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LWT 41 (2008) 331–336
www.elsevier.com/locate/lwt
Production of wara, a West African soft cheese using
lemon juice as a coagulant
V.O. Adetunjia, D.O. Alongea, R.K. Singhb, J. Chenc,
a
Department of Veterinary Public Health and Preventive Veterinary Medicine, University of Ibadan, Ibadan, Nigeria
b
Department of Food Science and Technology, The University of Georgia, Athens, GA 30602, USA
c
Department of Food Science and Technology, The University of Georgia, Griffin GA 30223-1797, USA
Received 11 July 2006; received in revised form 22 January 2007; accepted 8 February 2007
Abstract
As an important protein source for West African consumers, wara cheese made from the leave extract of Calotropis procera has
extremely short shelf life of only 2–3 days [Adegoke, G. O., Nse, E. N., & Akanni, A. O. (1992). Effects of heat, processing time, and pH
on the microflora, aflatoxin content, and storability of wara, a soft white cheese. Die Nahrung, 36(3), 259–264; Umoh, V. J., & Solomon,
O. (2001). Safety assessment and critical control point of milk product and some cereal beverages in Northern Nigeria. In: Proceedings of
USDA/USAID/NIGERIA international conference on food safety and security, August 1–3 (pp. 122–127). Ibadan, Nigeria: IITA; Belewu,
M. A., Belewu, K. Y., & Nkwunonwo, C.C. (2005). Effect of biological and chemical preservatives on the shelflife of West African soft
cheese. African Journal of Biotechnology, 4, 1076–1079; Adetunji, A. O., Alonge, D. O., & Chen, J. (Unpublished). Microbial quality of
wara, a southwestern Nigerian soft cheese]. Lemon juice was used in this study as a substitute coagulant during wara manufacture in
order to improve the microbial quality of wara. The cheese was manufactured from pasteurized milk inoculated with 101 or
102 CFU ml1 of Listeria monocytogenes. Samples of the milk or cheese were taken along the manufacturing steps and during a 5 d
storage period at 15 and 28 1C in order to determine the populations of L. monocytogenes, total aerobes, Enterobacteriaceae, and
psychrotrophs, as well as mold and yeast. On the 4th day of storage, portions of the un-inoculated control cheese from 28 1C were deep
fried in vegetable oil, mimicking the practice of West African local cheese processors. The results showed that L. monocytogenes, at both
inoculation levels, did not survive the manufacture of wara. In samples initially inoculated with 101 CFU ml1 of L. monocytogenes, the
Enterobacteriaceae counts decreased from the initial 1.78 to 1.00 Log10 CFU g1 with the addition of lemon juice, and became
undetectable (o1.00 Log10 CFU g1) at the curdling point as well as during the 5 d storage period at both temperatures. The total aerobic
counts increased from the undetectable level on the 1st day of storage to 7.65 and 3.39 Log10 CFU g1, respectively at 28 or 15 1C on the
5th day of storage. The psychrotrophic, as well as the yeast and mold counts increased from the undetectable levels on the 1st day of
storage to 7.11 and 5.03 Log10 CFU g1, respectively at 28 1C. At 15 1C however, the population of pyschrotrophs remained undetectable
throughout the 5 d storage period whereas, the yeast and molds count increased to 3.08 Log10 CFU g1 on day 3 before quickly
decreasing to the undetectable levels on the 5th day of storage. A similar trend was observed in cheese made from the milk with an initial
Listeria inoculation level of 102 CFU ml1. The results of this study showed that lemon juice significantly reduced the populations of the
sampled microorganisms, especially the populations of Enterobacteriaceae.
r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Wara cheese; Lemon juice; Coagulant; Listeria monocytogenes; Spoilage microorganisms
1. Introduction
Corresponding author. Tel.: +1 770 412 4738; fax: +1 770 412 4748.
E-mail address: jchen@griffin.uga.edu (J. Chen).
Wara is an un-ripened cheese consumed in several parts
of West Africa. The cheese is prepared by coagulating
fresh cow milk with the leaf extract of the Sodom
apple (Calotropis procera) or pawpaw (Carica papaya).
0023-6438/$30.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2007.02.012
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The preferred coagulant comes from C. procera because the
cheese made with this coagulant has a sweeter flavor versus
the cheese made with the other coagulant. The ingredient in
the leaves of C. procera that is useful in cheese production
is calotropin, an enzyme that curdles milk proteins (Belewu
& Aina, 2000).
Wara has an average shelf life of 2–3 d when stored in
whey at ambient temperature (approximately 28 1C)
(Adegoke, Nse, & Akanni 1992; Belewu, Belewu, &
Nkwunonwo, 2005; Umoh & Solomon, 2001) or 4–5 d
when placed in cool well water (approximately 15 1C). The
cheese is usually deep fried in vegetable oil near the end of
its shelf life in order to further preserve the cheese. Due to
the lack of household refrigeration facilities in Nigeria and
West Africa, attempts have been made in the recent past to
include starter cultures or various chemical preservatives
such as propionic acid, sodium benzoate, and sorbic acid in
the production of wara (Anonymous, 1995; Aworh &
Egounlety, 1985; Belewu et al., 2005; Joseph & Akinyosoye,
1997; Sanni & Onilude, 1999). Some of these preservatives have been shown to be effective in inhibiting
mesophilic and psychrotrophic bacteria as well as coliforms. However, these preservatives are not easily accessible to the local cheese processors in West Africa.
Therefore, the need for a useful and realistic alternative
must be met.
Previous laboratory studies have confirmed the presence
of Listeria monocytogenes in milk and milk products
processed in Nigeria including ice cream, fermented milk,
and local butter (Adetunji, Ikheloa, Adedeji, & Alonge,
2003). The pathogen was isolated from 20% of the 1-d-old
wara cheese and cheese storage whey collected from the
local cheese vendors in Southwestern Nigeria (Adetunji
et al., 2003).
Outbreaks of listeriosis have been linked to the consumption of cheese in many parts of the world (Gellin & Broome,
1989; Goulet et al., 2001; Makino et al., 2005; Wehr, 1989).
A Latin-style fresh cheese was found to be responsible for a
large listeriosis outbreak in California in 1985 (Linnan et al.,
1988), while another outbreak occurring in Japan in 2001,
was attributed to the consumption of a wash-type cheese
(Makino et al., 2005). Although the first confirmed case of
human L. monocytogenes infection in Nigeria was reported
in 1982 (Onyemelukwe, Lawande, Egler, & Mohammed,
1983), foodborne listeriosis has not been documented. It is
known that L. monocytogenes is able to survive the
manufacture and storage conditions of several cheeses
(Anonymous, 2006; Buazzi, Johnson, & Marth, 1992;
Carminati, Gatti, Bonvini, Neviani, & Mucchetti, 2004;
Cetinkaya, & Soyutemiz, 2004; Erkmen, 2001; Manfreda,
Acesare, Stella, Cozzi, & Cantoni, 2005; Yousef & Marth
1990). However, the fate of the pathogen during the
manufacture and storage of wara cheese has not yet been
investigated.
The leaf extract of C. procera has been shown to
introduce the microorganisms naturally associated with
them into wara cheese (Adetunji, Alonge, & Chen,
Unpublished), an alternative coagulant has therefore, been
sought in this study. Lemon fruit is readily accessible in
West Africa, and its use as a sanitizer and bactericidal
agent against bacterial pathogens has been reported
(Sengun & Karapinar 2004, 2005). In this study, lemon
juice was used as a coagulant to replace the leaf extract of
C. procera in wara cheese manufacture in order to observe
whether the lemon juice would improve the microbial
quality and shelf life of wara cheese.
2. Materials and methods
2.1. Preparation of L. monocytogenes inoculum
Five strains of L. monocytogenes were grown and subcultured on modified oxford agar base (MOX) supplemented with appropriate antibiotic supplements (Becton,
Dickinson and Company, Sparks, MD, USA). The
inoculated plates were incubated at 37 1C for 24 h.
A colony of each culture was transferred into test tubes
containing 10 ml tryptic soy broth, respectively then
incubated under the same conditions as described above.
The populations of Listeria in the broth cultures were
determined by plating appropriate serial dilutions of the
cell cultures on MOX agar supplemented with appropriate
antibiotics, followed by incubating the inoculated plates at
37 1C for 24 h. One milliliter of each broth culture was
placed into a sterile test tube to make a 5-strain mixture of
L. monocytogenes.
2.2. Preparation of milk for cheese manufacture
Pasteurized whole milk (32.5 g l1 fat) was purchased
from a grocery store in Griffin, Georgia, USA. The milk
was maintained at 4 1C in a cooler and transported to the
laboratory where it was stored at 4 1C until use. A volume
of 2.20 l of the milk was placed into three cooking pots: A,
B, and C. The milk in pots A and B were inoculated,
respectively with the 5 strain mixture of L. monocytogenes
at a level of 101 and 102 CFU ml1, respectively. The milk
in pot C was not inoculated with Listeria and was used as a
negative control in the study. Both the inoculated and uninoculated milk were then used for wara manufacturing.
2.3. Wara manufacture
The milk described above was heated to approximately
45–50 1C in about 30–40 min. The milk was stirred gently
during the heating process using magnetic stir bars. Freshly
squeezed lemon juice (49.5 ml) was added to the warm milk
(2.2 l), and the milk and lemon juice mixture was heated
with intermittent stirring until it reached 95 1C. The milk
with added lemon juice was kept at this temperature until it
coagulated and the separation of curd and whey became
visible. The milk pots were then removed from the heating
source, and the curds and whey were ladled or poured into
sterile egg separators (8 mm in diameter), which facilitated
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whey drainage and gave the cheese its characteristic shape
and size. When the cheese was firm enough to retain its
shape it was removed from the egg separator and placed in
a glass beaker with approximately 350 ml of whey. The
cheese was then stored at 28 and 15 1C, respectively, for 5 d.
2.4. Microbiological sampling
As shown in Fig. 1, samples were taken at each step
along the line of cheese manufacturing, as well as daily
during cheese storage (Fig. 1). At each sampling, 10 ml of
milk or 10 g of cheese samples were withdrawn and the pH
of the milk and cheese was measured using a pH meter
(Model 8000, VWR Scientific, West Chester, PA, USA).
The samples were then homogenized with 10 ml de-ionized
water in stomacher bags using a Stomacher 400 lab blender
(Seward Ltd., London, UK). Serial dilutions were made in
1 g l1 peptone water and appropriate dilutions were
surface plated on MacConkey agar plates for the enumeration of Enterobacteriaceae, on MOX agar plates for the
enumeration of L. monocytogenes, on tryptose soy agar
(TSA) plates for the enumeration of total aerobic and
psychrotrophic bacteria, and on potato dextrose agar for
the enumeration of yeast and mold. All of the plates were
incubated at 37 1C for 24 h except for the TSA plates for
psychrotrophs, which were incubated at 10 1C for 7–9 d.
Colonies on each agar plate were enumerated using a
333
colony counter (Model 3325, Leica Quebec Dark Field,
Buffalo, NY, USA).
2.5. Frying
On the fourth day of storage, 80 g of the control cheese
stored at 28 1C were deep fried in boiling vegetable oil for
5 min. The internal temperature of the cheese was
measured immediately after the frying using a Fisherbrand
traceable dual channel thermometer with offsets (Fisher
Scientific Inc., Pittsburgh, PA, USA). Additionally, portions of the fried cheese were sampled for 3 consecutive
days after the frying in order to determine the populations
of Enterobacteriaceae, L. monocytogenes, total aerobic and
psychrotrophic bacteria, as well as yeast and mold.
2.6. Statistical analysis
The study was performed in two separate trials, each
experiment in an individual trial was duplicated. All
microbiological
data
were
transformed
into
Log10 CFU ml1 or Log10 CFU g1 before comparison of
means. Separation of means was accomplished using the
Fisher’s least significant difference of means of bacterial
populations calculated with the General Linear Model
(GLM) procedure of SAS (a ¼ 0.05; SAS, 2000). The data
were analyzed as a completely randomized block design
Pasteurized milk (1 liter in pot A, B, and C)
Sampling point I---Heat the milk to 45-50°C
Sampling point II---Add lemon juice
Stir twice (few seconds each), the two stirs were approximately 10 min apart
Sampling point III---Heat the mixture of milk and lemon juice to 95°C until curd formation
Pour the milk curd into molds for whey drainage
Storage of cheese in whey at 28 or 15°C for 5 d and sampled daily
Fig. 1. Flow chart of wara cheese manufacture.
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334
within split plot structure, considering storage temperature
(two levels), sampling time (5 days), and Listeria inoculation level (three levels).
3. Results and discussion
3.1. The survival of the sampled microorganisms during
wara manufacture
The population of L. monocytogenes in the pasteurized
milk decreased from the initial inoculation levels to the undetectable level (o 1.00 Log10 CFU ml1) with the addition
of lemon juice (Table 1). The same treatment changed the
psychrotroph counts, in the pasteurized milk inoculated
with 101 or 102 CFU ml1 of L. monocytogenes, from the
initial 4.27 and 4.10 Log10 CFU ml1, respectively to the
undetectable level (o1.00 Log10 CFU ml1). The decreases
in the populations of L. monocytogenes and psychrotrophs
may be due to the corresponding changes in the milk pH
from the initial 6.38 to 5.76 after the addition of lemon
juice (Table 1). Yazici (2004) studied the effect of pH on
the microflora in civil, an acid curd, cheese and observed a
negative correlation between the pH of the cheese and the
populations of total aerobic bacteria, lactic acid bacteria,
as well as yeast and mold. The addition of lemon juice
also resulted in a decrease in the populations of total
aerobes and Enterobacteriaceae. The counts became
undetectable in the milk initially inoculated with
102 CFU ml1 of L. monocytogenes (Table 1). In the milk
initially inoculated with 101 CFU ml1 of L. monocytogenes
however, the cells of total aerobes, Enterobacteriaceae, and
mold and yeast remained detectable (Table 1).
In addition to the use of lemon juice, another possible
quality control point of wara cheese processing is the
heating step at the curdling point that raises the
temperature of milk to 95 1C. At this point, the populations
of total aerobes, Enterobactericeae, psychrotrophs, and
mold and yeast decreased to the undetectable level except
for the total aerobic counts in the milk inoculated with
101 CFU ml1 of L. monocytogenes which was approximately 1.30 Log10 CFU ml1 (Table 1). Adegoke et al.
(1992) observed a similar decrease in the population of
total aerobes at the cooking point during wara manufacture. The inoculated L. monocytogenes was not found at
the curdling point (Table 1) and throughout the 5 d storage
period at 28 and 15 1C in this study (data not shown).
Working with water-buffalo Mozzarella cheese, Villani,
Pepe, Mauriello, Moschetti, and Sannino (1996) noticed
an approximate 2 log reduction in the population of
L. monocytogenes after the cheese was stretched in hot
water with a temperature of 95 1C. The pathogen became
undetectable after 24 and 48 h of storage at room
temperature in the surface layer of water used to stretch
the cheese (Villani et al., 1996).
3.2. The survival of the sampled microorganisms and the pH
of wara cheese during storage
The pH of 1-d-old wara cheese manufactured by the
leaf extract of C. procera was approximately 5.70–6.90
(Adegoke et al., 1992) and 6.15–6.43 (Adetunji, Alonge,
& Chen, Unpublished), whereas the wara cheese manufactured with lemon juice in this study had a pH range of
5.53–5.68 (Table 2). Although the pH of the wara cheese
fluctuated throughout the 5 d storage period, storage
temperature and storage time did not seem to have a
significant influence on the pH of wara (Table 2).
The relatively lower pH of the wara cheese manufactured
with lemon juice effectively inhibited Enterobacteriaceae
and the counts remained undetectable throughout the 5 d
storage period at both 28 and 15 1C (Table 2). In wara
cheese manufactured with the leaf extract of C. procera, the
cells of Enterobacteriaceae survived the conditions used for
wara manufacture, with the counts increasing from 3.35 to
3.48 Log10 CFU g1on the 1st day of storage to 7.63–
8.46 Log10 CFU g1 at 28 1C and 5.41–6.36 Log10 CFU g1
at 15 1C after the 5 d storage (Adetunji, Alonge, & Chen,
Unpublished).
In general, the populations of total aerobes, psychrotrophs, and mold and yeast increased rapidly with the
extension of storage time at 28 1C (Table 2). A significant
difference (Po0.05) in the populations of the three types of
microorganisms was observed between the two storage
temperatures over the 5 d storage period. Storage of the
Table 1
Microbial counts and pH of the milk used for wara manufacture
Inoculation level (Log CFU ml1)
101
102
Processing steps
I
II
III
I
II
III
I
II
III
Microbial counts or pH
L. monocytogenes
Total aerobes
Enterobacteriaceae
Psychrotrophs
Molds and yeasts
pH of the cheese
1.00b
2.89b
1.78b
4.27b
o1.00b
6.38a
o1.00c
1.54c
1.00c
o1.00d
1.48a
5.76b
o1.00c
1.30cd
o1.00c
o1.00d
o1.00b
5.49b
2.70a
3.78a
3.63a
4.10c
o1.00b
6.40a
o1.00c
o1.00f
o1.00c
o1.00d
o1.00b
5.75b
o1.00c
o1.00f
o1.00c
o1.00d
o1.00b
5.37b
o1.00c
2.96b
o1.00c
4.40a
o1.00b
6.38a
o1.00c
1.24de
o1.00c
o1.00d
o1.00b
5.71b
o1.00c
o1.00f
o1.00c
o1.00d
o1.00b
5.63b
Control
Values not containing a common letter in the same row are significantly different.
Processing step I: heat milk to 45–50 1C; II: add lemon juice; and III: heat the mixture of milk and lemon juice to 95 1C.
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335
Table 2
Microbial counts and pH of wara cheese during a 5-d storage at 28 and 15 1C
Inoculation levels (CFU g1)
101
Storage temp (1C)
Microbial counts or pH
28
15
28
15
28
15
Total aerobes
Day 1
2
3
4
5
o1.00aC
6.23aB
6.75aB
7.57aA
7.65aA
o1.00aD
o1.00cD
3.16cB
1.54cC
3.39bA
o1.00aD
6.20aC
6.42bBC
7.04aAB
7.57aA
o1.00aD
o1.00cD
3.03cA
1.54cC
2.26cB
o1.00aC
6.61aB
6.48bB
7.14aAB
7.43aA
o1.00aD
1.54bC
3.17cA
2.91cB
3.16bA
Enterobacteriaceae
Day 1
2
3
4
5
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
o1.00aA
Psychrotrophs
Day 1
2
3
4
5
o1.00aC
4.39bB
6.71aA
6.72aA
7.11bA
o1.00aA
o1.00cA
o1.00bA
o1.00bA
o1.00dA
o1.00aC
4.29bB
6.87aA
6.99aA
7.36aA
o1.00aA
o1.00cA
o1.00bA
o1.00bA
o1.00dA
o1.00aC
4.56aB
6.84aA
6.85aA
6.93cA
o1.00aA
o1.00cA
o1.00bA
o1.00bA
o1.00dA
Molds and yeasts
Day 1
2
3
4
5
o1.00aD
o1.00bD
6.60bA
4.83aC
5.03bB
o1.00aC
o1.00bC
3.08eA
1.74cB
o1.00cC
o1.00aD
o1.00bD
6.83aA
4.85aC
5.09aB
o1.00aC
o1.00bC
3.06eA
1.59cB
o1.00cC
o1.00aC
6.36aA
6.45cA
4.90aB
5.03bB
o1.00aC
o1.00bC
3.21dA
3.06bB
o1.00cC
pH of the cheese
Day 1
2
3
4
5
5.53aC
5.64aBC
5.75bABC
5.96aA
5.77abAB
102
5.53aAB
5.72aA
5.79bA
5.49bAB
5.36bB
5.57aA
5.61aA
5.68bA
5.78abA
5.53abA
Control
5.57aA
5.61aA
5.65bA
5.47bA
5.29bA
5.68aA
5.61aA
6.03aA
5.66abA
5.91aA
5.68aA
5.75aA
5.66bA
5.63abA
5.60abA
Values not containing a common lowercase letter in the same row are significantly different with respect to storage temperature.
Values not containing a common uppercase letter in the same column are significantly different with respect to storage time for either a given type of
microbial count or the pH of the cheese.
cheese at 28 1C encouraged the growth of microbial cells,
while at 15 1C viable psychrotrophs were not detected
during the 5 d storage period (Table 2). The population of
mold and yeast increased significantly with storage time
(Po0.05) and the highest counts were observed between
days 1 and 3 (Table 2). Only at 15 1C the population of
mold and yeast dropped below the detectable level on day
5. The existence of mold and yeast in wara cheese observed
in this study was not unusual. Other researchers have
isolated Penicillium and Aspergillus spp. from wara in
earlier studies (Adegoke et al., 1992). The results of this
study suggest that without efficient preservation, the
recommended holding time of wara cheese should be
significantly shortened.
In wara cheese made with the leaf extracts of C. procera,
the psychrotrophic bacteria counts increased from
2.52–3.73 Log10 CFU g1 on the first day of storage to
7.16–8.18 Log10 CFU g1 at 28 1C and 6.75–7.00 Log10
CFU g1 at 15 1C by the 5th day of storage (Adetunji,
Alonge, & Chen, Unpublished). The total aerobic plate
counts were 3.38–5.03 Log10 CFU g1 in 1-d-old wara
whereas in the 5-d-old wara, the total plate counts
increased to 8.44–8.96 Log10CFU g1 at 28 1C and 4.85–
6.70 Log10 CFU g1 at 15 1C (Adetunji, Alonge, & Chen,
Unpublished). The yeast and mold counts changed from
2.86–5.07 Log10 CFU g1 on the first day of storage to
8.21–8.65 Log10 CFU g1 at 28 1C and 4.09–5.95 Log10
CFU g1 at 15 1C at the end of the 5 d storage period
(Adetunji, Alonge, & Chen, Unpublished). In the present
study, the populations of psychrotrophs, total aerobes, and
mold and yeast were 6.93–7.36, 7.43–7.65, and 5.03–
5.09 Log10 CFU g1 after the 5 d storage at 28 1C. At
15 1C storage however, the populations of total aerobes
were approximately 2.26 and 3.39 Log10 CFU g1, while
the cells of the psychrotrophs as well as the yeast and mold
became undetectable at the end of the 5 d storage period.
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A significant difference was found in populations of total
aerobes, psychrotrophs, and mold and yeast obtained
before and after the frying of wara. The populations of
total aerobes, psychrotrophs, and mold and yeast changed
from 7.14, 6.85, and 4.90 Log10 CFU g1, respectively
before frying to o1.00 Log10 CFU g1 after frying. The
frying process raised the internal temperature of wara to
60–62 1C, which was however, insufficient in eliminating all
microbial cells in wara cheese. The populations of total
aerobes, psychrotrophs, and mold and yeast were 7.00,
o1.00, and o1.00 Log10 CFU g1, respectively at the 3rd
day after the frying.
4. Conclusions
This study has shown that the conditions used for wara
can inactivate low levels of L. monocytogenes present in the
cow milk used to manufacture the cheese. However, this
does not necessarily mean that Listeria contamination is
not a problem in wara. The cheese can certainly support
the growth of the pathogen should post-processing
contamination occur. The manufacture and storage conditions used in this study eliminated the cells of Enterobacteriaceae in the cheese stored at both temperatures, and of
the psychrotrophs in the cheese stored at 15 1C. However,
the populations of total aerobes in the cheese stored at
both temperatures, as well as psychrotrophs and mold and
yeast in the cheese stored at 28 1C remained high. Frying
temporarily reduced the counts of total aerobes, but failed
to eliminate the microbial cells from wara. These results
indicate that additional control measures need to be
identified in order to further improve the microbial quality
of wara cheese.
Acknowledgments
This work was funded in part by FY-2005/2006 Junior
Staff Development Fulbright Scholarship of the United
States Institute of International Education. The authors
wish to thank Mr. Jerry Davis and Mr. Amit Ahaja for
statistical assistance.
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