Int.J.Curr.Microbiol.App.

Sci (2014) 3(2): 20-27

ISSN: 2319-7706 Volume 3 Number 2 (2014) pp. 20-27

http://www.ijcmas.com

Original Research Article

Microbiological quality assessment of bottled yogurt of different brands sold
in Central Market, Kaduna Metropolis, Kaduna, Nigeria

N.De*, T.M.Goodluck, and M.Bobai

Department of Microbiology, Kaduna State University, Kaduna, Nigeria

*Corresponding author

ABSTRACT

An investigation on microbiological quality of twenty commercial samples of ten

different brands of bottled plain yogurt sold in Central market, Kaduna was carried
out using standard microbiological procedures. Out of 10 different brands, five
were registered by NAFDAC and the other five were not registered by NAFDAC.
The pH of the registered yogurt samples were in the range of 4.01-4.79 while the
pH of the non-registered samples were in the range of 5.28-5.63. The total bacterial
Keywords counts (TBC) of registered and nonregistered samples were in the range of 3.0 x
103-10.5 x 104 and 8.2 x 104-28.4 x 105 respectively. Out of registered samples,
Yogurt; sample A21 had the highest count whereas sample A511 had the lowest count. In
NAFDAC; case of non- registered samples, sample a411 had the highest count and sample
S.aureus; a311 had the lowest count. Statistical test (t=-2.28 and F= 9.78) revealed that there
Starter is significant difference between the colony counts of registered and non-registered
culture; samples0.05level.Lactobacillusat bulgaricus and Streptococcus thermophilus, the
TBC. bacteria present in the starter culture used for manufacturing yogurt, were present
in all the samples as the manufacturers claimed on their labels. Bacillus sp. were
obtained from all the samples whereas Staphylococcus aureus was isolated from
A41, A411, a21 and a211. Aspergillus sp. was isolated from all the samples tested.
Mucor sp. was isolated from A11, A111, a11, a111, a41 and a411. Penicillium sp.
was isolated from samples A21, A211, A31, A311, a21, a211, a31 and a311.
Acremonium sp. was isolated from A41, A411, a11 and a111. Proper care should
be taken for storage and handling of yogurt due to the fact that fungi like
Aspergillus and bacteria like S. aureus were isolated from some NAFDAC
registered samples along with some non-registered samples.

Introduction
Yogurt is one of the most popular Streptococcus thermophilus and
fermented dairy products widely Lactobacillus delbruckii spp. bulgaricus.
consumed all over the world. It is obtained The role of these two genera in yogurt
by lactic acid fermentation of milk by the manufacture can be summarized as milk
action of a starter culture containing acidification and synthesis of aromatic

20
 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 20-27

compounds (Serra et al., 2009). In Nigeria, The other ten samples were collected
it is a popular drink due to its nutritional, without any registration no. from
probiotic and organoleptic characteristics. NAFDAC and the labeling on these
But yogurt can be contaminated by fungi samples did not have any manufacturing
able to modify organoleptic characteristics date and expiration date. For non-
and be a risk of human health. Yogurt can registered samples, the samples were
be easily subjected to microbial labeled as a1-a5. For each brand, 2
contamination, as especially by fungi samples were collected and labeled
which grow and reproduce in acid accordingly (a11-a111). After collection,
environment with feasible oxygen. Some the samples were put into a cooler packed
of the molds can produce secondary toxic with ice and then were small scale and
metabolites resisting to common were commercially available in
reclamation treatments. Aflatoxins are Faisalabad, Pakistan. They observed that
carcinogenic and toxic, which is a the coliform count of yogurt produced at
secondary metabolic product of some small scale was much higher than that of
Aspergillus sp. Issazadeh et al. (2012) branded samples (produced at large scale).
worked on 60 yogurt samples and out of Montagna et al. (1998) studied on
these 60 samples, 59 samples were found microbiological quality of 166 samples
to be contaminated with AFMI. Ahmad et and observed that 7.2% of the
al. (2013) worked on quality assessment of contaminated samples were positive for
yogurt produced at industrial and fungi. El-Malt et al. (2012) worked on
evaluation of yogurt products in Qena city, microbiological Nigeria. quality of yogurt
Egypt. They showed that out of 100 sold in different markets by hawkers. An
random samples purchased from various attempt was made here to investigate the
dairy shops, street vendors and microbiological quality of yogurt of
supermarkets located in Qena city, Egypt, different brands approved by NAFDAC
S. aureus were detected in 72% and 35% and also of some yogurts not approved by
of small and large scale yogurt samples. NASFDAC but readily available in the
Though bottled yogurt is a popular drink market for consumption in Kaduna.
in Kaduna Metropolis, Kaduna, Nigeria, NAFDAC is a governmental organization
very little research has done on which is in charge of maintaining the
microbiological quality of food products made in
transported to the Microbiology
Collection of yogurt samples Laboratory of Kaduna State University
within 3h for analysis purpose.
Twenty samples of ten different brands of
bottled plain yogurt were randomly Materials and Methods
purchased from different sellers hawking
in the market. Out of 20 samples, ten were The study period was between the months
purchased with registration no. approved of July 10, 2013 to August 27, 2013.
by NAFDAC and with manufacture date Analysis of samples
and expiration date on their labels. For
registered samples, the samples were Physio-chemical characteristics of
labeled as A1-A5. For brand A, two yoghurt samples:
samples were collected from two different
hawkers and were labeled as A11-A111. After collection of samples, the color,

21
 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 20-27

organoleptic properties, consistency of reinoculated onto appropriate medium in

each sample were recorded. Then the pH order to obtain pure isolates. These pure
value of each sample was also determined isolates were then kept at 4oC for
using the 3505 pH meter. For identification purpose (Benson, 2005).
determination of pH, 80ml of each yoghurt
sample was poured into 100ml of beaker Results and Discussion
and the pH of the sample was then
determined using pH meter. Physiochemical characteristics of
samples
Total bacterial count of yoghurt
samples All the twenty samples of yoghurt
purchased from Central Market were white
The total bacterial count was done using in color. Both A11 and A111 were solid in
pour plate method as described in Benson, nature, A21- A511 were semisolid in
2005. Serial dilutions for each sample nature whereas a11- a511 were watery
were made upto 10-4. One ml of each when the samples were purchased from
dilution was introduced onto sterile the market. The pH values of samples
petridish containing plate count agar were in the range of 4.01- 5.63. The results
(PCA) and the plates were incubated at are expressed in Table 1.
37oC for 24 h. The total bacterial count
was expressed as cfu/ml. Identification of Isolates obtained from
MRS agar and NA agar
Isolation of bacteria associated with
purchased yoghurt samples For isolates obtained from MRS agar and
NA agar, gram staining was done
For lactic acid bacteria, MRS Agar was according to the procedure as described in
used. Serial dilutions upto 10-4 were made Benson, 2005. Biochemical tests such as
and 1 ml of each suspension was catalase test, coagulase test, indole test,
introduced onto agar medium using pour urease test, Methyl red and other
plate method. The plates were incubated biochemical tests were also done as
anaerobically at 37oC, 45oC and 50oC for described in Benson, 2005 in order to
48 to 72 h to obtain lactic acid bacteria identify the isolates.
colonies. Discrete colonies were isolated
and reinoculated onto appropriate medium Isolation and identification of fungi
in order to obtain pure isolates. These pure associated with the collected samples
isolates were then kept at 4oC for
identification purpose (Michaylova, 2007) The isolation and identification of the
fungi was done using Potato dextrose agar
For isolation of bacteria other than lactic (PDA). Wet preparation was done using
bacteria, Nutrient agar (NA) and lactophenol cotton blue as mountant and
MacConkey agar (MCA) were used. Serial viewed under the low power objective lens
dilutions upto 10-4 were made and 1 ml of and then using 40 high power objectives
each suspension was introduced onto NA (Benson, 2005). The values of total
agar medium using pour plate method. The bacterial count of registered samples were
plates were incubated at 37oC for 24 hrs. in the range of 3.0 x 103-10.5 x 104
Discrete colonies were isolated and whereas the values for non registered

22
 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 20-27

samples were in the range of 8.2x 104-28.4 proper system of culture dosage in
x 105. The results are expressed in Table 1. unbranded yogurt, which largely affects
the acidity of the final yogurt (Abrar et al.,
Isolation of bacterial isolates from 2009). The total bacterial counts of the
purchased yoghurt samples registered and non-registered samples
were in the range of 3.0 x 103 9.0 x 104b
The cultural and morphological and 8.2 x 104- 28.4 x 105 respectively.
characteristics of isolates obtained from There is significant difference in TBC
nutrient agar medium were listed in Table among the registered and nonregistered
2. The isolates were labeled as NS1-NS24. samples (t= - 2.28 and F= 9.78 at 0.05
Biochemical characteristics revealed that level). Ifeanyi et al. (2013) carried out a
NS1-NS20 was Bacillus sp. and NS21- research on microbiological assessment of
NS24 was Staphylococcus aureus. four brands of yogurt samples collected
Bacillus sp. was isolated from all the from vendors in Onitsha market and found
samples whereas Staphylococcus aureus the values of these four brands were in the
was isolated from A41, A411, a21 and range of 1.9 x 105- 6.1 x 105. Obande and
a211. Azua (2013) worked on yogurt sold in
Makurdi, Benue state, Nigeria and
Two types of colonies were isolated from observed the mean value for yogurt
all the samples in MRS agar medium. The samples was 1.4 x 107cfu/ml. The results
colonies were labeled as MS1-MS20 and obtained in this study for yogurt samples
ms1 - ms20. All the colonies were agree with the range obtained for yogurt
subjected to gram reaction and the size and by other investigators. The high count for
shape of the cells were observed. The non-registered samples especially for
results are expressed in Table 3. The samples a41 and a411 is indicative of poor
results of catalase activity, sugar handling during processing and packaging
utilization tests are also listed in Table 3. of the product.

Isolation and identification of fungi Bacillus sp. was isolated from all the
from samples samples studied. The presence of Bacillus
sp. in all the yogurt samples implies post-
Aspergillus sp. was isolated from all the pasteurization contamination (Huck et al.,
samples tested. Mucor sp. was isolated 2008). S. aureus was isolated from
from A11, A111, a11, a111, a41 and a411. samples A41, A411, a21 and a211
Penicillium sp. was isolated from samples samples. El-Malt et al. (2012) also
A21, A211, A31, A311, a21, a211, a31 detected S. aureus in 72% of the 100
and a311. Acremonium spp. was isolated yogurt samples they analyzed in Qena
from A41, A411, a11 and a111. The City, Egypt. Presence of S. aureus usually
results are shown in Table 4. indicates contamination from food
handlers (Abdel Hameed et al., 2009).
The pH values of yogurt samples
registered by NAFDAC were in the range Aspergillus sp. was isolated from all the
of 4.01-4.79 but the values for non- samples analyzed for this research
registered samples were in the range of purpose. Mucor sp. was isolated from
5.28-5.63. The high pH and low acidity A11, A111, a11, a111, a41 and
may be due to the fact that there is no a411.

23
 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 20-27

Table.1 Total bacterial count (cfu/ml) and pH of different yogurt samples purchased from
different hawkers in Central Market, Kaduna.

S/N Sample code pH TBC

1 A11 4.79 4.5 x 104
2 A111 4.63 4.9 x 104
3 A21 4.23 9.0 x 104
4 A211 4.30 8.9 x 104
5 A31 4.52 6.4 x 104
6 A311 4.50 6.6 x 104
7 A41 4.62 10.4 x 104
8 A411 4.57 10.5 x 104
9 A51 4.01 3.3 x 103
10 A511 4.06 3.0 x 103
11 a11 5.31 22.1 x104
12 a111 5.28 22.6 x 104
13 a21 5.45 21.2 x 104
14 a211 5.42 21.0 x 104
15 a31 5.58 8.6 x 104
16 a311 5.60 8.2 x 104
17 a41 5.45 28.4 x 105
18 a411 5.48 28.2 x 105
19 a51 5.63 20.5 x 104
20 a511 5.59 20.5 x 104
Key: A11-A511are registered yogurt samples; a11-a511 are non-registered samples

Table.2 Cultural and Biochemical Characteristics of Isolates on NA Medium

S/N IN MC GR CS CA CO ID UR CI MR VP MO OR
1 NS1- Cream, circular, + Rod + + - + + + - + Bacillus
NS20 smooth, opaque sp.
and mucoid
2 NS21 Yellowish, + Coc + + + - - - - - Staphylo-
- circular, opaque ci coccus
NS24 and mucoid aureus
IN- Isolate number; GR- Gram reaction; CS- cell shape; CA-catalase test; Co- coagulase test;
ID-indole test; UR-urease test; CI- citrate utilization; MR- methyl red test; VP- Voges-
Proskauer test; MO- motility test, OR- isolated organism.

24
 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 20-27

Table.3 Cultural and Biochemical Characteristics of Isolates on MRS agar

Isolate Shape and color Glucose GR CS CA Organism

Number utilization
MS1-MS20 Yellowish, + + Growth at - L. bulgaricus
mucoid rounded No gas 45oC Rod
colonies Production
ms1-ms20 Growth at - S. thermophiles
50oC Cocci
Keys:
GR= gram reaction; CS= colony size; CA- catalase activity; += Positive; -= negative

Table.4 Cultural and morphological characteristics of fungi isolated from yogurt samples

Isolate
Colony morphology Microscopic morphology Organism
number

PS1- Growth begins as yellow Exhibits septate hyphae long Aspergillus niger
colonies that soon develop a conidiophores that support spherical
PS20
black, dotted surface as vesicle that give rise to mutulae and
conidia are produce within 2- phalides from which conidia are
6 days the colony becomes produce.
jet black and powdery and
the reverse remains cream
color.

PS21- Colonies characteristically The hyphae appear to be coarse and Mucorsp

produce a fluffy white fills the entire culture dish rapidly with
PS26
growth that diffusely covers hyphae dotted with brown or black
the surface of the agar within sporangia.sporangiosphores are branch
24-48 hours. and have at their tip a sporangium filled
with sporangiophores, no rhizoids or
stolons.

PS27- Green colonies, surface of Hyphae are septate and produce brush Penicilluimsp
colonies becomes powdery like conidiophores , conidiophores
PS34
due to presence of conidia. produce metulae from which phalides
producing chains of conidia arise.

PS35- White colonies. Septate hyphae that produce single Acremoniumsp

unbranched tube like phalides, phalides
PS38
give rise to cluster of conidia at the tip
of the phalide.

25
 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 20-27

Penicillium sp. was isolated from samples and commercially imported starter
A21, A211, A31, A311, a21, a211, a31 Milchwissensch 64(4): 392-395
and a311. Acremonium spp. was isolated Ahmad, I., Gulzar,M., Shahzad, F., Yaqub,
fromA41, A411, a11 and a111. Montagna M. and Zhoor, T. 2013. Quality
et al. (1998) has examined some different assessment of yogurt produced at large
yogurt to verify fungal contamination and (industrial) and small scale. The
potentially toxigenic moulds in Journal of Animal and Plant Sciences,
commercially available yogurt samples. Benson H. 2005. Microbiological
th
7.2% of the samples was positive for fungi Applications 9 ed. McGraw Hill
such as Penicillium and Cladosporium sp. Higher Education New York pp. 239-
Fungi in yogurt samples generally 266 and 299-304
correspond to poor cleaning practices and El-Malt, L.M., Abdel Hameed, K.G. and
the use of unhygienic techniques or Mohammed , A.S 2013.
inadequate storage conditions. Thus the Microbiological evaluation of yogurt
fungal contamination might to occur Products in Wena City, Egypt.Vet
during transportation process and/or World 6(7): 400-404
packaging, storage, transport and sales; Huck, J.R., Sonnen, M. and Boor, K.J.
besides it can survive in food by its ability 2008. Tracking heat resistant, cold
to adapt at differential conditions. thriving fluid milk spoilage Bacteria
from farm to packaged product
Though all the yogurt samples have L. Journal of Daily Science 91:
bulgaricus and S. thermophilus , the 1218:1228
organisms present in starter culture, the Ifeanyi, V.O., Ihesiaba, O. M. and Ikenga,
samples were contaminated with bacteria C. 2013. Assessment of
like Bacillus sp. and S. aureus. NAFDAC Microbiological quality of yogurt sold
registered samples are commonly products by street vendors in Onisha
of high standard but in this case these Metropolis, Anambra State, Nigeria.
products are not safe for people to British Microbiology Research
consume. Also the presence of fungi 3(2):198-205
especially those capable of producing Issazadeh, K., Darsanaki, R.K. and
aflatoxins is a great concern. So there Pahlaviani, K. 2012. Occurrence of
needs to be a HACCP program for aflatoxin M1 levels in local yogurt
transportation /packaging and storing of samples in Gilan Province,Iran Annals
yogurt in Nigeria. of Biological Research 3(8):3853-3855

References Montagana, M.T., Erroi, R., Sanapo, S.,

Caggiano, G., Bagordo A. and Donno
Abdel Hameed, K.G. and El-Malt , L.M. A. De 1998. Food products and fungal
2009. Public health hazard of contamination Note 1. Preliminary
Staphylococcus aureus isolated from investigation in commercial yogurt
raw milk and ice cream in Qena Journal of Preventive Medicine and
governorate. Assiut Vet. Med. J. Hygiene 39: 68-70
55(121): 191-200 Michaylova, M., Minkova, S., Kimura, K.,
Abrar, M., Anjum, F.R., Zahoor, T., Sasaki, T. and Isawa, K. 2007.
Hussain, S. and Ahmad, S. 2009. Isolation and characterization of
Chemical and sensory characteristics Lactobacillus delbruckii ssp.
of yogurt produced by locally isolated
26
 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 20-27

bulgaricus and Streptococcus

thermophilus from plants in Bulgaria
FEMS Microbiology Letters269(1):
160-169
Obande, A. G. and Azua, E. T. 2013.
Extent of microbial contamination of
nono, fresh cow milk and yogurt sold
in Makurdi, Benue State, Nigeria
Journal of Microbiology and
Biotechnology Research 3(3):6-14
Serra, M., Trujillo,J.A., Guamis, B. and
Ferragut, V. 2009. Flavor profiles and
survival of starter cultures of yogurt
produced from high pressure
homogenized milk. International
Dairy Journal, 19:106-109.

27