Ljubia . aria, , ,
Bojana M. aria,
Anamarija I. Mandia,
Aleksandra M. Torbicaa,
Jelena M. Tomia,
Dragoljub D. Cvetkovib,
ore G. Okanovia
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doi:10.1016/j.idairyj.2012.03.007
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Abstract
The aim of this study was to investigate the antibacterial properties and the
protein profile, with an emphasis on the lysozyme and lactoferrin, of raw donkeys
milk from an autochthonous Serbian breed. The antibacterial activity
against Escherichia coli andSalmonella enteritidis during 96 h of storage at
different temperatures and changes in the microflora during 6 d of storage at 4 C
were examined. Investigation of artificially contaminated samples indicated that
the most favourable temperature for the antibacterial activity against E. coli was
15 C and against S. enteritidis was 9 C.Clostridium perfringens, coagulase
positive staphylococci, fungi, Salmonella spp. andE. coli were not detected after
6 d of storage at 4 C, indicating strong antimicrobial activity of Domestic Balkan
donkeys milk against these microorganisms. Lysozyme and lactoferrin content in
the donkeys milk samples tested were 1.31 g L1 and 4.80 mg L1, respectively.
1. Introduction
Milk, as a highly nutritious food, presents a suitable environment for the growth of
many microorganisms (Varga, 2007). On the other hand, milk contains a number
of antimicrobial factors, especially milk proteins, which play a major role in milk
protection. The iron-binding glycoprotein lactoferrin and the enzymes lysozyme
and lactoperoxidase are marked as the main antimicrobial agents in milk. There
is a great variation in their concentrations in the milk of different species (Clare
et al., 2003 and Floris et al., 2003).
Despite the fact that literature sources on donkeys milk are very limited, there
are some reports of its strong antimicrobial properties (Tidona et al.,
2011 and Zhang et al., 2008). Lysozyme is considered as the main defence
factor in donkeys milk (Vincenzetti et al., 2008), followed by lactoferrin and other
naturally occurring inhibitory substances (Coppola et al., 2002 and Zhang et al.,
2008). Donkeys milk is regarded as a very rich source of lysozyme (1 g L1)
(Vincenzetti et al., 2008) and has significantly higher content of lysozyme than
e.g., human milk (0.4 g L1) and bovine milk (0.13 mg L1), while the level of
lysozyme in donkeys milk is quite similar to that in equine milk (0.41 g L1)
(Floris et al., 2003).
Research interest in the nutrient composition, health effects and antibacterial
activity of donkeys milk has increased in recent years, although its therapeutic
effects have been known since ancient times. Donkeys milk is favoured as a
potential new dietetic food and a good alternative for infant nutrition in the case of
bovine milk protein allergy (Iacono et al., 1992, Monti et al., 2007 and Vita et al.,
2007), because of its low casein content, high percentage of essential amino
acids and protein and lipid profiles similar to those of human milk (Fantuz et al.,
2001, Salimei et al., 2004 and Vincenzetti et al., 2008).
Currently, donkeys milk is mainly consumed in those countries where donkeys
are traditionally bred, especially in Africa, Asia and Eastern Europe (Blench,
1999, Kugler et al., 2008, Tadesse, 2010 and Zhang et al., 2008). Considering
the recent studies of potential health benefits, an increase in the production of
donkeys milk is expected.
The Domestic Balkan donkey is an autochthonous breed primarily farmed in the
Northern and Eastern regions of Serbia, with a total population size of about
1000 (FAO DAD-IS, 2009 and Kugler et al., 2008). The Special Nature Reserve
Zasavica, near Sremska Mitrovica, located in the northwest of Serbia, currently
has over 100 female donkeys belonging to the Domestic Balkan donkey breed.
The objective of this study was to investigate the changes in the natural
microflora of raw Domestic Balkan donkeys milk during storage at 4 C, as well
as its protein profile, with an emphasis on the major antimicrobial compounds,
such as lysozyme and lactoferrin. The natural antibacterial potential of the tested
milk at different storage temperatures, against some human pathogens that
belong to the family Enterobacteriaceae was also examined.
The antibacterial assay against two bacterial strains was carried out using E.
coli ATCC 10536 and Salmonella enteritidis ATCC 13076. Cultures were stored
on nutrient agar slants in a refrigerator at 4 C and subcultured on fresh slants
weekly.
Donkeys milk samples were artificially contaminated with these two bacterial
strains at a level of 105 cfu mL1. After overnight incubation on nutrient agar at
37 C, well-isolated colonies of each test microorganisms were selected and
transferred with an inoculating loop to a tube of sterile saline and vortexed
thoroughly. The density of the bacterial suspension was adjusted to a turbidity
equal to the 0.5 McFarland standard using a DEN-1 densitometer (Biosan, Riga,
Latvia).
Contaminated samples were dispensed into sterile Stomacher bags (25 mL
each) and stored at 3, 6, 9 and 15 C for 96 h. Ten-fold sequential dilutions of the
samples were made in 0.1% peptone saline. The enumeration of E. coli was
done by pour plating on tryptone bile X-glucuronide (TBX) agar (Himedia) and
incubating for 1824 h at 44 C.S. enteritidis was enumerated by pour plating on
xylose lysine desoxycholate (XLD) agar (Himedia) and incubation at 37 C for
24 h 3 h. The number of viable bacteria was expressed as log cfu mL1.
Nutrient broth (Himedia) and commercially available UHT bovine milk, inoculated
with 105 cfu mL1 of test microorganisms were used as positive controls whereas
non-inoculated donkey milk was used as a negative control. All experiments were
performed in triplicate.
2.3.1. Determination of protein profile
Sample preparation was carried out according to Tidona et al. (2011) with some
modifications. Milk samples were diluted in 1:1.5 (v/v), sample:buffer
(0.125 M TrisHCl, 4% SDS, 2% glycerol, 2% -mercaptoethanol, pH 6.8) and
heated at 100 C for 5 min.
The chip-based separations were performed on the Agilent 2100 bioanalyzer
(Agilent Technologies, Santa Clara, CA, USA) in combination with the Protein 80
Plus LabChip kit and the dedicated Protein 80 software assay on 2100 expert
software. Chips were prepared according to the protocol provided by the Protein
80 LabChip kit. Fractioning is size-based, and the profiles show the smallest
proteins emerging first in the profiles but at the bottom of the gel patterns,
according to the convention for SDS-PAGE (Torbica, ivanev, Nikoli, orevi,
& Nikolovski, 2010). Bovine serum albumin was used as the standard for
quantitation of the milk proteins. All samples were analyzed in triplicate.
2.4. Statistical analysis
Results were expressed as mean standard deviations, of triplicate analyses for
all measurements. Analysis of variance was followed by Duncans multiple
comparison tests using STATISTICA version 10 (StatSoft Inc., Tulsa, OK,
USA). P values < 0.05 were regarded as significant.
3. Results
3.1. Changes in the microflora during storage at 4 C
Total bacterial counts at day 0 and day 5 were not significantly different
(P < 0.05), while on day 6 this count reached 6.4 log cfu mL1 ( Table 1). Lactic
acid bacteria count increased slightly whereas bacterial endospores remained at
a low level (<2 log cfu mL1) during storage. In the first experiment (milk collected
in January 2011), Enterobacteriaceae and coliforms were not detected until day 2
and day 4, respectively. In the milk samples collected in October and November
2011 these bacteria were not detected during the entire period of
storage. Salmonella spp., E. coli, C. perfringens, coagulase positive
staphylococci, yeasts and moulds were not detected in any of the samples.
Table 1.
Microbial quality of donkeys milk during storage at 4 C.a
Storage period (days)
Microbial
groups/microorg
anismsb
0
1
2
3
4
a
a
a
a
TBC
4.57 (0.29 4.65 (0.30 4.66 (0.27 4.73 (0.31 4.91a(0.40
)
)
)
)
)
a
a
a
a
LAB
2.31 (0.39 2.34 (0.40 2.49 (0.36 2.54 (0.33 2.60a,b(0.1
)
)
)
)
6)
a
a
b
b
BS
1.30 (0.00 1.30 (0.07 1.59 (0.16 1.60 (0.16 1.73b,c(0.0
)
)
)
)
1)
a
Enterobacteriac n.d.
n.d.
n.d.
0.60 (0.16 0.78b(0.07
eae
)
)
Coliforms
n.d.
n.d.
n.d.
n.d.
n.d.
a
5
6
a,b
5.21 (0.5 6.41b(0.21)
5)
2.71a,b(0.1 2.82a,b(0.19
3)
)
c
1.83 (0.03)1.15c(1.06)
1.00c(0.02)1.04c(0.03)
0.85a(0.05 1.00b(0.07)
)
Results are expressed in log cfu mL1. Each value is the mean of three independent
experiments. Standard deviation values are given in parentheses. Means with different
superscript letters in the same row are significantly different (P < 0.05).
b
Abbreviations are: TBC, total bacteria count; LAB, lactic acid bacteria; BS, bacterial
endospores; n.d., not detected.
Table options
Fig. 1.
E. coli count changes in donkeys milk stored at different temperatures over 96 h: ,
15 C; , 9 C; , 6 C; , 3 C. Each value is the mean of three independent experiments,
with the standard deviation indicated by vertical error bars. Different letters in the same
bar indicate a significant difference (P < 0.05) between values according to the Duncans
multiple range test.
Figure options
and 6.0 log cfu mL1, respectively. The decrease in E. coli count was higher at
3 C and 6 C than at 9 C (Fig. 1), whereas the E. coli count remained at a
constant level in both positive controls.
S. enteritidis counts in donkeys milk stored at 15 C remained at a constant level
during the first day of storage, after which it increased to 7.1 log cfu mL1 ( Fig. 2).
The presence of an exceedingly large number of this microorganism after the
48 h of storage at 15 C indicates that donkeys milk did not possess any
remaining antibacterial activity, and therefore it was not further examined. In
bovine milk, the S. enteritidis count increased to 8.0 log cfu mL1 after 48 h while
in nutrient broth, it was 8.6 log cfu mL1. At 9 CS. enteritidis count decreased
during 72 h of storage, reaching the value of 4.2 log cfu mL1 ( Fig. 2). After 96 h
of storage at 9 C S. enteritidis count was 6.2 log cfu mL1 in bovine milk and
6.6 log cfu mL1 in nutrient broth.
Fig. 2.
S. enteritidis count changes in donkeys milk stored at different temperatures over 96 h:
, 15 C; , 9 C; , 6 C; , 3 C. Each value is the mean of three independent
experiments, with the standard deviation indicated by vertical error bars. Different letters
in the same bar indicate a significant difference (P < 0.05) between values according to
the Duncans multiple range test.
Figure options
At 3 and 6 C, the S. enteritidis count reached the lowest level after 96 h ( Fig. 2).
In both positive controls during the storage at 3 and 6 C the count of these
bacteria remained constant.
3.3. Donkeys milk protein characterization
The gel image of donkeys milk is presented in Fig. 3. The obtained lysozyme
concentration in tested milk samples was 1.31 g L1, whereas lactoferrin content
was 4.80 mg L1. According to Tidona et al. (2011), lysozyme and lactoferrin were
identified as proteins with molecular weight of 15 and 78.1 kDa, respectively.
Results of the chip-based separation show a pattern similar to reported data for
donkeys milk found in the literature (Salimei et al., 2004 and Tidona et al., 2011).
Fig. 3.
Lab-on-a-Chip gel images of: lane 1, molecular mass ladder (1.6, 3.5, 6.5, 15, 28, 46, 63
and 95 kDa); lane 2, donkeys milk sample. Arrows indicate the position of lactoferrin (LF)
and lysozyme (LZ).
Figure options
4. Discussion
The results of the total bacterial count in the tested donkeys milk were similar to
those obtained by other authors, i.e., 4.24.6 log cfu mL1 (Chiavari et al.,
2005, Coppola et al., 2002 and Zhang et al., 2008). Generally, raw donkeys milk
has lower total bacterial count compared with that in milk from other sources
5. Conclusions
Acknowledgements
This work is a part of the National Project (TR-31029) financially supported by
the Ministry of Education and Science, Republic of Serbia. We are grateful to Mr
Slobodan Simi (Special Nature Reserve Zasavica, Serbia) for providing the
milk samples.
References
1.
o
AOAC, 2002
AOAC
o
2.
o
AOAC, 2003
AOAC
3.
o
Blench, 1999
R. Blench
o
o
4.
o
Antimicrobial peptides and proteins of the horse insights into a wellarmed organism
5.
|
Full Text via CrossRef
|
Citing articles (26)
6.
o
Article
|
PDF (205 K)
|
8.
o
9.
o
EC, 2004
EC
o
10.
o
o
|
Article
|
PDF (142 K)
|
View Record in Scopus
|
Full Text via CrossRef
|
Citing articles (43)
12.
o
FAO DAD-IS
o
13.
o
Article
|
PDF (309 K)
|
View Record in Scopus
|
Citing articles (296)
14.
o
15.
o
16.
o
Grubb, 2005
P. Grubb
Order perissodactyla
D.E. Wilson, D.M. Reeder (Eds.), Mammal species of the world: A taxonomic and
geographic references (3rd ed.), Johns Hopkins University Press, Baltimore, MD, USA
(2005), pp. 629633
o
17.
o
18.
o
ISO, 2001
ISO
Microbiology of food and animal feeding stuffs Horizontal method for the
enumeration of beta-glucuronidase-positive Escherichia coli Part 2: Colony count
technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide
o
19.
o
ISO, 2003a
ISO
Microbiology of food and animal feeding stuffs Horizontal method for the
enumeration of microorganisms Colony count technique at 30 degrees C
o
20.
o
ISO, 2003b
ISO
Microbiology of food and animal feeding stuffs Horizontal method for the
enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other
species) Part 1: Technique using BairdParker agar medium
o
1.
ISO, 2004
ISO
Microbiology of food and animal feeding stuffs Horizontal method for the
enumeration ofClostridium perfringens Colony count technique
ISO standard 7937 International Organization for Standardization, Geneva,
Switzerland (2004)
o
2.
o
ISO, 2006
ISO
Microbiology of food and animal feeding stuffs Horizontal method for the
detection ofSalmonella spp.
ISO standard 6579: 2002/AC: 2006 International Organization for
ISO, 2008
ISO
Microbiology of food and animal feeding stuffs Horizontal method for the
enumeration of yeasts and moulds Part 1: Colony count technique in products with
water activity greater than 0.95
ISO standard 21527-1 International Organization for Standardization, Geneva,
Switzerland (2008)
o
4.
o
5.
o
o
6.
o
Protein and fat composition of mares milk: some nutritional remarks with
reference to human and cows milk
Article
|
PDF (136 K)
|
View Record in Scopus
|
Citing articles (106)
7.
o
8.
o
Article
|
PDF (94 K)
|
View Record in Scopus
|
Citing articles (38)
9.
o
10.
o
11.
o
Tadesse, 2010
T. Tadesse
Article
|
PDF (568 K)
|
View Record in Scopus
|
Citing articles (21)
13.
o
14.
o
Varga, 2007
L. Varga
and trends in applied microbiology, Formatex, Badajoz, Spain (2007), pp. 487494
View Record in Scopus
o
|
Article
|
PDF (796 K)
|
View Record in Scopus
|
Citing articles (47)
16.
o
Asss milk in children with atopic dermatitis and cows milk allergy:
crossover comparison with goats milk
17.
o
The antimicrobial activity of donkey milk and its microflora changes during
storage
Article
o
|
PDF (126 K)
|
View Record in Scopus
|
Citing articles (27)
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