Pakistan Journal of Nutrition 14 (10): 642-646, 2015

ISSN 1680-5194
© Asian Network for Scientific Information, 2015
Corresponding Author: Nurliyani, Department of Animal Product Technology, Faculty of
Animal Science, Universitas Gadjah Mada,
Jl. Fauna 3-Kampus UGM-Bulaksumur, Yogyakarta-55281, Indonesia
642

Change of Mare Milk Quality During Storage at Room Temperature

Nurliyani, Friska Aryuanti and Indratiningsih
Department of Animal Product Technology, Faculty of Animal Science, Universitas Gadjah
Mada,
Jl. Fauna 3-Kampus UGM-Bulaksumur, Yogyakarta-55281, Indonesia

Abstract:
Mare milk possesses similar biophysical and biochemical characteristics to breast milk.
Compared with cow and goat milk, mare milk also has higher lysozyme and lactoferrin, which
affect its shelf
life. This research was conducted to study the microbial, chemical and physical quality of fresh
mare milk
during 24 h storage at the room temperature. Milk sample which was taken from local mares in
Yogyakarta
stored in a sterile bottle and incubated at room temperature. Total count of bacteria, methylene
blue reduction
test (MBRT), acidity, pH, free fatty acid (FFA), alcohol test and clot on boiling (COB) test were
assessed every
4 h within 24 h incubation. Protein, lactose, fat, ash, total solid contents and density assessment
were
carried out as complementary data. Although storing mare milk in room temperature for 24 h
decreased the
pH of milk, it did not affect its acidity and FFA. Mare milk showed positive reaction on alcohol
and COB test
after 24 h of storage. After being stored for 16 h, mare milk was still in a range of standard
number of total
bacteria and reduction time of mare milk still within the range of quality standard, which proven
by its total
bacteria number (1.4 x 10 cfu/ml) and MBRT (3.67 h). In conclusion, fresh mare milk has 16 h
shelf life when
6
stored at room temperature.

Key words:
Mare milk, microbial quality, chemical quality, physical quality, room temperature

INTRODUCTION
Seventy percent of domestic milk consumption in
Indonesia is still rely on imported. One of efforts to
reduce this high dependency on import can be achieved
by optimizing milk utilization from the other sources,
such as goat, buffalo and mare milk. Mare milk in
Indonesia has not been widely used, even though the
horse population in Indonesia is quite large, especially
in South Sulawesi and Nusa Tenggara. In some parts of

MATERIALS AND METHODS

Indonesia, mare has been utilized as dairy, such as inThis study using three local mares from
Yogyakarta-
Brastagi (North Sumatra) and Sumbawa (West NusaIndonesia and then were stored at room
temperature for
Tenggara). However, the lack of knowledge on mare24 h. Every 4 h within 24 h, the sample of
mare milk
milk characteristics leads to miss interpretation inwere analyzed the total count of bacteria,
MBRT, acidity,
assessing mare milk quality. To reach their consumer,pH and FFA. The protein, lactose, fat, ash,
total solid and
mare milk in Indonesia is often went through a long time density were analyzed at the first time
(0 h of storage).
without any refrigeration facilities and only stored atThe density of mare milk was measured by
lactometer
room temperature. In addition, some people are used to(Draaiyer et al., 2009), whereas the pH
value was
consume raw mare milk, which may negatively affectmeasured by pH-meter (HANNA-HI
98103). Protein
their health since milk is an excellent medium forcontent of mare milk was determined using
Kjeldahl
bacterial growth and an important source of bacterialmethod (AOAC, 1995). Total solid was
calculated from
infection when consumed without pasteurizationmoisture content. Sample for moisture analysis
was
(Donkor et al., 2007). Furthermore, milk in its naturaldried at 105°C for 24 h and the water
content of the
state is a highly perishable material because it issamples was gravimetrically determined
(AOAC, 1995).
susceptible to rapid spoilage by the action of naturallyAsh content was analyzed according to
AOAC (1995),
occurring enzymes and contaminating microbeswith combustion of sample at 500°C for 12 h and
ash
(Perko, 2011). Milk is also an important vehicle forcontent was determined gravimetrically. The
fat content
transmission of pathogenic microorganisms to humanof mare milk was analyzed by Babcock
methods
beings unless it is produced and handled under good(Lampert, 1975), whereas the lactose content
was
hygienic conditions. Thus, hygienic production of milkanalyzed volumetrically by chloramine-T
method (Early,
has to get due attention in order to provide more and
better quality milk for the general public (Dehinenet
et al., 2013). However, the differences in chemical
composition between mare and cow milk would affect its
shelf life. The purpose of this study was to assess the
quality changes of fresh mare milk that stored at room
temperature.
Pak. J. Nutr., 14 (10): 642-646, 2015
643
1998). Mare milk acidity was analyzed by titratable acidity The density of mare milk in this
study was 1.033
as lactic acid percentage according to Hashim et al.(Table 1). This density similar to the result
study by
(2009). Free fatty acid of mare milk was analyzed bySummer et al. (2000), which is density of
mare milk at
adding of heated ethanol, titration with natrium hydroxide 15°C in 20-180 day lactation stage
was 1.0345-1.0361.
and expressed as oleic acid percentage according toThe density of milk decreases with increasing
FSSAI (2012). temperature mainly because water expands. Specific
The total count of bacteria in mare milk was evaluatedgravity is nearly constant, a small decrease
with
using Plate Count Agar (PCA) (Merck) medium byincreasing temperature is caused by the
expansion of fat
Standard Plate Count (SPC) method according to Devibeing larger than that of water and the
contraction on
and Sowmya (2012). MBRT of mare milk was evaluatedmixing of the other solids with water
slightly decreasing
according to Draaiyer et al. (2009), which shows that the (Walstra and Jennes, 1984). Changes in
density are
length of time milk takes to decolourise methylene bluestrongly related to changes in the amount
of
is a good measure of its bacterial content and hence ofcomponents. Fat, protein and LOS (lactose
and other
its hygienic quality. This interpretation of results in MBRT solids) compositions were the most
important elements
as follow: if time taken to decolourise <30 min (very badin density estimation (Ueda, 1999).
grade milk), 30 min-1 h (bad grade milk), 1-2 h (poor
grade milk), 2-4.5 h (fair grade milk) and >4.5 h (goodTotal count of bacteria and MBRT of
mare milk: The
grade milk). The methylene blue reduction test dependstotal count of bacteria of fresh mare milk
(0 h) in this
upon the ability of bacteria in milk to grow and tostudy was 6.1 x 10 cfu/ml (Table 2). This total
count was
consume the dissolved oxygen, which reduces thelower than the previous study from mare of
Wielkopolska
oxidation reduction potentials in the medium (Srujana breed within interval from 3.3 x 10 -5.1 x
10 cfu/ml
et al., 2011). (Dankow et al., 2006). At 8 h storage, the total count of
The alcohol test of mare milk was assessed by usingmare milk was still in the range of 10 cfu/ml.
According
70% alcohol and COB test by boiling of milk for 4to SNI (1998) the standard of minimal total
count of
minutes according to Draaiyer et al. (2009). bacteria in fresh milk was 1.0 x 10 cfu/ml, but
according

RESULTS AND DISCUSSION

Chemical composition of mare milk: The average of
total solid of mare milk in this study was 10.00%
(Table 1). This result was consistent with the previous
study by Cagalj et al. (2014) that is the total solid of
horse (mare) milk at 3-11 month of lactation in around
8.91-10.86% and according to Summer et al. (2000) the
total solid of mare milk at 20-180 day lactation stage
was around 9.21-10.39%.
The mare milk composition averages of 1.70% fat,
1.94% protein, 6.65% lactose. The concentrations of fat
and protein tended to decrease as a whole throughout
lactation, while those for lactose increased (Well et al.,
2012). The content of fat and lactose of mare milk in this
study were lower and the protein was higher than mare
milk in the previous study by Cagalj et al. (2014) that the
fat, protein and lactose of mare milk were in order to
1.23, 1.76 and 6.26%. The ash content of mare milk in
this study was higher than the ash content of mare milk
according to Martuzzi et al. (2004) that in the ranges of
0.30-0.50%. Differences in composition of mare milk
can be caused by many factors as stated by
Vishweswar and Krishnainah (2005) that the milk differ
in composition. However, all milk contains the same
kind of constituent, but in varying amounts. Milk fat
shows greatest daily variation, then come protein,
followed by ash and lactose. According to Pieszka et al.
(2011), many factors (genetic, physiological, nutritional,
environmental) affect milk composition of mammalian
species, of which that breed as the genetic factor affects
the composition of mare milk, especially protein, fat and
lactose.
2
44
4
6
to Dirjenak (1983), the minimal total count was 3.0 x
10 cfu/ml. Therefore, the mare milk stored at 16 h in the
6
room temperature was still quite a good quality.
Total count of bacteria in fresh bovine milk during 24 h of
storage at 30 C was 1.4 x 10 cfu/ml (Van den Berg,
o9
1988), whereas the total count of fresh mare milk in this
study was 4.9x10 cfu/ml at the same storage condition.
7
This indicates that mare milk has a good bacteriological
quality, because the content of lactoferrin and lysozyme
of mare milk were higher than bovine milk (Krol et al.,
2012; Benkerroum, 2008). Lactoferrin have antimicrobial
and antiviral activity (Zimecki and Kruzel, 2000), that
relatively stable to heat. As for lysozyme, their inhibition
activity not only against to cell wall peptidoglycan, but
against to virus and eukaryotic, through the mechanism
other than hydrolytic activity, which is may be through the
interaction with inner lipid membrane (Benkerroum,
2008).
The useful indicator for monitoring the sanitary
conditions present during the production, collection and
handling of raw milk is the “total” bacterial count or
standard plate count (SPC), which is of interest for
ensuring both quality and safety of raw milks. The
numbers and types of microorganisms in milk
immediately after production (i.e., the initial microflora)
directly reflect microbial contamination during
production, collection and handling. The microflora in the
milk when it leaves the farm is influenced significantly by
the storage temperature and the elapsed time after
collection (Robinson, 2002). Therefore, estimation of
microbial load in raw milk is highly recommended in
relation to its shelf life (keeping quality) (Bilkis et al.,
2013).
Pak. J. Nutr., 14 (10): 642-646, 2015
644
Table 1: Average of chemical composition and density of mare
milk
Chemical composition Average
Protein (%) 2.68
Lactose (%) 5.79
Fat (%) 0.30
Ash (%) 0.61
Total solid (%) 10.00
Density 1.033
Table 2: Average of total count of bacteria and reduction time of
mare milk during storage at room temperature for 24 h
Storage (h) Total count (cfu/ml) MBRT (h)
06.1 x 10 15.17
2a e
41.8 x 10 11.50
4a de
81.4 x 10 8.67
4a cd
12 9.7 x 10 6.33
5a bc
16 1.4 x 10 3.67
6a ab
20 4.9 x 10 1.08
7b a
24 5.5 x 10 0.67
7b a
Different superscript in the same column indicate significantly
different (p<0.05)
Table 3: Average of acidity, pH and free fatty acid of mare milk
during storage at room temperature for 24 h
Storage (h) Acidity (%) pH FFA (%)
00.07 6.92 0.38
aaa
40.07 7.00 0.38
aaa
80.08 6.92 0.41
aaa
12 0.07 6.94 0.42
aaa
16 0.08 6.93 0.44
aaa
20 0.08 6.90 0.44
aaa
24 0.11 6.62 0.50
aba
Different superscript in the same column indicate significantly
different (p<0.05)
Table 4: Average of alcohol and COB test of mare milk during
storage for 24 h at room temperature
Storage (h) Alcohol test COB test
0- -
4- -
8- -
12 - -
16 - -
20 - -
24 + +
-: (negative) and +: (positive) reaction
Showed on Table 2, during 24 h of storage at room
temperature the mare milk decreased in reduction time
on MBRT. At the first time (0 h) had reduction time 15.17
h, but after 24 h of storage decreased into 0.67 h. In the
previous studies showed that the Sumbawa mare milk
stored at room temperature for 1 day had reduction time
5.84 h, whereas after 5 days decreased into 4.62 h
(Saragih et al., 2013). The difference in reduction time
can be caused by differences in the amount of bacteria
which reduce methylene blue to colorless. The more
number of bacteria in milk, the faster of reduction time
(O'Connor, 1995). According to SK. Dirjenak No.concentration that destabilizes the milk
proteins in
17/1983, the standard of reduction time on MBRT in a
good fresh milk is 2-5 h. Reduction time of mare milk in
this study up to 16 h was still included in the standards
of good milk quality, whereas at 20 h of storage was not
included in the range of a good quality standard. There
are correlation between total count of bacteria in milk
and reduction time, in order to 1.5x10 cfu/ml vs.
4
>8h,1.5x10 cfu/ml vs. 2.83 h, 1.5x10 cfu/ml vs. 2.5 h,
56
1.5 x 10 cfu/ml vs. 1.6 h (Muhammad et al., 2009).
7
Compared with cow milk which a low in microbial quality
will be damage at 3-4 h on storage temperature of
30-35 C, whereas if the storage temperature at 18-22 C
oo
will damage later (Bayemi et al., 2005).
Acidity, pH and free fatty acid of mare milk: There were
no change of mare milk acidity and FFA during 24 h
storage at room temperature, whereas the pH decrease
significantly (p<0.05) at 24 h of storage (Table 3).
Titratable acidity test is use to screening, determine
suitability for processing (Draaiyer et al., 2009). The
acidity and pH of fresh mare milk in the initial storage
were 0.07% and 6.92, respectively. This results similar
to the previous study by Summer et al. (2000) that the pH
of mare milk at 20-180 day lactation was 6.88-7.11.
However according to Civardi et al. (2003), have not
detected the presence of lactic acid before and after
pasteurization of mare milk. Different from fresh cow
milk that has a higher acidity and lower pH than mare
milk i.e., 0.13-0.18% (Lampert, 1975) and 6.7 (Walstra
and Jennes, 1984), respectively. Normal milk acidity
ranges from 0.10 to 0.20% lactic acid (Draaiyer et al.,
2009). Therefore, based on its acidity, mare milk stored
for 24 h at room temperature was still in a good quality.
Lactoferrin in mare milk is higher than cow milk,
consequently the pH of mare milk was higher than cow
milk since the lactoferrin is rich in arginine within N
terminal (Steijns and van Hooijdonk, 2000), that
positively charged. The FFA in mare milk during 24 h of
storage was in the range of 0.38-0.50%. Because the fat
content in mare milk was low, almost no changes in FFA
during storage. In addition, the high lactoferrin and
lysozyme in mare milk (Krol et al., 2012; Benkerroum,
2008) could inhibit of microbial growth which produces
lactic acid and free fatty acids during storage.
Alcohol and COB test: Based on Table 4, the alcohol
test of mare milk showed positive reaction at 24 h of
storage in room temperature. By using 70% alcohol in
this study seemed small flakes of mare milk on the tube
wall, because the casein in the mare milk was lower
(50%) than cow milk (77.23%) (Potocnik et al., 2011). In
the previous study by Uniacke-Lowe (2011), showed that
assessment of the ethanol stability of equine milk that
observed precipitation on the bottom of the tube indicate
that an ethanol content of 45% is the minimum
equine milk. The high concentration of ionic calcium and
Pak. J. Nutr., 14 (10): 642-646, 2015
645
the low level of 6-casein in equine milk probablyEagalj, M., A. Brezoveeki, N. Mikulec and N.
Antunac,
contribute to its low ethanol stability.
Clot on boiling test of mare milk in this study showed
positive reaction at 24 h of storage in room temperature
(Table 4), although the acidity was 0.11% (Table 3). This
test is used to screening and rapid testing of increased
acidity, but has disadvantages because it is not very
sensitive to slightly sour milk. Therefore, the acidity of
milk that gives a positive test is generally above 0.22%
(as lactic acid) (Draaiyer et al., 2009). However, this COB
test can also be affected by the fat content of milk, since
the fat content of mare milk in this study was low i.e.
0.30%. According to Aberoumand (2010), sheep milk
was more stable than cow milk. The different resistance
of the samples in COB test depends on differences in
milk composition. Because fat contents of sheep milk,
goat milk and cow milk are about 7.2, 4.5 and 3.5%,
respectively and this different composition probably will
delay milk proteins coagulation.
Conclusion: There were no changes in acidity and FFA
of mare milk during 24 h of storage at room temperature.
Changes in pH, alcohol and heat stability of mare milk
occurred after 24 h of storage. However, based on the
total number of bacteria and MBRT of mare milk was
still in the range of quality standard after being stored at
16 h. Therefore, mare milk has 16 h shelf life when
stored at room temperature. The results of this study
recommend that transported of fresh mare milk should
not exceed 16 h without refrigeration facilities.
ACKNOWLEDGMENT
The authors thank Cuk Tri Noviandi, Ph.D for reading the
manuscript.
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