were fastest in fish oil emulsions, followed by sunflower
and soybean oil emulsions (data not shown) and the control samples. In digested
samples, bands of BLG and ALA were not seen in all lanes, and at the same time,
large aggregates, especially molecular larger than 250 kDa, were still visible in fish,
sunflower and soybean oil emulsions after 48, 96 and 144 h of incubation, but not in
control samples.
As shown in Figure 1, in all non-digested fresh samples the subunits BLG and
α-lactalbumin (ALA) of whey protein were observed clearly at 14 and 18 kDa,
respectively. Degradation of ALA was considerably fast and in all samples the bands
corresponding to ALA almost disappeared after 48 h of heat-treatment. While the loss
of BLG was slightly slower than ALA and it could still be seen in the control sample
after 144 h of incubation. The rate of basic bands smearing out and accumulation of
aggregates and fragments were fastest in fish oil emulsions, followed by sunflower
and soybean oil emulsions (data not shown) and the control samples. In digested
samples, bands of BLG and ALA were not seen in all lanes, and at the same time,
large aggregates, especially molecular larger than 250 kDa, were still visible in fish,
sunflower and soybean oil emulsions after 48, 96 and 144 h of incubation, but not in
control samples.
Fig.1
stand
5, 7
lane
and
fish
1. Electrophor
dards; lanes 2
and 9, digest
1, molecular
144 h, respec
oil emulsions
retic pattern o
, 4, 6 and 8, n
ted protein so
mass standar
tively. For pa
, 0, 24, 48, 96
of whey protei
non-digested p
lutions, 0, 48
rds; lanes 2, 3
nel C: lane 1,
6 and 144 h, re
21
in after auto-o
protein solutio
, 96 and 144
, 4, 5 and 6, n
, molecular m
espectively.
oxidation. In p
ons, 0, 48, 96
h, respectivel
non-digested
mass standards
panel A: lane
and 144 h, res
ly; lane 10, b
fish oil emuls
; lanes 2, 3, 4
1, molecular
spectively; lan
blank. For pan
sions, 0, 24, 4
4, 5 and 6, dig
mass
nes 3,
nel B:
48, 96
gested
Fig.2
stand
5, 7
mass
lanes
cont
high
αs1-
com
and
com
with
aggr
incr
sunf
sligh
emu
visib
2. Electropho
dards; lanes 2
and 9, digeste
s standards; la
s 3, 5, 7 and 9
Fig.2 pres
taining diff
hest degree
CN (23 kD
mpounds (≥
144 h (data
mpounds sto
h time of
regates and
reased with
flower oil e
htly faster
ulsions, fol
ble bands w
oretic pattern
2, 4, 6 and 8, n
ed control sam
anes 2, 4, 6, a
9, digested fish
sents the ele
ferent oils. A
e of casein
Da), αs2-CN
250 kDa)
a not shown
opped in the
incubation
d small pep
h the seque
emulsions (d
accumulati
lowed by
were found i
of casein af
non-digested c
mples, 0, 48, 9
and 8, non-dig
h oil emulsion
ectrophoret
After 24 h
degradatio
N (25 kDa)
and small p
n) of incuba
e staking ge
. The degr
ptides were
ence of con
data not sho
on of high
sunflower
in digested
22
fter auto-oxid
control sampl
96 and 144 h
gested fish oil
ns, 0, 24, 48 an
tic profiles
of incubati
on, indicate
and β-CN
peptides for
ation, the ba
el and small
radation of
also observ
ntrol, soybe
own) and fi
molecular
oil emulsio
control sam
dation. In pan
es, 0, 48, 96 a
, respectively.
l emulsions, 0
nd 96 h, respe
of casein w
on, the fish
d by the le
(24 kDa),
rmed. In fis
ands of case
l peptides in
f casein an
ved in othe
ean oil emu
sh oil emul
compounds
ons and so
mples.
nel A: lane 1
and 144 h, res
. For panel B:
0, 24, 48 and
ectively; lane 1
which varied
h oil emulsi
east intensi
and simult
sh oil emul
ein almost d
n the runnin
nd accumul
er samples,
ulsions (da
sions. In dig
s was obser
oybean oil
1, molecular
spectively; lan
: lane 1, mole
96 h, respecti
10, blank.
d from sam
ions exhibit
ity of band
ltaneously l
lsions at 48
disappeared,
ng gel incre
ulation of l
and the de
ata not sho
gested samp
rved in fish
emulsions.
mass
nes 3,
ecular
ively;
mples
ted a
ds of
large
8, 96
, and
ased
large
egree
wn),
ples,
h oil
No
23
4.1.4 Digestibility
The protein digestibility of protein solutions and protein emulsions upon
incubation for 0, 48, 96 and 144 h was shown in Table 3. In fresh samples containing
whey protein or casein, the addition of oil negatively influenced the protein
digestibility with 80% of control samples and about 74% of emulsions. Protein
digestibility decreased in all samples over time, but the fall was more obvious in
emulsions containing whey protein than in emulsions containing sodium caseinate.
Sunflower and soybean oil were observed to have the same impact on both whey
protein and casein digestibility upon incubation at 70 °C.
Table 3. Protein digestibility of protein solutions and protein emulsions (soybean, sunflower or fish oil)
after auto-oxidation under 70 °C1
Samples Protein digestibility (%)
0h 48h 96h 144h
Whey protein
Protein solution 80.0±0.4a 69.0±1.9b 65.5±0.8c 65.4±1.8c
Soybean oil emulsion 73.2±4.0a 60.4±1.4b 62.4±1.2b 58.0±2.2b
Sunflower oil emulsion 76.7±1.3a 64.5±0.2b 61.1±0.2bc 57.0±5.3c
Fish oil emulsion 74.2±1.1a 44.4±0.9b 45.4±3.3b 43.7±1.4b
Sodium caseinate
Protein solution 80.2±0.7a 75.3±0.1ab 73.3±1.6b 71.3±0.8b
Soybean oil emulsion 74.9±0.8a 72.8±0.9ab 71.9±0.5b 68.5±1.1c
Sunflower oil emulsion 74.7±0.1a 73.5±0.3b 71.0±0.4c 68.4±0.5d
Fish oil emulsion 73.8±1.0a 69.0±0.3ab 66.7±0.9b 61.3±3.8c
1Values represent mean values ± SD of three independent determinations
Different letters within the same row indicate statistically significant differences (p < 0.05)
In samples containing whey protein, fish oil emulsions showed the fastest
24
decrease of protein digestibility with 44% at 144 h, followed by sunflower and
soybean oil emulsions. The lowest loss of whey protein digestibility occurred in the
control group. In contrast, the decrease degrees over time of casein digestibility in all
samples were similar. After 144 h, casein digestibility in fish oil emulsions dropped to
61%, lower than 68% in soybean or sunflower oil emulsions and 71% in the control
group.
4.2 Photo-oxidation
4.2.1 Malondialdehyde (MDA)
The MDA production in all samples was evaluated after 0, 15 and 30 days upon
incubation (Table 4). In samples containing whey protein during the 30 days of
storage, MDA was below limit of detection in soybean oil emulsions under dark and
in protein solutions under light or dark (data not shown). Nevertheless, sodium
caseinate solution and soybean oil emulsions containing sodium caseinate were level
off at 15 and 30 days with 0.2 µg/mL MDA. Additionally, fish oil promoted the
accumulation of MDA, which increased over time in all fish oil emulsions.
Furthermore, light exposure also caused a faster production of MDA in both fish oil
emulsions and they were as much as 9 times of MDA in the same samples under dark.
But the two proteins did not have big different impact on the MDA production as in
samples with the same oil had similar MDA.
4.2.2 Hexanal
In addition to the MDA shown in Table 5, the level of hexanal was also measured
that are formed during oxidation of lipids (data not shown). In general, slightly less
hexanal was found in samples containing whey protein than in samples containing
sodium caseinate. The amount of hexanal was below limit of detection in all samples
without illumination (fresh samples and samples kept in the dark) and both of the two
control groups. For both fish oil emulsions under light exposure, an increasing trend
25
was obtained but the values were more than 10 times lower than those observed in
soybean oil emulsions under light.
Table 4. MDA in protein solution and protein emulsions (soybean or fish oil) under photo-oxidation at
4 °C1
Samples MDA (µg/mL sample)
Day 0 Day 15 Day 30
Whey protein
Soybean oil emulsion (light) ND 0.1±0.0a 0.1±0.0a
Fish oil emulsion (light) 0.1±0.0b 1.4±0.0a 1.8±0.5a
Fish oil emulsion (dark) 0.1±0.0a 0.2±0.0a 0.2±0.1a
Sodium caseinate
Protein solution (dark) ND 0.1±0.2a 0.2±0.3a
Soybean oil emulsion (light) ND 0.3±0.1a 0.2±0.0a
Soybean oil emulsion (dark) ND 0.1±0.1a ND
Fish oil emulsion (light) 0.1±0.0b 1.3±0.1a 1.8±0.2a
Fish oil emulsion (dark) 0.1±0.0b 0.2±0.1ab 0.4±0.1a
1Values represent mean values ± SD of three independent determinations ND: not detected Different letters within the same row indicate statistically significant differences (p < 0.05)
4.2.3 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE)
The electrophoretic pattern of the samples containing whey protein and samples
containing sodium caseinate is presented in Fig.3 and Fig.4 as function of the storage
time under illumination conditions.
SDS-PAGE pattern of fresh control sample containing whey protein (Fig.3C lane
4) presented characteristic bands for the subunits of BLG and ALA at, respectively, 14
26
and 18 kDa. No visible change of electrophoretic pattern was observed when control
containing whey protein was incubated for 15 (Fig.3C lane 8) and 30 (data not shown)
days in the dark, whereas incubation with light exposure for 15 and 30 days resulted
in gradual degradation of BLG and ALA. In light exposed solutions, compounds with
molecular weights above 25 kDa were also seen on the top of the separating gel.
Basic polypeptides of whey protein in soybean oil and fish oil emulsions also
became less intense with increasing time of illuminated incubation but more
significant than that in control samples with the sequence of control < soybean oil
emulsions < fish oil emulsions. The intensity change of BLG and ALA in all samples
in the dark was negligible. Yet in all incubated samples, high molecular complexes
were accumulated both in the staking gel and in the running gel, which intensity
increased with time of incubation.
Whey protein digested by gastrointestinal proteases exhibited distinctively
different electrophoretic patterns, and these patterns were also affected by the addition
of oil and time of incubation. Bands of BLG and ALA were not seen in all lanes, and
at the same time, large aggregates, especially molecular larger than 250 kDa, slightly
increased in fish oil emulsions and soybean oil emulsions along with time of
incubation regardless of light exposure, but not in control samples. A much more
quantity of low molecular peptides (< 10 kDa) appeared in all emulsions compared
with the control group, which also shown visible bands of the same molecular weight.
For all the samples containing sodium caseinate, the smearing out of the bands
for αs1-CN, αs2-CN and β-CN was very fast upon light exposition and almost
disappeared after 30 days of illumination. After 15 and 30 days of light exposition,
bands of higher protein aggregates (> 50 kDa) and lower small peptides (< 10 kDa)
increased. Also aggregates with molecular weight higher than 250 kDa formed in light
exposed soybean and fish oil emulsions, while such aggregation was not observed for
control samples. Similar changes were observed in samples stored in the dark, but it
was much less significant than that in light exposed samples.
Fig.3
mass
day
15 li
lanes
emul
soyb
1, m
respe
15 d
and d
3. Electrophor
s standards; la
30 light and d
ight, day 15 d
s 2, digested
lsions, day 0,
bean oil emuls
molecular ma
ectively, day 3
dark and day 3
day 15 dark, r
retic patterns
anes 2, 4, 6, 8
day 30 dark, r
dark and day
fish oil emu
day 15 light,
sions, day 0, d
ss standards;
30, dark; lane
30 light, respe
respectively.
of whey prot
and 10, non-d
respectively; l
30 light, resp
ulsions, day 3
day 15 dark a
day 15 light, d
lanes 2 and
es 4, 6, 8 and 1
ectively; lanes
27
teins after ph
digested fish o
lanes 3, 5, 7 a
pectively. For
30, dark; lan
and day 30 lig
day 15 dark an
d 3, non-dig
10, non-diges
s 5, 7 and 9, d
hoto-oxidation
oil emulsions,
and 9, digeste
r panel B: lan
es 3, 5, 7 an
ght, respective
nd day 30 ligh
ested and di
ted protein so
digested prote
. In panel A:
day 0, day 15
d fish oil emu
ne 1, molecula
nd 9, non-dig
ly; lanes 4, 6,
ht, respectivel
gested soybe
olutions, day 0
in solutions, d
lane 1, mole
5 light, day 15
ulsions, day 0
ar mass stand
gested soybea
8 and 10, dig
ly. In panel C
ean oil emuls
0, day 15 light
day 0, day 15
ecular
5 dark,
0, day
dards;
an oil
gested
: lane
sions,
t, day
light
Fig.4
stand
30 li
light
dige
day
emul
mass
dark
light
respe
4. Electropho
dards; lanes 2
ight and day 3
t, day 15 dark
sted fish oil e
15 light, day
lsions, day 0,
s standards; la
k; lanes 4, 6, 8
t, respectively
ectively.
oretic patterns
, 4, 6, 8 and 1
30 dark, respe
and day 30 li
mulsion, day
15 dark and
day 15 light,
anes 2 and 3,
8 and 10, non-
y; lanes 5, 7 a
of casein af
10, non-digest
ectively; lanes
ight, respectiv
30, dark; lane
day 30 light
day 15 dark a
non-digested
-digested prot
and 9, digeste
28
fter photo-oxi
ted fish oil em
s 3, 5, 7 and
vely. For panel
es 3, 5, 7 and
t, respectively
and day 30 lig
d and digested
tein solutions,
ed protein sol
idation. In pa
mulsions, day 0
9, digested fi
l B: lane 1, m
9, non-digeste
y; lanes 4, 6,
ght, respective
d soybean oil e
, day 0, day 1
utions, day 0
anel A: lane 1
0, day 15 light
sh oil emulsio
olecular mass
ed soybean oil
8 and 10, dig
ly. In panel C
emulsions, res
5 light, day 1
, day 15 light
1, molecular
ht, day 15 dark
ons, day 0, da
s standards; la
l emulsions, d
gested soybea
C: lane 1, mole
spectively, da
15 dark and da
t and day 15
mass
k, day
ay 15
anes 2,
day 0,
an oil
ecular
ay 30,
ay 30
dark,
29
After digestion of samples primarily containing sodium caseinate, bands
corresponding to subunits of casein disappeared but high molecular complexes and
small peptides were still visible in all samples, which were more intense in fish oil
emulsions, followed by soybean oil emulsions and control samples. The time of
storage but not light exposure had a positive impact on the accumulation of these
compounds.
4.2.4 Digestibility
The effect of photo-oxidation on protein digestibility is shown in Table 6. In all
samples, the digestibility of whey protein was higher than that of casein. Moreover,
the fortification of oil had a positive effect on protein digestibility in fresh samples
and light exposure impacted the protein digestibility in a negative way as evidenced
by the lower digestibility measured in illuminated samples compared with their
respective reference groups. In both illuminated fish oil emulsions containing whey
proteins and emulsions containing sodium caseinate, a dramatic time-dependent
decrease in protein digestibility was noted.
In samples containing whey proteins, at day 0, the highest digestibility occurred
in fish oil emulsion (77 %), which was a little higher than that in control solution
(74%). After 15 days illumination, the digestibility of whey protein increased in
soybean oil emulsion and in the control solution, followed by a slight decrease at day
30.
In illuminated samples, the digestibility of casein in soybean or fish oil
emulsions was decreasing from day 0 to day 30. At day 0, the digestibility in fish
emulsions was similar to that in control samples, which was lower than that in
soybean oil emulsions. After 15 days illumination, the digestibility dropped in both
emulsions but increased in control solution. After that the decrease continued in fish
oil emulsions but stopped in soybean oil emulsions.
30
Table 5. Digestibility of whey protein or casein in protein solutions and protein emulsions (soybean or
fish oil) after 0, 15 and 30 days of photo-oxidation at 4 °C1
Samples Protein digestibility (%)
Day 0 Day 15 Day 30
Whey protein
Protein solution (light) 73.9±0.9b 76.1±1.5a 73.9±0.0ab
Protein solution (dark) 73.9±0.9b 76.9±0.9a 76.1±0.2a
Soybean oil emulsion (light) 75.4±1.0ab 77.6±0.9a 70.0±1.5c
Soybean oil emulsion (dark) 75.4±1.0ab 76.2±1.0a 75.1±2.1ab
Fish oil emulsion (light) 76.9±0.0a 64.8±2.2b 56.4±1.2c
Fish oil emulsion (dark) 76.9±0.0a 76.4±0.2a 73.4±1.6ab
Sodium caseinate
Protein solution (light) 61.2±1.2b 67.9±0.9a 70.1±0.2a
Protein solution (dark) 61.2±1.2b 70.9±1.0a 72.1±0.3a
Soybean oil emulsion (light) 70.1±0.5a 61.5±1.0b 62.1±0.6b
Soybean oil emulsion (dark) 70.1±0.5a 70.2±1.4a 70±0.01a
Fish oil emulsion (light) 61.1±1.0a 53.4±0.2b 37.6±1.3c
Fish oil emulsion (dark) 61.1±1.0c 69.5±0.7a 63.3±1.0b
1Values represent mean values ± SD of three independent determinations
Different letters within the same row indicate statistically significant differences (p < 0.05)
4.3 Validation with milk
4.3.1 MDA in milk
Effect of photo-oxidation on MDA formation was evaluated in milk and milk
with 1.5% or 3.5% fish oil (Table 6). Compared with the control groups, fortification
of 1.5% and 3.5% fish oil kept under light and dark both promoted the production of
MDA, but the increased level of oil did not make a remarkable difference. In light
31
exposed emulsions, higher amount of MDA was accumulated compared with their
respective reference group in the dark. During the first 15 days of illumination, a
sharp increase was recorded in both emulsions. After 21 days of light exposition, a
slight decrease was observed compared with the 15 days illuminated emulsions. Light
exposure did not have a noticeable effect on the control group.
Table 6. MDA in milk and milk with fish oil (1.5% or 3.5%) after 0, 15 and 21 days storage at 4 °C in
the presence or absence of light1
Samples MDA (µg/mL sample)
Day 0 Day 15 Day 21
Milk (light) ND 0.3±0.1a 0.2±0.0ab
Milk (dark) ND 0.3±0.0a 0.2±0.0a
Milk with 1.5% fish oil (light) 0.4±0.0b 2.1±0.4a 1.8±0.2a
Milk with 1.5% fish oil (dark) 0.4±0.0c 0.8±0.0a 0.6±0.0b
Milk with 3.5% fish oil (light) 0.3±0.0b 2.3±0.0a 2.1±0.5a
Milk with 3.5% fish oil (dark) 0.3±0.0c 0.7±0.0a 0.6±0.1b
1Values represent mean values ± SD of two independent determinations ND: not detected Different letters within the same row indicate statistically significant differences (p < 0.05)
4.3.2 Hexanal in milk
Hexanal in emulsions without illumination and in the control groups was under
the limit of detection (data not shown). In light exposed emulsions, the level of fish
oil and prolong of incubation time did not exhibited a big influence on the hexanal
formation.
4.3.3 Amino acid composition
The amino acid composition of proteins in milk samples was determined after 0
and 21 days of storage (Table 7). At day 0, the total amount of amino acids increased
32
with the increasing level of oil and all amino acid content increased when fish oil
added. The same concentration of aspartate, histidine, glycine, threonine, alanine,
phenylalanine and proline was found in fresh milk with 1.5% fish oil and fresh milk
with 3.5% fish oil. Contents of all other detected amino acids showed increases with
the level of oil in fresh samples while the concentration of methionine decreased
when the level of fish oil increased to 3.5% from 1.5%.
Table 7. Selected amino acid composition of the proteins in milk samples after 0 and 21 days
of photo-oxidation
g amino acid
/100g protein
Day 0 Day 21
milk milk
with
1.5%
fish oil
milk
with
3.5%
fish oil
milk
(light)
milk
(dark)
milk
with
1.5%
fish oil
(light)
milk
with
1.5%
fish oil
(dark)
milk
with
3.5%
fish oil
(light)
milk
with
3.5%
fish oil
(dark)
Histidine 3.6 3.7 3.7 3.3 3.3 3.6 3.4 3.4 3.4
Threonine 5.3 5.5 5.5 4.9 5.1 5.4 5.2 5.2 5.2
Arginine 4.3 4.4 4.5 4.1 4.1 4.4 4.2 4.2 4.2
Tyrosine 4.9 4.9 5.0 4.7 4.5 4.9 4.7 4.6 4.7
Valine 8.7 9.0 9.1 8.6 8.3 9.0 8.5 8.5 8.5
Methionine 2.0 2.2 2.1 3.1 2.3 2.1 1.5 1.8 2.2
Lysine 9.9 10.5 10.7 9.9 9.7 10.4 9.8 9.8 9.9
Tryptophan 7.4 8.9 9.0 5.1 4.8 3.9 5.9 6.7 7.7
Total 46.1 49.1 49.6 43.7 42.1 43.7 43.2 44.2 45.8
1Values are mean values of two independent determinations
After 21 days incubation, loss of total amino acids was observed in all samples.
The light exposure impact the total amino acid content in the control sample and milk
with 1.5% fish oil in a positive way since the total amino acid contents were higher
than
trea
kept
in s
met
4.3
tryp
dark
loss
ligh
and
NFK
exp
addu
Fig.
sam
n those in
atments on
t in the dark
single amin
thionine con
.4 N-form
Tryptopha
ptophan alw
k, but the ill
s in all sam
ht exposure
At the sam
lipid-prote
K and add
erienced a
ucts exhibit
5. Impact o
mples measure
their respec
all amino a
k and with
o acid kept
ntent was ob
mylkynuren
an was mea
ways increas
lumination
mples over ti
than their re
me time, NF
ein adducts
ducts also
similar tre
ted lower am
of light expo
ed in PBS du
ctive refere
acids were
3.5% oil ex
t in step wi
bserved in t
nine (NFK
asured by f
ed with the
promoted th
ime and tha
eference gro
FK (Fig. 5),
(Fig. 6) we
increased
nd as funct
mount at da
osure and fi
uring the incu
33
ence group
obtained am
xposed to fl
ith total am
the control b
K) and lipi
fluorescenc
e level of fis
he degradat
at less trypt
oups.
as one of th
ere both me
with the l
tion of tim
ay15 than at
ish oil fortif
ubation of 0,
kept in th
mong samp
uorescent li
mino acids,
both kept in
id-protein
ce in PBS
sh oil additi
tion of trypt
tophan exis
he degradat
easured in P
level of oi
me and the
t day 0 and d
fication on
, 15 and 21 d
he dark. Sim
ples with bo
ight. Additi
except that
n the dark an
adducts
(Table 7).
on in sampl
tophan as ev
sted in samp
tion product
PBS. The c
l. Moreove
content of
day 21.
the NFK fo
days at 4 °C.
milar effect
oth level o
ionally, chan
t an increas
nd light.
The conten
les stored in
videnced by
ples kept u
ts of tryptop
concentratio
er, all sam
both NFK
ormation in
ts of
f oil
nges
se in
nt of
n the
y the
under
phan,
on of
mples
and
milk
Fig.
milk
4.3
(SD
corr
oil
form
illum
sligh
prot
sma
oil a
6. Impact of
k samples me
.5 Sodiu
DS-PAGE)
SDS-PAG
responding
and time o
mation of ag
minated sam
htly smeari
tein were n
all peptides
and time of
f light exposu
easured in PB
um dodec
) of milk
GE pattern
to high mo
of storage (
ggregates a
mples than
ing out of b
not visible.
with molec
f incubation.
ure and fish o
BS during th
cyl sulph
of milk sam
lecular com
(Fig. 7). Li
as the bands
n non-illum
basic protei
In digested
cular weight
.
34
oil fortificati
he incubation
hate-polya
mples show
mpounds (>
ight exposi
s between 7
minated sam
ins of milk
d samples,
t between 2
ion on the pr
n of 0, 15 and
acrylamide
wed the inc
250 kDa) w
tion had a
70 and 250
mples, which
k, as the ch
intensity o
and 5 kDa
otein-lipid ad
d 21 days at 4
e gel el
creased inte
with the inc
positive in
kDa were m
h could als
anges of ca
of bands co
increased w
adducts conte
4 °C.
lectrophor
ensity of b
creasing lev
nfluence on
more intens
so be proo
asein and w
orrespondin
with the lev
ent in
resis
ands
el of
n the
se in
of of
whey
ng to
el of
Fig.7
days
and
oil, l
respe
lane
respe
lane
and
light
lane
lanes
with
7. Electrophor
s (C and D). I
5, non-digeste
light and dark
ectively; lanes
1, molecular
ectively; lane
1, molecular
5, digested m
t and dark, res
10, non-dige
s 2 and 4, dig
h 3.5% fish oil
retic pattern o
In panel A: la
ed milk witho
k, respectively
s 8 and 10, dig
mass standar
s 3 and 5, dig
mass standard
milk, light and
spectively; lan
ested milk wit
gested milk w
l, dark.
of the milk sam
ane 1, molecul
out oil, light an
y; lanes 7 and
gested milk w
rds; lanes 2 an
gested milk w
ds; lanes 2 and
dark, respecti
nes 7 and 9, di
th 3.5% fish o
with 3.5% fish
35
amples after p
lar mass stand
nd dark, respe
d 9, non-dige
with 1.5% fish
nd 4, non-dige
with 3.5% fish
d 4, non-diges
ively; lanes 6
igested milk w
oil, light. For
oil, light and
hoto-oxidatio
dards; lanes 2
ectively; lanes
sted milk wit
h oil, light and
ested milk wi
h oil, light and
sted milk, ligh
and 8, non-d
with 1.5% fish
r panel D: lan
d dark respecti
n for 15 days
2, blank witho
s 4 and 6, dig
th 1.5% fish o
dark, respect
th 3.5% fish o
d dark, respec
ht and dark, re
digested milk w
h oil, light and
ne 1, molecula
ively; lane 3,
s (A and B) an
out sample; la
gested milk wi
oil, light and
tively. For pan
oil, light and
ctively. In pan
espectively; la
with 1.5% fis
d dark, respect
ar mass stand
non-digested
nd 21
nes 3
ithout
dark,
nel B:
dark,
nel C:
anes 3
sh oil,
tively;
dards;
d milk
36
4.3.6 Digestibility of milk
As shown in Table 8, milk protein digestibility during 21 days illumination was
evaluated when fortified with different levels of fish oil (1.5% and 3.5%). In all
samples, no considerable change in protein digestibility occurred over the 21 days of
storage. In fresh samples, the protein digestibility dropped with the increasing
concentration of fish oil. However, after 15 days storage, milk with 1.5% and 3.5%
fish oil had higher digestibility than the control group. And after 21 days illumination,
milk with 3.5% fish oil exhibited the highest digestibility compared with all other
samples, which was also higher than that in the same sample at the beginning of
incubation.
Table 8. Milk digestibility upon photo-oxidation at 4 °C1
Samples Digestibility (%)
Day 0 Day 15 Day 21
Milk (light) 63.9±1.9a 54.4±4.0b 66.7±3.6a
Milk (dark) 63.9±1.9a 52.9±4.1b 66.4±1.2a
Milk with 1.5% fish oil (light) 61.9±2.6a 59.3±3.0a 64.0±3.0a
Milk with 1.5% fish oil (dark) 61.9±2.6a 59.4±3.3a 63.0±2.1a
Milk with 3.5% fish oil (light) 59.7±1.4ab 57.6±3.4b 63.7±1.0a
Milk with 3.5% fish oil (dark) 59.7±1.4b 54.8±0.0b 68.5±0.5a
1Data points represent mean values of three independent determinations.
5 Discussion
Formation of MDA is a generic marker of lipid oxidation, and the susceptibility
of oils to oxidation increases with the unsaturation degree of the fatty acids in the oils
[66]. Under auto-oxidation condition, our results demonstrated that the generation of
MDA in fish oil emulsions was considerably faster and much more than in sunflower
37
oil emulsions (Table 1). Also the same results were obtained in light exposed samples
(Table 4). Given the fact that fish oil is rich in PUFAs (especially EPA and DHA,
18.41 and 7.89 g/100g fatty acids, respectively), it is most sensitive to oxidation.
While there are as much as respective 52 and 60 g/100 g fatty acids of linoleic acid
(18:2 ω-6) in soybean oil and sunflower oil. It is well reported that the oxidability of
EPA and DHA are 4 and 5 times greater, respectively, than that of linoleic acid [21].
This therefore explains the highest amount of MDA was found in fish oil emulsions
containing whey protein or sodium caseinate and less in sunflower or soybean oil
emulsions containing the same protein. Although soybean oil contains moreover 6 g
linolenic acid (C18:3 ω-3) per 100 g fatty acids which is around 100 times more than
that in sunflower oil, it was observed that sunflower oil is more sensitive to oxidation
when compared with soybean oil. This is most probably due to the lower content of
tocopherol, a natural antioxidant, in sunflower oil [21].
It was also reported that accumulation of MDA in the model systems was
significantly faster than in milk [67, 68], which can also be evidenced in our study
that in light induced samples, less MDA was found in milk samples than in samples
containing whey protein or sodium caseinate. This phenomenon was explained by the
fact that more extensive light scattering occurred in milk compared with the model
emulsions.
Besides MDA, hexanal was also used as an indicator of lipid oxidation. Since it
is a particular degradation product of ω-6 fatty acids [69], little hexanal was obtained
in fish oil emulsions either upon auto-oxidation or photo-oxidation and higher amount
of hexanal observed in sunflower oil emulsions under auto-oxidation. During thermal
treatment, soybean oil emulsions showed lower level of hexanal than sunflower oil
emulsions, which is in agreement with the result of MDA. This is partly due to the
less content of linoleic acid than sunflower oil. The higher content of natural
antioxidants in soybean oil might also contribute to this result [21].
Upon lipid oxidation in food systems, other food components can be attacked by
the oxidation products. Proteins are major targets [7] and this can lead to severe
38
molecular changes including backbone fragmentation, aggregation, oxidation of the
amino acid residuals and loss of functional properties and nutritional value [70].
Protein modification is highly dependent on the structure of the protein.
Dalsgaard et al. [28] reported earlier that compared with BLG and ALA, the random
coil α and β-CN were remarkably more prone to photo-oxidation as assessed by the
formation of protein carbonyls. This result was confirmed in a later study by Trine et
al. [71], who found that after exposure to light for 44 h, higher molar contents of
dityrosine, an often used biomarker for oxidation [72], existed in the protein
preparations of α-CN and β-CN. These findings are consistent with the results we
found in the present study that after 15 days light exposure more hexanal was detected
in fish oil emulsion containing sodium caseinate than in fish oil emulsion containing
whey protein. Meanwhile, we found that more aggregates were formed in samples
containing sodium caseinate (Fig.4) than in samples containing whey protein (Fig.3)
after 15 days of illumination. This result confirmed the the study conducted by
Dalsgaard and Larsen [71], who suggested that oxidative and conformational changes
were high in the casein and very limited in whey protein. Thus, proteins lacking
well-defined tertiary structures are more readily oxidized than globular proteins.
However, under 70 ºC, the globular whey protein can be unfolded followed by
formation of aggregates [73]. Whey protein is more sensitive to heat denaturation than
casein, as accessed by conformational changes of protein. After denaturation,
aggregation of whey protein is observed in protein solutions in the present study
(Fig.1), through covalent (not reversible) and noncovalent (possibly reversible)
interactions between protein molecules [74]. For example, disulfide bonds (covalent
compounds) can be formed between two molecules of BLG resulting from sulfhydryl
oxidation or sulfhydryl-disulfide interchange. These changes of protein structure
might facilitate its modification by lipid oxidation process.
Conformational and chemical changes of protein molecular can also occur upon
impact of lipid oxidation, owing to the reactions between lipid oxidation products
with nucleophilic side chain of cysteine, histidine, and lysine residues, especially the
39
ε-amino of lysine [42, 75]. The two reactive carbonyl groups in MDA molecular will
react with free amino groups of protein and form Schiff base, leading to the formation
of the MDA-modified protein. Therefore, the results observed in heated fish oil
emulsions that the amount of MDA reduced after 48 h might due to that the rate of
MDA formation is lower than the consuming to form MDA-protein adducts. And
similar reason can be used to explain the decreasing level of hexanal in 144 h heated
sunflower oil emulsion.
Furthermore, lipid oxidation products affected proteins resulting in protein
aggregation, and it is most likely because of increased hydrophobic interaction and
non-disulfide covalent cross-linkage between the oxidized proteins. Since the
SDS-PAGE was performed under reducing condition and in the presence of
mecaptoethanol, disulfide bonds will be broken down in the molecules. The
electrophoretic pattern of whey protein (Fig.1 and Fig.3) and casein (Fig.2 and Fig.4)
were significantly altered after incubation with oils after auto-oxidation and
photo-oxidation. The SDS-PAGE pattern of these two proteins presented that more
aggregation were observed where MDA or hexanal was higher. When whey protein or
sodium caseinate was incubated under 70 °C, the characteristic bands of them faded
faster in all emulsions than in the control samples, and more pronounced in fish oil
emulsions. At the same time, more aggregation was found in fish oil emulsions than in
sunflower or soybean oil emulsions, and lowest density of large molecular protein
bands was shown by control samples. Similarly in light induced samples, more
aggregation was observed in all emulsions and milk with 1.5% or 3.5% fish oil where
more MDA and hexanal were obtained. Our results indicated that the more MDA or
hexanal was produced, the more aggregation would be formed. Nannan Chen et al.
[76] studied the effects of MDA modification on the changes of soy protein. In their
research, soy protein isolate with different concentrations of MDA were prepared and
the results showed that increasing MDA concentration resulted in significant carbonyl
group generation and loss of free amino groups of soy protein. Meanwhile, during the
auto-oxidation of this study, the quantity of MDA reduced after long time of
40
incubation, and this phenomenon might due to the high reactivity of MDA and it
reacted with other compounds including protein. All these results indicated that lipid
oxidation products can react with protein and cause protein modification.
The aggregates can also be formed by direct protein oxidation and can be partly
digested by gastrointestinal enzymes. Compared with the SDS-PAGE pattern of
non-digested samples with the pattern of the same digested samples, much less
aggregates can be observed instead of the presence of small peptides in the lanes of
digested samples. This is because some part of the large molecules can also be
accessed to pepsin, trypsin or chymotrypsin, but the modified bonds are resistant to
proteolysis. In all the control samples, high molecular compounds can also be seen
stopped in the staking gel, but they are much less than the emulsions containing the
same protein. These results indicate that lipid oxidation products are not essential to
lead to protein oxidation, which can be directed oxidized by extreme conditions.
To bring the protein modification a step further, the lipid-protein complex are
produced because the lipid oxidation products, like hydroperoxides and MDA, are
prone to react with nucleophilic amino acids. It is reported that lysine, arginine and
histidine can form adducts with oxidized lipid degradation products [77]. Besides
adduct formation, singlet oxygen is also known to directly react with the amino acids
methionine, histidine, tyrosine and tryptophan. In some studies [28, 78, 79] the
sensitive amino acids showed remarkable loss after light exposition. They are in
agreement with the results found in our study that upon light exposition, almost all
amino acids in milk samples suffered loss when compared with the fresh samples.
However, more loss of amino acids was observed in samples kept in the dark for 21
days. This might be ascribed to that the lipid oxidation products played a more crucial
role than the direct reaction of singlet oxygen with these amino acids [20]. The less
degradation of the nucleophilic amino acids in illuminated milk samples can at least
partially be explained by the adduct formation with lipid peroxidation products, which
are resistant to be destroyed by direct oxidation or some other reaction. Because the
method used in our study to analyze amino acids did not protect lipid-protein adducts
41
from acid hydrolysis, the bond amino acids could also be detected in the present
study.
Tryptophan and one of its major degradation products of photo-oxidation, NFK,
were measured in PBS by direct fluorescence spectroscopy. Fast degradation of
tryptophan was observed in all samples exposed to light. In a previous study the
destruction of tryptophan was observed by Foettinger et al., who reported that the
indole nitrogen of the tryptophan side chain would react with MDA [78]. But
strangely the accumulation of NFK in PBS showed a different trend with tryptophan.
Similar results were also found by other researchers [20], and they explained it in the
way that NFK is only one of the degradation products of tryptophan and it might
further degrade or react with other compounds, e.g. protein nucleophiles. Other
researchers proposed that there was an underestimation of the tryptophan content by
fluorescence spectroscopy upon photo-oxidation [67] as a result of the shielding, due
to what a part of the tryptophan residues were not determined in the direct
fluorescence spectroscopy assay. Here the same reason could be used to explain the
difference of NFK and tryptophan in PBS.
In parallel to the tryptophan degradation and increase of NFK, accumulating of
fluorescent protein-lipid complexes was measured simultaneously in PBS. The
complexes were formed via Schiff base and they are conjugated fluorochromes with
an excitation maximum around 350 nm and an emission maximum around 460 nm
[79]. Previous studies found that with the increasing concentration of MDA, the
fluorescence from Schiff base increased [76, 80, 81]. The increasing fluorescence in
460 nm was attributed to the Schiff base formation. This further confirmed the
interaction between lipid oxidation products and free amino groups in protein.
Results of in vitro digestion shown that oxidation can influence the protein
digestibility either positively or negatively depending on the oxidation extent [82, 83].
The inhibition of proteolysis in oxidized lipid-damaged proteins was reported using
BSA modified by different concentrations of 4,5(E)-epoxy-2(E)-heptenal [60]. Also a
previous research indicated that plasmin hydrolysis was affected by photo-oxidation
42
due to a changed accessibility of plasmin to specific peptide bonds [71]. However,
another research group proposed that the aldehydes accumulated from lipid oxidation
can modify β-CN and thereby increase susceptibility of the proteins to proteolysis
[61]. Some other authors also reported a biphasic curve when protein digestibility was
measured in relation to oxidative modification [84, 85]. For these researches, low
levels of oxidation can cause unfolding of protein structure and expose the recognition
sites of enzymes leading to a higher proteolytic susceptibility initially increased with
oxidation. At higher level of oxidation, formation of aggregates and new bonds which
changed both chemical and physical properties of the recognition sites, and so the
protein digestibility decreased. This is in agreement with the increased protein
digestibility was observed after 15 days illumination in soybean oil emulsion
containing whey protein and increased digestibility occurred when fortified with
soybean oil or fish oil in fresh protein solutions.
6 Conclusion
To conclude, it is clear that the presence of PUFAs in food emulsions intensified
both the auto-oxidation and photo-oxidation processes by creating more reactive
oxygen species. Oxidizing lipids further interacted with the proteins especially as a
result of the reactions between the nucleophilic amino acid residues and secondary
oxidation products. Moreover, the interaction between oxidized lipids and the protein
also stimulated protein-protein cross-links. These modifications had a major impact
on the conformational and chemical structures of protein since the primary and higher
structures of protein were altered.
Protein modification is highly dependent on the structure of the protein and the
random coil casein was more reactive than globular whey protein. Upon oxidation,
high molecular weight aggregates were formed from both kinds of proteins, which
were known to be more resistant to digestion [60]. The slightly modification of
protein structure enhanced protein digestibility but higher oxidation degree impacted
proteolysis in a negative way. Thus proper protection of PUFAs-enriched foodstuffs,
43
through low temperature or protection from light exposition, is thus essential in order
to safeguard the nutritional benefits. To which extent of protein oxidation that differ
positive and negative impact on protein digestibility, should be investigated in further
study.
44
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