Hindawi

International Journal of Microbiology

Volume 2019, Article ID 7179514, 11 pages
https://doi.org/10.1155/2019/7179514

Research Article
In Vitro Evaluation of Probiotic Properties of Lactic Acid Bacteria
Isolated from Some Traditionally Fermented Ethiopian
Food Products

Guesh Mulaw ,1,2 Tesfaye Sisay Tessema,3 Diriba Muleta ,3 and Anteneh Tesfaye3
1
Department of Microbial, Cellular and Molecular Biology, College of Natural and Computational Sciences,
Addis Ababa University, Addis Ababa 1176, Ethiopia
2
Department of Biology, College of Natural and Computational Sciences, Aksum University, Axum 1010, Ethiopia
3
Institute of Biotechnology, College of Natural and Computational Sciences, Addis Ababa University, Addis Ababa 1176, Ethiopia

Correspondence should be addressed to Guesh Mulaw; guesh2001@gmail.com

Received 19 April 2019; Revised 28 June 2019; Accepted 16 July 2019; Published 25 August 2019

Academic Editor: Joseph Falkinham

Copyright © 2019 Guesh Mulaw 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.
Probiotics are live microorganisms which when consumed in large number together with a food promote the health of the
consumer. The aim of this study was to evaluate in vitro probiotic properties of lactic acid bacteria (LAB) isolated from traditional
Ethiopian fermented Teff injera dough, Ergo, and Kocho products. A total of 90 LAB were isolated, of which 4 (4.44%) isolates
showed 45.35–97.11% and 38.40–90.49% survival rates at pH values (2, 2.5, and 3) for 3 and 6 h, in that order. The four acid-
tolerant isolates were found tolerant to 0.3% bile salt for 24 h with 91.37 to 97.22% rate of survival. The acid-and-bile salt-tolerant
LAB isolates were found inhibiting some food-borne test pathogenic bacteria to varying degrees. All acid-and-bile-tolerant isolates
displayed varying sensitivity to different antibiotics. The in vitro adherence to stainless steel plates of the 4 screened probiotic LAB
isolates were ranged from 32.75 to 36.30% adhesion rate. The four efficient probiotic LAB isolates that belonged to Lactobacillus
species were identified to the strain level using 16S rDNA gene sequence comparisons and, namely, were Lactobacillus plantarum
strain CIP 103151, Lactobacillus paracasei subsp. tolerans strain NBRC 15906, Lactobacillus paracasei strain NBRC 15889, and
Lactobacillus plantarum strain JCM 1149. The four Lactobacillus strains were found to be potentially useful to produce
probiotic products.

1. Introduction approaches that involve human beneficial live microbes

(probiotics) in order to combat these pathogens and their
Worldwide, a variety of fermented food products are pro- associated health risks [3]. Several in vitro studies indicate that
duced, which contribute significantly to the diets of many the growth of food-borne pathogenic microbes is inhibited by
people [1]. Fermented food products are used to describe a probiotic lactic acid bacteria [4–6]. The consumption of a large
special class of the food products characterized by various number of probiotic live microorganisms together with a food
kinds of carbohydrate breakdowns in the presence of pro- fundamentally promotes the health of the consumers [7].
biotic microorganisms, but seldom is carbohydrate the only Lactic acid bacteria (LAB) are a diverse group of microor-
constituent acted upon [2]. Fermented food and beverage ganisms consisting of Gram-positive, aerotolerant, acid-tol-
products have emerged as not only the source of nutrition erant, usually nonsporulating and nonrespiring rod or cocci
but also as functional and probiotic foods, which besides microorganisms, and play an important role in the process of
nutritional value have health effects or provide protection fermentation of food by inhibiting spoilage/pathogenic bacteria
against food-borne diseases. and by producing excellent flavor, aroma, and texture of
The problem of food-borne diseases (FBD) is multifacto- fermented foods [8, 9]. Some LAB are probiotics, and others
rial, and their prevention and control require multidisciplinary may be potential probiotics or just fermentation cultures that
2 International Journal of Microbiology

are widely distributed in nature and can be used in the food sample of traditionally fermented foods (Teff dough, Kocho
industry [10]. and Ergo) was mixed with 225 ml of separate sterile peptone
LAB could be isolated from many kinds of sources such as water (0.1% W/V). Then, a sequential decimal dilution of the
milk products, fermented foods, animal intestines or fresh- homogenate was obtained. From the appropriate dilutions,
water fishes, soil samples, sugar cane plants, and poultry farms 0.1 ml aliquots were spread plated on duplicate predried
[11]. The most common types of probiotic LAB include dif- surfaces of MRS (de Man, Rogosa, and Sharp) agar (Oxoid,
ferent Lactobacillus spp. (Lb. acidophilus, Lb. johnsonii, Lb. Basingstok, Hampshire, England) plates. The inoculated
casei, Lb. rhamnosus, Lb. gasseri, and Lb. reuteri) and genus plates were incubated under anaerobic condition using an
Bifidobacteria (Bf. bifidum, Bf. animalis subsp. lactis, Bf. longum anaerobic jar (BBL, Gas Pak Anaerobic Systems) at 37°C for
subsp. longum, and Bf. longum subsp. infantis) [12, 13]. LAB 48 hours. Then, 10–20 distinct colonies were randomly
are also useful in the treatment of various diseases caused by picked from countable MRS plates for further purification.
drug-resistant pathogenic microbes [14]. Probiotic microbes The isolated colonies of LAB were transferred into about
may provide nutrients, enhance growth, produce enzymes, 5 ml MRS broth (Oxoid) and purified by repeated streaking
inhibit pathogens, and enhance immune responses [15]. on MRS agar. Pure cultures of LAB were then streaked onto
In Ethiopia, traditionally fermented food products are slants of MRS agar and stored at +4°C for further charac-
prepared at the household level and the consumption of terization [18].
these fermented food products is commonly practiced.
Therefore, some studies on isolation and screening of an-
2.3. Confirmation Tests of LAB Isolates
tibacterial producing lactic acid bacteria from traditionally
fermented foods were undertaken by many workers [16, 17]. 2.3.1. KOH Test. The KOH test was used to determine the
Likewise, Tesfaye et al. [4] have revealed the antagonistic gram reaction of LAB isolates. LAB cultures were grown on
effect of lactic acid bacterial strains either as pure or defined MRS agar at 37°C for 24 h under anaerobic conditions. A
mixed cultures against some food-borne pathogens during drop of 3% aqueous KOH was placed on a clean slide. Using
fermentation and storage of fermented milk. However, there a sterile loop, visible cells from fresh cultures were trans-
are still few research data available on the characterization of ferred to the drop of 3% KOH. The cells and KOH were
probiotic LAB. Most of the traditionally fermented products mixed thoroughly on the slide and stirred constantly over an
of Ethiopia are consumed without further heat processing area about 1-2 cm2. The isolates, which did not give a viscid
which can be considered as ideal vehicles to carry probiotic product, were selected since lactic acid bacteria (LAB) are
bacteria into the human gastrointestinal tract. known as Gram-positive cells [19].
Probiotic strains isolated from traditionally fermented
foods and drinks could have application as a starter culture
for large-scale production of the traditional product and 2.3.2. Catalase Test. Overnight cultures of isolates were
have a desirable functional property for their application as grown on MRS agar at +37°C for 24 h under anaerobic
probiotics against food-borne pathogens. Therefore, this conditions. The catalase test was conducted by dripping two
study attempts to evaluate the in vitro probiotic properties of drops of hydrogen peroxide (3%) on 24 h-old cultures on a
LAB isolated from three traditionally fermented Ethiopian glass slide. The catalase test showed positive reaction
food products such as Teff dough, Ergo, and Kocho with characterized by the formation of oxygen bubbles that in-
respect to their potential probiotic properties against some dicate the production of catalase enzyme by the test bac-
food-borne pathogens. terium. Therefore, the isolates, which did not give gas
bubbles, were selected for subsequent activities.

2. Materials and Methods

2.3.3. Spore Staining. Gram-positive and catalase-negative
2.1. Sample Collection. Traditionally fermented food prod- isolates were grown on MRS agar at +37°C for 24 h under
ucts (Teff dough, Kocho, and Ergo) were obtained from Addis anaerobic conditions. The spore-staining procedure was
Ababa and its surroundings, Ethiopia. Each sample (200 g) applied [20]. After the spore-staining technique, the en-
was aseptically collected by using sterilized containers. The dospore formulation was examined under light microscopy
samples were brought to the laboratory with an ice box and using oil immersion objectives. The isolates which did not
stored in a refrigerator at +4°C until further analysis was form endospores were selected for further analysis.
carried out. Ergo is a locally fermented milk product. Teff
dough is made by fermenting teff (Eragrostis tef ) flour which 2.4. In Vitro Characterization of Probiotic Properties. The
is used to prepare a thin pancake-like product with many common methods for in vitro analysis of probiotic prop-
eyes known as injera. Kocho is a product which is prepared erties include tolerance to low pH, tolerance against bile salt,
from decorticated and pounded pulp of enset plant (Ensete antibiotic susceptibility, antimicrobial activity, and bacterial
ventricosum), which is further mixed and kneaded into a adherence to stain steel plates.
mash and fermented in a pit.

2.4.1. Tolerance to Low pH. The isolates were grown sepa-

2.2. Isolation and Purification of LAB from Traditional Fer- rately overnight in 5 ml MRS broth at +37°C under anaerobic
mented Foods. For isolation of LAB, 25 ml or 25 g of each conditions. A volume of 1 ml of log 7 CFU/ml of each
International Journal of Microbiology 3

overnight-grown culture was inoculated into 10 ml of MRS 2.4.3. Antimicrobial Activity. Antibacterial activity of the
broth to give an initial inoculum level of log 6 CFU/ml. The acid-bile-tolerant LAB strains against some food-borne
culture was then centrifuged at 5000 rpm for 10 min at pathogens was determined using the agar-well diffusion
+4°C. The pellets were washed twice in phosphate buffer method with some modifications of the protocol indicated
(pH 7.2). The pellets were resuspended in 5 ml sterile MRS by Fontana et al. [22]. The test organisms (Staphylococcus
broth which was adjusted to pH values of 2.0, 2.5, and 3.0 aureus ATCC 25923, Listeria monocytogenes (clinical iso-
using 1 N·HCl to simulate the gastric environment. The test late), Salmonella enterica Typhimurium, and Escherichia coli
tubes were incubated for 3 and 6 hours at 37°C. After an ATCC 25922) were obtained from the Ethiopian Public
appropriate incubation period, 1 ml of the culture was Health Institute (EPHI), Addis Ababa, Ethiopia.
diluted in sterile 9 ml phosphate buffer (Sigma, St. Louis, The selected acid-bile-tolerant LAB isolates were in-
MO USA) prepared according to the manufacturer’s in- oculated from slants to fresh MRS broth containing 1%
struction (0.1 M, pH 6.2) in order to neutralize the medium glucose and incubated overnight at 37°C. The overnight
acidity. Briefly, a 100 μl aliquot of the culture and its 10-fold active culture broth of each isolate was centrifuged sepa-
serial dilutions were plated on the MRS agar medium. The rately at 5000 rpm for 10 min at 4°C. The cell-free super-
inoculated plates were incubated at 37°C for 24 to 48 h natant from each separate culture was collected as a crude
under anaerobic condition using an anaerobic jar (BBL, extract for the antagonistic study against selected food-borne
Gas Pack System). The grown LAB colonies were expressed pathogens. The pure cultures of food-borne pathogens were
as colony-forming units per milliliter (CFU/ml). A positive inoculated from slants to brain heart infusion broth. After
control consisting of regular MRS broth inoculated with 24-hour incubation at 37°C, a volume of 100 μl of inoculum
the culture was used [21]. The survival rate was calculated of each indicator bacteria was swabbed evenly over the
as the percentage of LAB colonies grown on MRS agar surface of nutrient agar plates with a sterile cotton swab. The
compared to the initial bacterial concentration: plates were allowed to dry, and a sterile cork borer (diameter
log CFUN1 5 mm) was used to cut uniform wells in the agar. Each well
survival rate(%) � × 100, (1) was filled with 100 μl culture-free filtrate obtained from each
log CFUN0
of the acid-bile-tolerant LAB isolates. After incubation at
where N1 is the viable count of isolates after incubation and 37°C for 24 to 48 hours, the plates were observed for a zone of
N0 is the initial viable count. inhibition (ZOI) around the well. The diameter of the in-
hibition zone was measured by calipers in millimeters, and a
clear zone of 1 mm or more was considered positive in-
2.4.2. Tolerance to Bile Salts. To estimate bile tolerance of hibition [23, 24]. The experiment was carried out in trip-
acid-tolerant LAB (those only were grown in pH 2.0, 2.5, licates, and the activity was reported as the diameter of
and/or 3.0), the isolates were separately grown overnight in ZOI ± SD.
MRS broth at 37°C under anaerobic conditions [21]. Each
culture with the initial concentration of 106 CFU/ml was
then centrifuged at 5000 rpm for 10 min at 4°C. The pellets 2.4.4. Antibiotic Susceptibility Tests. Each of acid-bile-tol-
were washed twice in the phosphate-saline buffer (PBS at pH erant and antagonistic lactic acid bacteria isolates was
7.2). Cell pellets were resuspended in sterile MRS broth assessed for its antibiotic resistance by the disc diffusion
supplemented with 0.3% (w/v) bile salt (Oxgall, USA). method as described by Zhang et al. [25] against some
Samples were taken at 24 h from the onset of incubation to antibiotics that included ampicillin (10 μg/ml), erythromy-
determine the survivability of cells as described previously cin (15 μg/ml), streptomycin (10 μg/ml), kanamycin (25 μg/
[21]. A positive control consisting of plain MRS broth ml), and tetracycline (30 μg/ml). Thus, a volume of 100 μl of
without bile salts inoculated with each separate culture was actively growing cultures of each acid-bile-tolerant and
simultaneously set up. After appropriate incubation, 1 ml of antagonistic lactic acid bacteria was swabbed evenly over the
each separate culture was diluted separately in sterile 9 ml surface of nutrient agar plates with a sterile cotton swab.
phosphate buffer (Sigma, St. Louis, USA) prepared After drying, the antibiotic discs were placed on the so-
according to the manufacturer’s instruction (0.1 M, pH 6.2) lidified agar surface, and the plates were left aside for 30 min
in order to neutralize the medium. Concisely, a 100 μl ali- at 4°C for the diffusion of antibiotics and then anaerobically
quot of the culture and its 10-fold serial dilutions were plated incubated at 37°C for 24 to 48 h. Resistance was defined
on MRS agar medium. Plates were incubated at 37°C for 24 according to the disc diffusion method by using the above
to 48 h under anaerobic condition using an anaerobic jar antibiotic discs, and the diameters of inhibition zones were
(BBL, Gas Pack System). LAB counts were expressed in measured using calipers [26]; the zone of inhibition (di-
colony-forming units per milliliter (CFU/ml). The survival ameter in mm) for each antibiotic was measured and
rate was calculated as the percentage of LAB colonies grown expressed as susceptible, S (≥21 mm); intermediate, I (16–
on MRS agar compared to the initial bacterial concentration: 20 mm), and resistance, R (≤15 mm).
log CFUN1
survival rate(%) � × 100, (2) 2.4.5. Bacterial Adhesion to Stainless Steel Plates. The ad-
log CFUN0
herence assay of acid-bile-tolerant, antagonistic, and anti-
where N1 is the viable count of isolates after incubation and biotic-sensitive lactic acid bacterial isolates was determined
N0 is the initial viable count. on stainless steel plates with some modifications of the
4 International Journal of Microbiology

protocol given by El-Jeni et al. [27]. Briefly, LAB were tubes with 5 ml amount and inoculated with each of 1%
cultured in sterile MRS broth. Thereafter, the overnight overnight cultures of LAB. Thereafter, tubes were incubated
bacterial culture (500 μl) was deposited in a test tube, which at +37°C for 24 h. A 100 µl of cultures were transferred onto a
was then filled with 450 μl of MRS broth, wherein the sterile white background. Thereafter, the same amount of Nessler’s
stainless steel plate was deposited, and the test tubes were reagent was pipetted to the cultures. The change in color to
then incubated for 24 h at 37°C. The stainless steel plate was bright orange indicates a positive reaction, while the yellow
removed under aseptic conditions, washed with 10 ml of color indicates the negative reaction. A negative control,
sterile 1% peptone water, and left for 5 min in a sterile 1% which does not contain arginine, was used as a negative
peptone water tube. The plate was then washed again in the control.
same conditions and vortexed for 3 min in a sterile 1%
peptone water tube (6 ml) consecutively to detach the
bacterial cells adhering to the steel plate surface. The cell 2.5.5. Gas Production from Glucose. In order to determine
number was determined by counting on MRS agar after 24 h the homofermentative and heterofermentative characteris-
of incubation at 37°C. Simultaneously, the total initial cell tics of LAB isolates, CO2 production from glucose was
numbers were estimated to calculate the percentage of ad- determined in modified MRS broth containing inverted
hered bacterial cells for each LAB. Durham tubes with 1% glucose. MRS broth (8 ml) in sep-
arate tubes containing 1% glucose with inverted Durham
tubes was prepared and inoculated separately with 50 µl of
2.5. Morphological, Biochemical, and Physiological Tests. 1% overnight fresh LAB culture. Then the test tubes were
The probiotic LAB isolates were identified according to their incubated at +37°C for 5 days. The presence of gas in
morphological, physiological, and biochemical characteris- Durham tubes during 5 days of observation indicates CO2
tics based on Bergey’s Manual [28]. production from glucose.

2.5.1. Cell Morphology. Overnight cultures were wet 2.6. Identification of Probiotic LAB Isolates Using 16S rRNA
mounted on microscopic slides and examined under a light Gene Sequencing
microscope using oil immersion objectives. Cellular mor-
phological criteria considered during the examination were 2.6.1. Genomic DNA Extraction. Genomic DNA was
cell shape and cell arrangements. extracted from pure cultures (n � 4) of potential probiotic
LAB. Separately, 1 ml of each pure liquid culture was
centrifuged for 3 min at 10000 rpm. The supernatant was
2.5.2. Growth at Different Temperatures. Each of the over-
removed, and the cells were resuspended in 300 µl buffer
night LAB cultures of 50 μl was transferred into four separate
(10 mM Tris-HCl, pH 8.0; 50 mM glucose, and 10 mM
tubes that contained 5 ml medium (modified MRS broth)
EDTA). To the suspension, 3 µl lysozyme (10 mg/ml) was
containing bromecresol purple indicator at a concentration
added, and cells were lysed at 37°C for 60 min under oc-
of 0.12 g/l. After inoculation, two of the inoculated test tubes
casional stirring of the tube content by overturning it. Lysing
were incubated for 7 days either at 15°C and the other two
buffer (20 mM Tris-HCl, pH 8.0; 75 mM NaCl; 1% SDS;
test tubes at 45°C. During this incubation time, growth at any
10 mM EDTA) of 300 µl and 3 µl RNAse (10 mg/ml) was
temperature was observed by the change of the growth
added to the mixture. The mixture was incubated at 37°C for
medium (cultures) from purple to yellow.
30 min and then cooled on ice for 1 min. Then, 100 µl so-
lution of ammonium acetate (7.5 M) was added to the
2.5.3. Growth at Different NaCl Concentrations. LAB iso- mixture, mixed on a vortex for 20 seconds and was
lates were tested for their tolerance to different NaCl con- centrifuged at 13000 rpm for 5 min. The supernatant was
centrations. For this purpose, 4% and 6.5% NaCl transferred into clean 1.5 ml tubes, and 300 μl isopropanol
concentrations were used for testing. Similarly, test tubes was added. Thereafter, the mixture was mixed by over-
with 5 ml of modified MRS broth containing bromecresol turning for 1 min and stored at − 20°C for 30 min. The
purple indicator were prepared according to the appropriate mixture was centrifuged at 13000 rpm for 5 min. The su-
concentrations. Four test tubes with 4% NaCl and the other pernatant was accurately decanted, and the tubes were
four test tubes with 6.5% NaCl were inoculated separately placed overturned on a clean filter. Then, 400 µl of 70%
with 50 μl of 1% of each overnight culture of LAB and in- ethanol was added and mixed several times by overturning
cubated at +37°C for 7 days. The change of the color from to wash the DNA sediment. Finally, the sediment was dried
purple to yellow was considered as proof of cell growth. at 37°C for 15 min till ethanol drops disappeared completely.
The dried sediment was dissolved in 30 µl TE buffer.

2.5.4. Arginine Hydrolysis Test. Arginine containing MRS

medium and Nessler’s reagent (HgI4K2), a 0.09 mol/L so- 2.6.2. PCR Amplification of 16S rDNA. For the amplification
lution of potassium tetraiodomercurate (II) (K2 [HgI4]) in of the 16S rDNA gene, the specific primers AMP_F 5′- GAG
2.5 mol/L potassium hydroxide, were used in order to test AGT TTG ATY CTG GCT CAG -3′ and AMP_R 5′- AAG
ammonia production from arginine. MRS containing 0.3% GAG GTG ATC CAR CCG CA -3′ were used. PCR reaction
L-arginine hydrochloride was transferred into duplicate mixture was prepared by mixing 25 µl of the Tag 2x Master
International Journal of Microbiology 5

mix (buffer, polymerase and dNTPs), forward primer 1 µl, were found Gram-positive, endospore-negative, and cata-
reverse primer 1 µl, and UPH2O 22 µl. Then, 49 µl of the lase-negative.
mixtures was added to a sterile PCR tube, and 1 µl of the
gDNA was used as a template; the amplification reactions
were carried out in a thermal cycler (Bio-Rad Mycycler). 3.2. Morphological, Biochemical, and Physiological
Characterization. The 4 acid-bile-tolerant LAB isolates were
identified through their morphological, biochemical, and
2.6.3. DNA Electrophoresis. PCR products were separated in physiological features (Table 1). All the isolates were straight
a 1% agarose gel and stained with ethidium bromide fol- rod-shaped and able to grow at 4% NaCl salt concentration.
lowed by examination on a UV illuminator, and images were From the 4 acid-bile-tolerant LAB isolates, 3 of the isolates
captured by a digital camera. were found growing at 6.5% NaCl salt concentration. Testing
the abilities of isolates growing at 15°C and 45°C indicated
that 3 of the isolates were able to grow at both 15°C and 45°C
2.6.4. Sequencing of the PCR Products. A 16S rRNA PCR
(Table 1). On the contrary, isolate E031 was not found
amplification and sequencing was performed by Eurofins,
growing at 45°C.
Novogene (Hong Kong). The V4 hypervariable region of the
Out of the 4 acid-bile-tolerant isolates, 2 isolates (T035
16S rRNA was amplified using specific primers 515F and
and K011) produced gas from glucose (Table 1). Thus, the
806R. All the PCR reactions were carried out with Phusion
High-Fidelity PCR Master Mix (New England Biolabs). The
® four isolates were found to be equally divided into homo-
fermentative and heterofermentative types (50 : 50) (Ta-
libraries generated with TruSeq DNA PCR-Free Sample
ble 1). Regarding the arginine hydrolysis test, isolates E031,
Preparation Kit were sequenced using paired-end Illumina
T035, and K011 were found positive for arginine hydrolysis,
sequencing (2 × 250 bp) on the HiSeq2500 platform (Illu-
whereas E052 was not able to hydrolyze (Table 1).
mina, USA).

3.3. In vitro Characterization of Probiotic Properties

2.6.5. Phylogenetic Analysis. Forward and reverse sequences
were assembled and edited using BioEdit Sequence Align- 3.3.1. Tolerance to Low pH. Out of 56 isolates, 4 isolates
ment Editor Version 5.0.9. Sequence similarity was esti- (7.14%), 4 isolates (7.14%), and 9 isolates (16.07%) tolerated
mated by searching the homology in the Genbank DNA pH values of 2, 2.5, and 3 for 3 h, respectively (Table 2). Upon
database using BLAST. Finally, the isolate was then iden- further extension of the incubation period to 6 h, the 8
tified based upon the sequence. isolates (each 4 tested at pH 2.0 and 2.5) survived, whereas
The evolutionary history was inferred using the neigh- only 5 survived from 9 with the extension of incubation
bor-joining method [29]. The optimal tree with the sum of period to 6 h at pH 3.0 (Table 2).
branch length � 0.19972900 is shown. The percentage of Therefore, out of the total 56 LAB isolates, 4 (7.14%)
replicate trees in which the associated taxa clustered together isolates survived pH 2.0, 2.5, and 3.0 upon exposure for 3 and
in the bootstrap test (1000 replicates) is shown next to the 6 hours, and the mean value of the treatments was signifi-
branches. The tree is drawn to scale, with branch lengths in cantly different at p < 0.05 (Table 3). Among them, 1 (25%)
the same units as those of the evolutionary distances used to was isolated from each Teff dough and Kocho sample. And
infer the phylogenetic tree. The evolutionary distances were also, 2 (50%) LAB cultures were isolated from Ergo samples.
computed using the maximum composite likelihood method In general, the survival rate of the isolates was ranged from
and are in the units of the number of base substitutions per 38.40 to 97.11% at different pH values for 3 and 6 h in-
site. All positions containing gaps and missing data were cubation periods (Table 3). Isolates, like E052, K011, and
eliminated. There were a total of 891 positions in the final T035 were found highly tolerant and persisted above 50% for
dataset. Evolutionary analyses were conducted in MEGA7. both 3 and 6 hours. On the contrary, isolate K031 was not
able to grow above 50% at pH 2.0 upon exposure for 3 and
2.6.6. Statistical Analysis. All the measurements were per- 6 h (Table 3). Though the survival rate of the isolates was
formed in triplicate, and the results were expressed as mean markedly reduced at pH 2.0 for 6 h exposure, all the isolates
standard deviation (SD). Data were analyzed by the one-way were taken to the next experiments.
ANOVA plus post hoc Duncan’s test by SAS software (ver.
9.2, Raleigh, NC). The phylogenetic tree was prepared using 3.3.2. Tolerance to Bile Salts. All of the 4 LAB isolates (E052,
MEGA7 (version 7.0). Statistical significance was de- E031, K011 and T035) were able to survive above 90% in the
termined at p < 0.05. presence of 0.3% of bile salt (Table 3). Isolate E031 was the
most tolerant with 97.22% survival rate followed by isolates
3. Results E052 and K031 with 93.62% and 93.38% survival rates,
respectively. However, isolate T035 showed 91.37% survival
3.1. Isolation of Potentially Probiotic Lactic Acid Bacteria from rate (Table 3).
Traditional Fermented Foods. A total of 90 (30 from each
sample) lactic acid bacteria were isolated from three different
traditionally fermented Ethiopian food products (Teff 3.3.3. Antimicrobial Activities. The diameter of inhibition
dough, Ergo, and Kocho). Among them, 56 (62.22%) isolates zones showed that crude extracts from each isolate had
6 International Journal of Microbiology

Table 1: Physiological, morphological, and biochemical characteristics of the isolates.

Salt
Temperature
Spore concentration Production
Isolate KOH test Catalase test Shape (°C)
staining (%)
15 45 4 6.5 CO2 NH3
E052 − ve − ve Rod − + + + − − −
E031 − ve − ve Rod − + − + + − +
T035 − ve − ve Rod − + + + + + +
K011 − ve − ve Rod − + + + + + +

Table 2: Acid tolerance patterns of probiotic LAB at different pH values after 3 and 6 h exposure.
No. of survived isolates (%)
Source No. of isolates 3h 6h
pH 2 pH 2.5 pH 3 pH 2 pH 2.5 pH 3
Ergo 21 2 (9.5%) 2 (9.5%) 4 (19.1%) 2 (9.5%) 2 (9.5%) 3 (14.3%)
Teff dough 19 1 (5.3%) 1 (5.3%) 3 (15.8%) 1 (5.3%) 1 (5.3%) 1 (5.3%)
Kocho 16 1 (6.3%) 1 (6.3%) 2 (12.5%) 1 (6.3%) 1 (6.3%) 1 (6.3%)
Total 56 4 (7.14%) 4 (7.14%) 9 (16.1%) 4 (7.14%) 4 (7.14%) 5 (8.93%)

Table 3: Percentage survival of probiotic LAB at different pH levels and 0.3% bile salt.
pH tolerance Bile tolerance
Isolates 3h 6h 24 h
pH 2 pH 2.5 pH 3 pH 2 pH 2.5 pH 3 0.3%
E052 57.36 ± 1.63c 80.31 ± 1.17b 93.02 ± 1.40bc 54.68 ± 1.63a 65.58 ± 1.68b 88.68 ± 1.88a 93.62 ± 2.19ab
E031 45.35 ± 1.08d 79.39 ± 1.59b 94.00 ± 1.62ab 38.40 ± 1.24b 71.50 ± 1.08a 83.39 ± 1.27b 97.22 ± 0.35a
K011 73.29 ± 1.44a 90.13 ± 1.10a 97.11 ± 2.17a 49.34 ± 1.27a 70.52 ± 1.99a 90.49 ± 1.46a 93.38 ± 0.70b
T035 60.15 ± 0.74b 77.98 ± 1.19b 90.28 ± 2.03c 51.41 ± 0.77a 70.11 ± 1.11a 83.89 ± 0.93b 91.37 ± 2.66b
a–d
Means within a column with different superscripts are significantly different (p < 0.05). Results expressed as average (n � 3) ± SD (standard deviation).

antimicrobial effect against each tested food-borne pathogen 3.3.6. Identification of Probiotic LAB Isolates by 16S rRNA
(Table 4). The average zones of inhibition by which the crude Gene Sequencing. The 16S rRNA gene sequences of the 4
extracts inhibited the growth of the test food-borne path- LAB isolates with the best potential probiotic properties
ogens were found ranging between 17 to 21 mm. Isolate showed the highest homology to the known species of
T035 displayed highest antagonistic activity against Staph- bacteria in the database (Figure 1). Accordingly, E052
ylococcus aureus ATCC 25923, Listeria monocytogenes showed 99% match with Lactobacillus plantarum strain JCM
(clinical isolate), E. coli ATCC 25922, and Salmonella 1149, T035 showed 99% homology with Lactobacillus
enterica Typhimurium with the inhibition zone ranged from plantarum strain CIP 103151, K011 showed 95% similarity
19.33 to 21 mm in diameters. However, E052 showed a with Lactobacillus paracasei subsp. tolerans strain NBRC
minimum inhibition zone of diameter ranging from 17 to 15906, and E031 showed 99% homology with Lactobacillus
19 mm against the indicator microorganisms (Table 4). paracasei strain NBRC 15889 (Figure 1).

3.3.4. Antibiotic Susceptibility Test. The adherence ability of 4. Discussion

the potential probiotic LAB isolates was found ranging
The study of probiotic activities, for application in preser-
between 32.75 and 36.30% (Table 4). Isolate K011 showed
vation of food products and human health, remains to be of
the highest (36.30%) adherence rate followed by isolates
great interest. Currently, interest in antagonistic feature of
T035 and E052 with 33.48% and 33.17% adherence rates,
probiotic LAB against food-borne pathogens has indicated
respectively. However, isolate E031 showed the least
their potential to be likely alternatives to chemical drugs
(32.75%) adherence rate (Table 4).
[30]. Therefore, a significant effort has been made to select
lactic acid bacteria originating from the traditional Ethio-
3.3.5. Bacterial Adhesion to Stainless Steel Plates. The an- pian fermented food products on the basis of the most
tibiotic susceptibility test of the selected LAB isolates to some important technological, functional, and safety criteria in
common antibiotics showed sensitivity to tetracycline, order to obtain potentially probiotic lactic acid bacteria.
ampicillin, and erythromycin. However, all the 4 potential All the selected four potential probiotic bacteria were
probiotic LAB isolates displayed resistance to kanamycin identified as LAB based on their morphological, bio-
and streptomycin (Table 5). chemical, and physiological characteristics that were found
International Journal of Microbiology 7

Table 4: Antimicrobial activities of LAB against some food-borne pathogens and adhesion of LAB to stainless steel plate.
Diameter of inhibition zone (mm)
Isolates Adherence (%)
L. monocytogenes S. aureus E. coli S. typhimurium
E052 19.00 ± 1.00b 17.00 ± 1.00b 17.33 ± 0.58b 18.67 ± 0.58ab 33.17 ± 1.45b
E031 19.67 ± 0.58ab 20.33 ± 0.58a 20.00 ± 1.00a 18.33 ± 0.58b 32.75 ± 2.11b
T035 20.67 ± 0.58a 21.00 ± 1.00a 19.33 ± 1.15a 20.00 ± 1.00a 33.48 ± 1.05b
K011 19.67 ± 0.58ab 20.33 ± 1.15a 20.67 ± 0.58a 19.67 ± 0.58ab 36.30 ± 1.36a
a,b
Means within a column with different superscripts are significantly different (p < 0.05). Results expressed as average (n � 3) ± SD (standard deviation).

Table 5: Antibiotic susceptibility profile of probiotic LAB isolates.

Diameter of inhibition zone
Isolates
Kanamycin Streptomycin Tetracycline Ampicillin Erythromycin
E052 R R S S S
E031 R R S S S
T035 R R S S S
K011 R R S S S
Zone of inhibition (diameter in mm) for each antibiotic was measured and expressed as susceptible, S (≥21 mm); intermediate, I (16–20 mm); and resistance,
R (≤15 mm).

Lactobacillus paracasei strain NBRC 15889

Lactobacillus paracasei subsp. tolerans strain NBRC 15906

Lactobacillus plantarum strain CIP 103151

100

Lactobacillus plantarum strain JCM 1149

0.020
Figure 1: Evolutionary relationships of the isolation to the known strains.

to be equally divided into homofermentative and hetero- plantarum OL12, L. plantarum OL9, L. plantarum OL15,
fermentative types. This finding is in accordance with Mamo and L. plantarum OL33 isolated from fermented olives
et al. [31] who isolated Lactobacillus species from Ergo and showed survival percentages of 55%, 49%, 65% and 57%,
found that they were grouped as homofermentative and respectively, when exposed to pH 2.0 for 2 h. However, these
heterofermentative types. Akalu et al. [17] have also reported results are not in accordance with those reported by Akalu
that the probiotic LAB isolated from Shamita and Kocho et al. [17] and Rajoka et al. [34] who have indicated that
were identified morphologically, biochemically, and physi- most strains of Lb. plantarum isolated from different
ologically and comprised of both heterofermentative and sources showed a survival rate above 80% at pH 2 for 3 h.
homofermentative types. Azadnia and Khan Nazer [32] Other reports revealed that 5 acid-tolerant Lactobacillus
reported that the lactic acid bacteria isolated from traditional strains showed above 89% survival rate after exposure to pH
drinking yoghurt in tribes of Fars Province were able to grow 2 for 3 h [35]. Akalu et al. [17] verified that 14/17 Lacto-
at 4% NaCl concentration but not at 6.5% NaCl bacillus strains isolated from traditionally fermented Sha-
concentration. mita and Kocho were found tolerant to pH 2 when
In the present study, out of 56 probiotic LAB isolates incubated for 6 h with a survival rate of 74–89%. However,
tested to pH 2 for 3 and 6h, only 4 (7.14%) isolates showed incubation at low pH resulted in a significant decrease in the
tolerance to pH 2 for 6 h (Table 1). Thus, the tolerance of the survival rate of all LAB isolates as noted in another study by
four Lactobacillus spp. to pH 2.0 for 6 h showed a survival Guo et al. [36]. The authors noted that the viable counts of
rate of 38.40 to 73.29%. Similar to this study, Mourad and all lactic acid bacteria were significantly affected by low
Nour-Eddine [33] have demonstrated that Lactobacillus acidity, especially at pH 2.
8 International Journal of Microbiology

In this study, out of the 56 probiotic LAB isolates, only 4 demonstrated a low level of tolerance to bile salts by dis-
(7.14%) of the isolates were tolerant to pH 2.5 for 3 and 6 h playing surviving percentage less than 50% when exposed to
(Table 1). Similarly, out of 56 LAB isolates, 9 isolates 0.3% bile salts after 24 h at 37°C.
(16.1%) and 5 isolates (8.93%) survived at pH 3 for 3 and The selected four potential probiotic lactic acid bacterial
6 h, respectively. Thus, the survival rate of the four Lac- strains (Lactobacillus plantarum strain JCM 1149, Lacto-
tobacillus strains at pH 2.5 and 3 for 3 and 6 h was found to bacillus plantarum strain CIP 103151, Lactobacillus para-
be tolerant with a different survival rate (77.98–97.11%) casei subsp. tolerans strain NBRC 15906, and Lactobacillus
and (65.58–90.49%), respectively (Table 2). Similarly, paracasei strain NBRC 15889) exhibited varying degree of
Akalu et al. [17] have reported that out of 30 LAB isolated antagonism against Staphylococcus aureus, Listeria mono-
from traditionally fermented Ethiopian beverage and food cytogenes, Salmonella Typimurium, and Escherichia coli
(Shamita and Kocho), 17 lactobacilli showed 82 to 97% and (Table 4). According to Handa (2012), isolates having
81 to 91% survival rate at pH 2.5 and 3 for 3 and 6 h, clearance zones ≤9 mm and ≥12 mm diameter against the
respectively. This is also similar to the report of Oh and test pathogens indicated poor and strong antimicrobial
Jung [35] who have shown a better survival rate (above activity, respectively. Accordingly, all the selected potential
88%) of Lactobacillus species in pH 2.5 and 3 for 3 h that probiotic LAB strains (n � 4) exhibited strong antimicro-
were isolated from Omegisool, a traditionally fermented bial activity against the food-borne pathogens, where T035
millet alcoholic beverage of North Korea. As reported by (Lactobacillus plantarum strain CIP 103151) displayed the
Haghshenas et al. [37], Lactobacillus strains isolated from highest antagonistic activity against Staphylococcus aureus,
Iranian fermented dairy products survived (71% to 76%) at Listeria monocytogenes, E. coli, and S. typhimurium with
pH 2.5 for 3 h. From the previous investigations, it has the inhibition zone ranged from 19.33 to 21 mm in
generally been accepted that an isolate with full tolerance to diameters.
pH 3.0 for 3 h can be considered as high-acid-resistant In accordance to the current study, Bassyouni et al. [42]
strain with promising probiotic properties [36, 38]. Very have demonstrated that all of the Lactobacillus isolates
recently, Tang et al. [39] have reported that all the 9 obtained from Egyptian dairy product had a strong anti-
Lactobacillus plantarum strains recovered from the faeces bacterial effect against E. coli and Salmonella typhimurium.
of breast-feeding piglets were found to be highly tolerant to However, among the results that were revealed by the same
pH 3 for 3 h. author, 3 isolates had the most potent antimicrobial activity
The present results are in contrast with those of Mamo against the tested pathogenic microorganisms with the
et al. [31] who have found low to high survival rates inhibition zone ranged from 17 to 21 mm in diameters. In
(1.03–100%) for the six Lactobacillus species at pH 2.5 and agreement to this study, Tadesse et al. [5] have verified that
3.0 for 3 h. The same authors indicated that the maximum all the LAB isolates (n � 118) originated from Borde and
survival rate of the six strains were 22.5% at pH 3 for 6 h. Shamita belonging to the genera Lactobacillus, Lactococcus,
Nevertheless, complete loss of viability of lactobacilli isolated Leuconostoc, and Streptococcus were found to inhibit the
from traditional Ethiopian ergo was recorded at pH 2.5 for growth of the test strains such as S. aureus, Salmonella spp.,
6 h [31]. In addition, the current survival rate was higher and E. coli O157 : H7 with inhibition zones that ranged
than that of previously reported strains such as Lactobacillus from 15 to 17 mm in diameters. In line with this, Choi et al.
plantarum at pH 3 for 2 and 6 h exposure [33]. The 4 acid- [43] have reported that out of the 4 strains of LAB, the
tolerant LAB isolates showed high tolerance to bile salt Lactobacillus strain has completely inhibited the growth of
conditions (91.37% to 97.22%) (Table 3). Bile salt tolerance is food-borne pathogens, E. coli O157 : H7 ATCC 35150,
also considered as an important selection criterion for Salmonella enteritidis KCCM 12021, Salmonella typhimu-
probiotic isolates in order to survive the conditions in the rium KCTC 1925, and S. aureus. Tigu et al. [16] have also
small intestine. Moreover, tolerance to a high bile salt revealed that out of the 11 probiotic LAB isolated from
condition is also straining specific as demonstrated earlier traditional Ethiopian fermented condiments, namely,
where different Lactobacillus species isolated from Omegi- Datta and Awaze, 2 Lactobacillus isolates inhibited the
sool, a traditionally fermented millet alcoholic beverage in growth of Salmonella typimurium and Escherichia coli with
Korea showed considerable tolerance to bile salt [35]. Similar inhibition zones ranging from 10.3 to 14.3 mm in di-
to the present findings, the results in other studies have ameters. In line with this, Haghshenas et al. [37] have
revealed that all the isolated strains displayed high tolerance reported that among the selected 8 LAB isolated from
to bile salt conditions and the survival rates of Lactobacillus Iranian fermented dairy products, Lactobacillus species,
strains ranged from 88% to 92% [37]. In a related study, particularly Lb. plantarum 15HN, showed the most efficient
Akalu et al. [17] have also shown that out of the 30 tested antagonistic activity against Staphylococcus aureus, Listeria
LAB isolates, 17 Lactobacillus isolates obtained from Ethi- monocytogenes, Salmonella typimurium, and Escherichia
opian traditionally fermented Shamita and Kocho showed coli with inhibition zones of 11.7, 13.7, 12.3, and 12.3 mm
remarkably high tolerance to an environment containing diameters, respectively. Likewise, Rajoka et al. [34] have
0.3% bile salt. On the contrary, Boke et al. [40] have in- verified that all the Lactobacillus rhamnosus isolated from
dicated that Lactobacillus strains B3, G12, A13, and 22 human milk inhibited the growth of Staphylococcus aureus,
exhibited low level of tolerance to 0.3% bile salts with Salmonella typimurium, and Escherichia coli using the agar-
survival rates of 36%, 33%, 3%, and 3%, respectively. In line well diffusion method with variable diameters (6 mm to
with this, Handa [41] has reported that all of the LAB isolates 14 mm).
International Journal of Microbiology 9

Disparity in the antagonism activity against different 5. Conclusion

pathogens indicates that probiotic strains are highly path-
ogen-specific and a prerequisite for probiotic potential. In In the present study, four Lactobacillus isolates from Ergo,
general, the antimicrobial activity of probiotics might be Kocho, and Teff dough were found to have potentially
caused by the production of antimicrobial compounds such probiotic characteristics. It is suggested that these strains can
as organic acids, ethanol, carbon dioxide, hydrogen per- be good candidates for food industries as prospective pro-
oxide, short-chain fatty acids, and bacteriocins. Therefore, biotic cultures with other human health benefits. However,
by producing these antimicrobial compounds, probiotic further research work is needed to evaluate the in vivo
microorganisms gain an advantage over other microor- probiotic characteristics of these potential lactic acid
ganisms to survive in the adverse conditions of the gas- bacteria.
trointestinal tract [41].
All of the tested four Lactobacillus strains were found to Data Availability
be resistant to streptomycin and kanamycin but sensitive
towards tetracycline, ampicillin, and erythromycin (Table 5). The data used to support the findings of this study are in-
These results were found in agreement with the results that cluded within the article and are available (the SAS data)
were obtained using Lactobacillus species [34]. Tigu et al. from the corresponding author upon request.
[16] have also reported that all of the LAB isolates obtained
from traditional fermented condiments such as Datta and Conflicts of Interest
Awaze were susceptible to ampicillin, erythromycin, and
The authors declare that they have no conflicts of interest.
tetracycline. Similarly, according to Amraii et al. [44], all of
the selected LAB isolates were sensitive towards ampicillin,
erythromycin, and tetracycline. On the contrary, Sukmarini Acknowledgments
et al. [45] have reported that out of 120 isolates of LAB from The authors acknowledge Microbial, Cellular and Molecular
four different Indonesian traditional fermented foods, 16 Biology Department of Addis Ababa University and De-
isolates were resistant to erythromycin. In line with this, Pan partment of Food and Environmental Sciences, Helsinki
et al. [46] observed that among the 12 Lactobacillus species University, Finland, for provision of all laboratory facilities,
obtained from Chinese fermented foods, 5 isolates were Department of Biology, College of Natural and Computa-
sensitive to kanamycin, 7 resistant to erythromycin, 9 re- tional Sciences, Aksum University, Ethiopia, for financial
sistant to ampicillin, and 8 isolates resistant to tetracycline. support, and Ethiopian Public Health Institute (EPHI),
Among the main important characteristics of probiotic Addis Ababa, Ethiopia, for providing test organisms. The
bacteria, adhesion to the intestinal mucosa is required. The authors are also thankful to Dr. Wesley Morovic, Scientist,
current results showed that the screened probiotic LAB Strain Discovery and Microbiome Science, DuPont Nutri-
isolates possessed in vitro adherence property to stainless tion and Biosciences, Finland, for his support in 16S rDNA
steel plates with the adhesion rate ranging from 32.75 to analysis.
36.30% (Table 4). In agreement with this study, El-Jeni et al.
[27] have reported that the adhesion rate of lactic acid
bacteria to stainless steel plates ranged from 32 to 35%.
Supplementary Materials
Winkelströter et al. [47] have also revealed that pure culture Three (Teff dough, Ergo, and Kocho) traditionally fermented
of Lb. sakei ATCC 15521 showed strong adherence to Ethiopian food products were used as the isolation source.
stainless steel surface. Generally, this suggests that our The isolates were designated with E for Ergo, T for Teff
potential probiotic LAB isolates may have a potential ca- dough, and K for Kocho, followed with different numbers. In
pacity to colonize the gastrointestinal (GI) tract mucosa. general, the abovementioned traditionally fermented food
The phylogenetic analysis and the 16S rDNA sequencing products are prepared at the household level and their
assigned all the LAB isolates with probiotic properties to consumption is commonly practiced throughout the
genus Lactobacillus, and the identified species were Lacto- country. (Supplementary Materials)
bacillus plantarum strain JCM 1149, Lactobacillus paracasei
strain NBRC 15889, Lactobacillus plantarum strain CIP References
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