Isolation, characterisation and in vitro evaluation of bacteriocins-producing lactic acid bacteria from fermented products of Northern Borneo for their beneficial roles in food industry

Abstract

In this study, lactic acid bacteria (LAB) isolated from traditional fermented foods namely
coco bean, fermented cabbage, salted vegetable, tempeh, tempoyak, tapai ubi and tapai nasi were
screened for production of bacteriocin. Characterisation and in vitro evaluation of them were carried
out to assess their potential use in food industry. Towards these objectives, the inhibitory spectra of the
isolates against Listeria monocytogenes ATCC13932, resistance to phenol, amylolytic and proteolytic
activities, ability to produce acid and coagulate milk, antibiotic susceptibility and tolerance in the presence
of various concentration of NaCl and at different temperatures were evaluated. Two out of 15 LAB strains
were able to inhibit the growth of food-borne pathogen, L. monocytogenes ATCC 13932 and produce
bacteriocin-like inhibitory substances. The strains were identified as Pediococcus acidilactici TN1 (from
tapai nasi) and Lactobacillus farciminis TY1 (from tempoyak). Biochemical and physiological tests
demonstrated that, both strains were able to grow at wide range of NaCl concentrations (0.5 - 5.0 %, w/v)
and temperatures (28 - 70 ˚C), and capable to degrade protein. They lowered the pH level and coagulate
milk after 24 h of incubation. Both strains showed intrinsic mechanisms of antibiotic resistance towards
streptomycin, norfloxacin, erythromycin, amikacin and nalidixic acid. They also were able to grow in
0.3% (w/v) of bile salts and tolerate up to 0.5% (w/v) phenol. The findings from this study revealed the
presence of LAB strains in fermented foods of Northern Borneo which have an antimicrobial activity
towards the food-borne pathogen. Even though this study had generated extensive information to validate
Pediococcus acidilactici TN1 and Lactobacillus farciminis TY1 as potential probiotic strains for
application in the food industry, the study is by no means comprehensive nor complete. More laboratory,
particularly in vivo studies, are needed before this product could be accepted by the food industry and
most importantly to explore its novel health promoting functions as well as its colonization behaviour in
the gut.

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Isolation, characterisation and in vitro evaluation of bacteriocins-
producing lactic acid bacteria from fermented products of Northern
Borneo for their beneficial roles in food industry
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12th Seminar on Science and Technology
Journal of Physics: Conference Series 1358 (2019) 012020
IOP Publishing
doi:10.1088/1742-6596/1358/1/012020
1
Isolation, characterisation and in vitro evaluation of
bacteriocins-producing lactic acid bacteria from fermented
products of Northern Borneo for their beneficial roles in food
industry
R Jawan1,3*, ME Kasimin1, SN Jalal1, AA Mohd. Faik1, S Abbasiliasi2 and A Ariff3,4
1Biotechnology Programme, Faculty of Science and Natural Resources, Universiti Malaysia Sabah,
Jalan UMS, 88400 Kota Kinabalu, Sabah
2Halal Products Research Institute, Universiti Putra Malaysia, UPM Serdang, 43400 Selangor,
Malaysia
3Bioprocessing and Biomanufacturing Research Centre, Faculty of Biotechnology and Biomolecular
Sciences, Universiti Putra Malaysia, UPM Serdang, 43400 Selangor, Malaysia
4Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences,
Universiti Putra Malaysia, UPM Serdang, 43400 Selangor, Malaysia
*Corresponding author: roslinaj@ums.edu.my
Abstract. In this study, lactic acid bacteria (LAB) isolated from traditional fermented foods namely
coco bean, fermented cabbage, salted vegetable, tempeh, tempoyak, tapai ubi and tapai nasi were
screened for production of bacteriocin. Characterisation and in vitro evaluation of them were carried
out to assess their potential use in food industry. Towards these objectives, the inhibitory spectra of the
isolates against Listeria monocytogenes ATCC13932, resistance to phenol, amylolytic and proteolytic
activities, ability to produce acid and coagulate milk, antibiotic susceptibility and tolerance in the presence
of various concentration of NaCl and at different temperatures were evaluated. Two out of 15 LAB strains
were able to inhibit the growth of food-borne pathogen, L. monocytogenes ATCC 13932 and produce
bacteriocin-like inhibitory substances. The strains were identified as Pediococcus acidilactici TN1 (from
tapai nasi) and Lactobacillus farciminis TY1 (from tempoyak). Biochemical and physiological tests
demonstrated that, both strains were able to grow at wide range of NaCl concentrations (0.5 - 5.0 %, w/v)
and temperatures (28 - 70 ˚C), and capable to degrade protein. They lowered the pH level and coagulate
milk after 24 h of incubation. Both strains showed intrinsic mechanisms of antibiotic resistance towards
streptomycin, norfloxacin, erythromycin, amikacin and nalidixic acid. They also were able to grow in
0.3% (w/v) of bile salts and tolerate up to 0.5% (w/v) phenol. The findings from this study revealed the
presence of LAB strains in fermented foods of Northern Borneo which have an antimicrobial activity
towards the food-borne pathogen. Even though this study had generated extensive information to validate
Pediococcus acidilactici TN1 and Lactobacillus farciminis TY1 as potential probiotic strains for
application in the food industry, the study is by no means comprehensive nor complete. More laboratory,
particularly in vivo studies, are needed before this product could be accepted by the food industry and
most importantly to explore its novel health promoting functions as well as its colonization behaviour in
the gut.
Keywords: Lactic acid bacteria, isolation, characterisation, fermented foods, bacteriocins
1. Introduction
Massive development in food industry such as high processed, chemically preserved, high fat content
and zero calories foods are available in the market that could lead to unhealthy lifestyle. An awareness
by the community has brings a demand for the functional fermented foods that offer a various benefit
hence triggered the researchers to search for a newly isolated lactic acid bacteria (LAB) from the
fermented products that will open the possibility to find new strains with health promoting
characteristics. Various findings reveal the presence of potential probiotic strains that had been isolated
from numerous fermented foods either from vegetables, fruits or animal-based products such as L.
plantarum RYPR1 isolated from raabadi (fermented beverage) [1], Lactobacillus plantarum Bom 816

12th Seminar on Science and Technology
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and Lactobacillus pentosus N3 from boza (cereal-based beverage) [2], various LAB strains from
pastirma (air-dried cured beef) [3] and several Lactobacillus strains from lukanka (fermented and air-
dried meat) [4]. Even though related findings with similar fermented foods had been published by
Kormin et al. [5], the individuality and variance in fermented products preparation/ raw materials
supported with dissimilarity in geographical areas and fermentation periods had become a key factor for
discovering the new potential LAB with beneficial traits.
LAB are favourable microorganisms contributing to various industrial applications, extending
from food trade for food and beverage fermentation, to pharmaceuticals and also nutraceuticals
manufacturing. Benefits of LAB in controlling the development of foodborne diseases on fermented
products were long before known caused by the production of various by-products such as lactic acid,
acetic acid, ammonia, bacteriocins, ethanol, reuterin, hydrogen peroxide, and diacetyl. All these
compounds are able to inhibit the growth of food spoilage and pathogenic organisms [6] as well as
increasing the shelf life of the product [7]. Besides acting as natural food preservatives that delay the
spoilage, LAB also contributed to the flavour and the aroma development in food and beverages [8] and
increases the nutritional value of the product possibly containing health benefits [9]. One of the
important compounds produced by LAB is bacteriocins. Bacteriocins are ribosomally synthesized
antimicrobial peptides which provide promising technological alternative for food bio preservation, as
they can avoid the growth of spoilage and pathogenic microorganisms [10]. The use of bacteriocin-
producing bacteria is significantly more effective to improve human gut health as antimicrobial peptides
produced by probiotic strains in the intestine, can interrelate directly with the sensitive pathogenic
organisms in the intestine [11, 12].
In food industry, the strains have been selected for commercial use in foods must retain their
characteristics for which they were originally selected including the characteristics of growth and
survival during manufacture and, after consumption, during transit through the stomach and small
intestine. Importantly, probiotic must retain these characteristics to provide various health benefits to
the consumers [13]. Advancement in food manufacturing technology inspired the continuous search of
newly isolated strains that able to endure the harsh conditions during food processing, coupled with the
increase in antibiotic resistance incidence among the food-related microorganisms. Therefore, the
objectives of this study are to isolate, characterise, and evaluate the bacteriocins-producing LAB from
fermented foods of Northern Borneo to be applied in food industry such as in production of functional
foods, starter cultures, bio preservatives, and flavour compounds.
2. Methods
2.1. Collection of food samples
Seven (7) types of locally fermented products namely coco bean, fermented cabbage, salted vegetable,
tempeh, tempoyak, tapai ubi and tapai nasi were purchased from wet market at Kota Kinabalu, Sabah,
Malaysia (Figure 1).
Figure 1. Fermented food samples. (a) Coco bean; (b) Cabbage; (c) Salted vegetable; (d) Tempeh; (e)
Tempoyak; (f) Tapai ubi; (g) Tapai nasi.
(a)
(b)
(c)
(d)
(e)
(f)
(g)

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2.2. Isolation of lactic acid bacteria
Isolation of LAB was carried out based on Abbasiliasi et al. [14] with a slight modification. Briefly, for
a solid sample, 5 g was mixed with 45 mL of NaCl solution (0.85%, w/v NaCl) and homogenised using
home blender to get a uniform sample. To enrich the growth of potential targeted LAB, 25 mL of food
sample was added into 225 mL de Man Rogosa Sharpe (MRS) (Oxoid LTD, Basingstoke, Hampshire,
England) and M17 (Oxoid LTD, Basingstoke, Hampshire, England) broth to obtain a 1:10 dilution and
incubated at 37 ˚C for 24 h. The culture was then diluted (10-fold) in 0.85% (w/v) NaCl solution and
100 µL of diluted sample was spread-plated on M17 and MRS agar supplemented with 0.01% (w/v)
sodium azide (as an inhibitor for growth of Gram-negative bacteria). The plates were incubated at 37 ˚C
for 24 h under anaerobic conditions (AnaeroGen, Oxoid). Fifty colonies were selected randomly (based
on their differences in colour, shape, elevation and size) and were streaked onto MRS and M17 agar
media. The single colony was then sub-cultured twice to ensure the purity of the culture.
2.3. Biochemical and physiological characteristics of lactic acid bacteria
2.3.1. Gram staining and cell morphology
The identification of the isolates was performed by standard staining procedure. The shape morphology
of the fresh grown cells was viewed under the light microscope.
2.3.2. Catalase activity and carbon fermentation test
A drop of 3% (v/v) hydrogen peroxide solution was placed on pure single colony. Immediate formation
of bubbles (gas production) were consider as positive. In carbon fermentation test, nutrient agar was
prepared with 1% (w/v) of glucose and 0.004% (w/v) of bromocresol purple (as a pH indicator). About
10 μL of culture was then spotted on the agar. After incubation at 37 ˚C for 24 h, positive result was
shown by changing in purple colour to yellow zone around the culture as a result of reduced pH by acid
production through the fermentation of glucose by the bacteria.
2.3.3. Effect of NaCl and temperature on growth of lactic acid bacteria
The isolate (1%, v/v) was inoculated into M17 or MRS broth containing different concentrations of
NaCl (0.5, 2.0, 5.0 and 10 %, w/v) and 0.004 % (w/v) bromocresol purple prior to incubation at 37 ˚C
for 24 h. In temperature test, culture and media preparation was same as previous (except no addition of
NaCl) and culture was incubated at different temperatures (-20, -40, 14, 28, 37 and 70 ˚C). The tested
temperatures representing the common range of temperatures in fermented food processing. After 24 h
of incubation the growth was assessed as indicated by media colour changes from purple to yellow
2.4. Antimicrobial activity against Listeria monocytogenes ATCC13932
The antimicrobial activity of the isolates against food-borne pathogens, Listeria monocytogenes
ATCC13932, was determined by the agar well diffusion assay [15]. To prepare the cell-free culture
supernatants (CFCS), the isolate was grown in M17 or MRS broth at 37 ˚C for 24 h and the cultures
were centrifuged at 10,000 rpm for 10 min at 4 ˚C. The CFCS (100 μL) was then placed into 6 mm wells
of agar plates that was earlier sowed with 1% (v/v) L. monocytogenes ATCC 13932. The plates were
placed at 4 ˚C for 2 h to ensure a better diffusion of the CFCS through the agar media before incubated
at 37 ˚C. After 24 h, the inhibition zones formed surround the wells were measured using electronic
caliper.
2.5. Determination of probiotic properties of lactic acid bacteria
The potential isolates with antimicrobial activity were selected and further characterised to evaluate their
potentials use in food industry. The tests involved were design to mimic the in vitro gastrointestinal
condition.

12th Seminar on Science and Technology
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2.5.1. Bile salts and phenol tolerance test
Bile salts tolerance of the isolates was determined by the viable count method [16]. The overnight
incubated culture (1%, v/v) was inoculated into 100 mL of M17 or MRS broth supplemented with 0.3%
(w/v) bile salts. Media without addition of bile salts was used as control. The culture was agitated at 100
rpm and incubated at 37 ˚C for 4 h. An enumeration of viable cells was performed using pour plate
technique at 0, 1, 2, 3 and 4 h of incubation. The colony number was count using a colony counter and
compared with the control (0 h) to determine the bile salt tolerance. In conducting phenol tolerance test,
the procedure was similar with that of bile salts test except the broth media added with different
concentrations of phenol that usually applied in screening of probiotic strains (0.1, 0.3 and 0.5%, w/v).
Inhibitory effect was determined by comparing the viable cells count of isolate at 0 and after 24 h of
incubation. Negative sign of the equated viable cells count value indicating no inhibition occurs
throughout the experiments.
2.5.2. Amylolytic and proteolytic activity tests
Starch hydrolysis test was conducted by inoculating a loop full of pure bacterial strains and streaking it
on agar plates containing 2% (w/v) of soluble starch powder. After an overnight incubation at 37 ˚C, a
small amount of iodine solution was poured onto the starch agar plates to detect starch hydrolysis. The
presence of a clear halo zone around a tested colony was taken as indication of starch degradation and
therefore the production of α- amylase. In proteolytic activity, the test was determined by inoculating
the culture on agar supplemented with 1% (w/v) of skim milk [17]. The appearance of transparent halo-
forming colonies was considered as positive reaction after an overnight incubation at 37 ˚C.
2.5.3. Acidifying activity
The acidification test was determined by the changes in pH of the skim milk solution and coagulation
ability [18]. The cultures (1 mL) were inoculated into 100 mL of 10% (w/v) of skim milk and incubated
at 37 ˚C for 24, 48 and 72 h. Skim milk without addition of inoculum was used as control. The physical
properties of the skim milk such as the pH, aroma, coagulation and appearance were as described by
Bodyfelt et al. [19]
2.5.4. Antibiotic sensitivity test
Antibiotic susceptibility was performed by the disc diffusion method of Abbasiliasi et al. [14]. A single
colony of the isolate was inoculated into 10 mL of M17 or MRS broth and incubated at 37 ˚C for 24 h.
Bacterial suspension was adjusted to 0.5 McFarland and swabbed evenly onto Müller-Hinton agar plate.
Commercially available disc (Oxoid) containing penicillin G, colistin sulphate, streptomycin,
chloramphenicol, erythromycin, ceftriaxone, amikacin, norfloxacin, tetracycline, nalidixic acid and
ampicillin were then placed on the surface of the dried agar plates. The experiment was performed in
triplicate. All plates were incubated at 37 ˚C for 24 h before measuring the inhibition zones including
the disc diameter. Isolates were categorized as sensitive (≥21 mm), intermediate (16-20 mm) or resistant
(≤15 mm). Absence or presence of inhibition zones were defined as sensitivity or resistance,
respectively.
2.6. Identification of isolates by16S rRNA sequencing and phylogenetic analysis
DNA extraction and sequencing of the amplified fragments were carried out by Apical Scientific Sdn.
Bhd. Selangor, Malaysia according to methods of [20]. Briefly, the bacterial 16S rDNA, full-length 1.5
kb, was amplified using universal primers 27F and 1492R. The total reaction volume of 25 uL contained
genomic DNA purified using in-house extraction method, 0.3 pmol of each primer, deoxynucleotides
triphosphates (dNTPs, 400 µM each), 0.5 U DNA Taq polymerase, supplied PCR buffer and deionised
water. The PCR was performed as follow: 1 cycle (94 ˚C for 2 min) for initial denaturation; 25 cycles
(98 ˚C for 10 sec; 53 ˚C for 30 sec; 68 ˚C for 1 min) for annealing and extension of the amplified DNA.
The PCR products were purified by standard method and directly sequenced with primers 785F and
907R using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The fragments of

12th Seminar on Science and Technology
Journal of Physics: Conference Series 1358 (2019) 012020
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doi:10.1088/1742-6596/1358/1/012020
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sequences were assembled and consensus sequences were compared with those deposited in the
GenBank DNA database using the Basic Local Alignment Search Tool (BLAST; http:
//blast.ncbi.nlm.nih.gov/Blast. cgi). Then, a phylogenetic tree based on 16S rRNA 137 genes was
constructed to determine the closest bacterial species by using Neighbour Joining (Unrooted Tree) by
NCBI Blast Tree Method using Molecular Evolutionary Genetics Analysis (MEGA) software version
10.0.5 [21]. Distances and clustering with the Neighbour-Joining method was determined using
bootstrap values based on 1000 replications. Bacillus subtilis NCDO 1769 and Escherichia coli strain
U 5/41 were used as an outgroup organism, respectively that serves as a reference group in evolutionary
relationships determination of the ingroup.
3. Results
3.1. Isolation, morphological and biochemical characterisation of lactic acid bacteria
A total of 25 strains were isolated from 7 types of locally fermented foods. After preliminary
identification, 15 out of 25 isolates were LAB as they were Gram-positive, catalase negative and able
to ferment glucose and produce acid. Two of these LAB isolates, namely TN1 from tapai nasi, and TY1
from tempoyak were able to produce antimicrobial substances as they showed inhibitory activity against
a food-borne pathogen, L. monocytogenes ATCC13932 with 9.0- and 10.0-mm diameter of inhibition
zone, respectively (Table 1). Therefore, strain TN1 and TY1 were selected for further study. TN1 is
cocci shape cell and appear as round, concave and white opaque colonies. TY1 has bacilli shape cell
and seem round, concave with yellowish-white in colour.
Table 1. Morphological, biochemical characteristics and antimicrobial activity of isolates.
Characteristics
Coco
bean
Fermented
cabbage
Tapai
nasi
Tapai
ubi
Salted
vegetable
Tempeh
Tempoyak
No. of LAB isolates
2
3
3
2
3
1
2
No. of isolates showing antimicrobial
activity against L. monocytogenes
ATCC13932
0
0
1
(TN1)
0
0
0
1
(TY1)
Cell morphology
Cocci
Bacilli
Cocci
Cocci
Bacilli
Cocci
Bacilli
Gram stain reaction
+
+
+
+
+
+
+
Catalase activity
-
-
-
-
-
-
-
Glucose fermentation
+
+
+
+
+
+
+
Note: Positive reaction (+), negative reaction (−)
3.2. Identification of isolates by 16S rDNA sequencing
The predicted size (1.5 kb) of exposed genomic DNA bands from PCR analysis for TN1 and TY1 are
shown in Figure 2. The respective phylogenetic trees of partial 16S rDNA sequences as presented in
Figure 3. The optimal tree with the sum of branch length for TN1 is 0.22820880 and 0.38133460 for
TY1. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap
test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in
the same units as those of the evolutionary distances used to infer the phylogenetic tree. This analysis
involved 12 nucleotide sequences and codon positions included were 1st+2nd+3rd+Noncoding. All
ambiguous positions were removed for each sequence pair (pairwise deletion option). In addition, there
were a total of 1606 (TN1) and 1574 (TY1) positions in the final dataset.

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Figure 2. Agarose gel electrophoresis-PCR amplification products of 16S rDNA genes of lactic acid
bacteria isolates. Lane M: 10kb GeneRuler DNA Ladder; Lane -ve: PCR without template control; Lane
+ve: Positive control, Bacterial gDNA, 10 ng; Lane 1: TN1; Lane 2: TY1.
Figure 3. Phylogenetic tree of TN1 and TY1 isolates and related taxa based on partial 16S rDNA
sequences. The phylogenetic tree was constructed by the Neighbour-joining method (MEGA X 10.0.5).
Numbers in parentheses are accession number of published sequences. The numbers at the nodes are
bootstrap confidence levels (percentage) from 1000 replicates. Bacillus subtilis NCDO 1769 and
Escherichia coli strain U 5/41 were used as an outgroup organism, respectively (A) TN1; (B) TY1.
The obtained phylogenetic tree proved that strains TN1 (A_1515bp) and TY1 (B_1502bp) were
most closely related to Pediococcus acidilactici and Lactobacillus farciminis strains supporting by 100
and 93% value from bootstrap analysis of the phylogenetic tree, respectively. Both strains show 99%
similarity in its 16S rDNA gene sequences (Table 2).
Table 2. Analysis of 16S rDNA sequencing analysis (BLASTN) of TN1 and TY1.
Sources
Strains
Species
% Similarity (BLASTN)
NCBI accession No
Fermented rice
TN1
Pediococcus acidilactici
99
NR042057.1
Tempoyak
TY1
Lactobacillus farciminis
99
NR114396.1
3.3. Effect of NaCl and temperature on growth of lactic acid bacteria
P. acidilactici TN1 and L. farciminis TY1 were able to grow in MRS broth supplemented with up to 5%
(w/v) of NaCl and there was no growth detected on 10% (w/v) of NaCl. Both strains can tolerate
temperature ranged from 28 - 70 ˚C.
3.4. Probiotic characterisation of lactic acid bacteria
In starch hydrolysis and proteolytic activity test, only P. acidilactici TN1 was able to hydrolysed soluble
starch by synthesising amylase indicating by a clear zone on starch agar. Both strains were capable to
degrade protein by producing protease as clear zone was spotted on milk agar. Other than that, P.
acidilactici TN1 and L. farciminis TY1 were able to acidify milk as they lowered the pH level (TN1:
A
TN1
TY1
B
10 000
1500
250
bp

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pH 6.62 to 4.27; TY1: pH 6.65 to 4.24) prior to milk coagulation during 0 h until 24 h of incubation
producing a rancid odour and cream colour curd (Table 3).
Table 3 Milk acidification activity of TN1 and TY1.
Strains
Incubation time
(h)
pH
Characteristics
Ability to coagulate milk
Aroma
Colour
TN1
0
6.62
No
Flat
White
24
4.91
Yes
Foreign
Cream
48
4.31
Yes
Rancid
Cream
72
4.27
Yes
Rancid
Cream
TY1
0
6.65
No
Flat
White
24
4.92
Yes
Foreign
Cream-white
48
4.32
Yes
Rancid
Cream-white
72
4.24
Yes
Rancid
Cream-white
P. acidilactici TN1 and L. farciminis TY1 were able to grow in 0.3% (w/v) of bile salts (Figure
4). TN1 had a higher survivability percentage (100.93-145.87%) (except at hour-3) as compared to
control (MRS medium without addition of bile salt) (95.83-122.63%). Whereas, TY1 (111.12-154.49%)
shows a comparable growth pattern as linked to control (105.31-149.76%) indicated by survivability
percentage.
Figure 4. Survivability of TN1 and TY1 after 4 h of exposure to difference concentrations of bile salts
Phenol tolerance test has no inhibitory effect on growth of P. acidilactici TN1 and L. farciminis
TY1, as indicated by negative inhibition value (Table 4). Negative value indicating an increase in cell
number during the incubation time period as compared to the initial cell number, demonstrating the
ability of the cells to grow with the presence of various concentrations of phenol in MRS medium. The
inhibitory effect was decreased as phenol concentrations increase (except for 0.5%, w/v of phenol for
TY1).
Table 4. Effect of phenol on growth of TN1 and TY1 in MRS broth media supplemented with various
concentrations of phenol
Strain
Phenol
(%, w/v)
Viable cell count (Log10 CFU/mL)
Inhibition*
Incubation time (h)
0
24
TN1
0
6.49 ± 0.78
7.95 ± 0.15
-1.46
0.1
6.72 ± 0.01
8.78 ± 0.04
-2.06
0.3
7.14 ± 0.71
9.0 ± 0.06
-1.86
0.5
7.82 ± 0.15
8.02 ± 0.18
-0.20
TY1
0
5.98 ± 0.01
8.91 ± 0.07
-2.93
0.1
6.94 ± 0.06
9.01 ± 0.19
-2.07
0.3
6.99 ± 0.19
7.85 ± 0.16
-0.86
0.5
8.04 ± 0.19
8.01 ± 0.03
0.03
Note: *Inhibition= Viable cell count at 0 h – Viable cell count at 24 h
A
B

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Antibiotic sensitivity test showed that both strains (TN1 and TY1) exhibit intrinsic mechanisms
of antibiotic resistance towards certain antibiotics such as amikacin, erythromycin, nalidixic acid,
norfloxacin and streptomycin (Table 5). Additionally, TY1 was also resistance to colistin sulphate. Both
strains were susceptible to β-lactams antibiotics (ampicillin and penicillin G) as well as ceftriaxone and
chloramphenicol. Only TN1 susceptible to colistin sulphate. TN1 and TY1 strains showed an
intermediate effect on tetracycline.
Table 5 Inhibitory response of TN1 and TY1 towards antibiotic sensitivity test
Antibiotic
Disc
content
Inhibition zone diameter (mm)
TN1
TY1
Ampicillin
25 µg
24.5 ± 0.01 (S)
27.0 ± 0.07 (S)
Amikacin
30 µg
0 (R)
0 (R)
Ceftriaxone
30 µg
24.0 ± 0.03 (S)
25.0 ± 0.02 (S)
Chloramphenicol
30 µg
22.0 ± 0.03 (S)
22.5 ± 0.01 (S)
Colistin sulphate
10 µg
20.5 ± 0.01 (S)
0 (R)
Erythromycin
10 µg
0 (R)
0 (R)
Nalidixic acid
30 µg
0 (R)
0 (R)
Norfloxacin
10 µg
0 (R)
0 (R)
Penicillin G
2 units
25.0 ± 0.02 (S)
21.5 ± 0.01 (S)
Streptomycin
10 µg
0 (R)
0 (R)
Tetracycline
10 µg
16.0 ± 0.04 (I)
17.0 ± 0.04 (I)
Note: Results of zone of inhibition are triplicate and expressed as Mean ± S.D. Resistance (R) ≤15 mm;
Intermediate (I) 16-20 mm; Susceptible (S) ≥21 mm.
4. Discussion
In this study, two isolates from fermented food products of Northern Borneo that able to produce
antimicrobial substances against L. monocytogenes ATCC13932 were identified. Based on phylogenetic
analysis, these two isolates were unambiguously recognised as P. acidilactici TN1 and L. farciminis
TY1. Both strains were undergoing a testing based on experimental design that mimicking the
gastrointestinal track (GIT) of human-being to guarantee they are metabolically active within the GIT
and biologically effective against the identified target. The ability of LAB to produce interesting
inhibitory compounds (organic acids, H2O2, bacteriocins) that are important both in preventing the
growth of spoilage and pathogenic bacteria fascinate their application in food industry. Bacteriocin-
producing species and strains have been identified among all the genera that comprise the LAB,
including Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus and Carnobacterium
as well as several Enterococcus spp. [22]. Bacteriocins produced by LAB have been the subject of
extensive studies in recent years due to their potential use as novel, natural food preservatives also may
play a role in the regulation of population dynamics within a fermenting ecosystem [23].
NaCl is one of the most extensively used additives in food manufacturing as it has a preservative
and antimicrobial effect due to reduction of water activity values. It also has flavour enhancement effects
by reducing or enhancing the enzymatic activity of some enzymes responsible for the development of
organoleptic parameters or as a consequence of its effect on different biochemical mechanisms [24].
The current result showed that P. acidilactici TN1 and L. farciminis TY1 were able to grow in media
supplemented with 0.5-5.0% (w/v) of NaCl. Our finding was in line with study conducted by Islam et
al. [25], which stated the optimal growth of Lactobacillus spp was observed at 1-5% (w/v) NaCl. The
notable effect of NaCl concentration was highlighted by Chin and Koehler [26]. They concluded that
higher amine levels were found in low-salt (5%, w/v NaCl) formulations than in high-salt (10%, w/v
NaCl). The variety in biogenic compounds production might relates to the inhibiting effect of higher
salt concentration on the growth of many microorganisms, which in turn greatly decreases the likelihood
of the production of decarboxylase enzymes responsible for the decarboxylation of amino acids to form
amines. In a contrary, some of Lactobacilli species are more resistant to harsh conditions like higher
NaCl concentration, anaerobic condition and reduced availability of nutrients [27].
The ability of the cultures to grow in a particular temperature range is an important physiological
characteristic used for the identification of LAB [4]. Other than that, thermotolerance capacity of LAB

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benefits in acceptance the thermal treatment as industrial production of probiotic foods generally
involves processes taking place at high temperatures [28]. In this study, P. acidilactici TN1 and L.
farciminis TY1 can tolerate temperature ranged from 28-70 ˚C. Thermotolerance trait of our strains may
be advantageous if the bacteriocin produced is to be used as an antimicrobial agent in fermented foods
or thermally processed foods as Mathew and Augustine [29] exposed that optimum level of bacteriocin
was associated with an optimum temperature of growth. Fossi et al. [30] incorporated calcium and
magnesium salts into the media to improve the thermotolerance of probiotic strains. The protective effect
of the mineral salts is due to the fact that calcium and magnesium ions have the property of stabilizing
certain structural proteins and even enzymatic ones thus may prevent the rapid denaturation of
membrane proteins.
To reach the GIT in a viable form, probiotic strains have to overcome several biological barriers
including the presence of lysozyme in the saliva, low pH in gastric juice and bile salts in the upper GI
tract [31, 32]. Bacteria inhabiting intestinal tract must have intrinsic resistance mechanisms to cope with
bile salts [33]. Result showed that P. acidilactici TN1 and L. farciminis TY1 can tolerate 0.3% bile salt
thus they may survive under high acidity in the stomach and high concentration of bile components in
human gastrointestinal tract. Our finding was concurred with the ability of Bifidobacterium and
Lactobacillus species to cope with bile stress [33]. In addition, 0.3% of bile was used, as it corresponded
to that found in the human intestinal tract and 0.3% bile is the maximum concentration that is present in
healthy men [34].
To satisfy the probiotic criterion, the strain has to endure the action of toxic metabolites
(primarily phenols) produced during the digestion process [35] because bacteria that are lenient to
phenols may have better possibilities of persistence in the GIT. Furthermore, some aromatic amino acids
derived from dietary or endogenously produced proteins can be deaminated in the gut by bacteria leading
to the formation of phenols which have bacteriostatic properties [36]. P. acidilactici TN1 showed an
exceptional tolerance to phenol up to 0.5% (w/v), whereas the tolerance of L. farciminis TY1 was limited
to 0.3% (w/v). Results from this study is contradictory to P. acidilactici Kp10 that inhibited by as low
as 0.2% (w/v) of phenol [37]. The mechanism of inhibitory effect of phenolic compound is simply by
their ability to diffuse into the bacterial cell membrane and subsequently cause leakage of the
intracellular membrane that destroys the bacterial cell membrane. In addition, most of the phenolic
compounds remain in the gastrointestinal tract after consumption and this compound exert their
inhibitory effects on the enzyme involved in degradation of proteins, lipids, and saccharides [38].
P. acidilactici TN1 and L. farciminis TY1 were able to express proteolytic and amylolytic
enzymes. Fermentation with LAB is considered as an effective way to reduce whey protein antigenicity.
Milk protein allergens can be degraded by a series of proteolytic enzymes produced during the microbial
fermentation [39] in following steps: (i) Proteinases initially cleave the milk protein to peptides, (ii)
Peptidases cleave the peptides into smaller peptides and amino acids, (iii) Small peptides and amino
acids shift in the cellular uptake by transport systems [40]. Since probiotics require free amino acids for
growth and survival, the release of essential amino acids and production of growth stimulators thereby
assisted the growth and viability of probiotic in functional products [41].
Acidification capability of potential strains is one of the most important technological properties
for potential starter cultures especially for milk-based probiotic products making. The reduction of pH
can control the growth of a large number of pathogenic or undesirable microorganisms that cause
spoilage of fermented products, and can improve the hygienic properties and storage of the final products
[4]. In the present study, the pH of the milk decreased during fermentation, indicating increased acidity
over the storage, but did not reach less than pH 4.0 (for both strains TN1 ad TY1) and resulted in a
cream colour curd with rancid aroma after 24 h of incubation. This finding was in line with Senaka et
al. [41], who claimed that pH below pH 4 is generally considered detrimental to the survival of probiotic
organisms, even though sensitivity of probiotics to lower pH in functional food is species and strain
specific. Theoretically, the acidification of milk directly impacts the stability of casein micelles,
reducing their charge, dissolving some of the insoluble calcium phosphate crosslinks and modifying
internal bonding between proteins. It is reported that the aroma and taste of soured milk products are

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characterised by numerous bacterial metabolites (volatile or non-volatile acids and carbonyl
compounds) such as acetaldehyde, acetone, acetoin, and diacetyl in addition to acetic, formic, butanoic,
and propanoic acids [42]. Furthermore, the strains that able to coagulate milk within 16 h were defined
as fast coagulating strains, while slow variants require a longer period of time (more than 36 h) [43].
The human GIT ports 1013–1014 bacterial cells in adults and this microbiota is often exposed to
a variety of antibiotics, due to their routine use in clinical settings [44]. Consequently, the human GIT
microbiota may serve as an important reservoir of antibiotic resistant strains that could act as
opportunistic pathogens or as donors of resistance genes to other bacteria [45]. Authentication of
virulence factors in LAB is necessary due to the risk of genetic transfer, since these genes are usually
located in conjugative plasmids [46] hence antibiotic resistance genes can speedily move through
bacterial populations and emerge in pathogenic bacteria via horizontal gene transfer [47]. Therefore, the
safety assessment of potential probiotics strains towards antibiotics is highly needed. Our strains, P.
acidilactici TN1 and L. farciminis TY1 show a resistance response towards amikacin, erythromycin,
nalidixic acid, norfloxacin and streptomycin. Differences in resistance phenotypes may be due to non-
functional and / or silent genes [48].
5. Conclusion
Results from this study revealed that bacteriocins producing-LAB were successfully isolated from
fermented products of Northern Borneo identified as P. acidilactici TN1 (from tapai nasi) and L.
farciminis TY1 (from tempoyak). Both strains showed an antimicrobial activity against food-borne
pathogen, L. monocytogenes ATCC13932, grow at wide range of NaCl concentrations (0.5 - 5.0 %, w/v)
and temperatures (28 - 70 ˚C). They also demonstrated a probiotic characteristic such as able to degrade
protein, acidify skim milk, endure 0.3% (w/v) of bile salts, tolerate to phenol up to 0.5% (w/v) and show
an intrinsic mechanism towards various antibiotic. In future, more in vitro assessment and in vivo
evaluation should be carried out to unfold the novel potential of these two strains in food industry.
Acknowledgment
The authors wish to acknowledge the Faculty of Science and Natural Resources, Universiti Malaysia
Sabah for providing research facilities and financial support (Undergraduate Scientific Project).
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