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Scholars
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African Journal of Pig Farming ISSN 2375-0731 Vol. 2 (7), pp. 001-009, July, 2014. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 

 

Oxygen radical-scavenging capacities of peptides 
from swine blood 

 
Jun Fang1*, Rao Li-qun1, Tian Yun1, Xiangyang Lu1 and Hongmei Jiang1

 
 

1
College of Bioscience and Biotechnology, Hunan Agricultural University, 410125 Changsha, Hunan, People’s 

Republic of China. 
 

Accepted 29 June, 2013 
 
In China, about five hundred million swine are slaughtered yearly, which represents about 45% of the world´s 
production. Swine blood is generally discarded except for the small amount that is used in soybean curd and 
other food products. This not only wastes resources, but also contaminates the environment. In this study, we 
found that peptides from swine blood had molecular masses of less than 2,100 Da and most were about 1,000 
Da. Furthermore, the contents of Glu, Val, Met, Ile, Leu, Phe and Lys were higher than those in dried swine 
blood. Peptides from swine blood most strongly scavenged ·OH among different oxygen species. This is the 
first reported study on the oxygen radical-scavenging capacities of peptides from swine blood, and the results 
suggest that swine blood may be promising for use in food or feed. 

 
Key words: Peptides of swine blood, components, scavenging capacities, oxygen radicals. 
 
INTRODUCTION 

 
The metabolic balance of oxygen radicals is a basic factor in 
maintaining life and health (Stadtman and Levine, 2003). 
In some pathological conditions, the abnormal production 
and scavenging of oxygen radicals leads to the accumu-
lation of such radicals in the organism. A high level of 
oxygen radicals will damage the organism at the molecular, 
cellular, and organ levels, which can accelerate the senile 
process of the organism and may induce cancer, 
inflammation, and cardiovascular disease (Gutteridge 
1994 and Tabatabaie et al. 2003). Oxidative stress also 
plays a crucial role in the development of complications in 
diabetes mellitus (DM) (Kapalla et al., 2005). Oxygen 
radicals include many kinds of oxygen compounds, such 

as superoxide anion free radicals (O2
.-
), hydroxy free 

radicals (
.
OH), hydrogen peroxide (H2O2), fat hydroper-

oxide, and singlet oxygen (
1
O2). The hydroxy free radical  

 
 

 
*Corresponding author. E-mail: fangjun1973@yahoo.com.cn. 

 
Abbreviations: PSB, Peptides of swine blood; DM, diabetes 
mellitus; BHA, butylated hydroxyanisole; BHT, butylated 
hydroxytoluene; VC, vitamin C; VE, vitamin E; CL, luminous 

intensity. 

 
 
 

 
is the most active and also the most damaging to 
organisms (Gutterridge 1994; Tabatabaie et al. 2003; 
Peng et al., 2005; Zhang et al., 2005). Common oxygen 
radical scavengers include antioxidant enzymes such as 
superoxide dismutase, glutathione peroxidase, catalase 
(Fang et al., 2003), and endogenous and exogenous 
antioxidants such as vitamin C (VC), vitamin E (VE) and 
β-carotene. Recent reports have indicated that the 
hydrolyzed product of soybean protein and casein had 
certain antioxidant activity (Hou et al., 2008).  

Swine blood is a valuable resource in China due to its 
high protein content. However, since the protein from 
dried blood powder is hard to digest and has poor pala-
tability most of it is thrown out, which poses a serious 
threat to the environment (Fang et al., 2004). To solve 
these problems, peptides from swine blood (PSB) was 
obtained by the fermentation of swine blood (Fang et al. 
2005) followed by micro-filtration, ultra-filtration, and 
spray-drying. These peptides have been shown to have 
some biological activities. In this study, the components of 
PSB were analyzed by a mass spectrometer, infrared 
spectrometer, and automatic amino acid analyzer. The 
ability of these peptides to scavenge ultra oxygen anion free 
radicals, hydroxy free radicals and hydrogen peroxide was 

examined by a chemical luminescence detection system. 



 
 
 

 
MATERIALS AND METHODS 
 
Materials 
 
Bacillus subtilis Strain A32 was obtained from a slaughterhouse 
(GenBank, Accession No. DQ631809), PSB was obtained by 
fermentation of swine blood with A32, micro-filtration, ultra-filtration, 
and spray-drying. Luminol (chromatography grade) and catechin 
were obtained from Sigma and Hunan Kinglong BIO-resources 
Products Industry Co., LTD, respectively. 

 

Methods 
 
The molecular weight distribution of PSB was measured by mass 
spectrograph (MS) 4307 (Bruker, Munich, German). Mode of 
operation: reflection; extraction mode: delayed; polarity: positive 
acquisition; manual; accelerating voltage: 200000 V; grid voltage: 
68%; acquisition mass range: 850-5000Da; number of laser shots: 
150/spectrum. The amino acid composition was measured by an 
automatic amino acid analyzer L - 8800 (Hitachi, Tokyo, Japan). 

Column: Na
+
 exchange column (4.6 ID × 40 mm); detector: UV-

visible light detector; reagent: ninhydrin/sodium acetate buffer; buf-
fer system: citric acid buffer B1 (pH3.2), B2 (pH3.0), B3(pH 4.0), 
B4(pH4.9), column temperature: 55°C, reaction temperature: 

135°C, flow rate: 0.4 ml/min. The ability to scavenge O2
.-
 was 

determined according to the method of Xu et al. (2001). In this 
procedure, pyrogallol (10 μl mM) and luminol-carbonate buffer (940 
μl and pH 10.2) were added to 50 μl samples and mixed 
completely, and the chemical luminescence power curve was 

measured for 20 s. The ability to scavenge 
.
OH was analyzed 

according to the method of Fan et al. (1998). Thus, 50 μl mM 
CuSO4, 50 μl mM 1,10-phenanthroline, and 700 μl boric acid-borax 
buffer (pH 9.0) were added to 50 μl samples with thorough mixing. 
Next, 100 μl mM antiscorbutic acid and 50 μl 0.1 mM H2O2 were 
added, and the chemical luminescence power curve was recorded 
for 150 s. The ability to scavenge H2O2 was determined according 
to the method of Xu et al. (2001). In this method, to 50 μl samples 
(distilled water as a control) were added 50 μl 0.15% H2O2 solution 
and 900 μl luminol-carbonate buffer (pH 10.2). After thorough 
mixing, the chemical luminescence power curve was measured for 
60 s. The ability to scavenge oxygen free radicals was determined 
according to the method of Wang et al. (2003). A certain level of 
luminous intensity (CL) is associated with the oxygen free radical-
scavenging capacity. Therefore, CL can be used to show the rela-
tive output of oxygen free radicals. Furthermore, materials that can 
scavenge oxygen free radicals can reduce CL, so a material’s 
capacity to scavenge oxygen free radicals could be measured acc-
ording to the decrease in CL: 
 
Led inhibition rate = (CL control - CL samples) / CL control 
 
A luminous inhibition curve was obtained with the sample concen-
tration as the abscissa and the LED inhibition rate as the ordinate. 
The IC 50 was the concentration at which the rate was 50%. A 
small IC 50 was associated with strong oxygen radical-scavenging 
capacity, and vice versa (Wang et al. 2003). The assays were con-
ducted in triplicate.  

The amino acid composition of PSB was measured by an 
automatic amino acid analyzer L-8800 as described by Li et al 
(2008) and Kong et al (2008). 

 

RESULTS 
 
Molecular composition of PSB 
 
Figures 1 and 2, respectively, show the molecular 

 
 
 
 

 

composition of swine blood powder and PSB. PSB was 
obtained from fermented swine blood by micro-filtration 
(0.05 μm ceramic membrane), ultra-filtration (1000 Da), 
and spray-drying. The molecular mass of PSB was less 
than 2000 Da, and in most cases about 1000 Da.  
 

 

Amino acid content of PSB 

 

Figures 3 and 4 show that he contents of glutamine, 
glutaminic acid, valine, methionine, isoleucine, tyrosine, 
leucine, pheny-lalanine, lysine, histidine in PSB were 
about 10 - fold greater than those in swine blood powder. 
Half-cystine was increased nearly four times, and 
threonine was about doubled. PSB had a good balance 
of amino acids and abundant essential amino acids, and 
the imbalance of leucine and isoleucine was improved 
compared to that in swine blood powder. At the same 
time, the contents of taste-related amino acids such as 
glutamine (fresh) and glycine (sweet) were very high, and 
this improved the nutritional value and flavor of PSB. 
 

 

Ability of PSB to eliminate free radicals 

 
Figure 5A shows that all of the antioxidants tested 
decreased both the peak value and the area of luminous 

curves for H2O2. PSB, catechin and VC could scavenge 

H2O2, with IC 50 values of 1.27, 0.45, and 1.31 mg·mL-1, 
respectively (Table1). The ability of catechin to scavenge 

H2O 2 was obviously higher than those of PSB and VC. The 

ability of PSB to scavenge H2O2 was slightly stronger than 
that of VC, but this difference was not significant.  

Figure 5B shows that all of the antioxidants tested 
could decrease both the peak value and the area of 

luminous curves for O2
.-
 PSB, catechin and VC could 

scavenge O2
.-

, with IC 50 values of 3.42, 1.19, and 0.45 
mg·ml-1, respectively (Table 1). The ability of VC to 

scavenge O2
.-
 was obviously higher than those of 

catechin and PSB. The scavenging capacity of PSB was 
weaker than that of catechin.  

PSB, catechin, and VC each reduced both the peak 
value and the area of luminous dynamics curves for ·OH. 
However, there were large differences in the ·OH-
scavenging capacities of these materials. The peak value 
and area of luminous dynamics curves of PSB were 
much lower than those of caffeine and VC (Figure 5C). 
Their respective IC 50 values were 0.66, 14.23, and 
16.74 mg·ml-1 (Table 1). The ·OH-scavenging capacity 
of PSB was nearly 22 times greater than that of catechin 
and 25 times greater than that of VC. 
 
 
DISCUSSION 

 
PSB had a molecular mass of less than 2000 Da, and most 

were about 1000 Da. When the molecular mass of peptides 

is less than 850 Da, the results obtained with the 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 1. The molecular weight distribution of swine blood which was spray-dried was measured by a mass spectrograph. 



    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. The molecular weight distribution of PSB was measured by a mass spectrograph; PSB was obtained by fermentation of swine blood with A32, micro-filtration, ultra-filtration, and 
spray-drying. 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

I
n
t
e
n
s
i
t
y
(
m
V
)

 
 
 

 
 

400  

M e t
 

L y s
 

350  

 

300  

 

 

250 

G l u
 

P h e
 

 

  
 

 

 

200  

 
 

150       
 

100  

A l a
 

  N H 3
  

 

     
 

      
 

50  

V
a
l

 

I
l
e
 
 

L
e
u

 

   
 

0 

T h r
  H i s
  

 

      
 

0 5 10 15 20 25 30 
 

 
 

Retention Time (min) 
 
Figure 3. The amino acid composition of PSB was measured by an automatic amino acid analyzer L-8800. PSB was obtained by fermentation of swine blood with A32, micro-filtration, ultra-
filtration, and spray-drying. 

 

 
 



 

 

80  
 

 

G
l
u
 

 
 

 

60  
 
 
 
 
 

 

 40 

M e t
 

P h e
 

L y s
 

 

  
 

I
n
t
e
n
s
i
t
y

(
m
V
)
 

A
l
a
 

V
a
l
 

  
  

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N H 3
 

 
 

T
h
r
 

I l e u
 

H i s
 

A r g
 

 

L e
 

  

 

 

 

0 5 10 15 20 25 30 

 

Retention Time (min) 

 
Figure 4. The amino acid composition of swine blood was measured by an automatic amino acid analyzer L-8800. Swine blood was spray-dried. 



  
 

A 

 

 350000        
 

 300000        
 

c
d

) 

250000        
 

         

no
u

s(
 

200000        
 

        
 

l u
m

i 150000        
 

100000        
 

 
50000 

A       
 

        
 

 0        
 

 A 
1 10 20 30 40 50 60 

 

  
   

Ti me( s) 
 

Figure 5A Scavenging of H2O2 by the various substrates used. 

 
 
 
 
 

 

 cont r ol   
 cat echi n 

 
PSB 

 
VC 

 

 
B 

 

 60000 
 

 50000 
 

c
d

) 

40000  

（ 

 

  
 

u
m

i n
o
u

s 

30000 
 

20000 
 l   
 

 10000 
 

B 0 
  

1 4 8 12 16 20 
 

Ti me（s） 
 

Figure 5B. Scavenging of O2
.-
 by the various substrates used. 

 
 
 
 
 
 
 
 
 

 cont r ol   
 VC  
 PSB  

 cat echi n 

 

 

Applied Bio-systems Voyager System 4307 are not 
precise, since they are influenced by the instrument itself. 
Thus, the optimum range for the molecular mass was 
chosen to be from 850 to 5000 Da. PSB with a different 
molecular weight distribution was also obtained in the 
ionization process, and the peak value of some peptides 
could not be scanned. Therefore, the PSB samples con-
tained some pieces of peptides.  

PSB could clearly scavenge O2
. -

, H2O2 and ·OH with 

IC 50 values of 3.42, 1.27, and 0.66 mg·mL-1. PSB is rich 
in hydrogen, and can provide a hydrogen proton, which 
can reduce highly oxidized oxygen radical. Thus, it can 
terminate the chain-reaction of oxygen radical and can 
scavenge or inhibit oxygen radical. The hydroxy free radi-
cal is the most active and dangerous among numerous 
oxygen radicals (Rong 2001). PSB had the strongest 

 

 

hydroxy free radical-scavenging activity, which suggests 
that PSB may be useful as an oxidation inhibitor.  

In addition, the oxygen radical-scavenging activities of 
catechin and VC in this study were the same as those 
reported by Hu (2004), but there was a difference in the 
IC 50 values. This discrepancy may have been due to the 
fact that the catechin and VC reagents were produced by 
different companies and the duration of recording the 
illumination curve also differed.  

At present, the use of chemical antioxidant additives 
like butylated hydroxyanisole (BAH) and butylated hydro-
xytoluene (BHT) has been limited due to considerations 
regarding food safety. The use of some natural anti-
oxidants such as VE and herbal extracts has also been 
limited due to their high cost, their effects on food flavor 
and color. Furthermore, oxidative stress can strongly 



 
 

 

  C  
 

 

70000 
    

 

     
 

 60000     
 

（
c
d

) 50000     
 

40000 
    

 

n
o

u
s     

 

30000 
    

 

l u
m

i     
 

20000 
    

 

     
 

 10000     
 

 
0 

    
 

 

1 

  
 

 C  
15  

     
  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
30 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
C 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 

60 75 90 105 120 135 150  
Ti me（s) 

 
 
 
 
 
 
 
 
 

 
 cont r ol   
 PSB  
 VC  
 cat echi n  

 

 
Figure 5C. Scavenging of ·OH by the various substrates used. 

 

 
Table 1. Radical-scavenging activities of different antioxidants. 

 

Antioxidants H2 O2 O2
.-
 ·O H 

PSB 1.27±0.13
a
 3.42±0.23

a
 0.66±0.14

a
 

Catechin 0.45±0.09
b
 1.19±0.14

b
 14.23±2.11

b
 

VC 1.31±0.11
a
 0.45±0.06

c
 16.74±2.60

c
 

 
abc

 Values with different lowercase letters are significantly different (P < 0.05). Values 

are given in IC 50(mg· ml
-1

). 

 
influence animal's growth. Thus, the identification of minimally toxic 
and yet highly effective natural antioxidants is important for 
maintaining health, promoting animal growth and enhancing animal 
performance.  

PSB may be produced by the fermentation of swine blood with 
strain A32. The production cost is very low, regardless of whether it 
is to be used as an antioxidant additive in food or feed. At present, 
there have been few reports on PSB, and this promises to be a 
fruitful field of research. 

 
ACKNOWLEDGEMENTS 
 
Dr. Jun Fang is grateful for financial support from the 
China Postdoctoral Science Foundation (20070410984), 
the Department of Science and Technology of Changsha  
City (k0904013-21 ） and Hunan Agricultural University 

(07YJ07). 
 

 

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