Immunoenhancing property of dietary un-denatured whey protein

Abstract

Data have demonstrated that whey protein (WP) enhances the immune system. The aim of this study was
to investigate and compare the effects of WP from three camel breeds on oxidative stress, blood lipid profile and the
cytokine levels. Seventy five male mice were randomly split into five groups. The first served as a control group. The second,
the third and the fourth groups were orally administrated the WP from Majaheim, Maghateer and Soffer camel breeds,
respectively, at a dose of 100 mg/kg mouse body weight. The fifth group was supplemented with bovine serum albumin
(BSA). Results showed similar electrophoretic patterns of the three whey proteins. WP was found to significantly inhibit
the hydroperoxide and the Reactive Oxygen Species (ROS) in leukocytes, liver and skin as well as the blood cholesterol
level in a time dependent manner. A significant enhancement of glutathione was revealed in WP groups. Furthermore, WP
was found to significantly elevate the IL-2 with a significant time dependent enhance of IL-8. On contrast, a significant
lowering effect of whey proteins on the pro-inflammatory cytokines, IL-1α, IL-1β, IL-6 and IL-10 was detected. Moreover, a
mitogenic activity of WP was observed on the lymphocytes. Non-significant changes were observed in AST, ALT, creatinine
and glucose level. These findings suggest that WP significantly improved the levels of the oxidative markers and the immune
functions without any difference in the bioactivities of the three studied whey proteins.

Full text

Page 1

Biologia 67/2: 425—433, 2012
Section Zoology
DOI: 10.2478/s11756-012-0014-0
Immunoenhancing property of dietary un-denatured whey protein
derived from three camel breeds in mice
Hossam Ebaid1,3, Gamal Badr1,4& Ali Metwalli2,5
1Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; e-mail:
hossamebaid@yahoo.com
2Food Science Department, College of Agriculture and Food Science, King Saud University, Saudi Arabia
3Zoology Department, Faculty of Science, El-Minia University, Egypt
4Zoology Department, Faculty of Science, Assiut University, Egypt
5Dairy Department, Faculty of Agriculture, El-Minia University, Egypt
Abstract: Data have demonstrated that whey protein (WP) enhances the immune system. The aim of this study was
to investigate and compare the effects of WP from three camel breeds on oxidative stress, blood lipid profile and the
cytokine levels. Seventy five male mice were randomly split into five groups. The first served as a control group. The second,
the third and the fourth groups were orally administrated the WP from Majaheim, Maghateer and Soffer camel breeds,
respectively, at a dose of 100 mg/kg mouse body weight. The fifth group was supplemented with bovine serum albumin
(BSA). Results showed similar electrophoretic patterns of the three whey proteins. WP was found to significantly inhibit
the hydroperoxide and the Reactive Oxygen Species (ROS) in leukocytes, liver and skin as well as the blood cholesterol
level in a time dependent manner. A significant enhancement of glutathione was revealed in WP groups. Furthermore, WP
was found to significantly elevate the IL-2 with a significant time dependent enhance of IL-8. On contrast, a significant
lowering effect of whey proteins on the pro-inflammatory cytokines, IL-1α, IL-1β, IL-6 and IL-10 was detected. Moreover, a
mitogenic activity of WP was observed on the lymphocytes. Non-significant changes were observed in AST, ALT, creatinine
and glucose level. These findings suggest that WP significantly improved the levels of the oxidative markers and the immune
functions without any difference in the bioactivities of the three studied whey proteins.
Key words: whey proteins; oxidative stress; glutathione; cytokines; lymphocytes
Abbreviations: Alanine Aminotransferase (ALT); Aspartate Aminotransferase (AST); Interleukin (IL); Peripheral Blood
Mononuclear Cells (PBMCs); Bovine serum albumin (BSA); Reactive Oxygen Species (ROS); Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE); Whey proteins (WP).
Introduction
Scientific efforts to search for natural bioactive sub-
stances for the amelioration of diseases have led to the
discovery of products with substantial bioactive prop-
erties. Components of milk including the whey protein
(WP) is easily available from different sources, espe-
cially camel in Saudi Arabia. WP is believed to be
the highest quality protein available, even when com-
pared to different proteins such as egg, casein, beef,
or soy (David 1999). Moreover, it was found that the
un-denatured WP was more effective when compared
with the denatured one (Ebaid et al. 2005), the WP
hydrolysates (Akhavan et al. 2010) or bovine collagen
hydrolysates (Castro et al. 2010).
WP contains all essential and non-essential amino
acids and is an excellent source of glutamine and the
branched-chain amino acids that are necessary for cell
growth (David 1999). In addition, WP contains a num-
ber of immunomodulatory peptides that are naturally
present or that are part of the primary sequence of whey
proteins. These peptide sequences can be released dur-
ing digestion in the gut and can also be produced by
in vitro enzymatic hydrolysis (Gauthier et al. 2006).
Normal processes to extract whey component from
the other constituents lead to significant denaturing of
the bioactive whey proteins components. Un-denatured
whey protein isolates utilize proprietary processes to at-
tain a protein containing over 90% un-denatured whey.
WP possesses many bio-active properties (Ballard
et al. 2009) and its peptide hydrolysates derived in vivo
and/or in vitro modulate various immune functions,
including lymphocyte activation and proliferation, cy-
tokine secretion, antibody production, phagocytic ac-
tivity, and granulocyte and natural killer cell activity
(Gauthier et al. 2006). Fractions of the WP stimu-
lated IL-1β, IL-8, IL-6, macrophage inflammatory pro-
tein (MIP)-1α, MIP-1β, and TNF-α (Rusu et al. 2010).
The pro-inflammatory cytokines IL-1β, IL-6 and TNF-
α play key roles within the cytokine network. They play
an important role in the mediation of inflammation and
trauma and could be useful for the determination of vi-
c ?2012 Institute of Zoology, Slovak Academy of Sciences

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H. Ebaid et al.
tality and wound age (Grellnera et al. 2000; Rivat et
al. 2005).
Data demonstrate that a cysteine-rich WP concen-
trate represents an effective cysteine delivery system for
glutathione (GSH) replenishment during the immune
response. The GSH antioxidant system is the principal
protective mechanism of the cell and is a crucial fac-
tor in the development of the immune response by the
immune cells. Animal experiments showed that the con-
centrates of whey protein also exhibit anticancer activ-
ity via the GSH pathway, the induction of p53 protein
in transformed cells and inhibition of neoangiogenesis
(Bounous & Molson 2003).
We have previously investigated the role of the un-
denatured WP in the wound healing in a non-diabetic
mouse model. Data of that work indicated an increased
capacity of the un-denatured WP-fed animals to heal
wounds comparing to those fed on casein or denatured
WP (Ebaid et al. 2005). In this work the un-denatured
WP was extracted from the milk of three different
camel species (Majaheem, Maghateer, Soffer) to com-
pare their immunomodulatory effects. Significant en-
hancement of the immune system was observed of these
three proteins with a little bioactivity difference among
them.
Material and methods
Preparation of whey proteins
Raw camel milk was collected from healthy she-camels
(three camel breeds, Majaheim, Maghateer, Soffer) from
Riyadh area, Saudi Arabia, and then centrifuged for cream
removing. The obtained skim milk was acidified to pH
4.3 using 1N HCl at room temperature and centrifuged at
10,000 g for 10 min. to precipitate casein. Resultant whey
containing whey proteins was saturated with ammonium
sulphate to final saturation 80% for whey proteins precipita-
tion. The precipitated whey proteins were dialyzed against
20 folds of distilled water for 48 h through molecular-porous
membrane MWCO. 6000–8000. The dialysate containing
un-denatured whey proteins was freeze-dried, then kept re-
frigerated till use.
Preparing diets for experimental mice
To prepare 500 g of the diet, 5 g vitamins, 25 g mineral
salts, 40 g fats, 50 g sucrose, 100 g protein (casein) and
280 g starch were mixed (20% protein by weight of total
diet). The diet was kept at 4◦C until use. All mice groups
fed this diet.
Animal groups
Seventy five male of laboratory mice weighing 25–30 g were
obtained from the Central Animal House, Faculty of Phar-
macy, King Saud University. All animal procedures were in
accordance with the standards set forth in the guidelines for
the care and use of experimental animals by the Commit-
tee Control for Purpose of Supervision of Experiments on
Animals (CPCSEA) and the National Institutes of Health
(NIH) protocol. The study protocol was approved by the
Animal Ethics Committee of the Zoology Department, Col-
lege of Science, King Saud University. Animals were allowed
to acclimatize in metal cages inside a well-ventilated room
for 2 weeks prior to the experiment. They were maintained
under standard laboratory conditions-temperature 23◦C,
relative humidity 60–70% and a 12-hour light/dark cycle,
and were fed a pellet diet prepared in the lab as mentioned
above, and water. Seventy five male mice were randomly
split into five groups. The first served as a control group.
The second, the third and the fourth groups were inde-
pendently orally supplemented with the un-denatured whey
protein from Majaheim (Second group, WPA), Maghateer
(Third group, WPB) and Soffer (Fourth group, WPC), re-
spectively, at a dose of 100 mg/kg body weight daily for
6 days. The fifth group was orally supplemented with the
bovine serum albumin (BSA) at the same dose (100 mg/kg
body weight daily for 6 days). The dose of the WP was
applied in the current study according to Bounous (2000).
Blood samples, plasma and PBMCs
Animals were anesthetized with pentobarbital (60 mg/kg
body weight) and samples (blood, liver and skin) were ob-
tained 2, 4 and 6 days post supplementation of whey pro-
teins. Whole blood was drawn from the abdominal aorta
in heparinized tubes. Heparinized venous blood was cen-
trifuged at 800 g for 10 min., and plasma was stored at
–20◦C until analysis. After plasma isolation, half of the
obtained blood cells were subjected to peripheral blood
mononuclear cell (PBMCs) isolation using a Ficoll gradient
method. Isolated mononuclear leukocytes were subjected to
the measurement of the ROS as mentioned below.
Estimation of glutathione
Glutathione (GSH) assay was carried out on tissue as previ-
ously described (Clark et al. 2010). Liver was removed and
gently rinsed in physiological saline. The fresh organ weights
was recorded, then was frozen until use. A 10% (w/v) ho-
mogenate of each frozen tissue was prepared. Glutathione
concentrations were measured by adding 100 µl of super-
natant to 400 µl PBS [containing 200 mM MCB and 2 U
ml−1glutathione S-transferase (per 100 µl)]. Glutathione
concentrations were then determined by measuring the ab-
sorbance of the reaction after 1 min at 340 nm using an UV
Visible Spectrometer (Ultrospec 2000, Pharmacia Biotech).
Glutathione standards were measured concurrently to ob-
tain a standard curve that was used to calculate GSH con-
centrations in samples. Results were expressed as µg GSH/g
tissue. Five replicates of each experiment were carried out
for performing statistical comparisons of GSH activities be-
tween controls and treatments in each case were performed
using Minitab statistical program as detailed below.
ROS measurement
ROS levels were determined using 2,7-dichlorodihydro-
fluorescein diacetate (H2DCF-DA) (Beyotime Institute of
Biotechnology, Haimen, China). Supernatant from skin and
liver homogenates, and the mononuclear leukocytes were di-
rectly treated with 10 µM H2DCF-DA dissolved in 1 ml
PBS at 37◦C for 20 min. The fluorescence intensity was
monitored using an excitation wavelength of 488 nm and an
emission wavelength of 530 nm (Shimada et al. 2011).
Hydroperoxide levels
Blood levels of hydroperoxide were evaluated using a free-
radical analytical system (FRAS 2, Iram, Parma, Italy).
This colorimetric test takes advantage of the ability of hy-
droperoxide to generate free radicals after reacting with
transitional metals (Wolff et al. 1994).

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Dietary un-denatured whey proteins enhances immune response
427
Plasma glucose and lipid profile
Blood glucose levels were measured with the glucose ox-
idase method (Chan et al. 2007) using BioMerieux kits
(France). Lipid profiles were determined colorimetrically
with BioMerieux kits and a standard assay method. Choles-
terol levels were evaluated using the cholesterol esterase
method (Allain et al. 1974).
Determination of serum ALT, AST, and creatinine levels
Aspartate aminotransferase (AST), alanine aminotrans-
ferase (ALT) and creatinine were measured using commer-
cial kits (Labtest Diagnostica, Brazil) according to the man-
ufacturer’s instructions.
ELISA assay for the plasma cytokine profile
The levels of IL-1α, IL-β, IL-10, IL-2, IL-6, and IL-8 were
estimated in sera of different groups according to the man-
ufacturers’ instructions (SABiosciences). The optical densi-
ties (OD) are measured using the molecular devices 405 nm.
The detection limits are set according to the log-log correl-
ative coefficient of the standard curve.
Polyacrylamide gel electrophoresis (SDS-PAGE)
Whey protein samples were separated using a polyacry-
lamide gel as previously described (Laemmli 1970). Briefly,
gel (final concentration): 12.5% acrylamide; 0.33% bisacry-
lamide; 0.37 M Tris-HCl buffer, pH 8.8; 0.1% SDS; 0.03%
ammonium persulphate; and 0.1% N,N,N?,N?-tetramethyl-
enediamine. Stacking gel (final concentration): 4.5% acry-
lamide, 0.12% bisacrylamide, 0.125 M Tris-HCl buffer pH
6.8; 0.1% SDS; 0.09% ammonium persulphate; and 0.1%
N,N,N?,N?-tetramethylenediamine. Running buffer: 50 mM
Tris, 0.196 M glycine and 0.1% SDS (wt/vol), pH 8.3.
After the electrophoresis run (18 mA/1.5 mm thickness
gel at 10◦C) for approximately 6 h the gels were marked
with Coomassie brilliant blue R-250 over night (0.2% in
45:45:10 methanol:water:acetic acid) and destaining with
water:methanol:acetic acid (65:25:10). Prestained molecu-
lar weight marker solution (broad range, Sigma) was used
contained: bovine albumin (66 kDa), egg albumin (45 kDa),
glyceraldehydes-3-p-dehydrogenase (36 kDa), carbonic an-
hydrase, bovine (29 kDa), trypsinogen, bovine pencrease
(24 kDa), soybean trypsin inhibitor (20 kDa), Bovine milk
α-lactalbumin (14.2 kDa).
Statistical analysis
Statistical analysis was undertaken using MINITAB soft-
ware (MINITAB, State College, PA, Version 13.1, 2002).
Data from experiments were first tested for normality using
Anderson Darling test, and for variances homogeneity prior
to any further statistical analysis. Data were normally dis-
tributed, and variances were homogeneous, thus, One-way
ANOVA was used to determine overall effects of treatments
followed by individual comparison using Tukey’s Pairwise
comparison.
Results
SDS-PAGE of three whey proteins shows similar elec-
trophoretic pattern
Nutritional and functional characteristics of whey pro-
teins are related to the structure and biological func-
tions of these proteins. In SDS-PAGE, the aim was
to determine the protein pattern-difference among the
three whey proteins. Difference should explain the vari-
ation in mode of action of each one. The SDS-PAGE
Fig. 1. Electrophoretic pattern of the whey proteins from three
camel breeds. M – protein marker (66, 45, 36, 29 24 20 14.2 kDa);
1 – Majaheim breed; 2 – Maghateer breed; 3 – Soffer breed; L
– Lactoferrin; SA – Serum Albumin, α-LA – α-Lactalbumin.
Mice were supplemented with WP (groups 2–4) at a dose of
100 mg kg−1of body weight. Electrophoresis shows no differ-
ence among the three whey proteins in the protein distribution
pattern. Five mice from each group were killed two days intervals
(2, 4 and 6 days post treatment).
analysis of these three whey proteins revealed a sim-
ilar electrophoretic pattern which indicated a relative
structure of the protein and carbohydrate components
(Fig. 1). At the whey protein from the three camel
breeds, lactoferrin, serum albumin and α-Lactalbumin
are distributed at molecular weights of about 80, 66
and 14 kDa, respectively, as previously described (Red-
wan & Tabll 2007; Farah 1986; Elagamy et al. 2008;
Elagamy et al. 1996).
WP supplementation improves oxidative stress and in-
creases glutathione
Similarity of the electrophoretic distribution clearly in-
dicated similar bioactivity as expected. A significant
inhibition of the oxidative stress parameters were ob-
served in similar behaviors for the three whey proteins
(Figs 2, 3). We monitored changes of the ROS in the
isolated leukocytes, liver and skin homogenates. We ob-
served a significant decrease of ROS in isolated leuko-
cytes, and liver and skin homogenates in the three WP
groups comparing to control and BSA groups. Notably,
the decreased effect in the three WP groups was ap-
proximately similar in a clear time dependent manner
(Fig. 2). Moreover, hydroperoxide level was measured
in the whole blood samples. Statistical analysis revealed
that the three mice groups (WPA, WPB, WPC) sup-
plemented with WP from three camel ecotypes showed
a significant decrease in the level of hydroperoxide com-
paring to the control mice. This decrease was time de-
pendent (Fig. 3). Interestingly, glutathione was signif-
icantly elevated (P < 0.05) in WP supplemented mice
in a time dependent manner comparing to the BSA and
control mice (Fig. 3).
WP improves blood lipogram, and liver and kidney
functions
Blood glucose, and liver and kidney functions have been
measured to monitor any adverse effects of the whey

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H. Ebaid et al.
Fig. 2. ROS levels in leukocytes, skin and liver from the five
groups of male mice (n = 15), the control group (N), three whey
protein groups (WPA, WPB, WPC) and BSA group. Five mice
from each group were killed in two days intervals (2, 4 and 6 days
post treatment). Results are expressed as means ± SEM. * P <
0.05.
proteins. Non-significant changes (P < 0.05) were ob-
served in AST, ALT and creatinine in whey protein
groups comparing to control and BSA groups (Table 1).
In addition, whey proteins regulated glucose level at a
range of approximately that of the control mice group.
WP was found to significantly (P < 0.05) decrease the
total blood cholesterol level compared with the control
and BSA groups (Table 1). The decrease effect on the
blood cholesterol level was in a time dependent manner
in almost all mice of the whey protein groups.
Whey proteins change the cytokine profile and increase
lymphocyte count
The effect of three whey components on cytokine ex-
pression profiles has been investigated. Significant en-
hancement of the immune system was observed by the
three WP. Results revealed a significant and a time
Fig. 3. Hydroperoxide of the whole blood and the liver total glu-
tathione levels from five groups of male mice (n = 15), the con-
trol group (N), three WP groups (WPA, WPB, WPC) and BSA
group. Five mice from each group were killed in two days inter-
vals (2, 4 and 6 days post treatment). Results are expressed as
means ± SEM. * P < 0.05.
dependent lowering effect of whey proteins on the pro-
inflammatory cytokines, IL-1α, IL-1β, IL-10 (Fig. 4)
and IL-6 (Fig. 5). In addition, WP was found to sig-
nificantly (P < 0.05) elevate the T cell growth factor
(IL-2) (Fig. 5). Moreover a significant elevation of IL-8
(P < 0.05) in a time dependent manner was recorded
in groups supplemented with WP (Fig. 5). A mitogenic
bioactivity of WP was observed on the total leukocytes
count, and in particular the lymphocyte count with lit-
tle changes in other blood parameters as shown in Ta-
ble 2.
Discussion
It is evident that amino acids incorporated in the WP
can influence the immune response (Bounous et al.
1989; Belokrylov et al. 1992). Bioactive components in-
clude lactoferrin, lactoperoxidase, glycomacropeptide,
BSA, various growth factors and immunoglobulins ex-
hibit anticancer, antiviral, antibacterial, and antifungal
activity (Kawasaki 1993; Marchetti 1994; Wong & Wat-
son 1995). Here, we found that WP clearly improved the
different immune functions compared with the BSA.
Oxidative stress takes place due to an imbalance
between the production of ROS and the protection by
cellular antioxidants (Shen et al. 2011). The blood level
of hydroperoxides and the ROS level in leukocytes, liver

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Dietary un-denatured whey proteins enhances immune response
429
Table 1. Liver (AST, ALT) and kidney (creatinine) functions, blood glucose level and plasma lipid profile of five groups of male mice
(n = 15), the control group, WP groups (WPA, WPB, WPC) and BSA group.
ParametersGroups2 days 4 days 6 days
AST (U ml−1) Control
WPA
WPB
WPC
BSA
44 ± 4.1
45 ± 4.6
43 ± 4.2
46 ± 4.4
46 ± 4.45
56 ± 4.6
54 ± 4.2
55.5 ± 5.1
57 ± 4.75*
53 ± 4.9
3.75 ± 0.27
3.71 ± 0.31
3.83 ± 0.36*
3.9 ± 0.38*
3.9 ± 0.33*
44 ± 3.9
45.6 ± 4.2
43.9 ± 4.3
41.2 ± 3.7*
46.4 ± 4.4
133 ± 9.6
139 ± 9.1*
143 ± 8.8*
137 ± 8.7*
143 ± 10.1*
44 ± 4.1
47.3 ± 4.9
46.4 ± 4.4
44 ± 3.8
43 ± 3.8
56 ± 4.6
57.6 ± 4.65*
59.1 ± 5*
54.4 ± 4.9
57 ± 5.9
3.75 ± 0.27
3.65 ± 0.30
3.7 ± 0.32
3.58 ± 0.33
3.6 ± 0.35
44 ± 3.9
40 ± 3.6*
39 ± 3.3*
37.3 ± 3.1*
43 ± 3.6
143 ± 9.2
150 ± 8.6*
148.6 ± 8.1
139 ± 7.9
135 ± 9.3
44 ± 4.1
43 ± 4.1
44.5 ± 4
46.7 ± 3.9
46 ± 4.55
56 ± 4.6
52 ± 5.1
55.1 ± 4.7
55.1 ± 4.5
58 ± 5.4
3.75 ± 0.27
3.5 ± 0.34
3.4 ± 0.34
3.45 ± 0.3
3.85 ± 0.36*
44 ± 3.9
36.1 ± 3.2*
33.6 ± 3.1*
34.3 ± 3.4*
46 ± 4.2
142 ± 8.6
145.6 ± 7.7
138.4 ± 7.1*
149 ± 8.2*
141 ± 8.7
ALT (U ml−1) Control
WPA
WPB
WPC
BSA
Creatinine (dg ml−1) Control
WPA
WPB
WPC
BSA
Blood Cholesterol (mg/100 ml)Control
WPA
WPB
WPC
BSA
Blood Glucose (mg dl−1)Control
WPA
WPB
WPC
BSA
Explanations: Results are expressed as means ± SEM; * P < 0.05.
Table 2. Blood parameters from five groups of male mice (n = 15), the control, WP (WPA, WPB, WPC) and BSA groups.
Parameters Groups2 days4 days 6 days
Hb (g dl−1) Control
WPA
WPB
WPC
BSA
13.6 ± 0.8
13.8 ± 1
13.8 ± 1
13.8 ± 0.7
33.9 ± 3.3
34.5 ± 2.1
34.55 ± 2.5
35 ± 3.1
34.9 ± 2.2
33.9 ± 3.3
5.9 ± 0.55
6.4 ± 0.6
6.5 ± 0.54
6.7 ± 0.7*
5.7 ± 0.51
9.9 ± 0.8
10.5 ± 1.1
10.7 ± 1.3*
10.6 ± 0.9*
10 ± 1.2
4 ± 0.34
4.5 ± 0.4
4.3 ± 0.42
4.4 ± 0.45
3.7 ± 0.37
5.6 ± 0.48
6 ± 0.53
6.4 ± 0.58
6.2 ± 0.6
5.5 ± 0.51
13.9 ± 0.2
13.9 ± 1
14.1 ± 1.1
14 ± 1.3
13.5 ± 1.2
35.5 ± 2.7
35.1 ± 3.2
35.4 ± 3.5
35.3 ± 2.9
34 ± 3.1
5.7 ± 0.6
6.6 ± 0.7*
6.7 ± 0.6*
6.8 ± 0.65*
5.8 ± 0.53
9.75 ± 0.7
11 ± 10*
10.9 ± 0.9*
9.85 ± 0.9
9.8 ± 0.8
4 ± 0.5
4.1 ± 0.35
4.1 ± 0.4
3.9 ± 0.37
3.9 ± 0.35
5.9 ± 0.6
5.8 ± 0.5
5.9 ± 0.45
6 ± 0.55
5.4 ± 0.4
14 ± 0.6
14.4 ± 1.2
14.3 ± 1.3
14.5 ± 1.5
13.7 ± 1.3
34.5 ± 3
36 ± 3.4
37.3 ± 3.6*
34.8 ± 3.1
35.4 ± 3.5
5.8 ± 0.7
7 ± 0.80*
7.3 ± 0.75*
6.9 ± 0.70*
6.1 ± 0.59
9.65 ± 0.5
12 ± 1.1*
12.6 ± 1.3*
13 ± 1.25*
10 ± 0.9
4 ± 0.65
5.1 ± 0.45
4.8 ± 0.4*
4.75 ± 0.5
4.2 ± 0.32
5.9 ± 0.85
6 ± 0.55
5.8 ± 0.5
6.2 ± 0.6
5.5 ± 0.48
Hematocrite (%) Control
WPA
WPB
WPC
BSA
Lymphocytes (× 103mm−3) Control
WPA
WPB
WPC
BSA
WBCs (× 103mm−3) Control
WPA
WPB
WPC
BSA
Neutrophils (× 103mm−3) Control
WPA
WPB
WPC
BSA
RBCs (× 103mm−3)Control
WPA
WPB
H WPC
BSA
Explanations: Results are expressed as means ± SEM; * P < 0.05.

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H. Ebaid et al.
Fig. 4. ELISA estimation of plasma cytokine levels (IL-1α, IL-β,
IL-10) from five groups of male mice, the control group (N), three
WP (WPA, WPB, WPC) and BSA groups. The three whey pro-
teins showed a significant down-regulation of all tested inflamma-
tory cytokines, IL-1α, IL-β, IL-10, in a time dependent manner.
Five mice from each group were killed in two days intervals (2,
4 and 6 days post treatment). Results are expressed as means ±
SEM. * P < 0.05.
and skin tissues were significantly suppressed by WP
what indicates a potential antioxidant role of WP. Pre-
vious studies confirmed the active roles of this pro-
tein in iron transport and in the cytotoxic defenses of
neutrophils, and in scavenging free radicals (Kawasaki
1993; Marchetti 1994; Wong & Watson 1995). Antiox-
idants play a vital role in maintaining immunity and
protection against free radical damage in the human
body. A significant enhancement of glutathione level
was recorded in WP supplemented mice in this study.
Un-denatured WP is a potent inducer of glutathione
Fig. 5. ELISA estimation of plasma cytokine levels (IL-2, IL-6,
IL-10) from male mice groups, the control group (N), three WP
(WPA, WPB, WPC) and BSA groups. WP was found to signif-
icantly up-regulate the levels of IL-2 and IL-8, and significantly
down-regulated the level of IL-6. Five mice from each group were
killed in two days intervals (2, 4 and 6 days post treatment).
Results are expressed as means ± SEM. * P < 0.05.
(Bounous 2000). Glutathione is an intracellular tripep-
tide vigorously binds damaging free radical molecules
that would otherwise harm the cell. Many studies con-
firmed the role of glutathione, which is increased by the
dietary WP, as a powerful antioxidant system (Lands
et al. 1999). The efficiency of cysteine in increasing the
glutathione level is greater when it is delivered in the
WP than as free cysteine (Bounous et al. 1989).
Accordingly, WP regulated and kept the blood glu-
cose level at normal values and significantly decreased
the total cholesterol levels. Akhavan et al. (2010) found

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Dietary un-denatured whey proteins enhances immune response
431
that dairy protein ingestion, when consumed with car-
bohydrates, reduces glucose. A significant increase in
arterial elasticity index and a significant improvement
in glucose and lipid metabolism were observed in pa-
tients who treated with antioxidants (Shargorodsky
et al. 2010). Additionally, the beneficial effect of an-
tioxidant supplementation on LDL oxidation has been
demonstrated (Plotnick et al. 1997; Devaraj et al.,
2000). An inverse correlation was demonstrated be-
tween blood lipid profile and T-cell proliferative capac-
ity in dogs, where the reduction in total blood choles-
terol, LDL and non-HDL-cholesterol levels was corre-
lated with an increase in T-cell proliferation (Nunes
et al. 2008). Another study revealed that the improve-
ment in T-cell function during diabetes was probably
partially a result of the reduction in total blood choles-
terol and LDL (Han et al. 2003).
A mitogenic effect of WP was observed on the to-
tal leukocytes, and in particular the lymphocyte count
in the present study. Similarly, it was previously found
that WP play the role of a lymphocytic mitogen (Be-
lokrylov et al. 1992; Middleton et al. 2004). This is
because WP contains substantially cysteine which is
the potential factor that controls the lymphocyte num-
ber in the blood. Moreover, Wong and Watson (1995)
found that response of T cells to the WP was signifi-
cantly higher than to the concanavalin A which is a T
cell mitogen. Mercier et al. (2004) showed that the in
vitro proliferation of murine spleen lymphocytes is stim-
ulated by WP which possesses many bio-active proper-
ties (Ballard et al. 2009). In addition, Rusu et al. (2010)
found that WP hydrolysates modulate various immune
functions including lymphocyte activation and prolifer-
ation.
More importantly, the plasma level of IL-2 (a T
cell growth factor required for the survival and pro-
liferation of T cells) was significantly increased in the
WP fed groups. Elevation of IL-2 led to the increase in
lymphocyte count which clearly appeared in the differ-
ential count of the current study. Most probably, the
increase in lymphocyte count was due to the prolif-
eration of T-cells but not B-cells. This suggestion is
partially accepted since, WP was found to significantly
decrease the IL-6 which is a major stimulator of B-
cell proliferation. On contrast to our results, Artym &
Zimecki (2005) found that lactoferrin promote the dif-
ferentiation of T and B cells from their immature pre-
cursors. IL-6 is mainly secreted by antigen-activated
T-cell and is originally identified as B-cell differentia-
tion factor (Akira et al., 1990). WP supplemented mice
did not challenged by an antigen in the current study,
and this can explain the contradiction of these results
with those obtained by Artym & Zimecki (2005). Thus,
WP did not promote the differentiation of B cells from
their immature precursors in the current study. An im-
provement of T cell-mediated immune functions, such
as increase in IL-2 production and the lymphocyte pro-
liferative response, provides important evidence of en-
hancement of immune functions (Han et al. 2003). This
maintained an efficient T cell immune response in the
WP groups. We have previously found that increase in
the levels of both IL-2 and IL-7 was accompanied by a
potential T cell function (Badr et al. 2011a, b).
WP was also found to significantly and time-
dependently elevate IL-8 in the current study, as it was
previously found by Ustunol & Wong (2010). Shinoda
et al. (1996) showed that lactoferrin stimulate the re-
lease of neutrophil-activating IL-8 from human poly-
morphonuclear leukocytes. Results revealed a signifi-
cant and a time dependent lowering effect of whey pro-
teins on the pro-inflammatory cytokines, IL-1α, IL-1β
and IL-10. Thus, WP showed an anti-inflammatory ef-
fect on the inflammatory cytokines. Similarly, Artym &
Zimecki (2005) proved an inhibition effect of lactoferrin
on the pro-inflammatory cytokines.
Our results indicated potential effects of the three
tested WP on immune functions without any consider-
able difference among their bioactivities. A significant
restoration of the oxidative markers with a stimula-
tion of glutathione by WP were proved in this study.
In addition, a significant time dependent up-regulation
of IL-2 and IL-8 levels was stimulated by WP. On
the other hand, a down-regulation of IL-1α, IL-1β, IL-
10 and IL-6 characterized the behavior of the three
whey proteins. Findings of this study suggest that whey
protein may serve as an alternative source of antioxi-
dants for prevention of many injuries caused by ROS
and/or mediated by the inflammatory cytokines. Thus,
this study may provide critical insight into future nu-
tritional intervention strategies designed to enhance
the anti-inflammatory and the anti-oxidants protection.
This strategy would show whether this WP has a signif-
icant clinical impact. Based on the current and previous
studies (Ebaid et al. 2005, 2011) we hypothesized that
un-denatured WP can have a significant role on the
progress of wound healing process in diabetic models,
and thus, such work has been initiated.
Acknowledgements
This work was supported by the National Plan for Sci-
ence and Technology (NPST) funded by King Abdul-Aziz
City for Science and Technology (KACST), Riyadh, KSA,
through the project number 10-BIO975-02.
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Received September 18, 2011
Accepted November 25, 2011