Advances in Microbiology, 2013, 3, 309-316 Published Online August 2013 (
In Vitr o Screening and Selection of Probiotic Lactic
Acid Bacteria Isolated from Spontaneously
Fermenting Kunu-Zaki
Solakunmi Omotunde Oluwajoba*, Felix Akinsola Akinyosoye, Victor Olusegun Oyetayo
Department of Microbiology, School of Science, Federal University of Technology, Akure, Nigeria
Email: *
Received April 8, 2013; revised May 8, 2013; accepted June 8, 2013
Copyright © 2013 Solakunmi Omotunde Oluwajoba et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
The present study was conducted to determine the pro-biotic properties in vitro of the lactic acid bacteria isolated from
spontaneously fermenting kunu-zaki. Kunu-zaki was processed using composite, non composite, germinated and
ungerminated Digitaria exilis (Fonio), Sorghum bicolor (Sorghum) and Pennisetum americanum (Millet) cereals. A
total of 150 LAB isolates were obtained from all the fermenting slurries. These 150 LAB isolates were screened for
their ability to grow at pH 3.0, resistance against bile salt and ability to inhibit reference test pathogens. Out of these
150 LAB isolates; 21 exhibited good probiotic properties. All the 21 isolates were further identified to specie and sub-
species level using standard API50CHL system with all 21 showing good survival (P < 0.05) in a pH 3.0 buffered me-
dium and subsequent resistance to 0.3% bile. The LAB isolates which survived these conditions consisted of 18 Lacto-
bacillus species, 2 Pediococcus species and 1 Lactococcus specie. These LAB species were further examined for an-
timicrobial activity against the growth of reference pathogens Staphylococcus aureus 25923, Escherichia coli 25922,
Pseudomonas aeruginosa 27853 and Enterococcu s faecalis 29212. All 21 LAB species exhibited good inhibition of all
test reference pathogens except Lactobacillus fructivorans, Lactococcus lactis sp lactis and L. fermentum which how-
ever, showed no zone of inhibition against the growth of E. faecalis. Kunu-zaki made from composite un-germinated
Sorghum bicolor (Sorghum) and Pennisetum americanum (Millet) cereal grains contained the highest percentage (52%)
of LAB species which showed good probiotic criteria in vitro. Non composite ungerminated cereals accounted for 33%
of the total probiotic LAB isolates whilst the germinated non composite and composite cereals recorded the lowest per-
centage (10%) and (5%) of probiotic LAB respectively. The results of this research study showed that the LAB spe-
cies isolated from wild fermentation of kunu-zaki beverage fulfilled the criteria for in vitro screening of probiotic char-
acteristics. These LAB species possed potential for further use as probiotic in human preparations and suggested the use
of kunu-zaki made from ungerminated composite sorghum and millet grains as a natural probiotic drink.
Keywords: Probiotic; Spontaneously Fermenting; Germination; Composite; Non Composite
1. Introduction
Lactic acid bacteria are integral to many African fer-
mented foods and a vast amount of literature is available
on them. However, only little is documented about the
pro biotic properties of these strains. Whether or not a
specific probiotic bacterium will have a beneficial effect
on health cannot be presumed only through determina-
tion of its genus or species [1]. Furthermore, that a par-
ticular genus or specie of bacterium mediating probiotic
properties in one substrate will continue to mediate such
effects in another substrate is only a speculation. Such a
speculation must be substantiated by research [1]. Kunu-
zaki is made from fermented cereals and taken as a re-
freshing drink in many parts of Nigeria today [2]. The
drink particularly presents an attractive research interest
for identifying organisms with probiotic potentials. This
is because the traditional production process of kunu-zaki
allows fermenting organisms to still remain viable inside
the drink even at the time of consumption. Those who re-
gularly take kunu-zaki have alluded to its seeming health
benefit effects. In Nigeria, the drink enjoys large patron-
age especially amongst low income earners who classify
*Corresponding author.
opyright © 2013 SciRes. AiM
the drink as “filling” and a meal in itself. Particularly,
regular consumers in Nigeria describe an overall feeling
of wellbeing attributed to long term consumption of ku-
nu-zaki. Scientific human studies are however yet to con-
firm these claims as there is little research detailing con-
firmation of the probiotic properties of the fermenting
organisms in kunu-zaki. “Acha” (Digitaria exilis) com-
monly referred to as Fonio, Finni,or Hungry rice [3], is
probably one of the oldest Africa cereals and classified as
one of the lost/forgotten crops of Africa. “Acha” crops
are exceptionally tolerant to a wide variety of conditions,
particularly drought and poor soil. The grains are widely
produced in the area of growth (Bauchi, Plateau, Kaduna
States of Nigeria), are uniquely rich in methionine and
cystine, and evoke low sugar on consumption, an advan-
tage for diabetics [4]. The use of Acha grains in the
making of kunu-zaki has not yet been satisfactorily ex-
plored, and literature is a little scanty on the subject. Acha
cereal grains offer an attractive interest not only because
of the nutritional attributes but also because it may serve
as a cheaper option to either the use of sorghum or millet
cereal grains in kunu-zaki. This research work therefore
sets out to isolate and identify the fermenting lactic acid
bacteria, screen the probiotic characteristics in vitro us-
ing sorghum and millet as conventional grains and acha
grains as a composite mix to both sorghum and millet.
2. Materials and Methods
2.1. Laboratory Production of Kunu-Zaki
Sorghum; Sorghum bicolor, Millet, Pennisetum ameri-
canum and Hungry rice (locally known as Fonio or Acha)
Digitaria exilis grains were obtained from the Nigeria
Cereal Research Institute in Ibadan. The grains were
cleaned, weighed and washed before steeping in distilled
water. 200 gms of cereal grains were used for the kunu-
zaki production. A control experiment was set up with
distilled water without the grains. For the kunu-zaki
made from composite grains, an equal weight of grains
was used for each part. The laboratory production me-
thod followed the traditional process of kunu-zaki fer-
2.2. Composition of the Kunun-Zaki Drinks
The following nine different formulations of the cereal
based kunu-zaki were used:
A = Acha (Hungry rice) Digitaria exilis
S = Sorghum (Sorghum bicolor)
M = Millet (Penniseteum americanum)
AS = Acha/Sorghum un-germinated composite grains
AM = Acha/Millet Ungerminated composite grains
SM = Sorghum/millet Ungerminated composite grains
ASG = Acha/Sorghum germinated composite grains
AMG = Acha/Millet germinated composite grains
SMG = Sorghum/Millet germinated composite grains.
The composite grains were used in the ratio of 1:1
2.3. Germination of Cereal Grains
200 g cereal grains were rinsed in distilled water and
drained. Steeping was carried out at a temperature of
48˚C to a moisture content of about 42% - 45%. Water
was drained and germination carried out by spreading the
steeped grains on a tray, in a room at temperature of
28+/2˚C. Seeds were sprayed intermittently with water.
The germinated grains were recovered when the radical
was about 1.5 mm in length.
2.4. Isolation of Lactic Acid Bacteria
Samples were obtained from the fermenting germinated
and ungerminated cereal grains at 0 hr, 24 hours, 48 hours
and from the fermenting slurry at 72 hours fermentation
time. Figure 1 depicts the traditional process method
used in this work for the kunu-zaki production. Samples
from these sources were diluted serially 10-fold in PSB
and then inoculated on deMan Rogosa and Sharpe (MRS,
Oxoid, England) agar plates by pour plate method [5]
MRS agar plates were incubated at 37˚C for 48 hours an-
aerobically. Morphologically distinct and well isolated
colonies were picked and transferred to new MRS agar
plates by streaking. Finally, pure colonies were obtained
and preserved for further study.
Sorghum grains Millet grains
Washin g
Soaking in water (1
Fermentatio n )
Wet milling
Sieving of the slurry
Cooking of 2/3 parts filtrate to gelatinization
(Cooling to 35˚C)
Addition of uncooked 1/3 part
fermentation (24 hrs)
Storage (Refrigeration temperature)
Figure 1. Flow diagram for the traditional processing of
kunu-zaki from composite grains.
Copyright © 2013 SciRes. AiM
2.5. Identification of Lactic Acid Bacteria
Macroscopic appearance of all the colonies was exami-
ned for cultural and morphological characteristics. Size,
shape, color and texture of the colonies were recorded.
Fresh cultures were used for the gram stain; the cultures
were aseptically transferred into tubes and centrifuged at
6000 rpm and the supernatant decanted. The cells were
then re-suspended in sterile water, gram stained and ob-
served under the light microscope. Bacterial isolates
were tested for catalase production by the catalase test
[6]. All the isolates were tested for growth at pH 3.0 and
subsequent resistance to 0.3% bile. Species which were
able to grow at pH 3.0 for 3 hours and resisted 0.3% bile
were identified. Identification of species was confirmed
using a standard commercial identification system, API-
50 CHL (Biomerieux®, France), according to the manu-
facturer’s instructions. Pure cultures were maintained in
MRS broth at 20˚C with 10% (v/v) glycerol.
2.6. Screening of isolated Lactobacillus Species
for Probiotic Properties
2.6.1. Resistance to Low pH
Resistance to pH 3 is often used in vitro assays to deter-
mine the resistance to stomach pH. Food usually stays in
the stomach for 3 hours and this time limit was taken into
account [7]. Active cultures were incubated for 16 - 18
hours in MRS broth. The cells were harvested by cen-
trifugation, washed once in phosphate-saline buffer (PBS
at pH 7.2), resuspended in PBS (pH 3) and incubated at
37˚C. Viable microorganisms were enumerated at the 0, 1,
2 and 3 hours with the pour plate technique. Dilutions
were done and the resulting plates were incubated at
37˚C under anaerobic conditions for 48 hours. The
growth was also monitored at OD 620 using a T70 UV:
VIS spectrometer PG Instruments Ltd.
2.6.2. Bile Tolerance
The mean intestinal bile concentration is believed to be
0.3% (w/v). The staying time of food in small intestine is
suggested to be 4 hours [7]. The experiment was applied
at this concentration of bile for 4 hours. MRS medium
containing 0.3% bile (Oxoid) was inoculated with active
cultures which had been incubated for 16 - 18 hours).
During the 4 hours incubation at 0.3% bile, viable colo-
nies were enumerated for every hour with the pour plate
technique and growth was also monitored at 620 Optical
Density-OD 620 (Thermo Multiskan EX).
2.7. Anti-Microbial Activity Using
Spot-on-Lawn Method
After 18 hours incubation, active cultures were spotted
on the surface of MRS agar plates; The plates were incu-
bated to grow cultures for 24 hours at 37˚C under an-
aerobic conditions. Overnight indicator pathogens were
inoculated into soft agar containing 0.7% agar. Staphy-
lococcus aureus 25923, Escherichia coli 25922, Pseu-
domonas aeruginosa 27853 and Enterococcus faecalis
29212 as test reference pathogens collected from the Ni-
gerian Institute of Medical research (NIOMER), Yaba,
Lagos were used. The inoculated agar was overlaid on
MRS plates and incubated at 37˚C which is appropriate
for human pathogens. At the end of the incubation, inhi-
bition zone diameters’ surrounding the spotted isolates
was measured. Isolates which gave an inhibition zone
bigger than 10 mm was determined to have antimicrobial
2.8. Identification of Microorganisms
Isolates which showed growth at pH 3.0 for 3 hours and
subsequent resistance to 0.3% bile for 4 hours, exceeding
10 mm inhibition zones of antimicrobial activity were the
isolates which were identified. The API 50 CH test kit
identification method was used. The API 50 CH medium,
intended for the identification of the genus Lactobacillus
and related genera is a ready-to-use medium which al-
lows the fermentation of the 49 carbohydrates on the API
50 CH strip to be studied. The last one is a blank which
serves as a control. A suspension was made on the me-
dium with the microorganism to be tested and each tube
of the strip was then inoculated with the suspension.
During anaerobic incubation, the carbohydrates are fer-
mented to acids which produce a decrease in the pH de-
tected by the change in color of the indicator. The results
make up the biochemical profile which was used by the
API web TM identification software (Ref 40011) Bio-
merieux to identify the strain.
3. Results and Discussion
Lactic acid bacteria (LAB) have been described as Gram
positive, catalase negative, cocci or rods non-spore for-
ming bacteria that are aero-tolerant, anaerobic or micro-
aerophilic [8]. They produce lactic acid as part or major
by product from fermentation of carbohydrates [8,9].
This group of bacteria includes the following genus Lac-
tobacillus, Pediococcus, Lactococcus, Leuconostoc and
Bifidobacterium. The bacteria are responsible for both
spontaneous and natural fermentation of sugars [10]. The
by-products of food fermentations mediated by LAB,
result in an improvement of the aroma, flavor, texture,
safety and shelf life of the food.
However, not all LAB are beneficial in foods as some
produce lipase and protease which degrade fats and pro-
teins leading to food spoilage [11]. In the present study,
Lactobacillus species, Lactococcus species and Pedio-
coccus species were isolated. Before evaluating as probi-
Copyright © 2013 SciRes. AiM
otic, important characteristics of these organisms were
studied. In order to be able to have beneficial effects on
the human gut, a candidate potential probiotic strain is
expected to have a number of properties. Probiotic strains
do not need to fulfill all of the selection criteria but the
most important ones are required [12,13]. One of the
major important criteria is that probiotic destined for
human usage should be of human origin [13]. Table 1
shows the species of lactic acid bacteria present in the
fermenting mash of kunu-zaki as identified by the API kit.
The lactic acid bacteria identified in this work have ful-
filled these criteria as all identified species are organisms
Table 1. Identification of isolated lab using the standard
API 50 CH test kit method.
Lab significant taxa % accuracy Remarks
Lactobacillus plantarum 1 98.8 Very good identification
Lactobacillus rhamnosus
(Lactobacillus casei
ssp. rhamnosus) 98.6 Good identification
Pediococcus pentosasceus 2 99.9 Excellent identification
Pediococcus damnosus 2 99.8 Very good identification
Lactobacillus fermentum 99.6 Very good identification
Lactobacillus cellobiosus 99.9 Excellent identification
Lactobacillus lindneri 99.9 Excellent identification
Lactobacillus pentosus 99.8 Very good identification
Lactobacillus plantarum 1 80.6 Good identification
Lactobacillus paracasei
ssp paracasei 2 99.3 Very good identification
Lactobacillus plantarum 1 99.9 Excellent identification
Lactobacillus paracasei
ssp paracasei 1 99.7 Very good identification
Lactobacillus plantarum 1 99.9 Excellent identification
Lactobacillus paracasei
ssp paracasei 3 99.8 Very good identification
Lactobacillus cellobiosus 99.9 Excellent identification
Pediococcus damnosus 99.9 Excellent identification
Lactobacillus paracasei
ssp paracasei 3 95.8 Good identification
Lactobacillus plantarum 1 99.9 Excellent identification
Lactobacillus acidophilus 1 91.7 Good identification
Lactococcus lactis ssp lactis 1 99.8 Very good identification
Lactobacillus crispatus 98.3 Good identification
Lactobacillus pentosus 99.9 Excellent identification
Lactobacillus fructivorans 99.9 Excellent identification
which are naturally present in the human gut. Probiotics
which are acceptable for food/medicine preparations for
humans are those which occur naturally in the intestinal
tract of healthy human and in foods. Another criteria is
that potential probiotic organisms should have acid and
bile tolerance which are conditions under which the LAB
will have to travel in order to reach the intestine which is
the site where any beneficial health effect can be made.
Traveling through the human digestive tract can be a
challenge for orally administered pro-biotic bacteria. The
high acid levels in the stomach and the pancreatic secre-
tions such as digestive enzymes and bile in the small
intestine can lead to the injury and death of a percentage
of the orally administered probiotic [14]. The mean stay-
ing time of food in the stomach is 3 hours, and in this
study Lactobacillus pentosus, Pediococcus damnosus,
Lactobacillus paracasei ssp paracasei 1, Lactobacillus
paracasei ssp paracasei 2 isolated from the fermenting
slurry of kunu-zaki showed the greatest resistance to ex-
posure at pH 3.0 with their counts actually increasing
after 3 hours exposure. Lactobacillus plantarum, Lacto-
bacilus acidophilus and Lactobacillus fermentum fol-
lowed closely even though there was a decline in all their
viable counts after 3 hours culturing in pH 3.0. Figure 2
shows the variation within each isolated LAB specie cul-
tured at pH 3.0 with respect to time. The Tolerance level
of all species to acidic environment was found signifi-
cantly (P < 0.05) variable. According to [15], enteric
lactobacilli are usually able to tolerate pH 3.0 for a few
hours, pH 2.0 for several minutes, while viable count will
be affected at slightly high acidic pH and at pH 1.0 all
the Lactobacillus species are destroyed. There was a
steady increase in viable counts of all species after cul-
turing in bile salt but L. paracasei ssp paracasei 1, Pedi-
ococcus damnosus, Lactococcus lactis ssp lactis, and
Lactobacillus rhamnosus could not maintain an appre-
ciable level of survival after the 3rd hour. These species
experienced a drop in their mean total viable counts be-
tween the 3rd and the 4th hours of exposure to 0.3% con-
centration of bile. Figure 3 shows the variation between
the mean total viable counts at exposure to 0.3% bile salt.
These results reveal that lactobacilli responsible for the
spontaneous fermentation of kunu-zaki are capable for
survival in the environment of the gastrointestinal tract
which has characteristic features of having acidic pH and
high concentrations of bile salts. [16] has recorded simi-
lar findings in another study: Bile resistance appears to
be mediated by bile salt hydrolysis and this results in
precipitation of cholesterol. In another research [17],
twenty nine Lactobacillus strains of dairy origin were
tested in vitro for their probiotic potential. The resistance
of the Lactobacillus strains to pH 1-3 was examined.
Tolerance to bile salt was tested against to 0.3% oxgall.
ll of the examined strains were resistant to pH 3 during A
Copyright © 2013 SciRes. AiM
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Figure 2. Growth pattern of lab isolates at pH = 3.0.
Figure 3. Growth pattern of LAB isolates on MRS agar supplemented with 0.3% bile.
3 h, but most of them lost their viability in 1 h in pH 1.
Also all of them tolerated 0.3% bile salts concentration in
4 h (Figure 3). All lactobacilli inhibited the growth of E.
coli, Staphylococcus aureus, P. aeruginosa and E. fae-
calisexcept P. damnosus, Lactococcus lactis ssp lactis1
and Lactobacillus fructivorans that showed significantly
(P < 0.05) low antimicrobial effect against E. faecalis.
The strongest antimicrobial effect was shown by L. aci-
dophilus and L. paracasei ssp paraca sei 3, Lactobacillus
rhamnosus, Lactobacilus fermentum and Lactobacillus
pentosus while antimicrobial effect of other lactobacilli
was similar against indictor bacteria (Table 2). The an-
timicrobial action is reportedly due to the potential of
LAB to produce lactic acid and bacteriocins [18]. It is
also reported that these bacteria produce peptides having
inhibitory properties [18]. LABs commonly produce
bacteriocins which are peptides with bactericidal activity
usually against strains of closely related species. Al-
though bacteriocins may enhance survival of LAB in
complex ecological systems interest has focused on pre-
vention of growth of harmful bacteria in the fermentation
and preservation of food products, it is more important
with respect to probiotic that individual strains may in-
hibit growth of or adhesion of pathogenic micro-orga-
nisms by secreted products, and not merely an effect of
acidic pH. Antimicrobial effects of lactic acid bacteria
are formed by producing some substances such as or-
ganic acids (lactic, acetic, propionic acids), carbon diox-
ide, hydrogen peroxide, diacetyl, low molecular weight
antimicrobial substances and bacteriocins [13,19]. The
isolated organisms as identified by the API 50 CHL Test
kit are reflected in Ta b le 1 . It also gives the percentage
of identification accuracy. Lactococcus lactis ssp lactis
had good identification only up to the genus level whilst
Lactobacillus acidophilus and Lactobacillus paracasei
ssp paracasei 3 recorded 91.7% and 95.8% identification
accuracy. All other LAB isolates recorded above 98%
identification accuracy by the API web TM identification
software (Ref 40011) Biomerieux system. Table 3 shows
that kunu-zaki made from ungerminated cereal grains
had a higher number of probiotic LAB compared to the
germinated cereals. Composite formulations also had a
higher community of probiotic LAB than the non compo-
site ones. Ungerminated Sorghum and Millet composite
formulation accounted for 53% of the total probiotic
LAB isolated. Ungerminated Sorghum non-composite
formulation accounted for 23.9% of the total probiotic
LAB isolated whilst ungerminated non composite Millet
formulation accounted for 14.25% of the total probiotic
LAB community. The fermenting germinated cereals
however recorded a poor score of probiotic LAB com-
munity as depicted in Table 4. Ungerminated cereals
have been shown by a few researchers to favour the
growth of probiotic bacteria. [20] investigated the growth
of probiotic Lactobacillus plantarum of human origin on
fermented brown rice. Ungerminated fermented brown
Table 2. Antimicrobial activity of LAB isolates against four selected pathogens.
LAB isolates from spontaneously fermenting kunu mash Inhibition (mm ± 0.2) against tested reference pathogens
E. coli S. aureus P. aeruginosa E. faecalis
L. plantarum (Millet) 20 25 23 18
L. plantarum 1 (Millet) 22 30 26 20
P. damnosus (Millet) 18 25 24 14
P. pentosaceus 2 (Millet Germinated) 28 22 23 18
L. lindneri (Millet Germinated) 29 21 17 23
L. plantarum 1 (Sorghum) 23 26 21 24
L. paracasei ssp para 3 (Sorghum) 24 20 29 14
L. acidophilus (Sorghum) 28 17 20 24
L. lactis ssp lactis 1 (Sorghum) 13 16 10
L. crispatus (Sorghum) 12 17 17 21
L. rhamn osus (Sorghum-Millet) 29 18 24 26
P. damnosus ( Sorghum-Millet) 18 21 25 -
L. fermentum (Sorghum-Millet) 28 24 26 18
L. plantarum 1 (Sorghum-Millet) 29 29 25 15
L. paracasei spp para 1 (Sorghum-Millet) 28 24 26 14
L. paracasei spp para 3 (Sorghum-Millet) 26 21 30 22
L. cellobiosus (Sorghum-Millet) 27 18 22 23
L. plantarum (Sorghum-Millet) 15 20 20 15
L. pentosus (Sorghum-Millet) 29 18 23 17
L. pentosus (Acha + Millet) 22 22 27 18
L.brevis (Acha + Millet) 23 18 30 14
L. fructivorans (Acha + Millet Germinated) 20 19 27 -
L. paracasei ssp para 2 (Acha + Sorghum) 11 22 24 13
= no inhibition.
Copyright © 2013 SciRes. AiM
Table 3. Probiotic LAB isolated from ungerminated com-
posite and non composite fermenting grains.
Source of isolation Significant Probiotic
LAB bacteria isolated
Fermenting non composite
ungerminated grain
Millet Lactobacillus plantarum 1
Lactobacillus plantarum
Pediococcus damnosus
Sorghum Lactobacillus plantarum 1
Lactobacillus paracasei ssp para 3
Lactobacillus acidophilus
Lactococcus lactis
Lactobacillus crispatus
Fermenting composite
ungerminated grain
Millet + Sorghum Lactobacillus rhamnosus
Pediococccus damnosus
Lactobacillus fermentum
Lactobacillu s paracasei spp para 1
Lactobacillu s paracasei spp para 3
Lactobacillus cellobiosus
Lactobacillus plantarum
Lactobacillus pentosus
Acha + Millet Lactobacillus pentosus
Acha + Sorghum Lactobacillus paracasei ssp pa ra 2
Table 4. Probiotic LAB isolated from germinated composite
and non composite fermenting grains.
Source of isolation Significant Probiotic LAB
bacteria isolated
Fermenting non composite
germinated grain
Millet Pediococcus pentosaceus
Lactobacillus lindneri
Fermenting composite
germinated grain
Acha + Millet Lactobacillus fructivorans
rice was found to support the growth of probiotic Lacto-
bacillus plantarum. Literature is quite scanty on the com-
parative effects of germination and fermentation on the
total microbial community of fermenting cereals. [21]
were able to demonstrate that the total LAB community
in malted cowpea fortified fermented cereal was time
related. Their work, however, did not compare with the
unmalted counterpart. This study has shown that germi-
nation does have an effect on the total probiotic LAB
4. Conclusion
It is concluded that the test species of Lactic acid bacteria
present in spontaneously fermenting kunu-zaki have the
potential ability to survive in the gastrointestinal tract of
human. Since the species isolated are also found as nor-
mal commensals of the human GIT, the LAB species
identified by this work can be used as probiotic in human
preparations. It can also safely be concluded that kunu-
zaki made from ungerminated Sorghum and Millet com-
posite cereal grains will have very good applications as a
natural probiotic drink.
5. Acknowledgements
The author acknowledges the following for their helpful
comments and contributions during the course of this
work: Olu Odeyemi, School of Bioscience and Biotech-
nology, University of Malaysia, Olu Malomo, Director,
Central Research Laboratory, Bells University of Tech-
nology and Biotechnology Division, Federal Institute of
Industrial Research, Oshodi. The author also acknowl-
edges the support of the grant received towards scientific
research from the Education Trust Fund through the Yaba
College of Technology.
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