See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/269093345
The Use of Lactic Acid Bacteria Starter Culture in the Production of Nunu, a
Spontaneously Fermented Milk Product in Ghana
Article  in  International Journal of Food Science & Technology · February 2014
DOI: 10.1155/2014/721067
CITATIONS READS
17 256
5 authors, including:
Fortune Owusu-Kwarteng James Owusu-Kwarteng
University for Development Studies University of Energy and Natural Resources
32 PUBLICATIONS   149 CITATIONS    21 PUBLICATIONS   247 CITATIONS   
SEE PROFILE SEE PROFILE
Kwaku Tano-Debrah Charles Parkouda
University of Ghana Centre National de Recherche Scientifique et Technologique
44 PUBLICATIONS   950 CITATIONS    96 PUBLICATIONS   437 CITATIONS   
SEE PROFILE SEE PROFILE
Some of the authors of this publication are also working on these related projects:
academic View project
Selection and assessment of the combination between waste of tropical fruits and probiotic strains in the modulation of intestinal microbiota of obese compared to
the normal weight using the simulator of the human intestinal microbial ecosystem View project
All content following this page was uploaded by James Owusu-Kwarteng on 03 December 2014.
The user has requested enhancement of the downloaded file.
Hindawi Publishing Corporation
International Journal of Food Science
Volume 2014, Article ID 721067, 11 pages
http://dx.doi.org/10.1155/2014/721067
Research Article
The Use of Lactic Acid Bacteria Starter Culture in
the Production of Nunu, a Spontaneously Fermented
Milk Product in Ghana
Fortune Akabanda,1,2 James Owusu-Kwarteng,2 Kwaku Tano-Debrah,1
Charles Parkouda,3 and Lene Jespersen4
1 Department of Nutrition and Food Science, University of Ghana, P.O. Box LG 25, Legon, Ghana
2Department of Applied Biology, Faculty of Applied Sciences, University for Development Studies, Navrongo Campus,
P.O. Box 24, Navrongo, Ghana
3 Food Technology Department (DTA/IRSAT/CNRST), BP 7074, Ouagadougou 03, Burkina Faso
4Department of Food Science, Food Microbiology, Faculty of Science, University of Copenhagen, Rolighedsvej 30,
1958 Frederiksberg C, Denmark
Correspondence should be addressed to Fortune Akabanda; fakabanda@gmail.com
Received 16 July 2014; Revised 10 November 2014; Accepted 10 November 2014; Published 2 December 2014
Academic Editor: Rosana G. Moreira
Copyright © 2014 Fortune Akabanda 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.
Nunu, a spontaneously fermented yoghurt-like product, is produced and consumed in parts of West Africa. A total of 373
predominant lactic acid bacteria (LAB) previously isolated and identified from Nunu product were assessed in vitro for
their technological properties (acidification, exopolysaccharides production, lipolysis, proteolysis and antimicrobial activities).
Following the determination of technological properties, Lactobacillus fermentum 22-16, Lactobacillus plantarum 8-2, Lactobacillus
helveticus 22-7, and Leuconostocmesenteroides 14-11 were used as single and combined starter cultures forNunu fermentation. Starter
culture fermented Nunu samples were assessed for amino acids profile and rate of acidification and were subsequently evaluated
for consumer acceptability. For acidification properties, 82%, 59%, 34%, and 20% of strains belonging to Lactobacillus helveticus, L.
plantarum, L. fermentum, and Leu. mesenteriodes, respectively, demonstrated fast acidification properties. High proteolytic activity
(>100 to 150𝜇g/mL) was observed for 50% Leu. mesenteroides, 40% L. fermentum, 41% L. helveticus, 27% L. plantarum, and 10%
Ent. faecium species. In starter culture fermentedNunu samples, all amino acids determined were detected inNunu fermented with
single starters of L. plantarum and L. helveticus and combined starter of L. fermntum and L. helveticus. Consumer sensory analysis
showed varying degrees of acceptability for Nunu fermented with the different starter cultures.
1. Introduction Africa, the production of Nunu is largely home-based and
the fermentation is spontaneous. Thus, starter cultures are
Nunu is a spontaneously fermented milk (yoghurt-like) pro- not available, but old stocks of previous ferments and fer-
duct in Ghana and other parts of West Africa including mentation containers are used to initiate fermentation in new
Nigeria and Burkina Faso. Unlike other African fermented batches. The dependence on such undefined and diverse
milk products where milk of goats, sheep, and camels is microbial consortium during Nunu fermentation may result
used, Nunu is solely prepared from cow milk. The traditional in product of variable quality and stability.
processing of Nunu involves collecting fresh cow milk into Currently, there is no information on the use of starter
containers and then allowing it to ferment for a day or two cultures for Nunu fermentation. However, few investigations
days at ambient temperature. Nunu is yoghurt-like in taste have been carried out on themicrobiology of Ghanaian tradi-
(a sharp acid taste) and it can be taken alone or with Fura tionally fermented milk products [2–4]. The predominant
[1, 2]. Like many other spontaneously fermented foods in microorganisms isolated from this traditionally fermented
2 International Journal of Food Science
milk should be developed into starter cultures that could as the difference between proteolytic activities in fermented
be used to produce fermented milk products of consistent milk to that of untreated milk.
quality and consumer acceptability. Thus, it should be pos-
sible to improve the quality and consumer acceptability of ∘
Nunu through controlled fermentation using starter culture. 2.1.3. Lipolytic Activity. Strains were grown overnight at 37 C
The culture should, however, be well-defined. Such starter in MRS broth. A loopful of fresh culture was placed on trib-∘
cultures must be developed with a clear understanding of the utyrin agar [7]. Plates were incubated at 37 C for 4 days and
ecology of the microbial species associated with the desirable observed daily for halo formation around the colonies. The
traditional fermentation process, and their contributions to radius of the halo formation (inmm) at the end of incubation
the products safety and quality are determined.The first stage was measured.
in designing such starter culture(s) is to characterize and
identify the technologically important microorganisms asso- 2.1.4. Antimicrobial Activities of LAB
ciated with the traditional fermentation of the product and
then to test the use of the identified organisms in fermenta- (1) Indicator Strains. The indicator strains included Bacillus
tion trials. cereus PA24, Staphylococcus aureus ATCC 19095, Escherichia
The objective of the present study was therefore to eval- coliO157:H7,Listeriamonocytogenes ScottA, Salmonella typhi
uate the technological potential of lactic acid bacteria isol- ATCC 13311, and Pseudomonas aeruginosa BFE 162. Bacillus
ated from spontaneously fermented Nunu in view of their cereus PA24, Escherichia coli O157:H7, and Pseudomonas
application as starter cultures in Nunu production. aeruginosa BFE 162 were obtained from the Department of
Applied Biology, DANIDA Microbiology Laboratory of the
University for Development Studies, while Staphylococcus
2. Materials and Methods aureus ATCC 19095, Listeria monocytogenes Scott A, and
Salmonella typhiATCC 13311 were obtained from the Depart-
2.1. Determination of Technological Properties of LAB. A ment of Nutrition and Food Science of the University of
total of 373 LAB isolated and identified from spontaneously Ghana.
fermentedNunu inGhana using a combination of phenotypic
and genotypic characteristics [2] were assessed for their tech- (2) Preparation of Cell-Free Supernatant (CFS). Each LAB
nological properties.The LAB species included 174 L. fermen- isolate was inoculated in 10mL of MRS broth and incu-
tum, 44 L. plantarum, 40 Ent. faecium, 41 L. mesenteroides, 35 bated at 30∘C for 48 hrs. After incubation, a cell-free super-
L. helveticus, and 39 Ent. italicus. natant was obtained by centrifuging the bacterial culture at
6000×g for 15min followed by filtration of the supernatant
through 0.20𝜇mpore size syringe filters (Sartorius, Minisart,
2.1.1. Acidification Properties. Acidification properties of the Göttingen, Germany).
LAB were measured by the change in pH with time [5]. The
strains were initially grown in MRS broth and then in sterile (3) Screening for Antimicrobial Activities. The agar-well dif-
reconstituted skim milk supplemented with yeast extract fusion method was employed in the screening of LAB for
(0.3%) and glucose (0.2%) for two successive subcultures. antimicrobial activities. Indicator lawns were prepared by
Sterile reconstituted skim milk (100mL) was inoculated with inoculating 20mL of BHI molten agar media with 100 𝜇L
1% of a 24 h activated culture and pH changes were deter- (approximately 107 cfu/mL) of an overnight culture of each
mined using pH meters (Crison Basic, Barcelona) during indicator organism and allowing them to solidify in a Petri
incubation at 30∘C. Measurement of pH was carried out in dish.Wells were cut into the agarwith a sterile 6mmdiameter
triplicate at 2 h intervals for 24 h. The acidification rate was cork-borer and sealed with two drops of sterile agar. Fifty
calculated as ΔpH; ΔpH = pHat time − pHzero time. The cultures microliters (50 𝜇L) of the filtered cell-free supernatant of test
were considered as fast, medium, or slow acidifying when a strains was separately placed into the wells. The plates, pre-
ΔpH of 0.4U was achieved at 3, 3–5, and >5 h, respectively. pared in duplicate, were kept at 4∘C for 24 h [8] to allow
prediffusion of the CFS into the agar and then incubated at
∘
2.1.2. Proteolytic Activity. The proteolytic activity of the iso- 37 C for 24 h.Theywere then observed for possible clearing of
lates during fermentation of milk was measured by assessing zones (inhibition zones). The antimicrobial activity was det-
the free amino groups using the o-phthaldialdehyde (OPA) ermined by measuring the diameter of the inhibition zones
method [6]. Briefly, 3mL aliquots of the samples were mixed around the well using caliper in mm. Results were recorded
with 3mL of 1% (w/v) TCA (trichloroacetic acid) and filtered as no inhibition (−), weak inhibition (+), moderate inhibition
using a filter paper (Dublin, CA, USA). The filtrate was (++), and strong inhibition (+++) when the diameter is <1–
collected and approximately 150 L was added to 3mL of 4mm, >4–8mm, and >8–12mm, respectively.𝜇
OPA reagent. The mixture was held at room temperature
( ∘∼20 C) for 2 minutes and the absorbance of each solution 2.1.5. Isolation, Purification, and Quantification of EPS Pro-
was measured by using a spectrophotometer (Melbourne, duced by the LAB. The screening of the isolates for EPSs pro-
Australia) at 340 nm. The proteolytic activity of the bacterial ductionwas carried out according to themethoddescribed by
cultures was expressed as the absorbance of OPA derivatives Guiraud [9].The isolates cultured onMRS agar were streaked
at 340 nm. A relative degree of proteolysis was determined onto LTV agar (0.5% (w/v) tryptone (Merck), 1% (w/v)
International Journal of Food Science 3
Table 1: Starter cultures used for Nunu fermentation.
Type of fermentation Starter cultures Codes
Lactobacillus fermentum LF-22-16
Single starters cultures Lactobacillus plantarum LP-8-2
Lactobacillus helveticus LH-22-7
Leuconostoc mesenteroides LM-14-11
Lactobacillus fermentum + Lactobacillus plantarum LF-22-16 + LP-8-2
Lactobacillus fermentum + Lactobacillus helveticus LF-22-16 + LH-22-7
Combined starter cultures Lactobacillus fermentum + Leuconostoc mesenteroides LF-22-16 + LM-14-11
Lactobacillus plantarum + Lactobacillus helveticus LP-8-2 + LH-22-7
Lactobacillus plantarum + Leuconostoc mesenteroides LP-22-7 + LM-14-11
Lactobacillus helveticus + Leuconostoc mesenteroides LH-22-7 + LM-14-11
No starter culture was added Spontaneously fermented SP
meat extract (Merck), 0.65% (w/v) NaCl (Merck), 0.8% (w/v) exopolysaccharides, and the possession of antimicrobial
potassium nitrate (Merck), 0.8% (w/v) sucrose (Merck), 0.1% properties in milk.
(v/v) Tween 80 (Merck), and 1.7% (w/v) agar (Merck), pH7.1±
0.2) and incubated at 30∘C for 48 h. The sticky aspect of the 2.2.2. Preparation of Starter Cultures and Fermentation of
colonies was determined by testing them for slime formation Milk. TheLAB strains to be used as inocula were prepared by
using the inoculated loop method [10]. The isolates were transferring a loopful of an overnight culture fromMRS agar
considered positively slimy producer if the length of slime into 10mL MRS broth and incubated at 35∘C for 24 h. One
was above 1.5mm. hundred microliters of the 24 h old culture was transferred
The positive isolates were confirmed growing them on into 10mL MRS broth and incubated at 35∘C for 16 h (over-
MRS sucrose broth and incubating them at 30∘C for 24 h. A night). Subsequently, cells were harvested by centrifugation
volume of 1.5mL of the 24 h culture was centrifuged at 5000 g at 5000 g for 10min (4∘C); washed three times with 20mL
for 10min (4∘C) and 1mL of the supernatant was put in a glass sterile diluent (Merck), pH 7.2 ± 0.2; and finally suspended
tube and an equal volume of ethanol 95% was added. An in 10mL of sterile diluent, and these served as the isolate
opaque link formed at the interface of the tube indicates the inocula. Flasks containing 500mLof freshmilk pasteurized at
presence of EPSs. Samples were centrifuged 2500×g for 60∘C for 30min in a water bath were inoculated in duplicate
20min and the pellets were dried at 100∘C. The total carbo- at 35∘C for 24 h. Single and combined starter cultures used
hydrate content of the EPS was determined using the phenol- for the fermentation of milk to produce Nunu are shown in
sulfuric acid procedure of Dubois et al. [11]. Briefly, two Table 1.
milliliters of sample solution was pipetted into a colorimetric
tube, and 0.05mL of 80% phenol was added. Then 5mL of
concentrated sulfuric acid was added rapidly, with the stream 2.2.3. Rate of Acidification during Nunu Fermentation with
of acid directed against the liquid surface rather than against Starter Cultures. The pH of fermenting Nunu was deter-
the side of the test tube in order to obtain good mixing. The mined using a digital pH meter (Crison basic 20, Barcelona).
tubeswere then allowed to stand for 10minutes, and then they The pH meter was calibrated using standard buffer solutions
were shaken and placed for 10 to 20minutes in a water bath at andmeasurements were taken at ambient temperature of 30±∘
25 to 30 C before readings were taken.The absorbance of the 2 C. All measurements were carried out in triplicate and∘ ∘
characteristic yellow orange color was measured at 490 nm. means and standard deviations were determined.
Distilled water was used as control. The amount of EPS was
expressed in 𝜇g/mL. 2.2.4. Determination of Amino Acids Profile
(1) Preparation of Samples. Ten milliliters of Nunu samples
2.2. Starter Culture Fermentation of Milk with Selected LAB was defatted with distilled petroleum ether (Labscan, Dublin,
Ireland) in a Soxhlet apparatus and stored in screw-capped
2.2.1. Selection of LAB Strains. Four (4) strains of LAB were plastic tubes at 20∘− C until they were required. Preparation
selected and used as starter cultures during the fermentation of each sample was carried out in triplicate.
of milk to produceNunu.These include Lactobacillus fermen-
tum 22-16 (LF-22-16), Lactobacillus plantarum 8-2 (LP-8-2), (2) Analysis of Free-Amino Acids Profile.The free-amino acids
Lactobacillus helveticus 22-7 (LH-22-7), and Leuconostoc profile analysis of the samples was assayed according to
mesenteroides 14-11 (LM-14-11). The strains were selected Ojinnaka and Ojimelukwe, [12] with minor modifications.
based on their predominance in traditional spontaneous First of all, the sampleswere hydrolyzed byweighing 0.5 g into
Nunu fermentation as well as their desirable technological 20mL volumetric flask.The volumetric flaskwas then filled to
properties including faster rates of acidification, high pro- the mark with 0.1M hydrochloric acid and was then shaken
teolytic and low lipolytic activities, ability to produce thoroughly to mix. The content was left over night to extract;
4 International Journal of Food Science
1mL of the overnight sample was filtered through a 0.45 𝜇m 120
filter. Ten microliters of the filtrate was then placed in a
sample vial for drying and redrying. The hydrolyzed dried 100
samples were derivatized automatically on the Waters HPLC 80
by allowing the samples to react, under basic situations with 60
phenylisothiocyanate (i.e., PITC) to get phenylthiocarbamide
(PTC) amino acid derivatives. The duration for this reaction 40
was 45 minutes per sample, as calibrated on the instrument. 20
A set of standard solutions of the amino acids were prepared
from Pierce Reference standards H (5 𝜇L) into autosampler 0
cups and they were also derivatized.These standards (200𝜇L, −20
250𝜇L, 300 𝜇L, and 400 𝜇L) were used to generate a calibra-
tion file that was used to determine the amino acid contents
of the samples. After the derivatization, a methanol solution
(1.5N), containing the PTC amino acids, was transferred to
a narrow bore (Waters 600) HPLC system for separation. Lactic acid bacteria species 
The separation and identification of amino acids were done
in reverse phase C18 silica column and the analytes were
detected at the wavelength of 254 nm. The elution of the Fast
whole amino acids in the samples took 12 minutes.The buffer MediumSlow
system used for separation was 140mM sodium acetate pH
6.40 as bufferA and 80% acetonitrile as buffer B.Theprogram Figure 1: Acidification properties of predominant LAB species
was run using a gradient of buffer A and buffer B concen- isolated fromNunu: fast, medium, and slow, when a pH of 0.4Uwas
tration and ending with a 55% buffer B concentration at the achieved after 3 h, 6 h, and 24 h, respectively.
end of the gradient. The intensity of the chromatographic
peaks areas were automatically and digitally identified and
quantified using Empowers 2 software data analysis system products’ appearance using a 9-point hedonic scale ranging
which was attached to the Waters 600 HPLC System. The from “dislike extremely” to “like extremely.” After judging
calibration curve or file prepared from the average values appearance, the panelists were then allowed to taste the
of the retention times (in minutes) and areas (in Au) of the samples and evaluate other sensory properties using a 9-point
amino acids in 5 standards runs was used. Since a known hedonic scale, once again ranging from “dislike extremely” to
amount of each amino acid in the standard was loaded into “like extremely.” The judges were made to wash their mouth
the HPLC, a response factor (Au/pmol) was calculated by with water after evaluating each product.
Empowers 2 software that was interphased with the HPLC.
This response factor was used to calculate the amount of each
of the amino acid (in pmol) in the sample. The amount of 2.3. Statistical Analysis. All analyses were carried out in
each amino acid in the sample was finally calculated by the triplicate. Data obtained were subjected to one-way analysis
software by dividing the intensity of the peak area of each by of variance (ANOVA) and means were separated by Tukey’s
the internal standard in the chromatogram and multiplying family error rate multiple comparison test (𝑃 < 0.05) using
this by the total amount of internal standard added to the the MINITAB statistical software package (MINITAB Inc.
original sample. Release 14 for windows, 2004).
3. Results and Discussion
2.2.5. Consumer Sensory Evaluation of Nunu. Nunu products
prepared by fermentation with different starter cultures were 3.1. Technological Properties of LAB Isolated from Nunu
served to 35 volunteered untrained panelists (drawn from the
Faculty of Applied Sciences of the University for Develop- 3.1.1. Acidification Properties. Rapid acidification is a prior-
ment Studies and the Navrongo Community) who are famil- ity for development of starter cultures for fermented milk
iar with Nunu. The panel independently, in separate sensory products. The acidification properties of predominant LAB
evaluation booths, evaluated the various products for their isolated from spontaneously fermented Nunu are shown in
sensory qualities including taste, colour/appearance, odour, Figure 1. Generally, the rate of acidification varied among
texture, and overall acceptability, using a nine-point hedonic the isolates tested. Eighty-two percent (82%) of Lactobacillus
scale (1, 5, and 9 represent dislike extremely, neither like nor helveticus were acidifying fast. Additionally, 34% of L. fer-
dislike, and like extremely, resp.). All eleven Nunu products mentum, 59% of L. plantarum, and 20% of L. mesenteroides
were presented to the panelists randomly placed side-by-side, demonstrated fast acidification properties as well. Idoui and
with each panelist receiving 2 rounds of each product and Karam [13] indicated that L. plantarum and L. curvatus
water for rinsing. Spontaneously fermented Nunu (without isolated from Jijel’s traditional butter made from cows’ milk
added known starter culture) served as control sample. Before were the fastest acid producers. In a study by Haddadin [14],
tasting the products, panelists were asked to evaluate the L. plantarum was the fastest acid producing isolated strain.
LAB species showing acidification (%) 
L. fermentum
L. plantarum
Ent. faecium
L. mesenteroides 
L. helveticus
Ent. italicus
International Journal of Food Science 5
80 Table 2: Lipolytic activities of predominant LAB isolated from
70 Nunu.
60
50 LAB species
Zones of inhibition
No inhibition 1–3 cm >3–5 cm
40 L. fermentum (𝑛 = 174) 56 100 18
30 L. plantarum (𝑛 = 44) 4 28 12
20 Ent. faecium (𝑛 = 40) 9 24 7
10 L. mesenteroides (𝑛 = 41) 9 24 8
0 L. helveticus (𝑛 = 35) 3 21 11
−10 Ent. italicus (𝑛 = 39) 7 30 2
−20
𝑛: number of organisms used.
cheese. The contrast in proteolytic activities may be due to
Lactic acid bacteria species the strains associated with the products. Peterson et al. [20]
reported that important differences exist between species of
Good LAB in terms of the types and quantities of peptidase activ-
Fair ities. The proteolytic activity of dairy lactic acid bacteria is
Poor essential for the bacterial growth in milk and is involved in
the development of organoleptic properties of different fer-
Figure 2: Pattern of proteolytic activities of predominant LAB mentedmilk products [21, 22].The production of high quality
isolated from Nunu. Good => 100–150 𝜇g tyrosine/mL, fair => 50– fermented dairy products depends on proteolytic systems of
100𝜇g tyrosine/mL, and Poor = 0–50𝜇g tyrosine/mL. starter bacteria, since peptidase and amino acids formed have
a direct impact on flavor or serve as flavor precursors in these
products.
Sixteen percent (16%) of Ent. italicuswere fast acidifiers while
no strain of Ent. faecium showed fast acidification. These 3.1.3. Lipolytic Activities. Lipolytic activities were recognized
results are in agreement with Sarantinopoulos et al. [15], who by the presence of a clear halo in the tributyrin agar plates.
indicated that Enterococcus strains were poor acidifiers in Out of the 174 strains of L. fermentum screened for lipolytic
milk and Ent. faecium and Enterococcus faecalis have been activities, 32% were negative (showed no clear zones on the
reported to degrade lactose in milk slowly [16]. From the agar plates). Similarly, L. plantarum and L. helveticus had 9%
results obtained, a majority of strains showed fast rate of and 8.6% strains not showing lipolytic activities, respectively
acidification as they were able to produce a ΔpH of 0.4U (Table 2). Generally, majority of the strains tested for lipolysis
after 3 h. A rapid decrease in pH is essential for coagula- were positive.
tion and prevention or reduction of growth of adventitious Very few studies on lactic acid bacterial lipases have
microflora in yoghurt production. The fast acidifying strains been carried out. Tsakalidou et al. [23] concluded that even
are therefore good candidates for dairy fermentation process though LAB are weakly lipolytic, the enterococcal strains
as primary starter culture, while poor acidification strains can showed a significantly higher activity than the strains of most
be used as adjunct cultures depending on other properties [5]. other genera of LAB. Microbial lipases are used in the dairy
Generally, the desirable characteristics for industrial LAB or industry extensively for the hydrolysis ofmilk fat, and current
starter are the abilities to rapidly and completely convert applications include acceleration of cheese ripening and
the raw materials into lactic acid with minimal nutritional lipolysis of butter, fat, and cream [24]. The ability of LAB to
requirements. A rapid acidification of the raw material show lipolytic activity in vitro is very promising. It is assumed
prevents growth of undesirable microorganisms and is also that such activity can be manifested by the isolates in vivo
essential for aroma, texture, and flavor of the end-product which will lead to the reduction of cholesterol level inhumans
(Nunu). It has been observed that the faster the decrease in if used as a starter or an adjunct culture [25].
pH to <4, the faster the growth inhibition of the fermenting
medium against pathogens such as Salmonella spp. [17]. 3.1.4. Exopolysaccharide Production. As shown in Figure 3,
all the groups of LAB strains tested produced exopolysaccha-
3.1.2. Proteolytic Activities. In general most of the strains ride to some extent. Strains of L. helveticus (38%), L. fermen-
tested showed proteolytic activities to varying degrees tum (30%), L. plantarum (27%) and L. mesenteroides (18%)
(Figure 2). Forty percent (40%) of L. fermentum, 27% of L. demonstrated good (>100–150𝜇g/mL) exopolysaccharide
plantarum, 10% of Ent. faecium, 50% of L. mesenteroides, and production abilities. Most of the Ent. faecium (95%) and Ent.
41% of L. helveticus showed good (>100–150 𝜇g tyrosine/mL) italicus (85%) strains were poor producers, producing below
proteolytic activities. The result is in contrast with those of 50 𝜇g/mL of exopolysaccharides. Exopolysaccharide pro-
Durlu-Ozkaya et al. [18] and Dagdemir and Ozdemir [19] duction is a desirable feature of bacteria applied in dairy
who reported high proteolytic activity for LAB isolated from products because EPSs act as natural biothickener leading to
LAB showing proteolysis (%) 
L. fermentum
L. plantarum
Ent. faecium
L. mesenteroides 
L. helveticus
Ent. italicus
6 International Journal of Food Science
7
120
100 6
80
60
5
40
20
4
0
−20
3
0 2 4 6 8 10 12 14 16 18 20 22 24
Fermentation time (h)
Lactic acid bacteria species Sp Lf + Lp
Lf Lf + Lh
Lp Lf + Lm
Good Lh Lp + Lh
Fair Lm Lp + Lm
Poor
Figure 4: pH of fermenting milk with different starter cultures.
Figure 3: Exopolysaccharides activities of predominant LAB iso- Spontaneously fermented (Sp), L. fermentum (Lf), L. plantarum
lated from Nunu. Good => 100–150 𝜇g/mL, fair => 50–100 𝜇g/mL, (Lp), L. helveticus (Lh), and L. mesenteroides (Lm), L. fermentum +
and poor = 0–50 𝜇g/mL. L. plantarum (Lf + Lp), L. fermentum + L. helveticus (Lf + Lh), L.
fermentum+ L.mesenteroides (Lf + Lm), L. plantarum+ L. helveticus
(Lp + Lh), L. plantarum + L. mesenteroides (Lp + Lm), L. helveticus+
higher consistency and viscosity of the product and reduced L. mesenteroides (Lh + Lm).
syneresis [26]. However, most of them are chemically or
enzymatically modified in order to improve their rheolog-
ical properties (e.g., cellulose, starch, pectin, alginate, and of various food borne pathogens by cell-free filtrates of
carrageenan) and, therefore, their use is strongly restricted LAB. Afolabi et al. [39] showed that antimicrobial producing
for food applications. An alternative source of biopolymers microorganisms had the ability to inhibit the growth of other
is microbial EPS. The EPSs of microbial origin have unique bacteria which included both Gram-negative and Gram-
rheological properties because of their capability of form- positive bacteria. Such antimicrobial activities were also
ing very viscous solutions at low concentration and their demonstrated in the works of other researchers such as Ades-
pseudoplastic nature [27]. Some strains of LAB have been okan et al. [40] where LAB species were tested against Staphy-
reported to produce EPS and gain increasing attention over lococcus aureus, Pseudomonas aeruginosa, Candida albicans,
the last few years because of their contribution to the rheology Escherichia coli, and Proteus vulgaris. Raccah et al. [41],
and texture of fermented milk and food products [28]. Smith and Palumbo [42], and Cintas et al. [43] have demon-
Most of the LAB producing EPS belong to the genera strated that the antimicrobial compounds produced by LAB
Streptococcus, Lactobacillus, Lactococcus, Leuconostoc, and can inhibit the growth of pathogenic bacteria of possible con-
Pediococcus [29]. EPS-producing LABhave a greater ability to taminants in fermented products. The ability to inhibit other
withstand technological stresses [30] and survive the passage organisms is due to the fact that LAB produces substances
through the gastrointestinal tract compared to their non- which are injurious to the indicator organisms depending
producing bacteria. Additionally, EPS may induce positive on the concentration or quantity produced.These substances
physiological responses including lower cholesterol levels serve as competitive advantage to LABwhen inmixed culture
[31, 32], reduce formation of pathogenic biofilms [33] and especially during fermentation and hence the dominance of
modulation of adhesion to epithelial cells [34], and increase LAB during fermentation of milk, cereals and vegetables.
levels of bifidobacteria showing prebiotic potential [35, 36]. Wakil andOsamwonyi [44] indicated that LAB isolates show-
Hence, the choice of EPS-producing starter culture seems to ing antimicrobial activity were discovered to produce antimi-
give several advantages over nonproducing ones. crobial substances like lactic acid, hydrogen peroxide, and
diacetyl, showing that the ability to inhibit other organisms
3.1.5. Antimicrobial Activities. The antimicrobial properties was directly related to the ability of these organisms to pro-
of predominant LAB isolated from Nunu are shown in duce these substances. Daeschel [45] reported the ability of
Table 3.The LAB strains were able to inhibit the selected indi- LAB to produce lactic acid, thereby reducing the pH of the
cator organisms to varying degrees and Figure 4 illustrates fermenting medium. The lactic acid produced serves to
the zones of inhibition. Similar to our findings, Kivanç [37] reduce the pH of themedium, therebymaking it acidic which
and Tadesse et al. [38] observed varying degrees of inhibition is not conducive for the survival of spoilage bacteria which
LAB showing exopolysaccharide activity (%)
L. fermentum
L. plantarum
Ent. faecium
L. mesenteroides 
L. helveticus
Ent. italicus
pH values
International Journal of Food Science 7
Table 3: Antimicrobial activities of predominant LAB against selected pathogenic microorganisms.
∗
Number of LAB tested Range of inhibition of pathogens
B. cereus E. coli L. monocytogenes S. typhi Staph. aureus P. aeruginosa
8 (+) 4 (+) 6 (+) 2 (+) 6 (+) 4 (+)
L. fermentum (174) 2 (++) 0 (++) 2 (++) 0 (++) 2 (++) 0 (++)
0 (+++) 0 (+++) 0 (+++) 0 (+++) 0 (+++) 0 (+++)
164 (ND) 170 (ND) 166 (ND) 172 (ND) 166 (ND) 170 (ND)
18 (+) 4 (+) 6 (+) 2 (+) 6 (+) 2 (+)
L. plantarum (44) 2 (++) 2 (++) 4 (++) 2 (++) 2 (++) 2 (++)
0 (+++) 0 (+++) 2 (+++) 0 (+++) 2 (+++) 0 (+++)
24 (ND) 38 (ND) 32 (ND) 40 (ND) 34 (ND) 40 (ND)
6 (+) 6 (+) 6 (+) 8 (+) 8 (+) 6 (+)
Ent. faecium (40) 4 (++) 4 (++) 6 (++) 4 (++) 2 (++) 6 (++)
2 (+++) 0 (+++) 0 (+++) 0 (+++) 2 (+++) 0 (+++)
28 (ND) 30 (ND) 28 (ND) 28 (ND) 28 (ND) 28 (ND)
4 (+) 5 (+) 4 (+) 6 (+) 4 (+) 3 (+)
L. mesenteroides (41) 1 (++) 0 (++) 0 (++) 0 (++) 3 (++) 2 (++)
0 (+++) 0 (+++) 0 (+++) 0 (+++) 0 (+++) 0 (+++)
36 (ND) 36 (ND) 37 (ND) 35 (ND) 34 (ND) 36 (ND)
6 (+) 11 (+) 4 (+) 6 (+) 10 (+) 6 (+)
Ent. italicus (39) 3 (++) 0 (++) 5 (++) 0 (++) 3 (++) 4 (++)
0 (+++) 0 (+++) 0 (+++) 0 (+++) 0 (+++) 0 (+++)
30 (ND) 28 (ND) 26 (ND) 33 (ND) 26 (ND) 29 (ND)
5 (+) 6 (+) 2 (+) 4 (+) 4 (+) 2 (+)
L. helveticus (35) 0 (++) 3 (++) 2 (++) 3 (++) 1 (++) 3 (++)
0 (+++) 0 (+++) 3 (+++) 0 (+++) 0 (+++) 0 (+++)
30 (ND) 26 (ND) 28 (ND) 28 (ND) 30 (ND) 30 (ND)
∗(+) <1–4 cm, (++) >4–8 cm, (+++) >8–12 cm and (ND) = Not detected. B.: Bacillus, E.: Escherichia, L.: Listeria, S.: Salmonella, Staph.: Staphylococcus and P.:
Pseudomonas.
may have found their way into the fermenting substrate prevents growth of undesirable microorganisms and is also
during spontaneous fermentation. Lactic acid is a natural essential for aroma, texture, and flavor of the end-product.
preservative that inhibits putrefying bacteria and is responsi-
ble for the improvedmicrobiological stability and safety of the 3.2.2. AminoAcid Profile. Table 4 summarizes the amino acid
food.The acidity also leads to the souring of the final product profile of Nunu prepared with various single and combined
which is characteristic of fermented products. Hydrogen starter cultures.The results from this study show that the free
peroxide produced adds to the antimicrobial activity of LAB amino acid profiles of Nunu produced with different starter
and in some cases is a precursor for the production of other cultures varied comparatively andwere significantly different.
potent antimicrobial compounds such as super oxide (O2−) All amino acids determined were detected in samples pro-
and hydroxyl (OH−) radicals [46, 47]. duced by fermenting milk with single starter cultures of L.
plantarum (LP) and L. helveticus (LH) as well as the sample
produced with a combined starter culture of L. fermentum
3.2. Impact of the Use of Starter Culture on Quality and and L. helveticus (LF + LH). Lysine, methionine, isoleucine,
Consumer Acceptability of Nunu proline, glutamine, asparagine, alanine, and leucine were
detected in allNunu samples irrespective of the starter culture
3.2.1. Acidification of Nunu. Therates of acidification ofNunu used. Except serine, all amino acids determinedwere detected
prepared with different single and combined starter cultures in spontaneously fermented Nunu at varying concentrations.
are shown in Figure 4. Generally, there was a fast decrease Many studies have shown that concentrations of most of the
in acidity with time of the milk using the starter cultures. amino acids slightly increase due to fermentation. Muradyan
The starter cultures were able to reduce the pH from approxi- et al. [48] reported that fermentation of milk by thermophilic
mately 6.46 to about 3.72 after 12 hours of fermentation, thus lactic streptococci or acidophilic rods enriched the final
reducing the fermentation time. Lf, Lf + Lp, and Lf + Lh were products with at least 4 amino acids (cysteine, valine, proline,
the fast acidifiers. and arginine). An obligatory dietary requirement exists
The rate of acid development is a critical factor in milk for tryptophan, leucine, isoleucine, valine, phenylalanine,
fermentation. The rapid acidification of the raw material methionine, lysine, threonine, and histidine. The last three
8 International Journal of Food Science
Table 4: Amino acids profile (mg/L) of Nunu prepared with various single and combined starter culture(s) of LAB.
Amino acids Starter culturesSp Lf Lp Lh Lm Lf + Lp Lf + Lh Lf + Lm Lp + Lh Lp + Lm Lh + Lm
Lysine∗ 2.53 ± 0.02h 4.60 ± 0.06a 3.80 ± 0.02b 4.65 a f e± 0.02 1.91 ± 0.01 2.43 ± 0.01 3.85 ± 0.01b 2.63 ± 0.02d 3.53 ± 0.06c 2.65 0.02d± 2.12 g± 0.01
Argentine 3.15 ± 0.01a 6.88 0.09b± 3.44 c± 0.01 4.32 ± 0.01d 1.93 0.01e 4.94 0.02f± ± 3.51 g± 0.01 ND 3.63 ± 0.01h 1.55 ± 0.01i ND
Histidine∗ 2.93 0.06a± 3.85 ± 0.09d 4.11 ± 0.01e 3.83 ± 0.03d 3.57 c± 0.03 3.00 ± 0.00b 2.96 ± 0.01b ND 3.97 ± 0.06f 3.10 0.10b± ND
Methionine∗ 1.75 a e f c b g h b i d d± 0.02 4.58 ± 0.02 3.99 ± 0.01 2.13 ± 0.02 1.88 ± 0.01 4.88 ± 0.01 5.73 ± 0.02 1.88 ± 0.01 6.77 ± 0.01 2.24 ± 0.02 2.24 ± 0.02
Isoleucine∗ 2.99 ± 0.01a 5.80 ± 0.11b 2.11 ± 0.01c 6.51 ± 0.02e 3.49 ± 0.01f 4.22 g a a h i j± 0.01 2.86 ± 0.01 2.95 ± 0.00 7.10 ± 0.10 1.16 ± 0.01 2.57 ± 0.02
Tryptophan 3.13 ± 0.01a 2.34 ± 0.02b 3.42 ± 0.02c 3.95 0.01d± 2.01 e± 0.01 4.86 ± 0.01f 2.51 ± 0.01g 3.66 ± 0.01h 5.27 0.06i± ND ND
Threonine∗ 3.33 ± 0.02a 3.21 0.01b± 2.82 c± 0.01 4.07 ± 0.15d ND 4.53 ± 0.01e 2.52 f a g± 0.01 3.32 ± 0.01 ND 1.46 ± 0.01 ND
Proline 2.63 ± 0.01a 2.68 ± 0.02a 3.77 ± 0.01c 4.51 0.02d± 0.80 e± 0.01 4.67 ± 0.01b 3.44 ± 0.02f 4.64 ± 0.01b 4.75 ± 0.04b 1.90 0.02g± 2.79 h± 0.02
Glutamine 4.02 ± 0.01a 4.22 ± 0.01g 2.68 0.00d± 7.82 i± 0.02 0.98 ± 0.01b 3.80 ± 0.01f 3.77 ± 0.02f 2.68 ± 0.01d 5.47 0.12h± 3.14 e± 0.02 1.97 ± 0.01c
Asparagine 3.21 ± 0.01f 2.45 ± 0.01c 2.55 ± 0.01d 4.70 0.01g± 1.58 a± 0.02 2.95 ± 0.01e 3.22 ± 0.01f 1.53 ± 0.01a 6.41 h d b± 0.01 2.61 ± 0.01 1.99 ± 0.01
Glutamic acid 3.14 f± 0.01 2.65 ± 0.02d 3.24 ± 0.05f 4.50 ± 0.01g 2.92 ± 0.02e ND 2.45 ± 0.02c 2.09 ± 0.01a 7.10 ± 0.01h 3.18 0.01f± 2.39 c± 0.02
Valine∗ 2.81 c± 0.02 ND 4.11 0.01e± 4.21 f± 0.01 4.87 ± 0.02g 2.47 ± 0.01b 2.46 ± 0.02b 2.96 ± 0.01d ND 2.16 ± 0.01a 2.95 ± 0.01d
Phenylamine∗ 1.18 0.01a 3.83 0.02e 4.07 0.16f± ± ± 3.62 d± 0.01 ND 4.74 ± 0.01h 1.94 ± 0.02b ND 4.45 ± 0.01g 3.96 ± 0.01f 2.92 ± 0.02c
Aspartic acid 1.25 ± 0.01a 3.54 ± 0.06d 3.71 0.02e± 4.07 f± 0.01 ND ND 2.81 ± 0.02b 3.27 ± 0.01c 4.45 0.01g± ND ND
Serine ND ND 2.66 ± 0.02a 4.74 ± 0.01c ND ND 6.33 ± 0.01b 3.28 0.01c± 5.32 ± 0.01g ND ND
Glycine 2.14 b h e e c± 0.02 4.23 ± 0.01 3.24 ± 0.02 3.22 ± 0.01 2.51 ± 0.01 ND 4.07 g± 0.01 3.42 ± 0.01f 5.57 0.01i± 3.12 d a± 0.01 1.77 ± 0.02
Tyrosine 1.16 ± 0.01a 2.78 ± 0.02d 5.34 h e f d g i c b± 0.02 3.03 ± 0.01 ND 3.70 ± 0.01 2.71 ± 0.01 4.22 ± 0.00 6.00 ± 0.10 2.16 ± 0.01 1.82 ± 0.02
Alanine 1.44 ± 0.01a 3.75 0.05h 7.96 0.01j 2.63 0.02e 2.47 0.01d± ± ± ± 3.70 ± 0.01h 3.10 ± 0.00g 2.43 c± 0.01 4.32 ± 0.01i 2.77 ± 0.02c 2.15 ± 0.02b
Cystine 2.10 ± 0.01b 2.81 ± 0.01e 2.70 0.01d± 5.60 g± 0.01 ND ND 6.18 ± 0.02h 2.41 ± 0.01c 4.51 f± 0.06 2.65 ± 0.02d 1.59 ± 0.01a
Leucine∗ 1.25 a b c d± 0.01 2.61 ± 0.02 3.92 ± 0.01 5.98 ± 0.01 0.76 ± 0.01e 5.77 0.01f± 4.56 g± 0.01 4.86 ± 0.02h 6.21 ± 0.01i 1.76 ± 0.01j 1.19 0.01k±
∗Essential amino acids. Values are means of three experiments; ND: not detected; ±: standard deviation. Values in the same row with different letters differ significantly from each other (𝑃 < 0.05). Spontaneously
fermented (Sp), L. fermentum (Lf), L. plantarum (Lp), L. helveticus (Lh), and L. mesenteroides (Lm), L. fermentum + L. plantarum (Lf + Lp), L. fermentum + L. helveticus (Lf + Lh), L. fermentum + L. mesenteroides
(Lf + Lm), L. plantarum + L. helveticus (Lp + Lh), L. plantarum + L. mesenteroides (Lp + Lm), L. helveticus + L. mesenteroides (Lh + Lm).
International Journal of Food Science 9
Table 5: Mean score of sensory attributes of Nunu prepared with various single and combined starter cultures. Evaluation is based on a
nine-point hedonic scale.
Starter cultures Taste Odour Colour/appearance Texture Overall acceptability
Sp (control) 6.53 ± 0.06d 5.00 ± 0.10a 6.40 ± 0.10b 6.53 b± 0.06 6.45 ± 0.06a
Lf 6.07 ± 0.15b 6.53 ± 0.06d 7.13 0.06c± 7.27 ± 0.06c,d 7.17 b± 0.12
Lp 7.53 g± 0.06 7.90 ± 0.10f 8.03 ± 0.15e 8.50 e± 0.20 7.20 ± 0.00b
Lh 6.40 ± 0.00c 8.43 ± 0.15g 7.43 ± 0.15d 8.20 ± 0.20e 8.00 ± 0.20e
Lm 5.00 a c b e b,d± 0.17 6.33 ± 0.06 6.53 ± 0.06 8.43 ± 0.15 7.40 ± 0.17
Lf + Lp 8.03 0.15h 8.50 0.20g 7.13 0.06c± ± ± 7.13 ± 0.12c 8.43 ± 0.15f
Lf + Lh 7.17 0.06e± 8.43 g± 0.06 8.03 ± 0.06e 8.43 ± 0.06e 8.17 e,f± 0.12
Lf + Lm 6.20 b± 0.10 6.10 ± 0.17b 7.27 ± 0.15c,d 7.43 ± 0.15d 7.43 ± 0.06d
Lp + Lh 7.03 ± 0.16e 8.63 ± 0.06g 7.87 ± 0.15e 8.50 0.20e± 7.70 ± 0.10d
Lp + Lm 7.30 0.00f± 5.17 a b e± 0.06 6.57 ± 0.15 8.30 ± 0.10 7.50 ± 0.26d
Lh + Lm 7.50 0.20g 7.20 0.10e 5.53 0.06a 6.23 0.15a± ± ± ± 6.50 ± 0.20a
Values are means of two experiments, ±: standard deviation. Values in the same column with different letters differ significantly from each other (𝑃 < 0.05).
Spontaneously fermented (Sp), L. fermentum (Lf), L. plantarum (Lp), L. helveticus (Lh), and L. mesenteroides (Lm), L. fermentum + L. plantarum (Lf + Lp), L.
fermentum + L. helveticus (Lf + Lh), L. fermentum + L. mesenteroides (Lf + Lm), L. plantarum + L. helveticus (Lp + Lh), L. plantarum + L. mesenteroides (Lp +
Lm), L. helveticus + L. mesenteroides (Lh + Lm).
of these indispensable groups of amino acids cannot be single or combined, showed improved acceptability as com-
transaminated and so they must be supplied in the diet as pared to the spontaneously fermentedNunu. However,Nunu
such [49]. It was also observed that the samples contained produced with single starter culture of L. helveticus or com-
the essential amino acids, namely, lysine, isoleucine, leucine, bined starter cultures of L. fermentum and L. helveticus (LF +
histidine, valine, threonine, methionine, and phenylalanine, LH) and L. fermentum and L. plantarum (LF + LP) showed
while the nonessential amino acids detected were arginine, significantly higher overall acceptability.
aspartic acid, serine, glutamic acid, proline, glycine, alanine,
cysteine, and tyrosine. Milk is essentially rich in essential 4. Conclusion
amino acids and branched chain amino acids. There is
evidence that these amino acids have unique roles in human Lactic acid bacteria isolated from Ghanaian traditional fer-
metabolism. In addition to providing substrates for protein mented milk product (Nunu) have demonstrated desirable
synthesis, suppressing protein catabolism, and serving as sub- technological properties and have subsequently been success-
strates for gluconeogenesis, they also trigger muscle protein fully used as starter cultures for Nunu fermentation. Lacto-
synthesis and promote protein synthesis [50]. The results bacillus fermentum, L. helveticus, and L. plantarum starter
suggest that Nunu produced with LAB starter culture can cultures (whether used alone or in combination) were able
serve as a good source of essential and nonessential amino to produce yoghurt with desirable consumer sensory charac-
acids required in metabolism. The amino acids indicated as teristics. Therefore, further development and application of
essential nutrients for infant growth [51], such as histidine, these cultures in commercial Nunu production will improve
arginine, cysteine, and tryptophan, were also found in Nunu. safety and consumer acceptability of the product.
Sulphur-containing amino acids (methionine and cysteine)
are the most critical essential amino acids because they were Conflict of Interests
easily lost from the body [52] and therefore their supplemen-
tation into food is greatly required. The results in this study The authors declare that there is no conflict of interestsregarding the publication of this paper.
however show thatmethionine was detected in allNunu sam-
ples, but cysteine was not detected in Nunu fermented with
L. mesenteroides (LM) starter culture and the combined Acknowledgment
starter culture of L. fermentum and L. mesenteroides (LF + This study is sponsored by the DANIDA-ENRECA project
LM). on capability building in research and quality assurance in
traditional fermented foods of West Africa.
3.2.3. Consumer Sensory Evaluation of Nunu. Consumer
sensory analysis showed varying degrees of acceptability References
for Nunu fermented with the different starter cultures in [1] J. Owusu-Kwarteng, F. Akabanda, D. S. Nielsen, K. Tano-
relation to their sensory attributes such as taste, odour, colour, Debrah, R. L. K. Glover, and L. Jespersen, “Identification of
texture, and overall acceptability (Table 5). Generally, Nunu lactic acid bacteria isolated during traditional fura processing in
fermented with lactic acid bacteria starter cultures, either Ghana,” Food Microbiology, vol. 32, no. 1, pp. 72–78, 2012.
10 International Journal of Food Science
[2] F. Akabanda, J. Owusu-Kwarteng, K. Tano-Debrah, R. L. K. bacteria,” Journal of Milk and Food Technology, vol. 35, pp. 482–
Glover, D. S. Nielsen, and L. Jespersen, “Taxonomic and 488, 1972.
molecular characterization of lactic acid bacteria and yeasts in [18] F. Durlu-Ozkaya, V. Xanthopoulos, N. Tunail, and E. Litopo-
nunu, a Ghanaian fermented milk product,” Food Microbiology, ulou-Tzanetaki, “Technologically important properties of lactic
vol. 34, no. 2, pp. 277–283, 2013. acid bacteria isolates from Beyaz cheese made from raw ewes’
[3] M. Obodai and C. E. R. Dodd, “Characterization of dominant milk,” Journal of AppliedMicrobiology, vol. 91, no. 5, pp. 861–870,
microbiota of a Ghanaian fermented milk product, nyarmie, 2001.
by culture- and nonculture-based methods,” Journal of Applied
Microbiology, vol. 100, no. 6, pp. 1355–1363, 2006. [19] E. Dagdemir and S. Ozdemir, “Technological characterizationof the natural lactic acid bacteria of artisanal Turkish White
[4] F. Akabanda, J. Owusu-Kwarteng, R. K. L. Glover, and K. Pickled cheese,” International Journal of Dairy Technology, vol.
Tano-Debrah, “Microbiological characteristics of Ghanaian 61, no. 2, pp. 133–140, 2008.
traditional fermented milk product,Nunu,”Nature and Science,
vol. 8, pp. 178–187, 2010. [20] S. D. Peterson, R. T. Marshall, and H. Heymann, “Peptidase
[5] E. H. E. Ayad, S. Nashat, N. El-Sadek, H. Metwaly, and M. profiling of lactobacilli associated with Cheddar cheese and its
El-Soda, “Selection of wild lactic acid bacteria isolated from application to identification and selection of strains of Cheese-
traditional Egyptian dairy products according to production ripening studies,” Journal of Dairy Science, vol. 73, pp. 1454–
and technological criteria,” Food Microbiology, vol. 21, no. 6, pp. 1464, 1990.
715–725, 2004. [21] L. Axelsson, “Lactic acid bacteria: classification and physiology,”
[6] O. N. Donkor, A. Henriksson, T. Vasiljevic, and N. P. Shah, in Lactic Acid Bacteria: Microbiology and Functional Aspects, S.
“Proteolytic activity of dairy lactic acid bacteria and probiotics Salminen and A. von Wright, Eds., pp. 1–72, Marcel Dekker,
as determinant of growth and in vitro angiotensin-converting New York, NY, USA, 1998.
enzyme inhibitory activity in fermented milk,” Lait, vol. 87, no. [22] J. E. Christensen, E. G. Dudley, J. A. Pederson, and J. L. Steele,
1, pp. 21–38, 2007. “Peptidases and amino acid catabolism in lactic acid bacteria,”
[7] R. G. Leuschner, P. M. Kenneally, and E. K. Arendt, “Method Antonie van Leeuwenhoek, vol. 76, no. 1–4, pp. 217–246, 1999.
for the rapid quantitative detection of lipolytic activity among [23] E. Tsakalidou, E. Manolopoulou, E. Kabaraki et al., “The com-
food fermentingmicroorganisms,” International Journal of Food bined use of whole-cell protein extracts for the identification
Microbiology, vol. 37, no. 2-3, pp. 237–240, 1997. (SDS-PAGE) and enzyme activity screening of lactic acid bac-
[8] J. A. Bonade, A. J. Dagnan, and M. J. Garver, “Production of teria isolated from traditional Greek dairy products,” Systematic
helveticin from Lactobacillus helveticus,” Letters in Applied and Applied Microbiology, vol. 17, no. 3, pp. 444–458, 1994.
Microbiology, vol. 33, pp. 153–158, 2001. [24] R. Aravindan, P. Anbumathi, and T. Viruthagiri, “Lipase appli-
[9] J. P. Guiraud, Microbiologie Alimentaire, Dunod Microsoft cations in food industry,” Indian Journal of Biotechnology, vol. 6,
Press, Paris, France, 1998. no. 2, pp. 141–158, 2007.
[10] E. P. Knoshaug, J. A. Ahlgren, and J. E. Trempy, “Growth asso- [25] V. Ramakrishnan, B. Balakrishnan, A. K. Rai, B. Narayan, and
ciated exopolysaccharide expression in Lactococcus lactissub- P. M. Halami, “Concomitant production of lipase, protease and
speciescremoris ropy 352,” Journal of Dairy Science, vol. 83, no. 4, enterocin by Enterococcus faeciumNCIM5363 and Enterococcus
pp. 633–640, 2000. duransNCIM5427 isolated fromfish processingwaste,” Interna-
[11] M. K. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. tional Aquatic Research, vol. 4, p. 14, 2012.
Smith, “Colorimetric method for determination of sugars and
Analytical Chemistry [26] P. Ruas-Madiedo, M. Gueimonde, A. Margolles, C. G. Derelated substances,” , vol. 28, no. 3, pp. 350– Los Reyes-Gavilán, and S. Salminen, “Exopolysaccharides pro-
356, 1956. duced by probiotic strains modify the adhesion of probiotics
[12] M. T. C. Ojinnaka and P. C. Ojimelukwe, “Study of the volatile and enteropathogens to human intestinal mucus,” Journal of
compounds and amino acid profile in Bacillus fermented castor Food Protection, vol. 69, no. 8, pp. 2011–2015, 2006.
oil bean condiment,” Journal of Food Research, vol. 2, pp. 191–
203, 2013. [27] A. Becker, F. Katzen, A. Pühler, and L. Ielpi, “Xanthan gumbiosynthesis and application: a biochemical/genetic perspec-
[13] T. Idoui and N. E. Karam, “Lactic acid bacteria from Jijel’s tradi- tive,” Applied Microbiology and Biotechnology, vol. 50, no. 2, pp.
tional butter: isolation, identification and major technological 145–152, 1998.
traits,” Grasas y Aceites, vol. 59, no. 4, pp. 361–367, 2008.
[14] J. S. Y. Haddadin, “Kinetic studies and sensorial analysis of [28] J. Cerning and V. M. E. Marshall, “Exopolysaccharides pro-
Lactic Acid Bacteria isolated from white cheese made from duced by the dairy lactic acid bacteria,” Recent Results and
sheep rawmilk,” Pakistan Journal of Nutrition, vol. 4, pp. 78–84, Developments, vol. 3, pp. 195–209, 1999.
2005. [29] A. Patel and J. B. Prajapati, “Food and health applications of
[15] P. Sarantinopoulos, C. Andrighetto, M. D. Georgalaki et al., exopolysaccharides produced by lactic acid bacteria,” Advances
“Biochemical properties of enterococci relevant to their tech- in Dairy Research, vol. 1, p. 107, 2013.
nological performance,” International Dairy Journal, vol. 11, no. [30] H. M. Stack, N. Kearney, C. Stanton, G. F. Fitzgerald, and R. P.
8, pp. 621–647, 2001. Ross, “Association of beta-glucan endogenous production with
[16] A. C. Freitas, A. E. Pintado, M. E. Pintado, and F. X. Malcata, increased stress tolerance of intestinal lactobacilli,” Applied and
“Role of dominant microflora of Picante cheese on proteolysis Environmental Microbiology, vol. 76, no. 2, pp. 500–507, 2010.
and lipolysis,” International Dairy Journal, vol. 9, no. 9, pp. 593– [31] M.-A. Levrat-Verny, S. Behr, V. Mustad, C. Rémésy, and C.
603, 1999. Demigné, “Low levels of viscous hydrocolloids lower plasma
[17] H. S. Park and E. H. Marth, “Behaviour of Salmonella typhi- cholesterol in rats primarily by impairing cholesterol absorp-
murium in skim milk during fermentation by lactic acid tion,”The Journal of Nutrition, vol. 130, no. 2, pp. 243–248, 2000.
International Journal of Food Science 11
[32] H.Maeda, X. Zhu, S. Suzuki, K. Suzuki, and S. Kitamura, “Struc- [47] E. L. Thomas and K. A. Pera, “Oxygen metabolism of Strepto-
tural characterization and biological activities of an exopolysac- coccus mutans: uptake of oxygen and release of superoxide and
charide kefiran produced by Lactobacillus kefiranofaciens WT- hydrogen peroxide,” Journal of Bacteriology, vol. 154, no. 3, pp.
2B T,” Journal of Agricultural and Food Chemistry, vol. 52, no. 17, 1236–1244, 1983.
pp. 5533–5538, 2004. [48] E. A. Muradyan, L. A. Erzhynkyan, and M. S. Sapondzhyan,
[33] Y. Kim, S. oh, and S. H. Kim, “Released exopolysaccharide (r- “Composition of free amino acids in fermented milk products,”
EPS) produced from probiotic bacteria reduce biofilm forma- Biologicheskii Zhurnal Armenii, vol. 29, pp. 111–112, 1986.
tion of enterohemorrhagic Escherichia coli O157:H7,” Biochem- [49] V. R. Young, “Protein and amino acids,” in Present Knowledge of
ical and Biophysical Research Communications, vol. 379, no. 2, Nutrition, B. A. Bowman and R.M. Russel, Eds., pp. 43–58, ILSI
pp. 324–329, 2009. Press, Washington, DC, USA, 8th edition, 2001.
[34] P. Ruas-Madiedo, M. Gueimonde, A. Margolles, C. G. de [50] A. Haug, A. T. Høstmark, and O. M. Harstad, “Bovine milk in
los Reyes-Gavilán, and S. Salminen, “Exopolysaccharides pro- human nutrition—a review,” Lipids in Health and Disease, vol.
duced by probiotic strains modify the adhesion of probiotics 6, article 25, 2007.
and enteropathogens to human intestinal mucus,” Journal of
Food Protection, vol. 69, no. 8, pp. 2011–2015, 2006. [51] FAO/WHO/UNU (Expert Consultation), “Protein and aminoacid requirements in human nutrition,” WHO Technical
[35] F. Dal Bello, J. Walter, C. Hertel, and W. P. Hammes, “In vitro Report, Food and Agriculture Organization/World Health
study of prebiotic properties of levan-type exopolysaccharides Organization/United Nations, Geneva, Switzerland, 2007.
from Lactobacilli and non-digestible carbohydrates using dena-
turing gradient gel electrophoresis,” Systematic and Applied [52] M. F. Fuller and P. J. Garlick, “Human amino acid requirements:
Microbiology, vol. 24, no. 2, pp. 232–237, 2001. can the controversy be resolved?” Annual Review of Nutrition,vol. 14, pp. 217–241, 1994.
[36] T. Hongpattarakere, N. Cherntong, S. Wichienchot, S. Kolida,
and R. A. Rastall, “In vitro prebiotic evaluation of exopolysac-
charides produced by marine isolated lactic acid bacteria,”
Carbohydrate Polymers, vol. 87, no. 1, pp. 846–852, 2012.
[37] M. Kivanç, “Antagonistic action of lactic cultures toward spoil-
age and pathogenicmicroorganisms in food.,”DieNahrung, vol.
34, no. 3, pp. 273–277, 1990.
[38] G. Tadesse, E. Ephraim, and M. Ashenafi, “Assessment of the
antimicrobial activity of lactic acid bacteria isolated fromBorde
and Shamita, traditional Ethiopian fermented beverages, on
some foodborne pathogens and effect of growthmedium on the
inhibitory activity,” Internet Journal of Food Safety, vol. 5, pp. 13–
20, 2005.
[39] O. R. Afolabi, O. M. Bankole, and O. J. Olaitan, “Production
and characterization of antimicrobial agents by Lactic Acid
Bacteria Isolated from Fermented Foods,” The Internet Journal
of Microbiology, vol. 4, p. 2, 2008.
[40] I. A. Adesokan, B. B. Odetoyinbo, and A. O. Olubamiwa, “Bio-
preservative activity of lactic acid bacteria on suya produced
from poultry meat,” African Journal of Biotechnology, vol. 7, no.
20, pp. 3799–3803, 2008.
[41] M. Raccah, R. C. Baker, J. M. Degenstein, and E. J. Mulnix,
“Potential application of microbial antagonism to extend stor-
age ability of a flesh type food,” Journal of Food Science, vol. 44,
pp. 43–46, 1979.
[42] J. L. Smith and S. A. Palumbo, “Use of starter cultures in meat,”
Journal of Food Protection, vol. 46, pp. 997–1006, 1983.
[43] L. M. Cintas, P. Casaus, H. Holo, P. E. Hernandez, I. F. Nes,
and L. S. Håvarstein, “Enterocins L50A and L50B, two novel
bacteriocins from Enterococcus faecium L50, are related to
staphylococcal hemolysins,” Journal of Bacteriology, vol. 180, no.
8, pp. 1988–1994, 1998.
[44] S. M. Wakil and U. O. Osamwonyi, “Isolation and screening of
antimicrobial producing lactic acid bacteria from fermenting
millet gruel,” International Research Journal ofMicrobiology, vol.
3, pp. 72–79, 2012.
[45] M. A. Daeschel, “Applications and interactions of bacteriocins
from lactic acid bacteria in foods and beverages,” inBacteriocins
of Lactic Acid Bacteria, pp. 63–91, Academic Press, New York,
NY, USA, 1993.
[46] S. Condon, “Aerobic metabolism of lactic acid bacteria,” Irish
Journal of Food Science and Technology, vol. 7, pp. 5–25, 1983.
International Journal of
Peptides
Advances in
BioMed Stem Cells Virolog y International Journal ofResearch International International Genomics
Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014
Journal of
Nucleic Acids
Z oInteornaltioonalg Joyurnal of
Hindawi Publishing Corporation Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014
Submit your manuscripts at
http://www.hindawi.com
Journal of The Scientific 
Signal Transduction World Journal
Hindawi Publishing Corporation Hindawi Publishing Corporation 
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014
Genetics  Anatomy International Journal of Biochemistry Advances in
Research International Research International Microbiology Research International Bioinformatics
Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014
Enzyme International Journal of Molecular Biology Journal of
Archaea Research Evolutionary Biology International Marine Biology
Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014
View publication stats