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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018
http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878
www.ijeab.com Page | 1308
Microbiological Quality of Kunnu-Zaki Drinks
Sold in Some selected Towns in Osun State,
Nigeria
Imoukhuede T. P.1, Adepeju A. B.2, Akinsuroju M. O.3
1Department of Science Laboratory Technology, Faculty of Science, Ekiti State University, Ekiti Nigeria
2Department of Food Science and Technology, Joseph Ayo Babalola University, Osun State, Nigeria
3Department of Microbiology, Joseph Ayo Babalola University, Osun State, Nigeria
Email: teniusng@yahoo.com
Abstract— The microbiological quality of freshly
processed and hawked kunnu-zaki drinks, a common
Nigerian non-alcoholic beverage was investigated in
some selected towns in Osun State, South Western
Nigeria. A total of nine (9) towns were assessed. Kunnu-
zaki drinks were purchased from these towns on twelve
different occasions for a period of six month. The samples
were microbiologically analyzed using standard methods.
pH values ranged from 3.09 - 4.21 in East and West
senatorial district. The TTA of the samples ranged from
0.32 - 0.49 in Central and East senatorial district. All the
screened drink samples had varying levels of bacterial
contamination ranging from 4.2 x 103
to 15.0 x 103
CFU/ml, 4.1 x 104
to 9.6 x 104
CFU/ml and 3.5 x 105
to
9.0 x 105
CFU/ml for total bacteria, coliform and E. coli
count respectively. Total coliform count on MPN ranged
from 3 MPN/ml to 93 MPN/ml. 77.78% of the total
sampled drinks(n=108) that is(84/108) had bacteria and
fungi count. While all the drinks sampled had total
coliform count on MPN exceeding the recommended safe
level of zero organisms detectable per 100ml. Seven (7)
bacteria species were isolated from the kunnu-zaki drink
sampled. The bacteria isolated were Escherichia coli,
Bacillus species, Staphylococcus species, Pseudomonas
species, Streptococcus specie, Enterobacter species, and
Klebsiella species. Fungal count ranged from 3.3 x 106
to
8.0 x 106
CFU/ml respectively. Four (4) fungi were also
isolated which include Aspergillus fumigatus, Penicilium
italicum, Aspergillus niger and Aspergillus flavus. It can
therefore be concluded that the presence of these isolated
organisms in kunnu-zaki samples analyzed in Osun State
could serve as indicator for the need to promote
awareness about the possible health hazards that could
arise due to the unhygienic ways of handling and
processing of the beverage.
Keywords— Physico-chemical analysis, Bacteria count,
Coliform count (MPN), Fungi count and Frequency of
Isolated organisms.
I. INTRODUCTION
Beverage is a liquid drink prepared for human
consumption (Robert, 2006). The word “beverage” has
been derived from the Latin word “bever” meaning rest
from work. After work, one tends to feel thirsty due to
fluid loss through perspiration and one is inclined to drink
water or other potable beverages to compensate fluid loss.
Beverages are potable drinks which have thirst-
quenching, refreshing, stimulating and nourishing
qualities (Achi, 2005). By refreshing, one means the
replenishment of fluid loss from the body due to
perspiration. Nourishment is provided by the nutrients in
the beverages, especially fruit juices.
Kunnu-zaki (Millet drink) also referred to as “sweet
kunnu-zaki” is a popular traditional and important cereal
based, non- alcoholic fermented beverage (Adejuyitan et
al., 2008) made from pearl millet (Pennisetum glaucum),
maize (Zea mays), or sorghum (Sorghum bicolor), grains
but the most commonly used basic ingredient is millet.
Maize (Zea mays), millet (Pennisetum glaucum), rice
(Oryza sativa) and sorghum (Sorghum bicolor) cereals
provide mainly carbohydrates and low quality protein
(Steinkraus, 1997). It is generally acceptable and widely
consumed throughout Nigeria by children and adults as
breakfast drink or food supplements and also a weaning
food for infants due to its refreshing qualities most
especially in the northern parts and it is consumed
throughout the year mostly during dry seasons
(Elmahmood and Doughari, 2007). Kunnu-zaki is a
refreshing drink usually used to entertain visitors,
appetizers and is commonly used or served at social
gatherings both rural and urban centers, hawked in the
motor parks, school premises and market places (Amusa
et al., 2005). It is a staple beverage that is relatively
nutritious and affordable when compared to other
carbonated and non-carbonated drinks (Adejuyitan et al.,
2008). It has low viscosity, a sweet-sour taste depending
on the level of fermentation and is milky cream in
appearance (Adeyemi and Umar, 1994). It has immense
International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018
http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878
www.ijeab.com Page | 1309
social, economic, nutritional and medicinal benefits to
most consumers (Akoma et al., 2006). The preparation of
locally fermented foods like kunnu-zaki has become a
technology in many homes particularly in rural
communities and more recently in the urban areas where
more women have developed the skill and commercial
production which has helped to alleviate poverty amongst
the people (Essien et al., 2011).
Kunnu-zaki is been prepared from local cereals which are
very common and part of our staple food substances. It is
relatively produced in low cost which makes it not
expensive because grains and other ingredients used for
production are locally sourced and are mostly grown
within the savannah region of Nigeria such as Bauchi,
Kano, Sokoto and Kastina states (Adebayo et al., 2010)
and almost throughout the years, most especially the
savannah belt of West Africa and the packaging materials
are also available, cheap and easily affordable.
Cereals are grains or edible fruits of the grass-monocot
family that may be cultivated and used as foods. In India
and Africa, cereals products comprise 80% or more of the
average diet, 50% in Central and Western Europe and
between 20-25% in the U.S. (Onwueeme and Finha,
1991). Millets are cereal crops or grains for foddler and
human food (Ray and Sivakumar, 2009). Millets are in
the family of cereals grown globally with differential
importance across continents and within regions of the
world. It is consumed as staple food and drink in most
areas. Across Africa several indigenous foods and drinks
are made from flour/meal and malt of millet. They are
nutritionally equivalent or superior to other cereals
(Obilana and Taylor, 2002). In the Northern area of
Nigeria Millet is known as Jero, ‘Oka baba’ in yoruba
land and in America, it is known as milo. It can be stored
for 6 months without any significant spoilage. Millet are
highly tolerant and are very nutritious containing low
phytic acid and are rich in dietary fiber, iron, phosphorus,
potassium, magnesium, zinc, thiamine, riboflavin calcium
and vitamin B (niacin, B6 and folic acid) which make
millet more superior than most cereals, are now being
enhanced through biofortification and micronutrient
research (Obilana and Taylor, 2002) which are essential
for body growth and development. It is easily digested
and gluten-free, an excellent choice for anyone with
gluten-intolerance (Oladele and Aina, 2007).
Kunnu-zaki processing is mostly done by women using
simple household equipment and utensil depending on
cereal availability. Kunnu-zaki is produced from malted
millet; a portion of the cereal is malted, dried, ground and
then mixed with the uncooked portion. The mixture is
then added to the cooked portion and stirred vigorously
and allowed to ferment (Akoma et al., 2012). Spices such
as ginger (Zingiber officinales), alligator pepper
(Afromonium melegueta), red pepper (Capsicum species),
black pepper (Piper guineense) and cloves (Syzygium
aromaticum) are commonly added to improve flavor and
taste while honey, sweet potatoes and sugar are also
added to act as a sweetener (Ahmed et al., 2003). The
processed kunnu-zaki is usually packed for sale either in
nylon or in a plastic container and can also be stored in
refrigerators. Some people in some regions prefer it with
pepper and sugar while some with little or no pepper and
sugar (Adeyemi and Umar, 1994). All these ingredients
perform one function or the other in the course of the
preparation. The most abundant constituent of kunnu-zaki
is water and it acts as the medium in which all other
constituents are dissolved and contain only traces amount
of inorganic substances. Spices are usually added in small
quantities to improve taste and flavor. Because these are
agricultural commodities, they may contain a high level
of microbial impurities (Adeyemi et al., 1994; Bibek,
2001). The quality and quantity of the products depend
largely on the quality of the ingredients and its proper
handling in the course of production by the producer.
Hence, kunnu-zaki samples for different market in Osun
state are purchased to determine the microbial and fungi
load.
II. MATERIALS AND METHODS
STUDY LOCATION
Osun State is located in the South Western part of
Nigeria. It covers an area of approximately 14,875 square
kilometers, lies between longitude 04 00E and latitude 05
558 and is bounded by Ogun in South, Kwara in North,
Oyo in West and Ondo States in the East. It total
population is approximately 3,416,959.
COLLECTION AREAS
Samples were collected and analyzed from nine (9) major
markets using Senatorial districts West, East and Central
to ensure adequate representation of samples in Osun
State Nigeria, which include three (3) popular towns from
each Senatorial district. The towns are: Gbongan, Ikire
(Sabo market) and Apomu all in Osun West Senatorial
districts, Ipetu-Ijesa, Ikeji-Arakeji and Owena-Ijesa all in
Osun East Senatorial district, Ada, Ikirun (Oja tuntun)
and Ororuwo all in Osun Central Senatorial district.
COLLECTION OF SAMPLES
Freshly prepared kunnu-zaki samples were purchased in
plastic containers (as packaged by the seller) from
different Senatorial market locations within Osun State.
The samples were labeled and held at 4 0C by placing
them in refrigerated coolers and conveyed in an ice
packed cooler and transported to Microbiology and
Biochemistry Department Laboratory of Joseph Ayo
Babalola University within three hours (3 hours) of
collection for microbiological and physico-chemical
analysis respectively. A total of 108 kunnu-zaki drinks
samples were purchased and sampled. The period of
International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018
http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878
www.ijeab.com Page | 1310
study was six months. All samples were subjected to the
same analysis.
PHYSICO-CHEMICAL ANALYSIS OF KUNNU-
ZAKI
The rate of fermentation by each bacteriumon the kunnu-
zaki sample was assessed by testing for pH and (TTA)
Titratable acidity (AOAC, 2005).
DETERMINATION OF pH OF THE SAMPLES
The pH of the samples was determined using a laboratory
pH meter (Jenway 3015, model 10). The pH was
measured by putting 10 ml of the kunnu-zaki into a
beaker and readings were taken by dipping the electrode
into the kunnu-zaki samples (rinsing out the electrode
with distilled water), and measurements taken from the
display screen when the readings stabilized (Ofori et
al.,1994).
DETERMINATION OF TOTAL TITRATABLE
ACIDITY (TTA)
This is to measure the acidity in a sample. The Total
Titratable acidity (TTA) calculated as percentage lactic
acid was determined following the method described by
(AOAC, 2005).
PROCEDURE
A 10 ml of kunnu-zaki sample was measured into a dried
conical flask, 3 drops of phenolphthalein indicator was
added and the flask thoroughly shaken. It was then
titrated against 0.1 M NaOH (Sodium hydroxide) to a
pink colour end-point (compared against a white
background) and the titre volume was noted and
calculated.
DETERMINATION OF MICROBIAL COUNT
This was carried out using standard microbiological
techniques following appropriate dilution on agar plates
of Nutrient Agar (NA) for bacteria count, MacConkey
Agar (MCA) for coliform count, Eosin Methylene Blue
agar (EMB) for E. coli and Potato Dextrose Agar (PDA)
for fungi count. The agar media were of oxoid grade
using pour plate method (Lateef et al., 2004).
PROCEDURE
Serial dilution was carried out using 1 ml of kunnu-zaki
samples into9 ml of water. Stocks were prepared in test-
tubes serially in rows containing 9.0 ml of distilled water
representing each sample from each location (APHA,
2001). Then from each suspension 1 ml of dilution was
pipette into a set of Petri-dishes and was overlaid with 20
ml of freshly prepared Nutrient agar (NA), MacConkey
(MCA) and Eosin methylene blue (EMB) while others
were overlaid with 20 ml of freshly prepared potato
dextrose agar (PDA) treated with chloramphenicol (1 per
100 ml) prior to sterilization. Colonies which develop
after incubation for 18 to 24 h at 37 0C on the cultured
plates were counted using Gallenkamp colony counter
and were sub-cultured to obtain pure isolates. The pure
isolates were aseptically transferred into Nutrient agar
slants and incubated. After 24 h incubation morphological
characterization and biochemical tests were carried out on
the isolates to aid identification to genus level on bacteria
(Cowan and Steel 1985). The purified fungal isolates
were identified on the basis of standard cultural,
morphological and microscopic characteristics as
described by (Samson et al., 1988). The fungal isolates
were sub-cultured on to a sterile fresh media and
incubated at room temperature for 3-5 days.
Morphological identification was based on colour,
textures spreading rate of each colony on the Potato
dextrose agar plate.
DETERMINATION OF MOST PROBABLE
NUMBER (MPN)
The multiple tube method was used to estimate coliform
numbers. The presumptive, confirmed and completed test
was done by adding varying quantities of kunnu-zaki such
as 0.1 ml, 1.0 ml and 10 ml to varying quantities and
strength of MacConkey broth in test tubes containing
Durham tube (APHA, 2001). The MPN of coliforms
present in the sample was estimated by making reference
to McCrady table or probability table (APHA, 2001).
III. RESULTS AND DISCUSSION
The results show that the pH of the samples ranged from
3.09 - 4.21 in East and West Senatorial district (Table 1).
The result of the analysis revealed that all samples used
have an acidic pH range of 3.09 - 4.21. The level of
acidity of kunnu has been reported by several researchers
including Efiuvwevere and Akoma, (1995) and Akoma et
al., (2006).
The TTA of the samples ranged from 0.0032 - 0.0049 in
central and east Senatorial district (Table 2). East
Senatorial district had the highest TTA values of kunnu-
zaki drink in the month of July according to the period of
samples collection.
All the kunnu-zaki drinks sampled were contaminated
with varying level of bacterial load ranging from 4.2 x 103
to 15.0 x 103 CFU/ml in West and East Senatorial district
(Table 3). This is in agreement with the findings of
Hatcher et al., (1992), Elmamood and Doughari, (2007),
Lawal (2012) and Aboh and Oladosu, (2014) who
reported a total bacterial count of 5.0 x104 to 2.0 x 106,
1.0 x 102 to 8.9 x 104 CFU/ml, 5.0 x 104 to 1.79 x 105
CFU/ml and 5.1 x 102 to 2.0 x 108 CFU/ml respectively.
The high bacteria count observed in this study might be
attributed to factors such as the environment, which
include exposure of the foods (kunun-zaki) to air, soil,
type of water used in processing, post production
operations and personal hygiene of the handlers (Kawo
and Abdulmumin, 2009; Aboloma, 2008). Exposure of
the foods to air or dust at the point of sale is likely to
increase the counts of the bacteria as virtually most of the
International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018
http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878
www.ijeab.com Page | 1311
bacteria are carried in aerosols by dust and air (Karagozlu
et al., 2007). Also, during production, most handlers
sometime dip their hands into the containers while
making kunun-zaki.
The total coliform bacterial count in the kunnu-zaki drink
samples using MPN method ranged from 3 MPN/ml to 43
MPN/ml in East, 3 MPN/ml to 93 MPN/ml in West and 3
MPN/ml to 39 MPN/ml in Central. All the samples
108/108 (100%) of the kunnu-zaki samples examined had
coliform count (Table 4). The result shows that West
district had the highest total coliform count.
Seven (7) bacteria genera were isolated from the kunnu-
zaki drink samples. These include Escherichia coli,
Pseudomonas species, Staphylococcus species,
Enterobacter species, Klebsella species, Streptococcus
species and Bacillus species. The detection of S. species
was equally isolated by Olasupo et al., (2002) from wara
and kunun-zaki, a cereal based, non-alcoholic beverage,
also by Aboh and Oladosu, (2014) from kunun-zaki. The
presence of organisms like Bacillus species,
Streptococcus has been reported by Adeyemi and Umar,
(1994) and Ayo, (2004) who also isolated these
organisms from kunun-zaki. The presence of Klebsiella
species as recorded in this study is usually associated with
faecal contamination. Being an enteric bacterium its
presence indicates poor practices among handlers. Due to
the significance of the faecal-oral route transmission for
many bacterial food-borne diseases, basic hygiene
measures assume a decisive importance in food safety
management (Uzeh et al., 2006). The high occurrence of
E. coli (77.78%) in kunnu-zaki is an indication of faecal
contamination. Ironically, most food handlers do not
practice good personal hygiene and do not follow good
manufacturing practices, which could reduce the
occurrence of such bacteria in foods (Bukar et al., (2009);
Kawo and Abdulmumin, (2009)). Egbere et al., (2007)
reported the presence of organisms like Bacillus cereus,
Staphylococcus aureus and Escherichia coli (PHLS,
1996). It is possible that contamination by these
pathogens could have occurred during sieving and
packaging, as most of the people involved in the
production, packaging and hawking do not take necessary
precautions, and as such contamination could be very
prominent (Elmamood and Doughari, 2007).Gadaga et
al., (2004) reported that pathogens have been isolated
from some fermented foods and some laboratory tests
have shown the possibility of pathogens to survive and
grow in some fermented foods. He said post processing
contamination is often cited as the major cause of food
poisoning, this agrees with the observation made as
regards the source of kunnu collected for this study which
is not really hygienic. Gadaga et al., (2004) equally said
that Escherichia coli, is the most commonly encountered
pathogens in African fermented food. This tally with the
types of organism isolated in this study. The occurrence
of bacterial pathogens in fermented foods suggests a need
for caution in the consumption of this beverage. The
source of contamination may also have come from the
spices used as additives (Essien et al., 2011 and Lawal,
2012).
Fungal count ranged from 3.3 x 106 to 8.0 x 106 CFU/ml
in West and Central district (Table 5). A total of Four (4)
different moulds were isolated from the kunnu-zaki drink
samples in the course of study. These were Aspergillus
fumigatus, Penicilium italicum, Aspergillus niger and
Aspergillus flavus.
The frequency of occurrence of the organisms isolated
from kunnu-zaki drink samples are shown in Table 6.
Escherichia coli and Streptococcus species were found in
77.78% (84/108) of samples examined, Pseudomonas
species (33%), Staphylococcus species (66.67%),
Enterobacter species (55.56%), Klebsella species
(44.44%), Bacillus species (33.33%), Aspergillus flavus
was found in 77.78% (84/108) of the samples examined,
Aspergillus fumigatus (44.44%), Penicilium italicum
(66.67%) and Aspergillus niger (55.55%). Aspergillus
species is ubiquitous, aerobic fungi that can survive in air,
dust. The presence of fungi may be attributed to the
acidic nature of the sample since it has been observed that
molds are capable of utilizing organic acids. Also the
presence of fungi in the food may lead to food poisoning
and contaminated fungi result in the production of
undesirable odour, colour changes and loss of taste of the
sample. The presence of these organisms in the sample
may be due to the nutritional composition of the millet;
these nutrients are present in different proportions.
Aspergillus flavus is a common mold in the environment,
and can cause storage problems in stored grains. It can
also be a human pathogen, associated with Aspergillosis
of the lungs and sometimes causing corneal, otomycotic
of aflatoxin and nasoorbital infections. Aspergillus flavus
spores are allergenic (Klich, 2007). Ijabadeniyi (2007)
and Omemu (2011) isolated Penicillium, Aspergillus,
Mucor and Rhizopus during the early stage of maize
fermentation for ogi production.
The bacterial and fungal counts showed that all the
kunnu- zaki sampled had a high prevalence of these
contaminants and spoilage organisms which of course is
of great concern. They are therefore considered
microbiologically unsafe for human consumption when
not hygienically prepared and utensils cleaned regularly.
Clusters of infection caused by the organisms have been
linked to poorly maintained utensils, poor personal
hygiene, improper storage facilities, and poor water
supply (Oyelana and Coker, 2012). Also it can be linked
to unhygienic human handlers, dispensing of the extract
into bottles, addition of flavors and sweeteners and
process of cooling of the extract (Omemu et al., 2007a).
International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018
http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878
www.ijeab.com Page | 1312
Many native African beverages are little known outside
the parent continent. Effort should be made to improve in
the quality, shelf-life and production process of these
indigenous beverages so that large- scale production for
export outside the continent can be carried out. Many
people now prefer imported and exotic beverages because
of their attractive form, long shelf life, ease of
transportation and other forms of utility which consumers
associate with them (Achi, 2005). As of now, there are no
industries involved in production of Kunnu-zaki. Kunnu-
zaki is widely believed to be of immense social, economic
and medicinal importance to its numerous consumers
(Akoma et al., 2006).
Table.1: pH Values of Kunnu-zaki Drink Sampled from Some Selected Towns in Osun State.
pH VALUES
MONTHS
JUL AUG SEP OCT NOV DEC
TOWNS
Gbongan 3.93 3.90 3.86 3.92 3.89 3.92 3.88 3.87 3.93 3.88 3.91 3.92
Ikire 4.03 3.98 4.00 3.99 4.01 4.03 4.02 4.01 4.00 4.01 3.97 4.00
Apomu 4.20 4.18 4.16 4.19 4.20 4.14 4.13 4.17 4.19 4.16 4.21 4.15
Ipetu-Ijesha 3.83 3.84 3.86 3.33 3.50 3.85 3.55 3.40 3.78 3.80 3.88 3.60
Owena 3.89 3.51 3.65 3.99 3.55 3.19 3.09 3.67 3.68 3.88 3.85 3.89
Ikeji-
Arakeji 3.60 3.50 3.58 3.63 3.45 3.52 3.57 3.48 3.56 3.59 3.60 3.58
Ada 3.94 3.90 3.91 3.93 3.80 3.79 3.88 3.91 3.93 3.69 3.89 3.91
Ikirun 3.75 3.69 3.72 3.74 3.68 3.75 3.73 3.66 3.69 3.72 3.71 3.77
Ororuwo 3.73 3.72 3.71 3.70 3.67 3.73 3.69 3.71 3.70 3.66 3.72 3.69
Table.2: Total Titratable Acidity Values for Kunnu-zaki Drink Sampled from Some Selected Towns in Osun State.
TITRABLE ACIDITY VALUES
MONTHS
JUL AUG SEP OCT NOV DEC
TOWNS
Gbongan 0.0038 0.0041 0.0036 0.0038 0.0039 0.0037 0.0035 0.0040 0.0039 0.0037 0.0039 0.0041
Ikire 0.0036 0.0040 0.0042 0.0037 0.0041 0.0041 0.0039 0.0036 0.0038 0.0040 0.0033 0.0037
Apomu 0.0035 0.0038 0.0039 0.0036 0.0038 0.0036 0.0040 0.0038 0.0041 0.0041 0.0038 0.0036
Ipetu-
Ijesha
0.0037 0.0041 0.0043 0.0039 0.0040 0.0038 0.0039 0.0041 0.0038 0.0037 0.0035 0.0039
Owena 0.0049 0.0047 0.0037 0.0040 0.0038 0.0036 0.0037 0.0038 0.0037 0.0039 0.0040 0.0038
Ikeji-
Arakeji
0.0040 0.0039 0.0038 0.0041 0.0036 0.0038 0.0037 0.0034 0.0041 0.0040 0.0039 0.0038
Ada 0.0037 0.0045 0.0038 0.0038 0.0039 0.0039 0.0037 0.0038 0.0039 0.0041 0.0040 0.0041
Ikirun 0.0036 0.0038 0.0040 0.0040 0.0041 0.0039 0.0032 0.0037 0.0044 0.0040 0.0039 0.0041
Ororuwo 0.0039 0.0041 0.0037 0.0039 0.0038 0.0040 0.0036 0.0041 0.0040 0.0039 0.0039 0.0038
International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018
http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878
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Table.3: Bacterial Count (CFU/ml) of Kunnu-zaki Drink Sampled from Some Selected Towns in Osun State.
BACTERIAL COUNT (CFU/ml)
MONTHS
TOWNS
JUL AUG SEP OCT NOV DEC
Gbongan
9.7
×103
9.0
×103
8.2
×103
9.5
×103
4.2
×103
8.0
×103
7.9
×103
7.5
×103
8.4
×103
8.6
×103
6.5
×103
7.0
×103
Ikire
6.5
×103
7.0
×103
7.5
×103
8.0
×103
8.0
×103
9.0
×103
7.6
×103
9.2
×103
9.3
×103
8.8
×103
10.0
×103
9.5
×103
Apomu
8.0
×103
8.8
×103
8.0
×103
9.1
×103
9.6
×103
8.9
×103
7.3
×103
7.4
×103
6.0
×103
9.2
×103
8.5
×103
6.4
×103
Ipetu-
Ijesha
6.6
×103
7.8
×103
6.8
×103
6.1
×103
9.5
×103
7.4
×103
8.6
×103
6.6
×103
8.8
×103
9.3
×103
7.7
×103
8.1
×103
Owena
15.0
×103
6.1
×103
5.3
×103
4.9
×103
7.6
×103
9.7
×103
6.0
×103
7.1
×103
6.6
×103
9.1
×103
5.5
×103
7.1
×103
Ikeji-
Arakeji
9.0
×103
7.5
×103
7.1
×103
5.2
×103
5.5
×103
7.3
×103
8.0
×103
8.5
×103
9.2
×103
7.3
×103
7.0
×103
8.1
×103
Ada
6.6
×103
4.7
×103
8.2
×103
6.7
×103
7.3
×103
8.4
×103
6.6
×103
9.1
×103
7.5
×103
6.2
×103
7.5
×103
7.5
×103
Ikirun
10.0
×103
8.2
×103
8.3
×103
9.9
×103
5.0
×103
8.9
×103
9.5
×103
9.0
×103
8.1
×103
9.7
×103
6.0
×103
9.1
×103
Ororuwo
7.5
×103
9.1
×103
8.6
×103
9.5
×103
8.0
×103
7.2
×103
9.5
×103
7.1
×103
7.2
×103
6.7
×103
9.0
×103
6.2
×103
Table.4: Coliform Count (MPN McCredy or Probability Table) of Kunnu-zaki Drink Sampled from Some Selected Towns in Osun
State.
COLIFORM COUNT MPN/ml
MONTHS
TOWNS
JUL AUG SEP OCT NOV DEC
Gbongan 9 3 7 3 3 43 11 28 75 93 3 4
Ikire 20 43 28 21 20 39 3 23 4 23 13 11
Apomu 7 3 4 7 7 4 3 11 9 11 23 28
Ipetu-Ijesha 11 23 21 7 3 4 4 20 28 43 3 3
Owena 4 7 4 11 13 3 21 21 28 3 4 7
Ikeji 11 21 13 7 4 3 7 20 11 43 28 39
Ada 3 4 11 9 20 21 4 3 7 7 3 3
Ikirun 7 9 13 4 3 11 21 11 13 3 4 4
Ororuwo 3 4 11 39 13 9 3 11 39 4 11 3
Legend:
MPN= (Most Probable Number)
International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018
http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878
www.ijeab.com Page | 1314
Table.5: Fungi Count of Kunnu-zaki Drink Sampled from Some Selected Towns in Osun State.
FUNGI COUNTS (SFU/ml)
MONTHS JUL AUG SEP OCT NOV DEC
TOWNS
Gbongan
4.5
×106
5.0
×106
3.3
×106
6.5
×106
4.0
×106
5.0
×106
4.1
×106
4.3
×106
6.0
×106
4.7
×106
5.1
×106
4.6
×106
Ikire
4.2
×106
5.8
×106
3.3
×106
5.9
×106
5.0
×106
6.0
×106
5.7
×106
6.7
×106
6.9
×106
6.4
×106
5.0
×106
6.0
×106
Apomu
5.0
×106
4.7
×106
6.0
×106
6.8
×106
6.9
×106
5.6
×106
5.0
×106
5.2
×106
6.3
×106
7.8
×106
6.5
×106
5.8
×106
Ipetu-
Ijesha
3.9
×106
5.8
×106
4.6
×106
4.0
×106
7.6
×106
4.8
×106
7.0
×106
5.0
×106
5.8
×106
5.0
×106
4.9
×106
5.5
×106
Owena
4.8
×106
4.3
×106
4.2
×106
3.8
×106
6.0
×106
6.8
×106
4.1
×106
5.3
×106
5.0
×106
6.4
×106
4.2
×106
5.9
×106
Ikeji-
Arakeji
4.9
×106
6.4
×106
5.8
×106
4.0
×106
3.5
×106
5.1
×106
6.3
×106
6.0
X106
4.9
X106
5.8
×106
4.9
×106
6.0
×106
Ada
5.3
×106
3.8
×106
6.0
×106
5.8
×106
6.0
×106
5.8
×106
4.8
×106
6.1
×106
5.7
×106
4.9
×106
6.0
×106
5.3
×106
Ikirun
5.0
×106
5.9
×106
5.1
×106
8.0
×106
4.3
×106
6.9
×106
5.7
×106
4.6
×106
6.0
×106
6.5
×106
4.5
×106
7.0
×106
Ororuwo
6.0
×106
5.7
×106
5.5
×106
5.9
×106
5.5
×106
5.8
×106
7.9
×106
5.5
×106
5.0
×106
4.8
×106
6.5
×106
5.0
×106
Legend:
Sfu/ml = Spore forming unit per ml
Table.6: Frequency of Occurrence of Organisms Isolated From Kunnu-Zaki Drink Sample From Some Selected Towns in
Osun State.
Isolates Gbongan Ikire Apomu Ipetu Owena Ikeji Ada Ikirun Ororuwo
%
Occurrence
Pseudomonas
species
- + - - + + - - - 33.33
Bacillus species
Klebsiella species
-
-
-
+
+
+
-
-
+
+
-
+
+
-
-
-
-
-
33.33
44.44
Aspergillus
fumigates - - - + + + - + - 44.44
Aspergillus Niger + + + - + - - + - 55.55
Enterobacter species + + - + - - - + + 55.56
Staphylococcus
species
+ - + - + + - + + 66.67
Penicilium italicum + - + - + + + + - 66.67
Escherichia coli + + + + + - + + - 77.78
International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018
http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878
www.ijeab.com Page | 1315
Streptococcus
species
- + + + - + + + + 77.78
Aspergillus flavus + + + + + - + - + 77.78
IV. CONCLUSION
It is well known that kunnu-zaki is one of the locally made
beverages that people purchase regularly for its cheap cost,
sweet taste and its nutritional values. The presence of these
isolated organisms in kunnu-zaki samples analyzed in Osun
State could serve as indicator for the need to promote
awareness about the possible health hazards that could arise
due to handling and processing of the beverage. The range
of microorganism isolated pose serious threat to food safety
and hence the need to ensure microbial safety during the
production and distribution of this drink that is widely
consumed in some towns in Osun State, Nigeria.
REFERENCES
[1] Aboh, M. I., Oladosu, P. (2014). Microbiological
assessment of kunun-zaki marketed in Abuja
municipal area council (AMAC) in Federal Capital
Territory (FCT), Nigeria. African Journal
Microbiology Resource, 8 (15): 1633 – 1637.
[2] Aboloma, R. I. (2008). Microbiological analysis of
bread samples from bakery to sale – point in Ado –
Ekiti, Ekiti State, Nigeria. Biology Environmental
Science Journal Tropical, 5 (3): 77 - 81.
[3] Achi, O. K. (2005). The potential for upgrading
traditional fermented foods through biotechnology.
African Journal of Biotechnology, 4 (5): 375 - 380.
[4] Adejuyitan, J. A., Adelakun, O. E., Olaniyan, S. A.
and Popoola, F. I. (2008). Evaluating the quality
characteristics of kunu produced by dry-milled
sorghum. African Journal of Biotechnology, 7 (13):
2244 - 2247.
[5] Adeyemi, I. A. and Umar, S. (1994). Effect of
manufacture on quality characteristics of kunu-zaki, a
millet based beverage. Nigeria Food Journal, 12: 34 -
40.
[6] Akoma, O., Agarry, O. O. and Nkama, I. (2013). A
study on the production and consumption pattern of
Kunun-zaki: A cereal based ethnic fermented beverage
of Northern Nigeria. British Journal Applied Science
Technology, 3: 1220 - 1227.
[7] Amusa, N. A. and Ashaye, O. A. (2009). Effect of
processing on nutritional, microbiological and sensory
properties of kunun-zaki (a sorghum based non-
alcoholic beverage) widely consumed in Nigeria.
Pakistan Journal Nutrition, 8 (3): 288 - 292.
[8] AOAC. (2005). Association of official analytical
chemists. Official methods of analysis (22nd
edition). Washington D. C, USA, 1 (5): 115 – 119.
[9] APHA. (2001). Compendium of methods for the
microbiological examination of foods, 4th edition, F.
L. Downes and K, Ito (editions), American public
health association, Washington, D. C., 34 - 170.
[10] Ayo, J. A. (2004). Microbiological evaluation of
“kunun-zaki” and “zoborodo” drink (beverages)
locally produced and sold in a polytechnic community
in Nigeria. Nigeria Food Journal 22: 119 – 126.
[11] Egbere, O. J., Adik, C., Inyang, C. U., Henry, U. I. and
Ogbonna, C. I. C. (2007). Microorganisms associated
with fermentation and spoilage of kunun zaki. Nigeria
Journal of Biotechnology, 18 (1-2): 49 – 55.
[12] Elmahmood, A. M. and Doughari J. H. (2007).
Microbial quality assessment of kunnu-zaki beverages
sold in Girei town of Adamawa State, Nigeria. African
Journal Science, 1: 011 – 015.
[13] Essien, E., Monago, C., and Edor, E. A. (2011).
Evaluation of the nutritional and microbiological
quality of kunun (a cereal based non-alcoholic
beverage) in Rivers State, Nigeria. The Internet
Journal of Nutrition and Wellness, 10 (2).
[14] Gadaga, T. H., Nyanga, L. K. and Mutukumira, A. N.
(2004). The occurrence, growth and control of
pathogens in African fermented food. African Journal
of Food Agriculture Nutrition and Development, 4:
5358 – 5374.
[15] Ijabadeniyi, A. O. (2007). Microbiological safety of
garri, lafun and ogiri in Akure metropolis, Nigeria.
African Journal of Biotechnology, 6 (22): 2633 - 2635.
[16] Klich, M. A. (2007). Aspergillus flavus: the major
producer of Aflatoxin. Molecular Plant Pathology, 8
(6): 713 - 722.

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Microbiological Quality of Kunnu-Zaki Drinks Sold in Some selected Towns in Osun State, Nigeria

  • 1. International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018 http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878 www.ijeab.com Page | 1308 Microbiological Quality of Kunnu-Zaki Drinks Sold in Some selected Towns in Osun State, Nigeria Imoukhuede T. P.1, Adepeju A. B.2, Akinsuroju M. O.3 1Department of Science Laboratory Technology, Faculty of Science, Ekiti State University, Ekiti Nigeria 2Department of Food Science and Technology, Joseph Ayo Babalola University, Osun State, Nigeria 3Department of Microbiology, Joseph Ayo Babalola University, Osun State, Nigeria Email: teniusng@yahoo.com Abstract— The microbiological quality of freshly processed and hawked kunnu-zaki drinks, a common Nigerian non-alcoholic beverage was investigated in some selected towns in Osun State, South Western Nigeria. A total of nine (9) towns were assessed. Kunnu- zaki drinks were purchased from these towns on twelve different occasions for a period of six month. The samples were microbiologically analyzed using standard methods. pH values ranged from 3.09 - 4.21 in East and West senatorial district. The TTA of the samples ranged from 0.32 - 0.49 in Central and East senatorial district. All the screened drink samples had varying levels of bacterial contamination ranging from 4.2 x 103 to 15.0 x 103 CFU/ml, 4.1 x 104 to 9.6 x 104 CFU/ml and 3.5 x 105 to 9.0 x 105 CFU/ml for total bacteria, coliform and E. coli count respectively. Total coliform count on MPN ranged from 3 MPN/ml to 93 MPN/ml. 77.78% of the total sampled drinks(n=108) that is(84/108) had bacteria and fungi count. While all the drinks sampled had total coliform count on MPN exceeding the recommended safe level of zero organisms detectable per 100ml. Seven (7) bacteria species were isolated from the kunnu-zaki drink sampled. The bacteria isolated were Escherichia coli, Bacillus species, Staphylococcus species, Pseudomonas species, Streptococcus specie, Enterobacter species, and Klebsiella species. Fungal count ranged from 3.3 x 106 to 8.0 x 106 CFU/ml respectively. Four (4) fungi were also isolated which include Aspergillus fumigatus, Penicilium italicum, Aspergillus niger and Aspergillus flavus. It can therefore be concluded that the presence of these isolated organisms in kunnu-zaki samples analyzed in Osun State could serve as indicator for the need to promote awareness about the possible health hazards that could arise due to the unhygienic ways of handling and processing of the beverage. Keywords— Physico-chemical analysis, Bacteria count, Coliform count (MPN), Fungi count and Frequency of Isolated organisms. I. INTRODUCTION Beverage is a liquid drink prepared for human consumption (Robert, 2006). The word “beverage” has been derived from the Latin word “bever” meaning rest from work. After work, one tends to feel thirsty due to fluid loss through perspiration and one is inclined to drink water or other potable beverages to compensate fluid loss. Beverages are potable drinks which have thirst- quenching, refreshing, stimulating and nourishing qualities (Achi, 2005). By refreshing, one means the replenishment of fluid loss from the body due to perspiration. Nourishment is provided by the nutrients in the beverages, especially fruit juices. Kunnu-zaki (Millet drink) also referred to as “sweet kunnu-zaki” is a popular traditional and important cereal based, non- alcoholic fermented beverage (Adejuyitan et al., 2008) made from pearl millet (Pennisetum glaucum), maize (Zea mays), or sorghum (Sorghum bicolor), grains but the most commonly used basic ingredient is millet. Maize (Zea mays), millet (Pennisetum glaucum), rice (Oryza sativa) and sorghum (Sorghum bicolor) cereals provide mainly carbohydrates and low quality protein (Steinkraus, 1997). It is generally acceptable and widely consumed throughout Nigeria by children and adults as breakfast drink or food supplements and also a weaning food for infants due to its refreshing qualities most especially in the northern parts and it is consumed throughout the year mostly during dry seasons (Elmahmood and Doughari, 2007). Kunnu-zaki is a refreshing drink usually used to entertain visitors, appetizers and is commonly used or served at social gatherings both rural and urban centers, hawked in the motor parks, school premises and market places (Amusa et al., 2005). It is a staple beverage that is relatively nutritious and affordable when compared to other carbonated and non-carbonated drinks (Adejuyitan et al., 2008). It has low viscosity, a sweet-sour taste depending on the level of fermentation and is milky cream in appearance (Adeyemi and Umar, 1994). It has immense
  • 2. International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018 http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878 www.ijeab.com Page | 1309 social, economic, nutritional and medicinal benefits to most consumers (Akoma et al., 2006). The preparation of locally fermented foods like kunnu-zaki has become a technology in many homes particularly in rural communities and more recently in the urban areas where more women have developed the skill and commercial production which has helped to alleviate poverty amongst the people (Essien et al., 2011). Kunnu-zaki is been prepared from local cereals which are very common and part of our staple food substances. It is relatively produced in low cost which makes it not expensive because grains and other ingredients used for production are locally sourced and are mostly grown within the savannah region of Nigeria such as Bauchi, Kano, Sokoto and Kastina states (Adebayo et al., 2010) and almost throughout the years, most especially the savannah belt of West Africa and the packaging materials are also available, cheap and easily affordable. Cereals are grains or edible fruits of the grass-monocot family that may be cultivated and used as foods. In India and Africa, cereals products comprise 80% or more of the average diet, 50% in Central and Western Europe and between 20-25% in the U.S. (Onwueeme and Finha, 1991). Millets are cereal crops or grains for foddler and human food (Ray and Sivakumar, 2009). Millets are in the family of cereals grown globally with differential importance across continents and within regions of the world. It is consumed as staple food and drink in most areas. Across Africa several indigenous foods and drinks are made from flour/meal and malt of millet. They are nutritionally equivalent or superior to other cereals (Obilana and Taylor, 2002). In the Northern area of Nigeria Millet is known as Jero, ‘Oka baba’ in yoruba land and in America, it is known as milo. It can be stored for 6 months without any significant spoilage. Millet are highly tolerant and are very nutritious containing low phytic acid and are rich in dietary fiber, iron, phosphorus, potassium, magnesium, zinc, thiamine, riboflavin calcium and vitamin B (niacin, B6 and folic acid) which make millet more superior than most cereals, are now being enhanced through biofortification and micronutrient research (Obilana and Taylor, 2002) which are essential for body growth and development. It is easily digested and gluten-free, an excellent choice for anyone with gluten-intolerance (Oladele and Aina, 2007). Kunnu-zaki processing is mostly done by women using simple household equipment and utensil depending on cereal availability. Kunnu-zaki is produced from malted millet; a portion of the cereal is malted, dried, ground and then mixed with the uncooked portion. The mixture is then added to the cooked portion and stirred vigorously and allowed to ferment (Akoma et al., 2012). Spices such as ginger (Zingiber officinales), alligator pepper (Afromonium melegueta), red pepper (Capsicum species), black pepper (Piper guineense) and cloves (Syzygium aromaticum) are commonly added to improve flavor and taste while honey, sweet potatoes and sugar are also added to act as a sweetener (Ahmed et al., 2003). The processed kunnu-zaki is usually packed for sale either in nylon or in a plastic container and can also be stored in refrigerators. Some people in some regions prefer it with pepper and sugar while some with little or no pepper and sugar (Adeyemi and Umar, 1994). All these ingredients perform one function or the other in the course of the preparation. The most abundant constituent of kunnu-zaki is water and it acts as the medium in which all other constituents are dissolved and contain only traces amount of inorganic substances. Spices are usually added in small quantities to improve taste and flavor. Because these are agricultural commodities, they may contain a high level of microbial impurities (Adeyemi et al., 1994; Bibek, 2001). The quality and quantity of the products depend largely on the quality of the ingredients and its proper handling in the course of production by the producer. Hence, kunnu-zaki samples for different market in Osun state are purchased to determine the microbial and fungi load. II. MATERIALS AND METHODS STUDY LOCATION Osun State is located in the South Western part of Nigeria. It covers an area of approximately 14,875 square kilometers, lies between longitude 04 00E and latitude 05 558 and is bounded by Ogun in South, Kwara in North, Oyo in West and Ondo States in the East. It total population is approximately 3,416,959. COLLECTION AREAS Samples were collected and analyzed from nine (9) major markets using Senatorial districts West, East and Central to ensure adequate representation of samples in Osun State Nigeria, which include three (3) popular towns from each Senatorial district. The towns are: Gbongan, Ikire (Sabo market) and Apomu all in Osun West Senatorial districts, Ipetu-Ijesa, Ikeji-Arakeji and Owena-Ijesa all in Osun East Senatorial district, Ada, Ikirun (Oja tuntun) and Ororuwo all in Osun Central Senatorial district. COLLECTION OF SAMPLES Freshly prepared kunnu-zaki samples were purchased in plastic containers (as packaged by the seller) from different Senatorial market locations within Osun State. The samples were labeled and held at 4 0C by placing them in refrigerated coolers and conveyed in an ice packed cooler and transported to Microbiology and Biochemistry Department Laboratory of Joseph Ayo Babalola University within three hours (3 hours) of collection for microbiological and physico-chemical analysis respectively. A total of 108 kunnu-zaki drinks samples were purchased and sampled. The period of
  • 3. International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018 http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878 www.ijeab.com Page | 1310 study was six months. All samples were subjected to the same analysis. PHYSICO-CHEMICAL ANALYSIS OF KUNNU- ZAKI The rate of fermentation by each bacteriumon the kunnu- zaki sample was assessed by testing for pH and (TTA) Titratable acidity (AOAC, 2005). DETERMINATION OF pH OF THE SAMPLES The pH of the samples was determined using a laboratory pH meter (Jenway 3015, model 10). The pH was measured by putting 10 ml of the kunnu-zaki into a beaker and readings were taken by dipping the electrode into the kunnu-zaki samples (rinsing out the electrode with distilled water), and measurements taken from the display screen when the readings stabilized (Ofori et al.,1994). DETERMINATION OF TOTAL TITRATABLE ACIDITY (TTA) This is to measure the acidity in a sample. The Total Titratable acidity (TTA) calculated as percentage lactic acid was determined following the method described by (AOAC, 2005). PROCEDURE A 10 ml of kunnu-zaki sample was measured into a dried conical flask, 3 drops of phenolphthalein indicator was added and the flask thoroughly shaken. It was then titrated against 0.1 M NaOH (Sodium hydroxide) to a pink colour end-point (compared against a white background) and the titre volume was noted and calculated. DETERMINATION OF MICROBIAL COUNT This was carried out using standard microbiological techniques following appropriate dilution on agar plates of Nutrient Agar (NA) for bacteria count, MacConkey Agar (MCA) for coliform count, Eosin Methylene Blue agar (EMB) for E. coli and Potato Dextrose Agar (PDA) for fungi count. The agar media were of oxoid grade using pour plate method (Lateef et al., 2004). PROCEDURE Serial dilution was carried out using 1 ml of kunnu-zaki samples into9 ml of water. Stocks were prepared in test- tubes serially in rows containing 9.0 ml of distilled water representing each sample from each location (APHA, 2001). Then from each suspension 1 ml of dilution was pipette into a set of Petri-dishes and was overlaid with 20 ml of freshly prepared Nutrient agar (NA), MacConkey (MCA) and Eosin methylene blue (EMB) while others were overlaid with 20 ml of freshly prepared potato dextrose agar (PDA) treated with chloramphenicol (1 per 100 ml) prior to sterilization. Colonies which develop after incubation for 18 to 24 h at 37 0C on the cultured plates were counted using Gallenkamp colony counter and were sub-cultured to obtain pure isolates. The pure isolates were aseptically transferred into Nutrient agar slants and incubated. After 24 h incubation morphological characterization and biochemical tests were carried out on the isolates to aid identification to genus level on bacteria (Cowan and Steel 1985). The purified fungal isolates were identified on the basis of standard cultural, morphological and microscopic characteristics as described by (Samson et al., 1988). The fungal isolates were sub-cultured on to a sterile fresh media and incubated at room temperature for 3-5 days. Morphological identification was based on colour, textures spreading rate of each colony on the Potato dextrose agar plate. DETERMINATION OF MOST PROBABLE NUMBER (MPN) The multiple tube method was used to estimate coliform numbers. The presumptive, confirmed and completed test was done by adding varying quantities of kunnu-zaki such as 0.1 ml, 1.0 ml and 10 ml to varying quantities and strength of MacConkey broth in test tubes containing Durham tube (APHA, 2001). The MPN of coliforms present in the sample was estimated by making reference to McCrady table or probability table (APHA, 2001). III. RESULTS AND DISCUSSION The results show that the pH of the samples ranged from 3.09 - 4.21 in East and West Senatorial district (Table 1). The result of the analysis revealed that all samples used have an acidic pH range of 3.09 - 4.21. The level of acidity of kunnu has been reported by several researchers including Efiuvwevere and Akoma, (1995) and Akoma et al., (2006). The TTA of the samples ranged from 0.0032 - 0.0049 in central and east Senatorial district (Table 2). East Senatorial district had the highest TTA values of kunnu- zaki drink in the month of July according to the period of samples collection. All the kunnu-zaki drinks sampled were contaminated with varying level of bacterial load ranging from 4.2 x 103 to 15.0 x 103 CFU/ml in West and East Senatorial district (Table 3). This is in agreement with the findings of Hatcher et al., (1992), Elmamood and Doughari, (2007), Lawal (2012) and Aboh and Oladosu, (2014) who reported a total bacterial count of 5.0 x104 to 2.0 x 106, 1.0 x 102 to 8.9 x 104 CFU/ml, 5.0 x 104 to 1.79 x 105 CFU/ml and 5.1 x 102 to 2.0 x 108 CFU/ml respectively. The high bacteria count observed in this study might be attributed to factors such as the environment, which include exposure of the foods (kunun-zaki) to air, soil, type of water used in processing, post production operations and personal hygiene of the handlers (Kawo and Abdulmumin, 2009; Aboloma, 2008). Exposure of the foods to air or dust at the point of sale is likely to increase the counts of the bacteria as virtually most of the
  • 4. International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018 http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878 www.ijeab.com Page | 1311 bacteria are carried in aerosols by dust and air (Karagozlu et al., 2007). Also, during production, most handlers sometime dip their hands into the containers while making kunun-zaki. The total coliform bacterial count in the kunnu-zaki drink samples using MPN method ranged from 3 MPN/ml to 43 MPN/ml in East, 3 MPN/ml to 93 MPN/ml in West and 3 MPN/ml to 39 MPN/ml in Central. All the samples 108/108 (100%) of the kunnu-zaki samples examined had coliform count (Table 4). The result shows that West district had the highest total coliform count. Seven (7) bacteria genera were isolated from the kunnu- zaki drink samples. These include Escherichia coli, Pseudomonas species, Staphylococcus species, Enterobacter species, Klebsella species, Streptococcus species and Bacillus species. The detection of S. species was equally isolated by Olasupo et al., (2002) from wara and kunun-zaki, a cereal based, non-alcoholic beverage, also by Aboh and Oladosu, (2014) from kunun-zaki. The presence of organisms like Bacillus species, Streptococcus has been reported by Adeyemi and Umar, (1994) and Ayo, (2004) who also isolated these organisms from kunun-zaki. The presence of Klebsiella species as recorded in this study is usually associated with faecal contamination. Being an enteric bacterium its presence indicates poor practices among handlers. Due to the significance of the faecal-oral route transmission for many bacterial food-borne diseases, basic hygiene measures assume a decisive importance in food safety management (Uzeh et al., 2006). The high occurrence of E. coli (77.78%) in kunnu-zaki is an indication of faecal contamination. Ironically, most food handlers do not practice good personal hygiene and do not follow good manufacturing practices, which could reduce the occurrence of such bacteria in foods (Bukar et al., (2009); Kawo and Abdulmumin, (2009)). Egbere et al., (2007) reported the presence of organisms like Bacillus cereus, Staphylococcus aureus and Escherichia coli (PHLS, 1996). It is possible that contamination by these pathogens could have occurred during sieving and packaging, as most of the people involved in the production, packaging and hawking do not take necessary precautions, and as such contamination could be very prominent (Elmamood and Doughari, 2007).Gadaga et al., (2004) reported that pathogens have been isolated from some fermented foods and some laboratory tests have shown the possibility of pathogens to survive and grow in some fermented foods. He said post processing contamination is often cited as the major cause of food poisoning, this agrees with the observation made as regards the source of kunnu collected for this study which is not really hygienic. Gadaga et al., (2004) equally said that Escherichia coli, is the most commonly encountered pathogens in African fermented food. This tally with the types of organism isolated in this study. The occurrence of bacterial pathogens in fermented foods suggests a need for caution in the consumption of this beverage. The source of contamination may also have come from the spices used as additives (Essien et al., 2011 and Lawal, 2012). Fungal count ranged from 3.3 x 106 to 8.0 x 106 CFU/ml in West and Central district (Table 5). A total of Four (4) different moulds were isolated from the kunnu-zaki drink samples in the course of study. These were Aspergillus fumigatus, Penicilium italicum, Aspergillus niger and Aspergillus flavus. The frequency of occurrence of the organisms isolated from kunnu-zaki drink samples are shown in Table 6. Escherichia coli and Streptococcus species were found in 77.78% (84/108) of samples examined, Pseudomonas species (33%), Staphylococcus species (66.67%), Enterobacter species (55.56%), Klebsella species (44.44%), Bacillus species (33.33%), Aspergillus flavus was found in 77.78% (84/108) of the samples examined, Aspergillus fumigatus (44.44%), Penicilium italicum (66.67%) and Aspergillus niger (55.55%). Aspergillus species is ubiquitous, aerobic fungi that can survive in air, dust. The presence of fungi may be attributed to the acidic nature of the sample since it has been observed that molds are capable of utilizing organic acids. Also the presence of fungi in the food may lead to food poisoning and contaminated fungi result in the production of undesirable odour, colour changes and loss of taste of the sample. The presence of these organisms in the sample may be due to the nutritional composition of the millet; these nutrients are present in different proportions. Aspergillus flavus is a common mold in the environment, and can cause storage problems in stored grains. It can also be a human pathogen, associated with Aspergillosis of the lungs and sometimes causing corneal, otomycotic of aflatoxin and nasoorbital infections. Aspergillus flavus spores are allergenic (Klich, 2007). Ijabadeniyi (2007) and Omemu (2011) isolated Penicillium, Aspergillus, Mucor and Rhizopus during the early stage of maize fermentation for ogi production. The bacterial and fungal counts showed that all the kunnu- zaki sampled had a high prevalence of these contaminants and spoilage organisms which of course is of great concern. They are therefore considered microbiologically unsafe for human consumption when not hygienically prepared and utensils cleaned regularly. Clusters of infection caused by the organisms have been linked to poorly maintained utensils, poor personal hygiene, improper storage facilities, and poor water supply (Oyelana and Coker, 2012). Also it can be linked to unhygienic human handlers, dispensing of the extract into bottles, addition of flavors and sweeteners and process of cooling of the extract (Omemu et al., 2007a).
  • 5. International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018 http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878 www.ijeab.com Page | 1312 Many native African beverages are little known outside the parent continent. Effort should be made to improve in the quality, shelf-life and production process of these indigenous beverages so that large- scale production for export outside the continent can be carried out. Many people now prefer imported and exotic beverages because of their attractive form, long shelf life, ease of transportation and other forms of utility which consumers associate with them (Achi, 2005). As of now, there are no industries involved in production of Kunnu-zaki. Kunnu- zaki is widely believed to be of immense social, economic and medicinal importance to its numerous consumers (Akoma et al., 2006). Table.1: pH Values of Kunnu-zaki Drink Sampled from Some Selected Towns in Osun State. pH VALUES MONTHS JUL AUG SEP OCT NOV DEC TOWNS Gbongan 3.93 3.90 3.86 3.92 3.89 3.92 3.88 3.87 3.93 3.88 3.91 3.92 Ikire 4.03 3.98 4.00 3.99 4.01 4.03 4.02 4.01 4.00 4.01 3.97 4.00 Apomu 4.20 4.18 4.16 4.19 4.20 4.14 4.13 4.17 4.19 4.16 4.21 4.15 Ipetu-Ijesha 3.83 3.84 3.86 3.33 3.50 3.85 3.55 3.40 3.78 3.80 3.88 3.60 Owena 3.89 3.51 3.65 3.99 3.55 3.19 3.09 3.67 3.68 3.88 3.85 3.89 Ikeji- Arakeji 3.60 3.50 3.58 3.63 3.45 3.52 3.57 3.48 3.56 3.59 3.60 3.58 Ada 3.94 3.90 3.91 3.93 3.80 3.79 3.88 3.91 3.93 3.69 3.89 3.91 Ikirun 3.75 3.69 3.72 3.74 3.68 3.75 3.73 3.66 3.69 3.72 3.71 3.77 Ororuwo 3.73 3.72 3.71 3.70 3.67 3.73 3.69 3.71 3.70 3.66 3.72 3.69 Table.2: Total Titratable Acidity Values for Kunnu-zaki Drink Sampled from Some Selected Towns in Osun State. TITRABLE ACIDITY VALUES MONTHS JUL AUG SEP OCT NOV DEC TOWNS Gbongan 0.0038 0.0041 0.0036 0.0038 0.0039 0.0037 0.0035 0.0040 0.0039 0.0037 0.0039 0.0041 Ikire 0.0036 0.0040 0.0042 0.0037 0.0041 0.0041 0.0039 0.0036 0.0038 0.0040 0.0033 0.0037 Apomu 0.0035 0.0038 0.0039 0.0036 0.0038 0.0036 0.0040 0.0038 0.0041 0.0041 0.0038 0.0036 Ipetu- Ijesha 0.0037 0.0041 0.0043 0.0039 0.0040 0.0038 0.0039 0.0041 0.0038 0.0037 0.0035 0.0039 Owena 0.0049 0.0047 0.0037 0.0040 0.0038 0.0036 0.0037 0.0038 0.0037 0.0039 0.0040 0.0038 Ikeji- Arakeji 0.0040 0.0039 0.0038 0.0041 0.0036 0.0038 0.0037 0.0034 0.0041 0.0040 0.0039 0.0038 Ada 0.0037 0.0045 0.0038 0.0038 0.0039 0.0039 0.0037 0.0038 0.0039 0.0041 0.0040 0.0041 Ikirun 0.0036 0.0038 0.0040 0.0040 0.0041 0.0039 0.0032 0.0037 0.0044 0.0040 0.0039 0.0041 Ororuwo 0.0039 0.0041 0.0037 0.0039 0.0038 0.0040 0.0036 0.0041 0.0040 0.0039 0.0039 0.0038
  • 6. International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018 http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878 www.ijeab.com Page | 1313 Table.3: Bacterial Count (CFU/ml) of Kunnu-zaki Drink Sampled from Some Selected Towns in Osun State. BACTERIAL COUNT (CFU/ml) MONTHS TOWNS JUL AUG SEP OCT NOV DEC Gbongan 9.7 ×103 9.0 ×103 8.2 ×103 9.5 ×103 4.2 ×103 8.0 ×103 7.9 ×103 7.5 ×103 8.4 ×103 8.6 ×103 6.5 ×103 7.0 ×103 Ikire 6.5 ×103 7.0 ×103 7.5 ×103 8.0 ×103 8.0 ×103 9.0 ×103 7.6 ×103 9.2 ×103 9.3 ×103 8.8 ×103 10.0 ×103 9.5 ×103 Apomu 8.0 ×103 8.8 ×103 8.0 ×103 9.1 ×103 9.6 ×103 8.9 ×103 7.3 ×103 7.4 ×103 6.0 ×103 9.2 ×103 8.5 ×103 6.4 ×103 Ipetu- Ijesha 6.6 ×103 7.8 ×103 6.8 ×103 6.1 ×103 9.5 ×103 7.4 ×103 8.6 ×103 6.6 ×103 8.8 ×103 9.3 ×103 7.7 ×103 8.1 ×103 Owena 15.0 ×103 6.1 ×103 5.3 ×103 4.9 ×103 7.6 ×103 9.7 ×103 6.0 ×103 7.1 ×103 6.6 ×103 9.1 ×103 5.5 ×103 7.1 ×103 Ikeji- Arakeji 9.0 ×103 7.5 ×103 7.1 ×103 5.2 ×103 5.5 ×103 7.3 ×103 8.0 ×103 8.5 ×103 9.2 ×103 7.3 ×103 7.0 ×103 8.1 ×103 Ada 6.6 ×103 4.7 ×103 8.2 ×103 6.7 ×103 7.3 ×103 8.4 ×103 6.6 ×103 9.1 ×103 7.5 ×103 6.2 ×103 7.5 ×103 7.5 ×103 Ikirun 10.0 ×103 8.2 ×103 8.3 ×103 9.9 ×103 5.0 ×103 8.9 ×103 9.5 ×103 9.0 ×103 8.1 ×103 9.7 ×103 6.0 ×103 9.1 ×103 Ororuwo 7.5 ×103 9.1 ×103 8.6 ×103 9.5 ×103 8.0 ×103 7.2 ×103 9.5 ×103 7.1 ×103 7.2 ×103 6.7 ×103 9.0 ×103 6.2 ×103 Table.4: Coliform Count (MPN McCredy or Probability Table) of Kunnu-zaki Drink Sampled from Some Selected Towns in Osun State. COLIFORM COUNT MPN/ml MONTHS TOWNS JUL AUG SEP OCT NOV DEC Gbongan 9 3 7 3 3 43 11 28 75 93 3 4 Ikire 20 43 28 21 20 39 3 23 4 23 13 11 Apomu 7 3 4 7 7 4 3 11 9 11 23 28 Ipetu-Ijesha 11 23 21 7 3 4 4 20 28 43 3 3 Owena 4 7 4 11 13 3 21 21 28 3 4 7 Ikeji 11 21 13 7 4 3 7 20 11 43 28 39 Ada 3 4 11 9 20 21 4 3 7 7 3 3 Ikirun 7 9 13 4 3 11 21 11 13 3 4 4 Ororuwo 3 4 11 39 13 9 3 11 39 4 11 3 Legend: MPN= (Most Probable Number)
  • 7. International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018 http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878 www.ijeab.com Page | 1314 Table.5: Fungi Count of Kunnu-zaki Drink Sampled from Some Selected Towns in Osun State. FUNGI COUNTS (SFU/ml) MONTHS JUL AUG SEP OCT NOV DEC TOWNS Gbongan 4.5 ×106 5.0 ×106 3.3 ×106 6.5 ×106 4.0 ×106 5.0 ×106 4.1 ×106 4.3 ×106 6.0 ×106 4.7 ×106 5.1 ×106 4.6 ×106 Ikire 4.2 ×106 5.8 ×106 3.3 ×106 5.9 ×106 5.0 ×106 6.0 ×106 5.7 ×106 6.7 ×106 6.9 ×106 6.4 ×106 5.0 ×106 6.0 ×106 Apomu 5.0 ×106 4.7 ×106 6.0 ×106 6.8 ×106 6.9 ×106 5.6 ×106 5.0 ×106 5.2 ×106 6.3 ×106 7.8 ×106 6.5 ×106 5.8 ×106 Ipetu- Ijesha 3.9 ×106 5.8 ×106 4.6 ×106 4.0 ×106 7.6 ×106 4.8 ×106 7.0 ×106 5.0 ×106 5.8 ×106 5.0 ×106 4.9 ×106 5.5 ×106 Owena 4.8 ×106 4.3 ×106 4.2 ×106 3.8 ×106 6.0 ×106 6.8 ×106 4.1 ×106 5.3 ×106 5.0 ×106 6.4 ×106 4.2 ×106 5.9 ×106 Ikeji- Arakeji 4.9 ×106 6.4 ×106 5.8 ×106 4.0 ×106 3.5 ×106 5.1 ×106 6.3 ×106 6.0 X106 4.9 X106 5.8 ×106 4.9 ×106 6.0 ×106 Ada 5.3 ×106 3.8 ×106 6.0 ×106 5.8 ×106 6.0 ×106 5.8 ×106 4.8 ×106 6.1 ×106 5.7 ×106 4.9 ×106 6.0 ×106 5.3 ×106 Ikirun 5.0 ×106 5.9 ×106 5.1 ×106 8.0 ×106 4.3 ×106 6.9 ×106 5.7 ×106 4.6 ×106 6.0 ×106 6.5 ×106 4.5 ×106 7.0 ×106 Ororuwo 6.0 ×106 5.7 ×106 5.5 ×106 5.9 ×106 5.5 ×106 5.8 ×106 7.9 ×106 5.5 ×106 5.0 ×106 4.8 ×106 6.5 ×106 5.0 ×106 Legend: Sfu/ml = Spore forming unit per ml Table.6: Frequency of Occurrence of Organisms Isolated From Kunnu-Zaki Drink Sample From Some Selected Towns in Osun State. Isolates Gbongan Ikire Apomu Ipetu Owena Ikeji Ada Ikirun Ororuwo % Occurrence Pseudomonas species - + - - + + - - - 33.33 Bacillus species Klebsiella species - - - + + + - - + + - + + - - - - - 33.33 44.44 Aspergillus fumigates - - - + + + - + - 44.44 Aspergillus Niger + + + - + - - + - 55.55 Enterobacter species + + - + - - - + + 55.56 Staphylococcus species + - + - + + - + + 66.67 Penicilium italicum + - + - + + + + - 66.67 Escherichia coli + + + + + - + + - 77.78
  • 8. International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-4, Jul-Aug- 2018 http://dx.doi.org/10.22161/ijeab/3.4.23 ISSN: 2456-1878 www.ijeab.com Page | 1315 Streptococcus species - + + + - + + + + 77.78 Aspergillus flavus + + + + + - + - + 77.78 IV. CONCLUSION It is well known that kunnu-zaki is one of the locally made beverages that people purchase regularly for its cheap cost, sweet taste and its nutritional values. The presence of these isolated organisms in kunnu-zaki samples analyzed in Osun State could serve as indicator for the need to promote awareness about the possible health hazards that could arise due to handling and processing of the beverage. The range of microorganism isolated pose serious threat to food safety and hence the need to ensure microbial safety during the production and distribution of this drink that is widely consumed in some towns in Osun State, Nigeria. REFERENCES [1] Aboh, M. I., Oladosu, P. (2014). Microbiological assessment of kunun-zaki marketed in Abuja municipal area council (AMAC) in Federal Capital Territory (FCT), Nigeria. African Journal Microbiology Resource, 8 (15): 1633 – 1637. [2] Aboloma, R. I. (2008). Microbiological analysis of bread samples from bakery to sale – point in Ado – Ekiti, Ekiti State, Nigeria. Biology Environmental Science Journal Tropical, 5 (3): 77 - 81. [3] Achi, O. K. (2005). The potential for upgrading traditional fermented foods through biotechnology. African Journal of Biotechnology, 4 (5): 375 - 380. [4] Adejuyitan, J. A., Adelakun, O. E., Olaniyan, S. A. and Popoola, F. I. (2008). Evaluating the quality characteristics of kunu produced by dry-milled sorghum. African Journal of Biotechnology, 7 (13): 2244 - 2247. [5] Adeyemi, I. A. and Umar, S. (1994). Effect of manufacture on quality characteristics of kunu-zaki, a millet based beverage. Nigeria Food Journal, 12: 34 - 40. [6] Akoma, O., Agarry, O. O. and Nkama, I. (2013). A study on the production and consumption pattern of Kunun-zaki: A cereal based ethnic fermented beverage of Northern Nigeria. British Journal Applied Science Technology, 3: 1220 - 1227. [7] Amusa, N. A. and Ashaye, O. A. (2009). Effect of processing on nutritional, microbiological and sensory properties of kunun-zaki (a sorghum based non- alcoholic beverage) widely consumed in Nigeria. Pakistan Journal Nutrition, 8 (3): 288 - 292. [8] AOAC. (2005). Association of official analytical chemists. Official methods of analysis (22nd edition). Washington D. C, USA, 1 (5): 115 – 119. [9] APHA. (2001). Compendium of methods for the microbiological examination of foods, 4th edition, F. L. Downes and K, Ito (editions), American public health association, Washington, D. C., 34 - 170. [10] Ayo, J. A. (2004). Microbiological evaluation of “kunun-zaki” and “zoborodo” drink (beverages) locally produced and sold in a polytechnic community in Nigeria. Nigeria Food Journal 22: 119 – 126. [11] Egbere, O. J., Adik, C., Inyang, C. U., Henry, U. I. and Ogbonna, C. I. C. (2007). Microorganisms associated with fermentation and spoilage of kunun zaki. Nigeria Journal of Biotechnology, 18 (1-2): 49 – 55. [12] Elmahmood, A. M. and Doughari J. H. (2007). Microbial quality assessment of kunnu-zaki beverages sold in Girei town of Adamawa State, Nigeria. African Journal Science, 1: 011 – 015. [13] Essien, E., Monago, C., and Edor, E. A. (2011). Evaluation of the nutritional and microbiological quality of kunun (a cereal based non-alcoholic beverage) in Rivers State, Nigeria. The Internet Journal of Nutrition and Wellness, 10 (2). [14] Gadaga, T. H., Nyanga, L. K. and Mutukumira, A. N. (2004). The occurrence, growth and control of pathogens in African fermented food. African Journal of Food Agriculture Nutrition and Development, 4: 5358 – 5374. [15] Ijabadeniyi, A. O. (2007). Microbiological safety of garri, lafun and ogiri in Akure metropolis, Nigeria. African Journal of Biotechnology, 6 (22): 2633 - 2635. [16] Klich, M. A. (2007). Aspergillus flavus: the major producer of Aflatoxin. Molecular Plant Pathology, 8 (6): 713 - 722.