International Dairy Journal

Volume 25, Issue 2, August 2012, Pages 142146

Antibacterial properties of Domestic Balkan donkeys milk

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.

2. Materials and methods

2.1. Sample collection
Donkeys milk samples were collected on three occasions, i.e., in January,
October and November 2011. Each time, donkeys milk samples were collected
immediately after morning milking from Zasavica Special Nature Reserve. The
milk from 8 manually milked donkeys was collected, mixed and kept in an ice box
at 4 C during transport. The milk samples were divided into two equal parts. Half
of the collected milk was frozen at 20 C until analysis of antimicrobial activity
against added bacteria. The rest of the milk was divided into seven sub-samples
of 50 mL each, designated to study the variability in microbial population during
6 d of storage at 4 C. All analyses of sub-samples from three sampling times
were performed in triplicate.
2.2. Determination of changes in microbial population during the storage
Changes in microflora during 6 d of storage at 4 C were monitored on donkeys
milk samples by enumeration of total count of bacteria (ISO, 2003a), yeasts and
moulds (ISO, 2008), coagulase positive staphylococci (ISO, 2003b), betaglucuronidase-positiveEscherichia coli ( ISO, 2001) and Clostridium
perfringens ( ISO, 2004). Enumeration of bacterial endospores was performed by
incubation of previously heated (100 C, 5 min) milk samples on nutrient agar
(Himedia, Mumbai, India) at 30 C for 72 h. The lactic acid bacteria count was
determined by incubation (30 C, 72 h) of inoculated Man, Rogosa and Sharpe
(MRS) agar (LabM, Bury, UK). Coliform bacteria and Enterobacteriaceae were
determined according to AOAC (2002) and AOAC (2003), respectively. Detection
ofSalmonella spp. was carried out according to ISO (2006). All experiments were
performed in triplicate.
2.3. Antibacterial assay

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

3.2. Antibacterial activity of donkeys milk

After the first day of storage at 15 C, the E. coli count significantly decreased
( Fig. 1). The lowest value was obtained on day 3, while the increase
in E. coli count was observed at day 4.

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

E. coli counts increased constantly in tested positive controls. After 96 h the

number of these bacteria was 8.1 log cfu mL1 in bovine milk and
7.2 log cfu mL1 in nutrient broth.
During storage at 9 C (Fig. 1), the E. coli count decreased constantly, and after
96 h reached the value of 5.0 log cfu mL1. In bovine milk and nutrient broth the
count of this pathogen slowly increased and after 96 h reached the values of 5.9

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

(Chye et al., 2004 and Morgan et al.,

2003). Salmonella spp., E. coli, C. perfringens, coagulase positive staphylococci,
yeasts and moulds were not detected during storage at 4 C. Data in the
literature vary with respect to the numbers of yeasts and moulds in donkeys
milk. Coppola et al. (2002)reported 4.5 log cfu mL1 while Zhang et al.
(2008) reported significantly lower count of these microorganisms, i.e.,
0.7 log cfu mL1.
The antimicrobial activity of the tested milk samples can also be illustrated by the
fact that bacterial endospores and Enterobacteriaceae were not detected during
the storage period or their counts were very low (Table 1).
The lactic acid bacteria count reported in this study was considerably lower in
comparison to data reported by Zhang et al. (2008) obtained under comparable
storage conditions. The strong antimicrobial activity of the tested donkeys milk
against lactic acid bacteria could possibly indicate its limited potential for the
production of fermented milk products. Zhang et al. (2008) reported high number
of coliforms during 4 d storage at 4 C (3.7 log cfu mL1) while in the milk samples
tested here, these microorganisms were not detected.
The results of in situ studies indicate that 15 C is the most favourable
temperature for antibacterial activity against E. coli. Enzymes, as the main
antimicrobial agents in donkeys milk, also show their activity on indigenous
microflora ( Gaya, Medina, & Nunez, 1991). Therefore, low antimicrobial potential
of the tested milk against E. coliafter 72 h at 15 C could possibly indicate
intensive growth of the other microorganisms at this temperature and their
consumption of enzymes with antimicrobial activity.
At 39 C, the E. coli count showed a decreasing trend with differences in final
value of the bacterial counts. The higher decrease in E. coli count at 3 and 6 C
compared with 9 C can possibly be attributed to the increased sensitivity of
bacterial cells to lysozyme at lower temperatures. The smaller size of bacterial
cells at lower temperatures causes lower amount of biomass for the same
number of cells, making them more sensitive to lysozyme ( Johansen, Gram, &
Meyer, 1994).
Analyses of artificially contaminated samples at 15 C indicated lower
antibacterial activity of the milk against S. enteritidis compared with that
against E. coli, which could possibly be explained by a higher growth rate
of S. enteritidis compared with E. coli, leading to higher consumption of

antimicrobial compounds present in milk. The highest antibacterial potential of

the milk against S. enteritidis was observed at 9 C.
Protein characterization of the tested donkeys milk by electrophoresis indicates
lysozyme as the main antimicrobial agent. The obtained concentration of
1.31 g L1 is in accordance with the results of Vincenzetti et al. (2008). Recent
studies also marked lysozyme as the most abundant antimicrobial agent in
donkeys milk (Coppola et al., 2002, Vincenzetti et al., 2008 and Zhang et al.,
2008).
Although Gram-negative bacteria are generally resistant to lysozyme due to their
lipopolysaccharide (LPS) layer (Ellison and Giehl, 1991 and Farnaud and Evans,
2003), the activity of donkeys milk against them could possibly be explained by
the specific structure of lysozyme. The donkey is a domesticated member of the
Equidae family (Grubb, 2005), so the structure of donkey lysozyme could be
similar to that of equine lysozyme. The ability of equine lysozyme to bind calcium
ions makes the enzyme more stable and improves its antibacterial activity
against some Gram-negative bacteria including E. coli ( Bruhn, Grtzinger,
Cascorbi, & Jung, 2011). Pellegrini, Waiblinger, and Von Fellenberg
(1991) reported a slight bactericidal activity of equine lysozyme
towardsS. enteritidis.
A synergistic activity of lysozyme and lactoferrin could also contribute to
antibacterial activity of donkeys milk. Interaction of lactoferrin with LPS can
cause disruption of the outer membrane and enhance susceptibility of Gramnegative bacteria to lysozyme by increasing in membrane permeability (Ellison
and Giehl, 1991 and Farnaud and Evans, 2003). Literature data about lactoferrin
content in donkeys milk are very limited. According to Salimei et al. (2004) its
relative percentage in total whey protein fraction of donkeys milk is 4.8%. The
highest content of lactoferrin (12 g L1) is found in human milk (Floris et al.,
2003), followed by equine milk (0.99 g L1) (Malacarne, Martuzzi, Summer, &
Mariani, 2002) and bovine milk (0.020.3 g L1) (Floris et al., 2003). Lactoferrin
content in the milk analyzed in this study was 4.80 mg L1. Since lysozyme
inhibits a growth of large number of Gram positive bacteria (Clare et al.,
2003 and Floris et al., 2003) it would be useful to investigate the antibacterial
activity of Domestic Balkan donkeys milk against them.

5. Conclusions

During 5 d of storage at 4 C, the microbiological quality of raw Domestic Balkan

donkeys milk was in accordance with the requirements of European Regulation
for raw milk from species other than bovine (EC, 2004). The antibacterial activity
against lactic acid bacteria could be a limiting factor for usage of Domestic
Balkan donkeys milk in the production of fermented products. Domestic Balkan
donkeys milk showed antibacterial activity against E. coli and S. enteritidis at all
tested temperatures. The most favourable temperatures for antibacterial activity
towards E. coli and S. enteritidis were 15 C and 9 C, respectively. Antimicrobial
potential and long shelf-life of the raw donkeys milk could possibly be attributed
to the presence of milk proteins and other antimicrobial agents that probably act
in synergistic manner. Present in high concentration, lysozyme could be marked
as the main antimicrobial agent in donkeys milk.

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.

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