See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/-
Microbial Analysis and Proximate Composition of Boiled and Fried Local Cheese
(WARA)
Article in International Journal of Scientific and Research Publications · December 2018
DOI:-/IJSRP-.p8491
CITATIONS
READS
3
2,091
4 authors, including:
Olufunmilola Ajayi
Olalekan Balogun
Joseph Ayo Babalola University
Joseph Ayo Babalola University
11 PUBLICATIONS 57 CITATIONS
26 PUBLICATIONS 147 CITATIONS
SEE PROFILE
All content following this page was uploaded by Olalekan Balogun on 20 January 2019.
The user has requested enhancement of the downloaded file.
SEE PROFILE
International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018
ISSN-
710
Microbial Analysis and Proximate Composition of
Boiled and Fried Local Cheese (WARA)
Ajayi, O. O1., Balogun, O. B1., Oriowo-Olaleye, M1. and Faturoti M. O1.
Department of Biological Sciences, Joseph Ayo Babalola University, Ikeji Arakeji, Osun State, Nigeria.
DOI:-/IJSRP-.p8491
http://dx.doi.org/-/IJSRP-.p8491
Abstract- Fried cheese locally called Wara gets spoiled when
attacked by pathogenic bacteria, as a result of poor hygiene
practice by the producer and handlers. Wara samples were
bought in Ilesha at Atakumosa market square Osun-state. This
research was designed to isolate and enumerate microorganisms
using standard microbial methods, proximate composition and
the nutritional composition of boiled local and fried cheese
(wara). Proximate analysis was carried out to determine
nutritional composition. Klebsiella species and Esherichia coli
Staphylococcus epidermidis, Bacillus species, Escherichia coli,
Salmonella species, Streptococcus species, Clostridium species
and Lactobacillus species were isolated using standard technique.
The highest bacteria count for the fried cheese was
2.1×105cfu/ml the lowest bacterial count was 1.5×105cfu/ml unit
highest bacterial count of the boiled local cheese was
4.4×105(cfu/ml), lowest bacterial count was 1.8×105(cfu/ml)
while the range of the values was 2.6×10 5. Fungi isolated were
Penicillium and Gymnoase. Fat content for the boiled cheese has
the highest mean value of 44.78 ±0.028cfu/ml and Ash had
lowest mean value of 2.46±0.014cfu/ml. The moisture content of
fried cheese had the highest mean value 55.32±0.014 while the
carbohydrate had the lowest mean value 0.63±0.02 for fried
chesse. Fibre content was not detected in both samples. The
microbial contamination might generally occur based on lack of
standardization, fecal contamination and poor personal hygiene
in the production, handling and storage. Therefore, all cheese
producers and consumers should take care during processing and
storage of the cheese to prevent contamination.
Index Terms- Pathogenic bacteria, Microorganisms, Proxinate
composition and Microbial methods
are not standardized (Adetunji, 2011). It could also be coagulated
with the juice extract of leaf of sochom apple (Colotropis
procera) or pawpaw ). (Augusti, 1996; Adetunji and Alonge,
2009; FAO, 2009).
Cheese is usually stored in its whey and consumed fresh,
but this can only last for 3-5 days after which spoilage occurs. It
is sometimes fried and used as a meat-substitute in stews and
soups, or smoke-dried to enhance its keeping qualities. However,
all these increase its shelf life by only a few extra days or few
weeks at best (Belewu et al., 2005).
Cheese is made mostly from the milk of cows, buffaloes,
sheeps or goats, and it is an important food component in the
healthy diet of human’s highly nutritious rich food and source of
protein, peptides, aminoacids, vitamins, salt, and essential
minerals including calcium (Augusti, 1996; Belewu and Aina,
2000). Cheese however, has been shown to have antimicrobial
properties that prevent disease, it has been used as drugs for
certain infection when common antimicrobial agents failed it
had also been found that soft cheese has growth inhibitory
activity against common bacteria that caused diarrhoea in South
West Nigeria (Ibrahim and Falegan, 2013).
Microorganisms such as Streptococci, Lactobacilli,
Coliform bacteria and some fungi are associated with milk and
milk products (Baba et al., 2004; Raheem and Saris, 2009). The
consumption of cheese is of utmost public health importance and
high consumption rate in Nigeria calls for the periodical
examination of cheese produced in different locality.. The study
was designed to isolate and identify microorganisms found in
both fried and boiled cheese; to determine the proximate
composition and assess the best form of cheese for humans
consumption.
I. INTRODUCTION
C
heese (wara) it is a Nigerian soft, white, unsalted and
unripened cheese usually processed from cow milk by the
Fulani tribes who are mainly cattle rearers in Nigeria (Raheem,
2006; Ogunbanwo et al., 2011). Wara making started with the
Fulani’s but as a result of their nomadic lifestyle, it spread to
other parts of the country including Kwara, Oyo, Ogun, Ondo
and the Benin Republic (Raheem, 2006).
It is obtained by coagulating the casein with rennette or
similar enzymes in the presence of lactic acid produced by
adding microorganisms (Adegoke et al., 1992). However in
many cases starter culture are not used as processing condition
http://dx.doi.org/-/IJSRP-.p84XX
II. MATERIALS AND METHOD
2.1
Study Area
The cheese sample (Wara) used for this study was
purchased from Ilesa West Local Government at Atakumosa
market square in Osun state, Nigeria. The market was sited close
to the place of the king of the town (Ereja square) on the outskirt
of the city of Ilesha. The total population according to 2006
census was 103,555 having the total area as 63km2 (24 sq mi)
and the coordinate of 7°39N 4°43’E.
2.2 Techniques for preparation of media
2.2.1
Sterilization Technique
www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018
ISSN-
The glassware used was thoroughly washed with
detergent rinsed with distilled water and air dried. The glassware
were wrapped with aluminum foil paper and sterilized in an
autoclave at 121°C for 15 minutes.
2.2.2
Collection of Samples
Wara sample processed traditionally by Fulani woman
were bought in highly populated location in Ilesha at Atakumosa
Market Ilesa Osun State, Nigeria. The Fried cheese was bought
into a sterile plastic plate and transported to Microbiology
laboratory for analysis within 2 hours of purchase.
III. PREPARATION OF MEDIA
3.1
Nutrient agar: This was prepared according to
manufacturer’s description. Seven (7g) of nutrient agar powder
was weighed and dissolved into 250 ml of distilled water. It was
heated to get it homogenized and allowed to change to golden
yellow before it was autoclave at 121º C for 15 minutes.
3.2
MacConkeyAgar: This was prepared
according to manufacturer’s description. 14g of MacConkey agar
powder was weighed and dissolved in 250ml of distilled water
and homogenized using heat to boil. This was then sterilized by
autoclaving at 121ºC for 15minutes.
3.3
Agar Slant Agar slant were prepared by
dispensing 15ml of nutrient agar into different MacCartney
bottles. The bottles were placed in a slanting position and
allowed medium to gel.
3.4
Serial dilution of Samples Five grams (5g)
of the cheese was meshed using the crucible and pestle, serial
dilution was done by measuring 9ml of sterile water into first test
tube till the tenth test tube. 1ml of the sample was then added
into each labeled and properly covered test tube using a sterile
syringe. 1ml was then taken from 10 5 or the fifth test tube into a
petridish and gently swirled and allow to set. The petridishes
were then incubated at 37°C for 24 hours. After the incubation,
the colonies that developed on nutrient agar plate were counted
and used to determine the total bacteria count of the sample
(cfu/ml). The representative colonies on the plates were subcultured on a fresh nutrient agar to obtain a pure culture of
isolates. The pure culture was then transformed into nutrient agar
slant for biochemical test.
3.5
Isolation and identification of the bacteria
Distinct and well isolated colonies were sub-cultured and
examined for various sizes, shapes, colours and texture, a series
of tests such as Catalase, Oxidase, Indole, Nitrate reduction, and
sugar fermentation were carried out to identify the bacteria
according to (Cheesbrough, 2006).
3.6
Isolation of Fungi
Nine point eight gram (9.8g) of Potato Dextrose Agar
(PDA) powder added with antibiotics (to inhibit the growth of
http://dx.doi.org/-/IJSRP-.p84XX
711
bacteria) was dissolved into 250ml of sterile water and
autoclaved at 121°C for 15 minutes and allowed to cool before
pouring aseptically into the petridishes. The prepared potato
dextrose agar (PDA) was aseptically dispensed into the Petri dish
containing the sample and swirled gently before cooling. The
plate was then incubated in an incubator at 26°C for five (5) days
before observation (Onyeagba, 2004).
3.8
Proximate
Analysis
of
Nutritional
Composition of Fried Cheese
This refers to the determination of the major constituents
of food sample and it is used to assess if a sample is within it’s
normal compositional parameters or somehow been adulterated.
This method partitioned nutrient in food sample into six
components: ash, crude protein, fat, crude fiber, moisture
content, and carbohydrate (FAO, 2008).
IV. RESULT
Table 1: It shows the total bacterial count in fried cheese
calculated in colony forming units per ml, the sample four had no
bacterial population .
Table 2: Total bacterial load of boiled local cheese
(wara) samples calculated in colony forming units per ml, all the
sample had bacterial population which varies from sample one
till sample five
Table 3: It shows the morphological characteristics and
biochemical test of bacteria isolate from boiled local cheese
(wara) based on size, texture, colour, opacity, surface, elevation
and margin.
Table 4: It shows the morphological characteristics and
biochemical test of bacteria isolate from fried local cheese (wara)
is based on size, texture, colour, opacity, surface, elevation and
margin.
Table 5: It shows the count for fungi isolated in the fried
cheese sample. The fungi isolate were: penicillium spp and
Gymnoaseus sp.
Table 6: Proximate analysis showing the nutrition
evaluation of cheese, which is based on Moisture content, Fat,
Fibre, Crude protein and Carbohydrate.
Table 7: Statistical analysis of proximate composition
shows mean Standard deviation, Variance and Coefficient of
variation.
Figure 1: Bacterial count in fried cheese which is
calculated in colony forming units per ml. .
Figure 2: Bacterial load of local cheese (wara) samples.
Figure 3: The boxplot of the statistical analysis of
proximate composition
www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018
ISSN-
712
TABLE 1: Total bacterial load of Fried Cheese (wara) sample
Replicate Sample 1(Cfu/ml) Sample 2(Cfu/ml) Sample 3(Cfu/ml) Sample 4 (Cfu/ml)
RI
1×105
R2
2×105
Mean
1.5×105
SD
0.70710
CV
47.40
VAR
0.5
Keys:
SD: Standard deviation
VAR: Variance
CV: Coefficient of variance
2×105
1×105
1.5×-
2×105
2×105
2 ×105
0
0
0
0
2×105
1×-
Table 2: Total bacterial load of boiled local cheese (wara) samples.
Replicate
Sample
(Cfu/ml)
1
Sample
(Cfu/ml)
2
Sample
(Cfu/ml)
3
Sample
(Cfu/ml)
4
Sample
(Cfu/ml)
5
Sample 6
(Cfu/ml)
R1
4.5×105
3.3×105
2.7×105
1.8×105
2.1×105
1.7×105
R2
4.3×105
3.7×105
3.0×105
2.3×105
1.5×105
2.6×105
Mean
4.4×105
3.5×105
2.85×105
2.05×105
1.8×105
2.15×105
SD
-
-
-
-
-
-
CV
3.2%
8%
7.4%
17%
24%%
29%
VAR
2
8
4.5
12.5
18
40.5
KEYS:
SD: Standard deviation
CV: Coefficient of variation
VAR: Variance
R1:Replicate 1
http://dx.doi.org/-/IJSRP-.p84XX
R2: Replicate 2
www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018
ISSN-
713
Sucrose
Probabale organism
G
Escherichia coli
-
-
G
G
Salmonella spp
Round
Cream
Smooth
Small
Transluscent
-
-
+
+
-
+
+
d
Entire
Cream
Smooth
Small
Opaque
-
+
+
+
+
+
d
Entire
Cream
Dry smooth
Big
Opaque
-
-
+
-
+
d
Round
cream
Dry smooth
Small
Translucent
-
-
+
-
+
Motility
d
Hydrogen sulphide
G
Indole
-
VP
-
Methyl red
Lactobacillus spp
Gram staining
G
Spore staining
G
Optical
-
Size
Klebsiellia spp
Surface
G
Colour
G
Edge
Gluose
TABLE 3: Morphological characteristics and biochemical test of Bacterial isolates from fried local cheese (wara)
Shape
Size
Texture
Color
Opacity
Edge
Gram stain
Spore stain
Methyl red
catalase
Indole
Motility
VP
H2S
Lactose
Glucose
Sucrose
Small
rough
Cream
opaque
entire
+
+
+
+
+
-
A
A
A
Small
rough
Cream
opaque
entire
+
Small
smooth
Yellow
opaque
center
+
Small
smooth
Cream
opaque
entire
Lactobacillus
spp
Rod
Clostridium
spp
+
Cocci
Streptococcus
spp
+
Rod
Bacillus
spp
Cocci
Probable
Organisms
Table 4: Morphological characteristics and biochemical test of bacterial isolates from boiled local cheese (wara)
+
http://dx.doi.org/-/IJSRP-.p84XX
-
+
+
+
-
-
-
-
A
A
+
+
+
+
-
+
+
-
AG
AG
-
-
+
-
-
-
+
G
AG
AG
www.ijsrp.org
Cream
opaque
center
opaque
center
smooth
rough
+
Pink
Small
Small
Escherichia
coli
Cocci
Staphylococcus
epidermidis
Rod
International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018
ISSN-
-
714
-
-
+
-
-
+
+
-
AG
AG
-
+
+
+
+
-
-
AG
AG
AG
Error bar ±1 SE
Figure 1: Bacteria count in fried cheese.
http://dx.doi.org/-/IJSRP-.p84XX
www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018
ISSN-
715
Error bar ±1 SE
Figure 2: Bacterial load of local boiled cheese (wara) samples.
Table 5: Total fungi count in fried cheese (Wara)
Sample-
Cfu/ml
6×103
4×105
Fungi organism
Penicillium spp
Gymnoase spp
Table 6: Proximate analysis result showing nutritional evaluation in fried cheese
S/N
%Ash
% MC
%CP
%Fat
%Fibre
%CHO
R1
2.45
10.33
21.11
44.8
0
21.31
R2
2.47
10.31
21.1
44.76
0
21.36
Mean
2.46
10.32
21.105
44.78
0
21.335
SD
-
-
-
-
0
-
VAR
0.002
0.002
5E-05
0.008
0
0.0125
CV
0.05749
0.0137
0.335
0.0632
0
0.01657
Sample
2.46±0.014
Keys:
MC: Moisture Content
SD: standard deviation
VAR: variance
CV: coefficient of variance
10.31±0.014
21.1±0.007
44.76±0.02
0
21.36±0.03
CP: Crude protein
CHO: Carbohydrate
Table 7: Statistical analysis of proximate composition in boiled cheese
Replicate
ASH
MC
CP
FAT
R1
1.16
55.31
18.11
24.78
0
0.64
R2
1.18
55.33
18.12
24.76
0
0.61
Mean
1.17
55.32
18.115
24.77
0
0.625
SD
-
-
-
-
0
-
CV
1.41%
0.03%
0.04%
0.1%
0
3.4%
VAR
0.0002
0.0002
5E-05
0.0002
0
0.00045
http://dx.doi.org/-/IJSRP-.p84XX
FIBRE
CHO
www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018
ISSN-
1.17±0.014a
Sample
55.32±0.014b
18.2±0.007c
716
24.8±0.014d
0e
0.63±0.021f
Data are presented as Mean±SD (n=2) from duplicate determination. Different superscripts in the same column are significantly
different (P<0.05).
Keys:
MC: Moisture content
CP: Crude protein
CHO: Carbohydrate
ND: Not detected
Boxplot of ASH, MC, ...
60
50
Data
40
% 30
20
10
0
ASH
MC
CP
FAT
FIBRE
CHO
Figure 3: The boxplot of the statistical analysis of proximate composition
V. DISCUSSION AND CONCLUSION
The results obtained from the microbial analysis of fried
cheese (wara) show that the products were contaminated with
microorganisms of public health concern. The bacterial count in
the sample may be a consequence of low level of hygiene
maintained during the processing and sale of the products. This
includes the handlers, quality of water used and the utensils.
During the sale of fried cheese (wara), dirty hands and spoons are
dipped into the bowl for product selection by both hawkers and
consumers.
The exposure of wara and while they are displayed for
sale in bowls can serve as source of contamination. The detection
Klebsiella spp and E.coli in wara may indicates possible faecal
contamination because the fulani’s do not disinfect the teats and
udders prior to milking despite the fact that the cow lies in a
muddy barnyard and dirty environment which inevitably
contaminate the milk and could increase the microbial load. The
http://dx.doi.org/-/IJSRP-.p84XX
presence of Staphyloccus epidermidis, as supported by the study
of Ajayi et al. (2016) may lead to contamination of food and
eventually affects the health of the consumers. The presence of
Bacillus species which produces several toxins, being isolated in
this study probably is an indication of poor hygienic habit of the
milker milking the cow. Clostridium species isolated which are
important cause of diarrhea inhabits the soil and intestinal tract of
animals including humans and can cause food intoxication which
is congruent to the findings of Ajayi et al. (2016). Being enteric
bacteria, their presence indicates poor hygienic practices among
handlers of wara. Due to the significance of the faecal-oral route
transmission for many bacterial food-borne diseases, basic
hygienic measures assume a decisive importance in food safety
management (Alalade and Adeneye, 2006).
The detection of Lactobacillus species isolated from
sample show that they are organism used for the production of
cheese. Lactobacillus species allows the production of gas in
lactose, sucrose and glucose Aworh and Egounlety (1985). They
are motile, gram positive bacteria which tests positive for
www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 8, Issue 12, December 2018
ISSN-
catalase test. The fungal isolates: Gymnoase and Penicillium,
species which were isolated are known as spore formers, which
therefore means that they can easily contaminate the dairy
products which are usually exposed during processing, storage,
and hawking. They are major spoilage organisms of carbohydrate
foods (Rhodes and Fletcher, 1966). However, their growth can
result in the production and accumulation of mycotoxins which
are of public health and economic importance (Burnett and
Beuchat, 2001). The nutritional analysis of fried cheese shows
that they are of appreciable nutritional status especially in the
protein and fat content. The dairy products particularly cheese
are good sources of protein. Higher fat content was observed in
fried cheese wara. This could be due to vegetable oil used in
frying of the cheese.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
Alalade, O.A. and Adeneye, J.A. (2006). The effects of short term frozen
storage on chemical composition and coliform and coliform microflora of
Wara cheese “Wara cheese under frozen storage. Advance Journal of Food
Technology. 2(1): 44-44
Augusti, K.T. (1996). Therapeutic values of onion and garlic. Indian J
ExpBiol 64: 334- 336.
Aworh, O.C. and Egounlety, M. (1985). Preservation of West African soft
cheese by chemical treatment. Journal of Dairy production Research. 52:
189-195.
Baba, K.M., Orire, A.O. and Suleiman, H W. (2004). Profitability of cheese
production and marketing among rural women in Moro local Government
area of Kwara state. Proc. American Journal of Food and Nutrition., 3(1):
13-21
Belewu M.A and Aina (2000). Microbial evaluation of indigenous milk
products with special reference to the bacterial flora of public health
importance in Nigeria. African Journal of Experimental Microbial analysis.
1 (1): 13-19.
Belewu, M.A., Belewu, K.Y and Nkwunonwo C.C. (2005). Effect of
biological and chemical preservatives on the shelflife of West African soft
cheese. African Journal of Biotechnology. 4:-.
Belewu, M.A., Belewu, K.Y. and Ganiyu, R.O. (2011). Evaluation of lemon
Grass Cymbopogon citrates) oil and pure natural honey on the shelf life of
cheese. Electronic Journal of Environmental, Agricultural and Food
Chemistry (EJEAFChe), 10(4),-.
Burnett and Beuchat, (2001). Emerging infectious Diseases. Produce
Handling and Processing Practices; 5:6
http://dx.doi.org/-/IJSRP-.p84XX
View publication stats
[16]
717
Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries.
Part 2 Press Syndicate of the university of Cambridge, United Kingdom
pp. 63-70.
FAO: Food and Agricultural organization.(2008). Milk for health and
wealth .FAO, Rome, Italy. Journal of agricultural, 19:34-12.
Ibrahim, T.A. and Falegan C.R. (2013). Anti-bacterial activities of crude
cell free supernatants of Lactic acid bacteria from wara (Nigeria soft
cheese). Research and Reviews..Journal of food and Dairy Technology, 1
(2):1-4.
Ogunbanwo, S. T., Sanni, A. I. and Onilude, A. A. (2011). Effect of
bacteriocinogenic Lactobacillus spp. On the shelf life of fufu, a traditional
fermented cassava product. World Journal of Microbiology and
Biotechnology, 20:57-63.
Oladipo I.C., Onyeruke I.C. and Adebiyi, A.O. (2009); Microbiological
analysis of some Vended cheese in Osun, Nigeria. Afrrican Journal of Food
Science. 3 (12) pp: 406-412.
Onyeagba , A. (2004) Laboratory guide for microbiology. Crystal
Publishers, Okigwe, Imo State.
pp 95-97.
Raheem, V.D. and Saris, D.E.J. (2009). Characterization and Application of
Calotropisprocera, a coagulant in Nigeria wara cheese. International
Journal of Food Science and Technology. 20:23-12.
Sanni A.I., Onilude A.A. and Momoh M.O. (2011). Selection of starters
and a Starter-mediated novel procedure for production of wara, a West
African soft cheese. International Journal of Food Science Technology, 34:
325-333.
AUTHORS
First Author – Ajayi, O. O, Department of Biological Sciences,
Joseph Ayo Babalola University, Ikeji Arakeji, Osun State,
Nigeria.
Second Author – Balogun, O. B, Department of Biological
Sciences, Joseph Ayo Babalola University, Ikeji Arakeji, Osun
State, Nigeria.
Third Author – Oriowo-Olaleye, M, Department of Biological
Sciences, Joseph Ayo Babalola University, Ikeji Arakeji, Osun
State, Nigeria., Corresponding Author e-mail:-
Fourth Author – Faturoti M. O, Department of Biological
Sciences, Joseph Ayo Babalola University, Ikeji Arakeji, Osun
State, Nigeria.
www.ijsrp.org