Journal of Biosciences and Medicines, 2014, 2, 8-13
Published Online November 2014 in SciRes. http://www.scirp.org/journal/jbm
How to cite this paper: Heit a, L.N. and Cheikhyoussef, A. (2014) Dominant Lactic Acid Bacteria and Their Antimicrobial Pro-
file from Three Fermented Milk Products from Northern Namibia. Journal of Biosciences and Medicines, 2, 8-13.
Dominant L actic Acid Bacteria and Their
Antimicrobial Profile from Three Fermented
Milk Products from Northern Namibia
L. N. Heita, A. Cheikhyoussef
Science, Technology and Innovation Division, Multidisciplinary Resea rch Cent re, Uni ver si t y of Namibia, Private
Bag 13301, 340 Mandume Ndemu fay o Avenue, Pionierspark, Windhoek, Namibia
Email: firstname.lastname@example.org, email@example.com
Received Oct ober 2014
The present study focused on the isolation, identification and an timicrob ial profile of the d omi-
nant l ac tic a cid bacteria from three t rad i tio nal fer m ented mil k products namely: Omashikwa, Ma-
bisi and Mashini Gh amush ikwa f rom th e n or th-c en tral and nor th-e aste rn p arts of Na mibia. The
microbiolo gical a nd anti m icrobial activi ties of th es e products fluctuate from o ne regi on to an othe r
depending on the local indigenous mic rofl or a. Omash ikwa and Mashini Gha mushikw a fermenta-
tion proces ses i nvolve s th e addition of Boscia albitrunca root ( Omunku zi) and butte r ch urnin g.
The root c ontribu tes to the flavor of the product, incre asing the milk fer ment ation rate and
churning. M abisi is produced by letting th e mil k to ferment n atural ly unt il th e water is sep arated
from the wh ey . The wat e r is th en d ecant ed, a nd th e whey is sh ake n until it is sm oo th wi thou t re-
moval of fat . A total of 180 isolates of Lactic acid bact eria (LAB) we re obta ined and id entified
based on thei r p hen otypic al cha racteriz ation. Cell fr ee superna tants (CFS) of the 180 LAB is olates
were ev al u ated for antim icrobi al activities ag ains t s elected fo od borne pathogens; Escherich ia c oli
ATCC 25,922, Staphylococcus aureus ATCC 25,923, Bacillus cereus ATTC 10,876, Candida famata
and Ge ot ric hu m klebahnii usin g the well d iffus ion assay. Twenty LAB isol ates having the h ighest
inhibitory effec ts were sel ected for biochem ical identif icatio n using API 50 kit and these were
identified as being; Lactobacillus plantarum (53%), Lactobacillus rhamnosu s ( 29%), Pediococcus
pentosaceus (6%), Lactobacillus parac asei ssp . p ar aca sei ( 6%) and L actococcus lac tic ssp . l ac tis
(6%). Pediococcus pent osaceus sh owed th e high est inh ibito ry effect on all th e ind icat or st rains.
This study provide s an ins ight int o LAB diver sity of unstud ied Namibi an fe rm ented milk products
and rep or ts a potent ial p rod u cti on of anti microbi al compounds which is sig nific ant in the stan-
dardiz ati on of pr ote cti v e starter cultures which c an be used to co nt rol fe rme nta tio n p rocess and
shelf life ex tension of d ai r y products in Namibia .
Namibia, T r adi ti on al Ferm ented Milk, Lactic Acid B ac teri a, Om ashikw a, Mab is i, Mashin i
L. N. Heita, A. Cheikhyoussef
Fermented Milk forms a maj or component of the traditional diet in many regions in Africa. Most of the milk
produced is consumed in the house mostly by children and excess is infreq uently sold in open markets to gener-
ate an in come to the ho us eho ld. Due to limitation of cold stora ge facilities in many rural areas in African coun-
tries, milk is stor e d at ambient temperatures which usual ly allow the m to beco me fermented rapidly by the nat u-
ral flor a. The fermentation process occurs spontaneously by back-sloping; i.e. inoculation of the raw material
with a small quantity of the pre vi ousl y per formed successful fermentation. Therefore, back-slopping results in
domi nance of the best adapted strains . Modern techniques of milk fermentation, on the o t her hand , use start-
er cultures with known characteristics . T he a dvantage of modern techni ques over the traditional methods is
the production of consistent products that are less likely to spoil and are relatively safe.
Lactic acid bacteria (LAB) are widely distributed in natur e, they are characterized as group of gram-positive,
non-spore forming, cocci or rods, which produce lactic acid as the major end pro duct during t he fermentation of
carbohydrates. The gro up con sists of several genera, which inc lude Enterococcus, Lactobacillus, Lactococcus,
Leuconostoc, Pediococcus, and Streptococcus . LABS are widely known to carry out safe metabolic activities
in food and are most ly used for food preservation and flavor development . Bio-preservation using lactic acid
bacteria (LAB) and/or their antimicrob ial metabolites are considered to be an alternative strategy for improving
food safety . As a result, there is an increased interest in the preservation through LAB because of the i r safe
association with fermented foods. These antimicrobial pr operti e s of LAB are derived from competition for nu-
trients and the production of one or more antimicrobial active metabolites suc h as organic acids (mainly lactic
and acetic acid), hydrogen peroxide and also o t he r compounds, suc h as bacteriocins and antifungal peptides .
Specific attention has been give n to bacteriocengenic LAB because it is believed that they provide an extr a pro-
tection during the abuse of temperature conditions; decrease the risk for tr a ns missi on of foodborne pat hogens
thro ugh the food chain; red uce chemical preservatives; redu ce heat treatment without compromising food safe ty:
better preservation of nutr i ents a nd vita mins, as well as sensoria l properties of food . In addition to that , the y
permit the marketing of “novel” food (less acidic, with a lower salt conte nt and with a higher water co ntent) to
satisfy the demands of both the consumers and industry .
Earlier studies on Namibian fermented milk have concentrated on the sensory evaluat i on , technological
properties  a nd microbiological  of Omashikwa . To our knowle dge, little i nformation exists on the tradi-
tional fermentative microflora that Omashikwa, Mabisi and Mashini Ghamushikwa use to produce these types of
milk a nd their antimicrobial p rofile. T he objecti ve of this study was to isolate a nd identify the dominant LAB
associated with Omash ik wa, Mabisi, and Mashini Ghamushikwa, and to provide information on the potential
production of antimicrobial compounds by these LAB for possible strain selection and development of starter
cult ure for enhanced quality and safety of the three traditional fermented milk products.
2. Materials and Method s
2.1. Sample Collection
Thirteen (13) fermented milk samples of Omashikwa were collected from northern Namibia from the Oshana,
Oha ngwena , Omusa t i, and Oshikoto regions. T he vi lla ges a nd house hold s were randomly selected. Four (4)
samples of Mabisi were collected fr om Zambezi region and three (3) Mashini Ghamushikwa from Kavango re-
gion. The samples were collected aseptically in sterile 250 ml plastic bottles and kept in an ice-box and trans-
ported to the Fo od Biotechnology Laboratory at the Department of Chemi stry and Biochemistr y of the Univer-
sity of Namibia.
2.2. Microbiological Analysis
Samples (1 ml ) of fermented milk were inoculated into 9 ml of buffe red peptone water (BPW). One ml of these
aliquots was directly inoculated in triplicates on the following media: a) de Man Rogosa Sha r p (MRS) agar in-
cubated for 24 to 48 hours at 37˚C fo r the isolation of Pediococcus and Streptococcus. b) M17 agar incubated
aerobically at 30˚C for 48 hours for enumeration of Lactococcus. c) Rogosa agar incubated for anaerobically at
35˚C for 48 hours for the enumeration of Lactobacillus. Si x colonies from MRS (37˚C) M17 (30˚C) and Rogosa
agar (35˚C) representative of all the sa mples were ran doml y p i cked based on their colony morphology from dif-
ferent section s of plates cont a i ning 10 - 300 colony forming units ( CF U) . Isolates (180) were grown in
L. N. Heita, A. Cheikhyoussef
MRS broth. The LAB isolates were purified by 4 consecutive sub-culturing in MRS agar and MRS broth (Oxoid)
before identification. The i solates were gram stained and morphologically identified usi ng a light microscope for
the pur i ty, cell shapes and arrangements. The isola tes were transferred to MRS broth, incubated for 24 and were
preserved in 25% of glycerol at −80˚C until furthe r analys i s .
2.3. Antimicrobial Assay of LAB
2.3.1. Micro organism s and Culture Conditio ns
Escherichia coli ATCC 25,922, Staphylococcus aureus ATCC 25,923, Bacillus cereus ATTC 10,876, Ca ndida
famata (IKST F. Lab. isolate ) and Geotrichum klebahnii (IKST F. Lab. isolate) were used as test microorgan-
isms or indicato r strains. The indicator strains used fo r determination of inhibitory activity spectra of studied
isolates were purchased fro m the American Type C ul ture C ollection (ATCC) Stock c ultur e s. Bacteria were kept
in a refrigerator (4˚C) on nutri e nt agar slants. Geotrichum klebahnii was isolated from Namibian fermented food
and beverages samples collected from/Karas regio n in the south of Namibia .
2.3.2. Agar Well Di ffusi on Method
Cell-fr ee s upernata nts (CFS) for antibacterial assay was prepared by growing t he LAB isolates in MRS broth at
37˚C and centrifuged at 13,000 × g for 10 min at 4˚C then pH was adjusted to 7 by 1M NaOH to exclude the an-
timicrobial effect of orga ni c acid . T he antimicrobial activity of the cell-free culture sup e rnatants of isolated
LAB aga i nst t he indicator organisms was determined by the agar well diffusion method . Aliquots of super-
natants (100 μL) were placed in wells (6 mm diameter) cut in cooled soft nutr i ent aga r plates using a cock bore,
pre vi ousl y seeded with 100 μL of the appropriate indicator strai n s. T he plates were firs t store d in the refrigerator
for to allow LAB s uper na t ant to set the n incubated under optimal conditions for growth of the target microor-
ganisms after which they were examined for clear zones aro und the wells. The diameters of the growth inhibi-
tion zones were measured and record ed in millimeter (mm).
2.4. Biochemical Identification of LAB
The car bohydrate fermentation profiles of the selected 20 LAB isolates were investigated using API 50 CH
strips and API CHL medium according to manufacturer’s instruction (API system, BioMèrieux, France). Over-
night cultures of the isolates grown in 10 ml MRS broth at 30˚C were washed twice with sterile peptone water
and the pe llets were re-suspended in API 50 CHL medium, usi ng sterile pas ture pipettes. With sub s e que nt mix-
ing, homo ge ni z ed s uspe nsi ons of the cells in the medium where transferred into each of the 50 wells on the API
50 CH strips. Strips were covered as recommended and inc ubate d at 30˚C. Change s in color were monitored af-
ter 24 hrs of incub a t i on. Results were represented by positive sign (+) while a nega t ive sign (−) was des ignated
for no change. The APIWeb™ V1.2.1. Sof tware (BioMèr ieux, France) was used according manufactures in-
struction in interpretation of the results.
2.5. Statistical Analysis
All the a na lys i s was done in triplicates. Val ues of different tests were e xp r e sse d as the mean ± standar d devi a-
tion (x ± SD). Statistical Package for Social Science (SPSS Inc., Chi c ago , IL) packet program for Windows
(SPSS V. 12.0) was used for the statistical analysi s. The di fferent samples treat ments were co mpared by per-
forming one -way analysi s of variance (ANO VA) on the rep licates at 95% level of significance.
3. Results and Discussion
3.1. Identification of Lactic Acid Bacteria
A total of 180 Gram-positive and catalase negative bacterial isolates wer e isolated from 20 samples of fermented
milk products from north east Namibia. They were gro uped a nd preliminary identified to the gen us le ve l based
on their cell shape a nd arrangement (Table 1).
Out of the 180 LAB isolates from the three types of fermented milk, most of the genera b elonge d Lactobacil-
lus 88%, Lactococcus 6%, and Pediococcus 6%. T he se results showed the hetero ge neity in the three types of
fermented mil k Omashi kwa, Mabisi and Mashini ghakushika from different regions or agro-climatic zones ei-
ther in its chemical or microbial composition and quality attributes.
L. N. Heita, A. Cheikhyoussef
Table 1. Morphological and biochemical characterization of bacteria isolates from Mabisi, Omashikwa and Mashini ghaku-
shika that were collected from north-east in Namibia.
Total number of
Cell shape Cellular arrangement C atalase Gram
Lactobacillus Rods Short rods, straight edges − + 88
Lactobacillus Rods Long rods, rounded edges − + 45
Strept oc o c cus Cocci Chains , pai rs − + 27
tetrads and pairs,
− + 20
3.2. Antimicrobial Activities of Lactic Acid Bacteria
All the 180 isolates of LAB isolated from the thr e e types of fermented milk were subjected to inhibitory activity
test using well diffusion method (Figure 1). All LAB-CF S showed inhibition effect a gainst Candida famata
with an average of at least 6.08 mm. In contrast, Bacillus cereus was the most resi stant strain as only fe w CFS
showed inhibition effects with an aver age of 1.07 mm. The formation of small zones of inhibition using the
well-diffusion-assay technique may be explained by the insufficient concentration of antimicrobial subst ances in
the s upernata nt of t he c ultur es  .
3.3. Identification of the Inhibitory Subs tance (s) Producing LAB
Twenty strains a mong 180 lactic acid bacteria isolated fr om the thr e e t ypes of milk “Oma sh ik wa , Mashini gha-
kushika, and Mabisi” were selected according to their max imu m antimicrobial activity aga i nst indicator str ai n s.
The y were the n identified fro m the production of acids from carbohydrates and related compounds using an API
50 CHL syste m (Biomérieux, France). Table 2 shows LAB species identified in five different species. The pre-
domi nant species were Lactobacillus plantarum (9 strains) , Lactobacillus rhamnosus (5 strai ns) , Pediococcus
pentosaceus 2 (1 strain), Lactococcus lactic ssp. lactis 1 (1 strain s) and Lactobacillus paracasei ssp. paracasei 1
Mabisi showed diversity of gener a with four type s of LAB species L. plantarum, L. paracasei ssp. paracasei,
L. rhamnosus and Pediococcus pentosaceus. Oma shikwa has o nly three species L. plantarum, L. rhamnosus and
L. lactis ssp. lactis; these were in agreement with t he repo r ted study , when he found that the ma in dominant
lactic acid bacteria belonged to Lactobacillus plantarum (25%) and 15% Lactococcus lactis ssp. lactis. The only
difference was the Lactobacillus rhamnosus which was found in this stud y which was no t the case with the re-
ported study . This may be attributed to high envi ro nmental temperatures during processing and probably the
use of Boscia albitrunca (Omunkuzi) root. The do mi na nc e of Lactobacillus plantarum strains in Omash i kwa and
Mashini ghakushika could be associated with the root which is added dur i ng ferment ati on. Reference  stated
that the presence of L. plantarum is known to be c ommon ly associa ted wi th plant based food fermentations.
Reference  reported that different stra ins of L. plantarum have been isolated fro m vari ous nic hes, such as
fermented mil k, chee se, fermented cucumber, fermented olives , pasta, pineapple, grapefruit juice , sorghum beer
and barley, molasses, boza, k efir and amasi. Lactobacillus plantarum ca n be considered as the mos t important
and versatile species among LAB. It can adapt to various niche s due to its abilit y to ferment a wide range of
carbohydrates. Additionally, it can be used as starter culture in food fermentations and as an ingred i ent fo r pro-
biotic foods, contributing to the organoleptic characteristics of foods at the s ame time prolonging the shel f-li fe
and sa fet y of these products . Reference  emphasized that the preliminary identificatio n of the inhibitory
activity of LAB isolated from foods is essentia l for further studies about their antimicrobial activity. After a nt a-
goni s t activity is detected, complementary tests can be implement ed to co nfirm the proteinaceous nature of the
produced subst a nc es, to identify the spectrum of other sensit ive mic roorga nis ms , to verify the factors that inter-
fere with the antimicrobial activity and to verify the p a t hogenici ty of the isolated cultur e s [ 14]  .
Microorganisms invol ve d in Omash ik wa , Mabasi and Mashini Ghakushika fermentation were found to consist
of LAB from the three ge ne ra Lactobacillus, Lactococcus and Pediococcus. T he do mi nan t ge nus was Lactoba-
L. N. Heita, A. Cheikhyoussef
Figure 1. Average of Inhibition zones toward the indicator strains.
Table 2. Distribution of LAB species in omashikwa, ma bisi and mashini ghakushika.
LAB species Type of fermented milk
Omashikwa Mabisi Mashini ghakushika
Lactobacillus rhamnosus 5 1 2
Lactococcus lactis ssp . lactis 1 1 − −
Lactobacillus plantarum 1 6 1 1
Lactobacillus paracasei ssp. paracasei 1 − 1 −
Pediococcus pentosaceus 2 − 1 −
cillus while the mo st dominant species were Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus
pentosaceus, Lactococcus lactic ssp. lactis and Lactobacillus paracasei ssp. Paracasei. Most inhibitory sub-
stances produced by lactic acid bacteria are safe and effective natural inhibitors of pathogenic and food spoilage
bacteria in various foods. These microor ganisms should be tested for their technologi cal p roperties, microbial
interactions and possible probiotic characterization. This will enable product development and innova t i on for
prolonging the shelf-life and safety for dairy products in Namibia.
The a uthors would like to thank the Directorate of Research Science and Technology (DRST) of the Ministry of
Education (Now called National Commis sion on Research Science and T echnology (NCRST ) , the Research &
Publication office of the Pro-Vice Chanc e l l or: Academic Affairs & Research of the University of Namibia, and
the Science, Technology and Innovat i on Division (ST&ID) of Multidisciplinary Research Centr e for their finan-
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