Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Biotyping of cultivable lactic acid bacteria isolated from

donkey milk
D. Carminati, F. Tidona, M. E. Fornasari, L. Rossetti, A. Meucci and G. Giraffa
Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (CRA-FLC),
Lodi, Italy

Significance and Impact of the Study: There is increased interest in using donkeys milk as a source of
human nutrition. The large amounts of antimicrobial components and defence factors present in donkeys milk provide protection from microbial infections and distinguish donkeys milk from the milks of
other mammals. However, the microbiota in donkeys milk has so far been poorly characterized, specifically with regard to the lactic acid bacteria (LAB). This study has identified cultivable, acidifying and
thermoduric LAB that could be used to develop starter cultures. This is the first study to investigate the
culturable LAB microbiota present in donkeys milk.

Keywords
dairy, diversity, lactic acid bacteria.
Correspondence
Domenico Carminati, CRA-FLC, Via A.
Lombardo 11, 26900 Lodi, Italy.
E-mail: domenico.carminati@entecra.it
2013/2357: received 26 November 2013,
revised 24 March 2014 and accepted 11 April
2014
doi:10.1111/lam.12275

Abstract
The diversity of lactic acid bacteria (LAB) species in donkeys milk was
analysed by culture-dependent microbial techniques. Dominant strains were
isolated on agar media generally used for enumerating LAB. To enrich the
number of acidifying LAB present, the milk samples were incubated at 37C
for 24 h (cultured milk samples, CM). One of the CM samples was heattreated at 63C for 10 min before incubation at 37C (heat-treated and
cultured milk sample, TCM) to select thermophilic LAB. The microflora in
these CM and TCM samples was then compared to that of the raw milk
samples (RM). Among the 129 LAB isolates, 10 different species (four
Enterococcus, five Streptococcus and one Pediococcus) were identified by
molecular methods. Although the 10 LAB species were present in the RM
samples, only three and two isolates were found in CM and TCM samples,
respectively. Despite the selection protocol being set up to favour the isolation
of all LAB isolates present in donkey milk, relatively few species and biotypes
were isolated. No LAB isolates belonging to the most technologically important
dairy starter species were detected. The possible factors related to the limited
LAB diversity in donkeys milk have been discussed below.

Introduction
Recently, donkeys milk has attracted increasing research
interest due to its nutritional and functional properties.
Its chemical composition is similar to that of human
milk, and therefore, donkeys milk can be used as an
alternative food source for infants who cannot tolerate
bovine milk (Monti et al. 2007). The enzymes in donkeys
milk possess some unique characteristics, such as
bactericidal activity, which make this milk different from
the milk of other mammals (Malacarne et al. 2002;
Tidona et al. 2011). Specifically, lysozyme and lactoferrin
are the most important naturally occurring inhibitory

components present at high concentration in donkeys

milk and play a role in the preservation of its hygienic
quality (Coppola et al. 2002; Chiavari et al. 2005; Zhang
et al. 2008; Saric et al. 2012). An antiviral activity of donkeys milk protein fractions has also recently been demonstrated (Brumini et al. 2013). However, the direct
relationship between the high levels of antimicrobial components and the reduced bacterial load in donkeys milk
is not well established and requires further studies (Conte
et al. 2012). Despite the high content of lysozyme, the
abundance of lactose seems to favour the growth and survival of adapted probiotic lactobacilli, which has been
reported previously by some authors (Coppola et al.

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Lactic acid bacteria in donkeys milk

D. Carminati et al.

2002; Chiavari et al. 2005). Although the chemical composition and nutritional properties of donkeys milk have
been widely investigated, the microbiota in this milk is
less well characterized (Quigley et al. 2013). A few studies
have focused on the detection of pathogens in or the evaluation of the hygienic quality of donkeys milk (Salimei
et al. 2004; Naccari et al. 2009; Pilla et al. 2010; Conte
et al. 2012; Sarno et al. 2012), but studies on donkeys
milk lactic acid bacteria (LAB) are even fewer. The LAB
content as evaluated by plate counts on specific agar
media was found to range between <1
04
2 and <1
03
2
log CFU ml1 on MRS and M17 agar, respectively
(Coppola et al. 2002; Chiavari et al. 2005; Zhang et al.
2008; Tadesse 2010; Salerno et al. 2011; Saric et al. 2012).
Only few LAB isolates present in donkeys milk have been
identified. The LAB isolates presumed to be present in
Ethiopian donkey milk samples were later confirmed as
not belonging to LAB species (Tadesse 2010). Recently,
potentially probiotic and bacteriocinogenic mesophilic
lactobacilli have been described (Nazzaro et al. 2008;
Ashokkumar et al. 2011; Murua et al. 2013).
The aim of this study was to investigate the LAB
microbiota composition of donkeys milk. A stepwise procedure was adopted to estimate LAB content, isolate LAB
strains and assess the diversity of viable and cultivable
LAB present in donkeys milk samples. To circumvent the
difficulties of isolating LAB because of its low numbers in
the microbiota and to select both acidifying and thermoduric LAB isolates, enrichment procedures consisting of
(i) milk culturing and (ii) pasteurization plus milk
culturing (Delgado et al. 2013) were utilized.
Results and discussion
The concentrations of LAB in the donkey raw milk samples (RM) as estimated from the bacterial counts on M17,
MRS and LBD agar media were 3
97 (0
60), 3
57
(0
79) and 3
81 (0
48) log CFU ml1, respectively
(Table 1). These values are within the average range
reported in the literature (Coppola et al. 2002; Chiavari
et al. 2005; Zhang et al. 2008; Tadesse 2010; Salerno et al.
2011; Saric et al. 2012). When the RM samples were incubated at 37C for 48 h (cultured milk samples, CM) to
allow the enrichment of naturally occurring acidifying
bacteria, the pH of CM samples decreased from 7
13
(0
09) to 4
85 (0
49) (Table 1), thus confirming the
presence of lactose-fermenting bacteria. The LAB concentration in CM samples reached about 8
0 log CFU ml1
on M17 and LBD agar media, while they were about 2
logs lower on MRS agar media (Table 1). An increase of
c. 2 logs was also observed by Zhang et al. (2008) after
storing donkeys milk samples at 20C for 24 h, and they
suggested that the natural antimicrobials such as lysozyme
300

Table 1 Lactic acid bacteria count* and pH values of different milk

samples
Samples

pH

M17

LBD

MRS

RM
CM
TCM

7
16 (0
09)
4
85 (0
49)
5
23 (0
35)

3
97 (0
60)
7
72 (0
33)
7
90 (0
18)

3
81 (0
48)
8
27 (0
12)
8
22 (0
13)

3
57 (0
79)
6
16 (0
12)
5
66 (0
26)

*Results expressed in log CFU ml1. Data are means of three

samples.
Standard deviation is given in parentheses.
RM, raw milk; CM, cultured milk; TCM, heat-treated cultured milk.

or lactoferrin did not control the growth of LAB. Conversely, Salerno et al. (2011) observed that upon storing
donkeys milk at 4C, there was a progressive decrease in
lactococci and lactobacilli from 3 to 2 log CFU ml1,
which was attributed to the high lysozyme content. Similar to the CM samples, the heat-treated (63C for
10 min) and cultured milk sample (TCM) became acidified and showed a final pH of 5
23 (0
35) with LAB
counts on different media comparable to that of the CM
samples (Table 1). Raw milk was heat-treated to enable
the selection and recovery of thermoduric and acidifying
LAB species (Delgado et al. 2013). A similar methodology
(i.e. thermization of raw milk at 6265C for 1015 min
followed by incubation at 3745C) has traditionally been
used to prepare artisanal milk-starter cultures that generally contain large numbers of thermophilic streptococci,
enterococci and other LAB species (Carminati et al.
2010).
A total of 150 colonies were isolated from the seven
samples (3 RM, 3 CM and 1 TCM). Each isolate corresponded to a distinct morphotype recognized per plate
and, according to the culture conditions applied, allowed
recovery of the highest diversity among the cultivable
LAB populations. After purification, 129 catalase-negative,
nonmotile and nonsporulating isolates were selected for
biotyping and identification. The majority of isolates were
derived from the RM and CM samples (55 and 57,
respectively) and only 17 from the TCM sample. M17
was the medium where the highest number of colonies
was isolated (63 isolates, 48
8%); this number was significantly higher compared with LBD (36 isolates, 27
9%)
and MRS (30 isolates, 23
3%) (Table 2). The genotypic
diversity evaluated by RAPD-PCR, resulted in grouping
the isolates into 18 clusters (Table 3). Nine clusters were
represented by a single isolate (i.e. clusters B, D, F, O, P,
Q, R, S, T), while the two major clusters, A and N,
included 31 and 57 isolates (with intracluster similarity
coefficient >80%), respectively (Table 3).
The RAPD-PCR analysis led to a preliminary identification of the isolates, thereby allowing investigation of LAB
diversity beyond the species level, that is, different

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D. Carminati et al.

Lactic acid bacteria in donkeys milk

comparing the unknown DNA patterns with patterns

existing for specific groups (each corresponding to a given
taxonomic unit) collected in the CRA-FLC database of
RAPD fingerprints of different LAB species. Although this
approach is generally considered reliable resulting in
>95% identification accuracy (Rossetti and Giraffa 2005),
a few isolates representing each RAPD cluster were
selected for confirmatory identification by 500-bp 16S
rRNA sequencing. This method provided reliable identification of the isolates (henceforth considered as individual
strains in this manuscript) belonging to the genera
Enterococcus, Streptococcus and Pediococcus. Consequently,
all isolates clustering with the representative strains mentioned above were indirectly assigned to the corresponding species.
Most of Enterococcus were isolated from M17 agar
plates (37 strains, 56%), while 16 (24%) and 13 (20%)
strains were isolated from LBD and MRS agar plates,
respectively. Similarly, Streptococcus strains were mainly
isolated from M17 (26 strains, 42%), while 20 (32%) and
16 (26%) were isolated from LBD and MRS, respectively.
The single Pediococcus pentosaceus strain was recovered

biotypes belonging to the same species could be discriminated (Rossetti et al. 2008). Using the BIONUMERICS
software, presumptive identification was obtained by
Table 2 Source of bacterial isolates according to the different types
of milk samples and culture media
Culture media (No. isolates)
Samples
RM
Bulk milk
Martina Franca milk
Romagnolo milk
Total RM
CM
Bulk milk
Martina Franca milk
Romagnolo milk
Total CM
TCM
Bulk milk
Total

M17

LBD

MRS

Total

14
5
4
23

0
7
8
15

10
4
3
17

24
16
15
55

16
5
6
27

0
10
11
21

4
5
0
9

20
20
17
57

13
63

0
36

4
30

17
129

RM, raw milk; CM, cultured milk; TCM, heat-treated cultured milk.

Table 3 Diversity of cultivable lactic acid bacteria isolated from donkeys milk samples. Biotyping of strains belonging to each species was
performed by cluster analysis of the RAPD profiles
Origin of isolates

Species (No. genotypes)*

Enterococcus faecalis (4)

Total Ent. faecalis

Enterococcus faecium/hirae/durans (5)

Total Ent. faecium

Enterococcus gilvus (1)
Enterococcus casseliflavus (1)
Streptococcus macedonicus (2)
Total Strep. macedonicus
Streptococcus equinus/bovis (1)
Streptococcus equi subsp. zooepidemicus (1)
Streptococcus criceti (1)
Streptococcus spp. (1)
Pediococcus pentosaceus (1)
Total

RAPD
genotypes
A
B
C
D
E
F
G
H
I
L
M
N
O
P
Q
R
S
T
18

Sample

Isolation media

Total

RM

CM

TCM

M17

MRS

LDB

31
1
3
1
36
4
1
8
6
2
21
5
4
57
1
58
1
1
1
1
1
129

25
0
3
0
28
1
0
5
0
2
8
5
4
5
0
5
1
1
1
1
1
55

6
1
0
1
8
3
1
3
3
0
10
0
0
39
0
39
0
0
0
0
0
57

0
0
0
0
0
0
0
0
3
0
3 (17
7)
0
0
13
1
14 (82
3)
0
0
0
0
0
17 (100)

10
0
2
1
13
3
1
6
6
2
18
2
4
24
1
25
0
1
0
0
0
63

6
0
1
0
7
1
0
2
0
0
3
3
0
13
0
13
1
0
1
1
1
30

15
1
0
0
16 (44
4)
0
0
0
0
0
0
0
0
20
0
20 (55
6)
0
0
0
0
0
36 (100)

(50
9)

(14
5)
(9
1)
(7
4)

(9
1)
(1
8)
(1
8)
(1
8)
(1
8)
(1
8)
(100)

(14
1)

(17
5)

(68
4)

(100)

(20
6)

(28
6)
(3
2)
(6
3)

(39
7)
(1
6)

(100)

(23
4)

(10
0)
(10
0)

(43
4)
(3
3)
(3
3)
(3
3)
(3
3)
(100)

*Number of different RAPD-PCR genotypes recognized for each species.

Total number of isolates from each sample or isolation medium (percentage in parenthesis) of the different species identified.
RM, raw milk; CM, cultured milk; TCM, heat-treated cultured milk.

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D. Carminati et al.

from MRS agar (Table 3). Although this observation is

not surprising because the culture media used do not
actually have selective properties, it is important to note
that only coccus-shaped LAB were found. LAB populations that exist in donkeys milk are quite unknown
(Quigley et al. 2013), and the few data available refer to
the isolation and characterization of single strains
(Nazzaro et al. 2008; Ashokkumar et al. 2011; Murua
et al. 2013) or to the isolation of some presumed but
unidentified LAB, most of which are coccus-shaped bacteria (Tadesse 2010). Based on studies exploring the ability
of LAB microflora to grow in donkeys milk, Zhang et al.
(2008) speculated that enterococci could be the major
portion of the growing bacteria.
The presence of only coccus-shaped species could be
due to the high lysozyme content in donkeys milk (average value of 1
32  0
12 g l1 in the samples studied,
data not shown). It has been reported that LAB cocci are
more resistant to lysozyme than lactobacilli, and among
lactobacilli, the lysozyme sensitivity is species specific or
strain specific (i.e. thermophilic species are more sensitive
than hetero-fermentative mesophilic lactobacilli) (Neviani
et al. 1991). Our observations are consistent with the
limited studies available on the characterization of
donkeys milk where the only lactobacilli isolated and
identified belong to the mesophilic species Lactobacillus
paracasei, Lactobacillus brevis, Lactobacillus salivarius and
Lactobacillus plantarum (Nazzaro et al. 2008; Ashokkumar
et al. 2011; Murua et al. 2013). Regarding cocci species,
Enterococcus faecalis is known for its extraordinary lysozyme-resisting capacity, although variations in the lytic
response of enterococcal cells have been observed (Hebert
et al. 2007). In Gram-positive bacteria such as Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus
aureus, other mechanisms of lysozyme resistance have
been reported (Le Jeune et al. 2010). Despite the selection
protocol adopted to facilitate the isolation of all cultivable
LAB present in the donkey milk, relatively few species
and strains were isolated. Moreover, none of the isolated
strains were found to belong to the most technologically
important dairy starter LAB species.
Streptococcus macedonicus (recently reclassified as Streptococcus gallolyticus subsp. macedonicus, Schlegel et al.
2003) was found to be the most frequently recovered
species (58 isolates) followed by Ent. faecalis (36) and
Enterococcus faecium/hirae/durans (21) (species not differentiated on the basis of sequence alignment of the first
500 bps of the 16S rRNA). These were the only species
found in CM and TCM samples, while other enterococci
(i.e. Enterococcus gilvus and Enterococcus casseliflavus) and
streptococci were also isolated from the RM samples
(Table 3). Among the coccus-shaped species, Strep. macedonicus is known to be a moderate acidifier in milk,
302

although strains isolated from Italian cheeses and natural

milk cultures are also good acidifiers (Lombardi et al.
2004). The acidifying activity of many Strep. macedonicus
strains isolated in this study was evaluated. Only few
strains were able to coagulate cows milk in 6
5 h and
most did so in 89 h (data not shown), thus confirming
the moderate acidifying capability of this species. These
characteristics make Strep. macedonicus unsuitable as a
primary starter. However, strains that are known to be
bacteriocin producers or possess intracellular peptidases
have been proposed as adjunct cultures in cheese production (Anastasiou et al. 2007). Elucidation of the potential
abilities of the strains recovered needs further investigation. A variety of species belonging to the genus
Enterococcus is found in dairy products, but Ent. faecium
and Ent. faecalis are the most important ones (Giraffa
2003). Enterococci are usually found to exhibit low milk
acidifying capability, but strains of Ent. faecalis and
Ent. faecium with high acidifying capability have been isolated from artisan Italian cheeses (Giraffa 2003). In addition, their recovery from the TCM samples confirmed the
thermotolerance of these enterococcal species. They are
present in natural milk cultures traditionally obtained by
incubating a previously heat-treated raw milk to promote
the selection of thermophilic and heat-resistant LAB (Giraffa et al. 1997; Giraffa 2003; Lombardi et al. 2004).
Among the streptococci isolated from the RM samples, opportunistic pathogens for both humans and
animals such as Streptococcus equi subsp. zooepidemicus
and Streptococcus equinus/bovis (Timoney 2004) were
found. The presence of undesirable Streptococcus species,
such as Strep. equi, Streptococcus equisimilis and other
potentially pathogenic bacteria in donkeys milk, has
been reported previously (Pilla et al. 2010; Salimei and
Fantuz 2012).
The relatively poor diversity of the LAB population
observed in this study could be attributed to several
factors: (i) the origin of the samples that were collected
from a single farm, (ii) the selection process, which
primarily favoured dominant cultivable LAB isolates,
(iii) the higher lysozyme resistance of coccus-shaped
LAB than lactobacilli. Other than these factors, the
enrichment/selection procedures applied to the raw milk
samples could have reduced the recovery of bacterial
diversity significantly. Our data confirmed that although
donkeys milk has low bacterial content, the presence of
some potentially pathogenic species underlines the
importance of providing a suitable heat treatment
before it is used for human consumption or for the
production of fermented milk products. This study
reports the first approach to studying the cultivable
LAB community in donkeys milk. A more representative sampling and the use of culture-independent

Letters in Applied Microbiology 59, 299--305 2014 The Society for Applied Microbiology

D. Carminati et al.

DNA-based techniques will be needed to study the

entire LAB microbiota also including noncultivable and
nondominant populations present in donkeys milk.
Material and methods
Sampling and treatment of milk
Two donkeys milk samples from single-breed animals
(Martina Franca and Romagnolo breeds) and one bulk
milk sample were kindly provided by the farm Azienda
Montebaducco (Salvarano di Quattro Castella, Reggio
Emilia, Italy). After milking, the samples were immediately cooled, transported to the laboratory under refrigerated conditions and stored at 4C for 1224 h before use.
The samples were divided into two equal parts, one
analysed immediately as raw milk (RM), and the other
incubated at 37C for 48 h (CM). Another aliquot of the
bulk milk sample was subjected to heat treatment at 63C
for 10 min and then incubated at 37C for 48 h (TCM).
Taken together, a total of seven samples were analysed.
Microbiological analysis
Serial dilutions of RM, CM and TCM samples were prepared in Ringer solution (Oxoid, Basingstoke, UK). Dilutions were plated on MRS agar, with a final pH of 5
7,
and M17 agar (Merck, Darmstadt, Germany) and on LBD
agar (Lactic Bacteria Differential; HiMedia, Mumbai,
India). To enumerate LAB, RM samples on M17 and
LBD plates were incubated under aerobic conditions at
37C for 48 h, and MRS plates were incubated under
anaerobic condition at 37C for 72 h. CM and TCM samples, plated and incubated on the different media as
described above, were used only for strain isolation after
application of enrichment and selective treatments. Colonies representative of all morphologies and sizes were randomly picked from the three different media and purified
twice in the same isolation medium. Each single colony
was then inoculated in the corresponding liquid medium
and incubated for 24 h at 37C. These cultures were
stored at 80C after addition of 15% sterile glycerol.
Identification of bacteria
The morphology of the bacteria in the purified cultures
was examined by phase-contrast optical microscopy, and
they were tested for catalase activity. The bacterial DNA
was extracted from all the catalase-negative strains for
further genotyping and identification using the Chelex
method described in the MicroSeq protocol following the
manufacturers instruction (Life Technologies, Monza,
Italy).

Lactic acid bacteria in donkeys milk

RAPD (randomly amplified polymorphic DNA) PCR

All isolates were preliminarily identified by RAPD-PCR
fingerprinting according to Rossetti and Giraffa (2005).
The isolated bacterial DNA was used as a template in
PCR fingerprinting using the M13 minisatellite core
sequence as the primer (Huey and Hall 1989) with the
sequence 50 -GAGGGTGGCGGTTCT-30 (Biotez ChemProgress, Milano, Italy). After amplification, PCR products
were analysed by electrophoresis on 1
5% (w/v) agarose
gels (Sigma-Aldrich, Milano, Italy) at 100 V for 2 h in
19 TAE buffer and visualized by GelRed (Biotium, Hayward, CA) staining. One-kb plus DNA Ladder (Invitrogen, Milan, Italy) was used as the DNA molecular weight
marker. Gel images from RAPD analysis were captured
using the Kodak Electrophoresis Documentation and
Analysis System 290 (EDAS 290; Celbio, Milan, Italy) and
processed with the pattern analysis software package
BIONUMERICS (ver. 6.6; Applied Maths, Sint-Martens-Latem,
Belgium). Similarities were calculated based on Pearson
correlation coefficients. Dendrograms were deduced from
the matrices of similarities by the unweighted pair group
method using arithmetic average (UPGMA) clustering
algorithm (Vauterin and Vauterin, 1992). The reproducibility of the RAPD-PCR fingerprinting patterns was evaluated by repeated running of DNA samples from
duplicate amplifications of a control strain. RAPD-PCR
fingerprints were compared with previously mapped
genotypic libraries to obtain preliminary strain identification on the basis of RAPD-PCR profile similarity
(Rossetti and Giraffa 2005).
16S rRNA gene sequence determination
Molecular identification was performed by sequencing a
500-bp fragment of the 16S rRNA of a subset of strains
including strains with a unique RAPD profile and representative strains among those that clustered together for
each cluster. The 500-bp 16S rRNA fragment was amplified from the 50 end of the gene using the forward primer
GCYTAACACATGCAAGTCGA (46 Escherichia coli numbering) and reverse primer GTATTACCGCGGCTGCTGG
(536 E. coli numbering). PCR was carried out in a 50 ll
volume containing 200 lmol l1 of each dNTP, 5
0 ll of
109 Taq reaction buffer, 0
5 lmol l1 each of the two
primers (Biotez, Berlin, Germany), 1
5 mmol l1 of
MgCl2, 1
25 U of AmpliTaq Gold DNA polymerase (Life
Technologies) and 50 ng of genomic DNA extracted as
described above. PCRs consisted of an initial denaturation
step at 95C for 10 min followed by 30 cycles of: 95C
for 30 s, 59C for 30 s and 72C for 45 s and a final
elongation step of 72C for 10 min. Prior to sequencing,
the amplified products were purified using Exo-SAP-IT
(GE Healthcare, Milan, Italy) according to the manufacturers recommendations. The PCR extension reactions

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D. Carminati et al.

were performed in 20 ml with 3
2 pmol l1 of each primer, 4 ml of sequencing mix (Life Technologies) and
7 ml of purified, amplified product. PCR extension consisted of 25 cycles at 96C for 10 s, 50C for 5 s and
60C for 4 min, including rapid thermal ramps (1C s1)
between steps. Amplified products were purified using
Big-Dye X-Terminator (Life Technologies) according to
the suppliers instructions. The DNA sequencing was performed using an ABI PRISM 310 automated DNA
sequencer (Life Technologies) as previously described
(Suarez et al. 2009). The sequence data were processed,
and the alignments were performed using the Sequence
Navigator software and CLUSTALW algorithm (Life
Technologies). Strain identification was performed using
the online BLAST software (www.ncbi.nlm.nih.gov/BLAST).
Acknowledgements
This work was funded by the Italian Ministry of Agriculture through the CANADAIR Project (D.M. MIPAF
27240/7303/2011).
Conflicts of Interest
The authors have no conflicts of interest to declare.
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