<i>In Vitro</i> Screening and Selection of Probiotic Lactic Acid Bacteria Isolated from Spontaneously Fermenting Kunu-Zaki

doi:10.4236/aim.2013.34044

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Advances in Microbiology, 2013, 3, 309-316

http://dx.doi.org/10.4236/aim.2013.34044 Published Online August 2013 (http://www.scirp.org/journal/aim)

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: *[email protected]

Received April 8, 2013; revised May 8, 2013; accepted June 8, 2013

Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

properly cited.

ABSTRACT

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.

S. O. OLUWAJOBA ET AL.

310

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-

mentation.

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)

Sweetening

Bottling

Storage (Refrigeration temperature)

Figure 1. Flow diagram for the traditional processing of

kunu-zaki from composite grains.

S. O. OLUWAJOBA ET AL. 311

2.5. Identification of Lactic Acid Bacteria

Species

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

activity.

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-

S. O. OLUWAJOBA ET AL.

312

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

S. O. OLUWAJOBA ET AL.

313

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-

S. O. OLUWAJOBA ET AL.

314

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.

S. O. OLUWAJOBA ET AL. 315

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

isolated.

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.

REFERENCES

[1] T. R. Klaenhammer, “Probiotic Bacteria: Today and To-

morrow,” Journal of Nutrition, Vol. 130, Suppl. 2S, 2000,

pp. 415S-416S.

[2] T. Gaffa, I. A. Jideani and T. Nkama, “Traditional Pro-

duction, Consumption and Storage of Kunu—A Non-

Alcoholic Cereal Beverage,” Plant Foods for Human Nu-

trition, Vol. 57, No. 1, 2002, pp. 73-81.

doi:10.1023/A:1013129307086

[3] J. A. Jideani and J. O. Akingbala, “Some Physiochemical

Properties of Acha (Digitaria exilis Stapf) and Iburua (Di-

gitaria iburua Stapf) Grains,” Journal of the Science of

Food and Agriculture, Vol. 63, No. 3, 1993, pp. 369-371.

doi:10.1002/jsfa.2740630317

[4] J. A. Ayo, I. Nkama, U. S. Haruna, Y. Bitrus and F. Ona-

jaife, “Effect of Dough Improvers on the Physical and

Sensory Quality of Acha (Digitaria exilis) Flour Bread,”

Nigerian Food Journal, Vol. 26, No. 1, 2008, pp. 102-

110.

[5] J. A. Awan, and S. U. Rahman, “Microbiology Manual,”

Unitech Communications, Faisalabad, 2005, pp. 49-51.

[6] W. F. Harrigan and M. E. McCance, “Methods in Food

and Dairy Microbiology,” Academic Press, London,

1976.

[7] J. Prasad, H. Gill, J. Smart and P. K. Gopal, “Selection

and Characterization of Lactobacillus and Bifidobacte-

S. O. OLUWAJOBA ET AL.

316

rium Strains for Use as Probiotic,” International Dairy

Journal, Vol. 8, No. 12, 1998, pp. 993-1002.

doi:10.1016/S0958-6946(99)00024-2

[8] B. J. B. Wood and W. H. Holzapfel, “The Genera of Lac-

tic Acid Bacteria in the Lactic acid Bacteria,” In: B. J. B.

Wood and W. H. Holzapfel Eds., The Genera of Lactic

Acid Bacteria Blackie, Academic and Professional, Lon-

don, 1995, p. 398. doi:10.1007/978-1-4615-5817-0

[9] K. H. Steinkraus, “Fermentations in World Food Proc-

essing,” Comprehensive Reviews in Food Science and

Food Technology, Vol. 1, No. 1, 2002, pp. 23-32.

doi:10.1111/j.1541-4337.2002.tb00004.x

[10] V. Lei and M. Jacobsen, “Microbiological Characteriza-

tion and Probiotic Potential of Koko and Koko Sour Wa-

ter African Spontaneously Fermented Millet Porridge and

Drink,” Journal of Applied Microbiology, Vol. 96, No. 2,

2004, pp. 384-397.

doi:10.1046/j.1365-2672.2004.02162.x

[11] C. M. Kalui, J. M. Mathara and P. M. Kutima, “Probiotic

Potential of Spontaneously Fermented Cereal Based

Foods—A Review,” African Journal of Biotechnology,

Vol. 9, No. 17, 2010, pp. 2490-2498.

[12] A. C. Quwehand, P. V. Kirjavainen, C. Shortt and S. Sal-

minen, “Probiotics: Mechanisms and Established Ef-

fects,” International Dairy Journal, Vol. 9, No. 1, 1999,

pp. 43-52. doi:10.1016/S0958-6946(99)00043-6

[13] İ. Çakir, “Determination of Some Probiotic Properties on

Lactobacilli and Bifidobacteria,” PhD Thesis, Ankara

University, Ankara, 2003.

[14] P. R. Marteau, M. Minekus, R. Havenaar, and J. H. J.

Huis Int’l Veld, “Survival of Lactic Acid Bacteria in a

Dynamic Model of the Stomach and Small Intestine.

Validations and the Effect of Bile,” Journal of Dairy Sci-

ence, Vol. 80, No. 6, 1997, pp. 1031-1037.

doi:10.3168/jds.S0022-0302(97)76027-2

[15] W. P. Charteris, P. M. Kelly, L. Morelli and J. K. Collins,

“Selective Detection, Enumeration and Identification of

Potentially Probiotic Lactobacillus and Bifidobacterium

Species in Mixed Bacterial Populations,” International

Journal of Food Microbiology, Vol. 35, No. 1, 1997, pp.

1-27. doi:10.1016/S0168-1605(96)01222-6

[16] T. R. Klaenhammer and M. J. Kullen, “Selection and

design probiotic,” International Journal of Food Micro-

biology, Vol. 50, No. 1, 1999, pp. 45-57.

doi:10.1016/S0168-1605(99)00076-8

[17] P. A. Maragkoudakis, G. Zoumpopoulou, C. Miaris, G.

Kalantzopoulos, B. Pot and E. Tsakalidou, “Probiotic po-

tential of Lactobacillus Strains Isolated from Dairy Prod-

ucts,” International Dairy Journal, Vol. 16, No. 3, 2005,

pp. 189-199. doi:10.1016/j.idairyj.2005.02.009

[18] M. Strus, K. Pakosz, H. Gościniak, A. Przondo-Mor-

darska, E. Rozynek, H. Pituch, F. Meisel-Mikołajczyk

and P. B. Heczko, “Antagonistic Activity of Lactobacillus

Strains against Anaerobic Gastrointestinal Tract Patho-

gens (H. pylori, C. coli, C. jejuni, C. difficile),” Medycyna

Doswiadczalna i Mikrobiologia, Vol. 53, No. 2, 2001, pp

133-142.

[19] A. C. Quwehand and S. Vesterlund, “Antimicrobial Com-

ponents from Lactic Acid Bacteria. Lactic Acid Bacteria

Microbiological and Functional Aspects,” Marcel Dekker

Inc., New York, 2004.

[20] P. Saman, P. Fuciños, J. A. Vázquez and S. S. Pandiella,

“Fermentability of Brown Rice and Rice Bran for Growth

of Human Lactobacillus Plantarum NCIMB 8826,” Food

Technology & Biotechnology, Vol. 49, No. 1, 2011, p.

128.

[21] S. L. Wakil and A. A. Onilude, 2011 “Time Related Total

Lactic Acid Bacteria Population Diversity and Domi-

nance in Cowpea-Fortified Fermented Cereal-Weaning

Food,” African Journal of Biotechnology, Vol. 10, No. 6,

2011, pp. 887-895.