CHAPTER III 
ISOLATION AND SCREENING OF LACTIC ACID BACTERIA FROM 
DIFFERENT FOOD SOURCES WITH MILK CLOTTING ACTIVITY 
1.1 INTRODUCTION 
Lactic acid bacteria refer to a large group of beneficial bacteria that have similar 
properties and produce lactic acid as an end product of the fermentation process. They 
are widespread in nature and are found in our digestive systems. Although they are 
best known for their role in the preparation of fermented food, LABs produce 
proteolytic enzymes and many of the investigations focused on the degradation of 
milk proteins. The ability to produce extracellular proteinases having MCE is an 
important feature of LAB in biochemical. Uses of animal rennet became limited for 
religious reasons. Most Muslims considered that cheese that is produced by an 
enzyme which comes from an animal slaughtered on the non- Islamic way is not halal 
(Nagodawithana & Reed, 1993). In fact, LABs also possess proteolytic enzymes that 
widely used for produce bioactive compound such as for antimicrobial activity (Belal 
et al., 2012), antifungal activity, bio-preservation (Gobbettia et al., 2002) and 
antioxidant activity (Osuntoki & Korie., 2010). The proteolytic behavior of LAB on 
casein varies according to the bacterial species and environmental conditions (Argyle 
et al., 1976; Giori et al., 1985; Nour et al., 2014). 
28 
Research on production of MCEs from microbial and plant sourcess are reported by 
several researchers. For example MCE were detected present in A. niger (Moosavi- 
Nasab et al, 2010) and R. miehei N (Reps et al., 2006) and Thermomucor indicae- 
seudaticae (Merheb et al., 2010) and A. oryzae (Shata, 2005). Furthermore MCE from 
plants such as Bromelia hieronymi (Mariela et al., 2010) and Solanum dubium (Isam 
et al., 2009) and artichoke flowers Cynara scolymus (Soledad et al., 2007). Attempts 
to study the production of MCE from bacteria have focused on B. subtilis natto (Shieh 
et al., 2009) and B. sphaericus (El-Bendary et al., 2007), Streptomycetes such as 
Streptomyces sp. (Kathiresan & Manivannan, 2007) and S. clavuligerus (Keila et al., 
2001). LAB is the predominant microbial group in fermented food and is important 
because of the key role it plays in fermentation processes. Makarova et al, (2006) 
reported that LAB can be sub-classified into seven groups based on fermented 
functional properties that include Lactobacillus, Lactococcus, Streptococcus, 
Pediococcus, Enterococcus, Leuconostoc and Oenococcus. Limited work has been 
reported on the potential of using LAB as a source of MCE except those reported by 
Hebert et al. (2001) on L. helveticus and Sato et al. (2004) on E. faecalis TUA2495L. 
Characterization of LAB from different sources can be identified by combining the 
phenotypic and genotypic methods (Hebert et al., 2001). Genotypic identification has 
been used as substitute or complement to the established phenotypic methods (Tang et 
al., 1998). Molecular methods have proven to have more specific than the phenotypic 
methods by distinguishing the differences between the two closely related species 
(Busch & Nitschko 1999). The phenotypic identification of LAB by using API 50 
CHL Kit and genotype-based methods such as 16S rDNA has been extensively 
29 
reported (Mohd Adnan et al., 2007; Abd El Gawad et al., 2010; Ali Baradran et al., 
2012). Therefore, the purpose of this study was to evaluate proteolytic activity and 
milk clotting activity of enzyme preparation from LAB isolated from food sources for 
possible application in dairy products. 
1.2 MATERIAS AND METHODS 
1.2.1 Isolation of Lactic Acid Bacteria 
Lactic acid bacteria were isolated from different sources (Malaysian fermented food 
such as Budu, Belacan, Pekasam, fermented cocoa, fermented buffalo milk, and 
yoghurt also from different fruit obtained from local markets and soil). A total of 10 g 
of sample was homogenized in peptone water (Oxoid) using stomacher (Stomacher R 
400 Circular Seward) for 2 min. For fermented fish (Pekasam) and fermented shrimp 
(Belacan) samples were diluted in 6.5% (w/v) NaCl. The samples were appropriately 
diluted and spread plated on modified MRS agar (De Man-Rogosa). The plates were 
incubated anaerobically at 37°C for 48 h. 
1.2.2 Gram Staining 
The method as described in food microbiology (Ahmed & Carolyn, 2004). A sterile 
wire loop was used to slightly touch a colony of each isolate and emulsified in a drop 
of distilled water on a clean slide to make a thin smear. This was air dried, then heat 
fixed. The smear was flooded with crystal violet stain and left for 30 sec, after rising 
under a gently running tap. Two drops of Gram's iodine solution was then added to 
30 
act as a mordant. This was left for 30 sec and washed off under a gently running tap 
95% ethanol was used to decolorize the smear for 10 sec. or until it appeared free of 
crystal violet stain. It was then rinsed under a running tap. Counter staining was done 
with 2 drops safranin for 30 sec and then rinsed off under a running tap. The smear 
was blotted dry and left to air-dry before viewing microscopically under oil immersion 
objective lens. 
1.2.3 Catalase Test 
The method as described in food microbiology methods. A sterile loop was used to 
touch a colony of overnight isolates and transferred onto a clean glass slide. Then a 
drop of 3% hydrogen peroxide (H202) was then added and a reaction was observed. 
Production of gas bubbles as white froth indicates a catalase positive reaction while 
the absence of the troth indicates a negative reaction. All the isolates were maintained 
in 15% glycerol stock at -20°C. 
1.2.4 Screening of Lactic Acid Bacteria for Milk Clotting Activity 
Lactic acid bacteria were sub-cultured in MRS agar was anaerobically incubated at 
37°C for 24 h, anaerobically. Each strain was inoculated into a test tube containing 5 
ml of litmus milk medium (Oxoid) and cultured statically at 30°C for 7 d. Strains 
which formed good clot of casein with clear and little whey were selected for further 
study. 
31 
1.2.5 Characterization of Lactic Acid Bacteria with Proteolytic Activity 
3.2.5.1 Bacterial Growth in Different Concentrations of Sodium Chloride 
Different concentrations 1.5%, 3.5%, 6.5%, and 7.5% of NaCI were dissolved in 100 
ml of MRS broth (Oxoid), respectively, dispensed into screw cap bottles and 
sterilized. The bottles were inoculated with LAB and incubated at 37°C for 48 h. 
Turbidity of the medium indicates growth, while un-inoculated bottles served as 
control. 
3.2.5.2 Bacterial Growth at Different Temperature 
MRS broth was sterilized and inoculated with the LAB isolates and incubated at 10, 
37 and 45°C anaerobically respectively for 72 h. Turbidity of the medium was 
compared with un-inoculated control bottles and served as indicator of growth by the 
isolates. 
3.2.6 Detection of Extracellular Proteinase 
The production of extracellular proteinase was detected following the method 
described by Pailin et al. (2001) and Aween et al. (2012) with some modification as 
follows: 25g of skim milk was reconstituted with 250 ml of distilled water, stirred 
thoroughly and autoclaved at 110°C for 10 min. Agar No. 3 (Oxoid) 500 ml of 2.5% 
(w/v) was sterilized at 121 °C for 20 min then mixed with skim milk and poured into 
petri dish. Lactic acid bacteria was spot inoculated on prepared skim milk agar media 
32 
then incubated at 37°C for 48 h in an anaerobic jar followed by cooling room at 4°C 
for 3 d. Protein digestion was observed by the production of clear haloes surrounding 
the colonies. 
3.2.7 Identification of Lactic Acid Bacteria 
3.2.7.1 Carbohydrate Fermentation Profile using API50 CH of Selected 
Lactic Acid Bacteria Isolates 
The API 50 CHL strips (API systems, Biomerieux, France) consisting of 50 
microtubes were used to identify the LAB isolates based on carbohydrate fermentation 
profile. Overnight bacteria cultures were incubated according to the manufacturer's 
instructions and the bacterial suspension was prepared with the turbidity equivalent to 
McFarland 2 standard. The strips of the API 50 CHL test were filled with the 
inoculated API suspension and overlaid with mineral oil. The inoculated strips were 
incubated at 37°C and the reactions were observed after 24 and 48 h of incubations. 
The results were analyzed by API software version (BioMerieux). 
3.2.7.2 Identification of Lactic Acid Bacteria using PCR 
3.2.7.2.1 DNA Extraction of Lactic Acid Bacteria Isolates 
Genomic DNA of LAB isolates was extracted using (Genomic DNA Extraction Mini 
Kit, Yeastern Biotech CO., LTD, Taiwan) as with manifesting instructions with some 
modifications. Briefly, 1500 gl of overnight culture was harvested by centrifuging at 
33 
14,000 xg for 1 min using (Eppendorf centrifuge 5804 R) and the supernatant was 
discarded. The cell pellet was re-suspended in 200 gl of lysozyme and then incubated 
at room temperature 25°C for 10 min with shaking the tubes every 2 to 3 min. 200 µl 
of GB buffer was added to the sample, and mixed by vortexing for 5 sec. The LAB 
samples were incubated at 37°C for 20 min until the sample lysate was clear. During 
incubation, the sample tubes were upturned every 3 mins. At that time, elution buffer 
(200 pl per sample) incubated at 60 °C (for DNA elution). Following that, a volume of 
200 gl of the ethanol was added to each sample list and mixed immediately by 
vortexing for 10 sec. GD column was placed on a2 ml collection tube. Then all the 
mixture from the previous step was applied to the GD column, and was then 
centrifuged at 14000 xg for 2 min. The flow-through was discarded and the GD 
column placed in a new 2 ml collection tube. 400 pl of W1 buffer (ethanol added) into 
the GD column, and then centrifuged at 14000 xg for 30 sec. the flow-through was 
discarded and the GD column placed back in the 2 ml collection tube, the wash step 
was repeated with 600 µl of wash buffer (ethanol added) with centrifuge at 14000 xg 
for 30 Sec. the flow-through was discarded and the GD column placed back in the 2 
ml collection tube, then centrifuged at 14000 xg for 3 min to dry the column matrix. 
The dried GD column was transferred into a clean 1.5 ml micro-centrifuge tube, and 
was then preheated with 100 µl elution buffer was added in the centre of the column 
matrix. Stand for 3 to 5 min until elution buffer absorbed by the matrix. The purified 
DNA was eluted by centrifuging at 14000 xg for 30 sec. Finally, the DNA sample 
was kept at -20°C for analysis. 
34 
3.2.7.2.2 PCR Amplification of 16S rDNA gene Sequence of Lactic Acid Bacteria 
Isolates 
The extracted DNAs were used directly in PCR reactions to amplify the 16S rDNA 
gene from LAB. The 16S rDNA region was amplified by using primers (5- 
AGAGTTTGATCCTGGCTC-3) and 16S reverse: (5-CGGGAACGTATTCAC-CG- 
3). The PCR reaction mixture contained 5 gl of 1X reaction buffer (NgCL2) (Yeastren 
Biotech CO., LTD. Taiwan), 1 µl of 0.2 mM dNTPs mix (Yeastren Biotech CO., 
LTD. Taiwan), 1 µl of 10 µM 16S forward and 1 µl of 10 pl 16S reverse primers, 1 µl 
of template DNA, and 0.5 µl of (1.25 U/ µl) YEAtaq DNA polymerase (Yeastren 
Biotech CO., LTD. Taiwan). A negative control without DNA template was included 
in parallel. Each sample toped up with 40.5µl ddH2O until 50 µl. The PCR were as 
follows: (LID 105°C), initial at 95°C for 3 min, denaturation at 95°C for 30 sec. 
annealing at 57°C for 15 sec. and extension at 73°C for 1 min, with 35 cycles for each 
step. Final elongation was at 73°C for 5 min; and then held at 4°C. From each 
amplification mixture 10 gl were mixed with 2 gl of 6X DNA loading dye 
(Fermentans), and then subjected to electrophoresis in 1.0% (w/v) agarose gels (Conda 
S. A, Spain) in lx TAE buffer (Bio Basic Canada INC, Canada) for 40 min and 80 V. 
6 pl of l Kb DNA ladder (250 to 10000 bp) from (1 s` Base, Malaysia) was used as 
standard. After electrophoresis the gel was stained in ethidium bromide (Merck, 
Germany) for 30 min and then washed with distil water for another 30 min. The gel 
was visualized and photographed with UV transilluminator (BIORAD). 
35 
3.2.7.2.3 PCR Product Cleanup 
The PCR product was purified using PCR clean up kit from (Yeastem Biotech, 
Taiwan). Initially 40 pl of the PCR product added into a micro-centrifuge tube with 
200µl of DF buffer and mixed by vortexing the tube. The sample was applied into DF 
column with collection tube, and then centrifuged at 6000 xg for 30sec. The flow- 
through was discarded and the DF column placed back in the collection tube. The 
centrifugation was repeated at 14000 xg for 2 min. the dried DF column was 
transferred to a new micro-centrifuge tube. 15µl of the elution buffer was added in the 
centre of the DF column matrix, stand for 2 min until the elution buffer absorbed by 
the matrix. Purified DNA was eluted at 14000 xg for 2 min. 
3.2.7.2.4 PCR Product Sequencing 
The partial 16S-S DNA and 16 S-R DNA PCR product of isolates was sequenced by 
sending to I" Base, Malaysia and sequences were compared (analyzed) by the BLAST 
program on the NCBI website databases (http: //www. ncbi. nlm. nih. gov/). (Gen- Bank). 
3.2.8 Production Media for Milk Clotting Enzyme 
The enzyme production media consisted of lOg tryptone soya, lOg glucose, 2g 
CH3000Na"3H2O, 0.2g MgS04.7H20,0.01g MnSO. 4H20,0.01g FeSO4.7H2O, 
0.01g NaCl , dissolved 
in 1000 ml of distilled water and adjust the pH to 6.8 using 
NaOH (0.1 M). One ml of the 24 h pre-cultured broth of LAB was inoculated into 
36 
Erlenmeyer flask containing 100 ml of the enzyme production medium with or 
without 1% CaCO3 previously sterilized by autoclaving at 121 °C for 15 min. The 
enzyme production, media with CaCO3 was gently shaken in a shaker incubator 
(SASTEC- MODEL-ST-200R) at 150 kept at 30°C for 48 h, followed by centrifuging 
at 9,000 xg for 20 min at 4°C. The supernatant was collected and filtrated regenerated 
cellulose membrane filter (filter size 0.22 µ (SARTORNS STEDIM BIOTECH. ) for 
MCA and PA measurement, and determination of MCA/ PA ratio (Sato et al., 2004). 
3.2.9 Milk Clotting Enzyme Assay 
Enzyme assay was carried out according to the method described by Arima et al. 
(1968) and Sato et al. (2004). Milk clotting activity was expressed as Soxhlet units 
(SU) in which 1 SU is defined as the amount of enzyme that clots 1 ml of a solution 
containing 0.1 g skim milk powder and 0.00111 g CaC12 in 40 min at 35°C. Under 
this assay condition, 400 units of activity (SU) is defined as the amount of enzyme 
that clot the milk solution in 1 min. Soxhlet unit is defined as the amount of enzyme 
(400 unit of activity) which clotted milk solution in 1 min. 
Skim milk (10% w/v, Oxoid) containing 10 mM CaC12 was used as a substrate, pre- 
incubated at 35°C for 5 min. Enzyme extract 0.5 ml was added to 5 ml of substrate 
solution, mixed well and incubated at 35°C. The time t (sec), the time period starting 
from the addition of the enzyme to the first appearance of clots of milk solution was 
recorded. MCA was calculated using the following formula. 
SU = 2400/Tx SIE whereas, 
37 
T= clotting time (sec), S= substrate solution (ml), E= enzyme solution (ml) 
3.2.10 Proteolytic Activity of Milk Clotting Enzyme 
The method described by Sato et al. (2004) was used to determine the proteolytic 
activity of the cell free enzyme production media. Hammerstein casein 1% was 
dissolved in 0.1 M Tris - Hcl buffer pH 7.5 as substrate, and 5 ml of this substrate 
solution was incubated with lml of crude enzyme solution at 45°C for 30 min; then 
the enzyme reaction was stopped with 5 ml of trichloroacetic acid mixture (0.11 M 
CCI3000H, 0.22 M CH3COONa and 0.33 M CH3COOH). After incubation for 30 
min the mixture was filtered using Whatman filter paper No. 1, and 2 ml of the filtrate 
was added to 5 ml of 0.55M Na2CO3 and 1 ml of Folin's reagent (diluted 1: 3). 
The reaction mixture was held at 30°C for 30 min and the optical density at 660 nm 
was measured using UV-Visible Spectrophotometer (4001/4, Thermo Spectronic, 
USA). One unit of proteolytic activity is defined as the amount of enzyme which 
released 1 pg of amino acid expressed as the tyrosine concentration per min under the 
above condition. The change in absorbance in test samples was determined by 
calculating the difference between test samples absorbance and absorbance of test 
blank. Inserting the absorbance value for one of the test samples into the slope 
equation, micromoles of tyrosine liberated during the proteolytic reaction was 
determined as follows. 
Units/ml enzyme = (: t mole tyrosine equivalents released) x (VI) 
(V2) (T) (V3) 
38 
Where: - 
VI = total volume (in ml) of assay 
T= time of assay in min as per the unit defined 
V2 = volume of enzyme (in ml) used 
V3 = volume (in ml) used in colorimetric determination 
3.2.11 Statistical Analysis 
A one way analysis of variance (ANOVA) using MINITAB version 16 was carried 
out and the P value was set 0.05 (P<0.05) for significant differences. Tukey's pairwise 
comparison was used to compare mean differences on the effects of different LABs 
isolates and their interactions on clotting time, milk clotting activity, proteolytic 
activity and ratio of milk clotting activity to proteolytic activity. 
3.3 RESULTS 
3.3.1 Isolation and Characterization of Lactic Acid Bacteria 
A total of 135 LAB was isolated from different sources of fermented food. The 
isolates were classified based on their morphology and biochemical characters all 
colonies exhibiting clear halos on MRS agar with 0.8% CaCO3 the isolates were Gram 
positive and catalase negative (Table 1). 
39 
Tablel: Characterization of LAB Isolates 
Isolate Isolate Number Gram Catalase Morphology 
code sources of isolates staining test 
FFB1 Budul 7+- Cocci 
Budul 4+- Short rod 
FFA I Budu2 3+- Cocci 
Budu2 6+- Short rod 
Budu2 4+- Bacilli 
Belacan 5+- Cocci 
SH Belacan 4+- Short rod 
Belacan 3+- Bacilli 
CFI Pekasam 7+- Cocci 
Pekasam 5+- Short rod 
Pekasam 3+- Bacilli 
COl Fermented 2+- Cocci 
cocoa 
Fermented 4+- Short rod 
cocoa 
Fermented 4+- Bacilli 
cocoa 
YOl Yoghurt 2+- Cocci 
Yoghurt 5+- Short rod 
Yoghurt 4+- Bacilli 
AS. A Ginger 3+- Cocci 
Ginger 2+- Short rod 
Ginger 5+- Bacilli 
G007 Guava 2+- Cocci 
Guava 6+- Short rod 
Guava 5+- Bacilli 
H6M Buffalo 8+- Cocci 
milk 
Buffalo 4+- Short rod 
milk 
Buffalo 9+- Bacilli 
milk 
SO Soil 4+- Cocci 
Soil 6+- Short rod 
Soil 5+- Bacilli 
Total 135 
40 
Figure 3: Lactic acid bacteria on MRS agar with CaCO3 
3.3.2 The Effect of Temperature and Sodium chloride on Growth of Lactic Acid 
Bacteria Isolates 
All the isolates grew at 10,37 and 45°C, but growth of FFA1, SH, SO was not 
observed at 10°C (Table 3). Growth at 1.5% NaCl was observed for all LAB isolates, 
however, their growth was inhibited at higher NaCl concentrations except FFA, SH 
and CFI (Table 2) which were isolated from Malaysian fermented food (Belacan and 
Pekasam). 
Table 2: The Effect of Temperature and NaCl on Growth of LAB Isolates* 
Isolate code Growth temperature (°C) Growth in NaCI (%) 
10 37 45 1.5 3.5 5.5 6.5 7.5 
FFA I-+++++++ 
SH -+++++++ 
CF1 -+++++++ 
Col ++++---- 
YO1 ++++---- 
AS. A ++++---- 
G007 ++++---- 
H6M ++++---- 
SO -+++---- 
*(+) growth (-) No growth 
41 
3.3.3 Detection of Extracellular Proteinase and Screening for Milk Clotting 
Activity by Litmus NEW Test 
Ten of 135 LAB that produces extracellular proteinase enzyme on skim milk agar 
were evaluated for their ability to clot milk based on litmus milk test. Result found 
that, the isolates SH and CF1 isolates milk clot that was large, soft with very little 
amount of whey (Table 3). 
Table 3: Proteolytic Activity on Skim Milk Agar and Litmus Milk Test 
Isolate code Casein hydrolysis Litmus milk test 
FFB 1+ Yellow weak curd 
FFA I+ Yellow weak curd 
SH + Purple soft curd 
CFI + Purple weak curd 
CO1 + No change 
YOI + No change 
AS. A + Pink weak curd 
G007 + No change 
H6M + Pink weak curd 
SO + No change 
Figure 4: Plate Showing Figure 5: Screening of LAB 
Proteolytic Activity for MCA by Litmus Milk Test 
42 
3.3.4 Identification of Lactic Acid Bacteria Isolates 
3.3.4.1 Identification of Lactic Acid Bacteria Isolates by (API 50 CH KIT) 
The results in Table 4 show that the identification of the isolates was performed 
through the fermentation of the carbohydrates in an API CH 50 kit (BioMeurix 
France). 
Table 4: Isolates Identification using the API 50 CHL kit 
Source Code Strain 
of isolates 
%ID T 
Belacan SH Pediococcus acdilactici 99.9 0.80 
Pekasam CF1 Lactobacillus lactis cremoris 93.7 0.66 
3.3.4.2 Identification of Lactic Acid Bacteria by (16s rDNA gene Sequences) 
Result of sequencing analysis of purified PCR showed that the isolates SH and 
CF 1 were isolated from belacan and pekasam identified as Pediococcus acdilactici and 
Lactobacillus paracasei (Table 5). 
Figure 6: 16S. S: (5-AGAGTTTGATCCTGGCTC-3) and 16S. R: (5- 
CGGGAACGTATTCACCG-3), (1) Lanelkb, DNA ladder; (2) SH (3) CF1. 
1400 bp 
43 
Table 5: Lactic Acid Bacteria Identified using 16S rDNA Sequencing 
Code of isolates Identification identification. 
SH Pediococcus acidilactici 99.7% 
C. F1 Lactobacillus paracasei 99.0% 
3.3.5 Milk clotting Activity and Proteolytic Activity of crud enzyme of Lactic 
Acid Bacteria Isolates 
Ten from 135 LAB isolated from different food sources exhibited extracellular 
proteolytic enzyme on skim milk agar were further evaluated for their MCA. It was 
observed that only six of the ten isolates namely SH, CF I, FFA, FFB, H6M, and ASA 
showed MCA (Table 6). The isolates SH and CFI showed lowest milk clotting time 
(72 and 82 Sec, respectively), and the highest MCA (50 SU/ml and 43 SU/ml, 
respectively) also low PA (1.9 ml and 3.5 ml, respectively). The ratio of MCA/PA of 
SH and CF1 was higher than other isolates. Other isolates, FFA, FFB, AS. A, and 
H6M showed longer clotting time (104,109,96, and 118 sec. respectively and PA 
(3.6 ml, 3.8 ml, 3.8 ml and 3.5 ml, respectively. MCA of these LAB isolates was low 
(34 SU/ml, 32 SU/ml, 32 SU/ml, and 30 SU\ml, respectively). Higher MCA/PA ratio 
is desirable for milk clotting enzyme and it generally indicates the restricted 
degradation of casein. Therefore, SH and CF1 isolates were selected for further 
experiments because of its high MCA/PA ratio 26.3 and 12.6, respectively. 
44 
Table 6: MCA, PA of Crude Enzyme of LAB Isolated from Different Sources 
Code of isolates 
AS25 
CFI 
SH 
Col 
FFA 
FFB 
so 
G007 
H6M 
ASA 
Clotting time(s) 
0.009 
82.0' 
72.0E 
0.009 
104.0° 
109.0b 
0.009 
0.009 
96.0d 
118.0a 
MCA(SU/ml) PA(U/ml) MCA/PA 
0.00 3.20ab 0.00` 
44.06 3.50ab 12.6b 
50. Oa 1.90c 26.3a 
0.00d 3.10b 0.00C 
34.0° 3.60ab 9.40b 
32. Oc 3.80ab 8.40b 
0.00d 3.20ab 0.00C 
0.00d 3.00b 0.00° 
32.0° 3.90a 8.40b 
30.0c 3.50ab 8.50b 
*(a-g) Means in the same column followed by different letters were significantly different (P<0.05). 
3.4 sDISCUSSION 
Lactic acid bacteria play an important role in successful fermentation of foods such as 
milk, fish, meat and vegetables. The production of organic acids and other compounds 
contribute to the antimicrobial activity of LAB and thus make fermented food safe 
(Saranraj et al., 2013). LABs also influence desirable flavor and texture changes in the 
product (Rhee et al., 2011). In yogurt for example, addition of LAB starter cultures 
reduce the pH, resulting in acidification of milk and precipitation of casein and whey 
protein as well as for flavor development (Jaiswal et al., 2014). In cheese however, 
rennin is added to coagulate milk casein and non-starters LAB are added for flavor 
and modifying the texture of cheese during aging (McSweeney & Sousa 2000). 
Proteolysis has been widely used as a basis for classification of cheese (Sousa & 
McSweeney, 2001). In present study ten of 135 LAB were able to produce 
extracellular proteinase as indicated by clear zone surrounding the colonies on skim 
45 
milk agar plate. Six of the ten isolates namely SH, CF1, FFA, FFB, H6M, and AS. A 
have the ability to produce MCE with milk clotting activity. Thus the MCE obtained 
from strain SH produced high milk clotting activity and, ratio of MCA/PA was (50 
SU/ml) and 26.3, respectively compared with other strains (Table 6). The MCA 
obtained from SH strain was higher than that E. faecalis 2495L, E. faecalis IAM10065 
(28.3 SU/ml) and E. faecalis 156 (22.7 SU/ml) with MCA/PA 3.8 as reported by Sato 
et al. (2004). However, Merheb et al. (2010) reported that MCA and MCA/PA 
obtained from T. indicae-seudaticae N31 were (160.3 SU/ml) and 76, respectively. 
Higher MCA/PA ratio is desirable for milk clotting enzyme and it generally indicates 
the restricted degradation of casein (Sato et al., 2004). 
Milk clotting time is affected by the enzyme, concentration of the enzyme, pH, 
temperature and Ca 2+ and species of LAB also depending on the proteolytic system of 
LAB (Smit et al., 2002; Leitner et al., 2006). In this study the time required to 
coagulate milk for the extracellular enzyme produce by SH isolate was 72 sec. less 
than the other isolates, but similar to S. esculentum 73 sec. as reported by Guiama et 
al, (2010). LAB with proteolytic activity and MCA were reported by several 
researchers, for example, L. helveticus (Hebert et al., 2001), L. paracasei (Haq-ul & 
Mukhtar, 2006) and E. faecalis 2495L, E. faecalis IAM10065 and E. faecalis 156 
(Sato et al. (2004). Milk clotting activities of microorganism have different 
characteristics and values. Fungi especially Mucor, Rhizopus, Endothia, Rhizomucor 
and Aspergillus have been used for production of MCE. Some Bacillus spp. notably B. 
subtilis, B. cereus, B. licheniformis (D'Souza & Pereira, 1982; Dutt et al., 2008) and 
others like Myxococcus xanthus strain 422 (Poza et al., 2003) and Nocardiopsis 
46 
(Cavalcanti et al., 2005) were reported to produce enzyme with MCA. LAB has also 
been reported as MCE producers such as L. helveticus (Hebert et al., 2001). Four from 
157 LAB (E. faecalis 2495L, E. faecalis IAM10065 and E. faecalis 156) were isolated 
from different fermented food produces MCE with milk clotting activity (MCA) (Sato 
et al., 2004). 
The ability to produce extracellular proteinases is a very important feature of LAB. 
This is because most of LAB isolated from fermented products has multiple amino 
acid auxotrophy and in order to grow in the protein rich medium they depend on the 
expression of a complex proteolytic system for the degradation of protein (Kok, 1993; 
Visser, 1993). In addition, proteinase play significant role in the formation of cheese 
texture due to protein degradation (Olson, 1990). Increasing demand for dairy product, 
low availability of rennet has led to the replacement of calf rennin by microbial milk 
clotting enzymes (Kurutahalli et al., 2010). A crude enzyme from SH and CF1 isolates 
showed the highest milk-clotting activity and lowest proteolytic activity. In contrast, 
the other isolate FFB FFA, ASA and H6M showed high proteolytic activity but low 
MCA. Fungi especially Mucor, Rhizopus, Endothia, Rhizomucor, and Aspergillus 
have been used for production of MCE. Some Bacillus sp. notably, B. subtilis, B. 
cereus, B. licheniformis (D'Souza & Pereira 1982; Dutt et al., 2008) and others like 
Myxococcus xanthus strain 422 (Poza et al., 2003) and Nocardiopsis (Cavalcanti et al., 
2005) were reported to produce an enzyme with MCA. 
High proteolytic activity normally leads to lower yield, soft body and bitter taste of 
milk curd (Chazarra et al., 2007; Vishwanatha et al., 2010). The cheeses obtained by 
47 
curdling milk with molds produced rennet, develop bitterness when maturation period 
is long, this being a constraint in the use of this microbial rennet (Samson et al., 2004). 
Therefore, the ratio of MCA to PA is used as an indicator for selecting the potential 
strain. 
Many LABs have been isolated from fermented seafood such as belacan, budu, 
cincaluk, pekasam, trassi, kicap, tauco, tompoyak and sambal belachan (Ohhira et al., 
1990). Varying concentrations of salt are added to modify the environment in 
fermentation of traditional fermented foods. The salt concentration in belacan is 1.7% 
budu 8.66% and pekasam 1.5% (Ohhira et al., 1990). This will influence the growth 
requirement of LAB present in these products. It was observed that SH isolated from 
belacan. LAB has ability to grow in NaCl concentration ranging from 1.5 to 10% salt 
concentration (Nanasombat et al., 2012). Salt is normally added in fish fermentation. 
The LAB isolated from pekasam, belacan and budu tolerated 7.5% NaCl compared to 
other LAB isolates. Papamanoli et al. (2003) reported that seven L. planetarum strains 
isolated from naturally fermented dry sausage have the ability to grow in 6.5,8.0, and 
10% (w/w) of NaCl. Similarly, Cho and Ki, (2006) isolated halo-tolerant strains of 
Leuc. mesenteroides and Str. salivarius from Korea fermented food. Jeotgal is made 
from raw seafood and fish by traditional methods with 6.5% NaCl. The salt tolerance 
gives them an advantage over other less tolerant species and allows the lactic acid 
fermenters to begin metabolism, which produces acid, which further inhibits the 
growth of non-desirable organisms (Oyewole & Isah, 2012). Growth of LAB at 
specific temperature and NaCl concentrations are used to group between thermophillic 
cocci and enterococci LAB that grow at 4°C and 6.5% NaCl are considered as 
48 
thermophilic cocci, while those that grow at 10°C and 45°C in the presence of 6.5% 
NaCl were considered as enterococci (El-Soda et al., 2003). 
The identification of LAB based on the physicochemical and biochemical 
characteristics is often considered as unreliable, since different species may have 
similar morphology and nutritional requirements (Ben-Amor et al., 2007). Therefore, 
the phenotypic characterization based on carbohydrates fermentation profile may be 
used as a possible identification, but it may not provide trusty identification. The 
identification method, genotype-based methods such as 16S rDNA are strong to 
identify bacteria as a complement or alternative to phenotypic methods (Weisburg et 
al., 1991). Genotype methods are independent from variation of growth conditions if 
species-specific primers or probes are available; these offer a very fast way to detect 
the target organism. It is clear from the above discussion that a careful consideration 
of numerous factors has to be made for the correct identification of specific species of 
LAB (Gyu Sung et al., 2006; Mohd-Adnan et al., 2007; Abd El Gawad, el al., 2010; 
Ali Baradran et al., 2012). 
Based on the biochemical and API CHL 50 Kit, the isolates of SH and CF1 showed 
99.9 % and 93% homology to the Pediococcus acidilactici, and Lactobacillus lactis 
crenroris, respectively. However, with 16S rDNA sequence analysis methods, the 
isolated LAB was 99.3% and 99% was identified be Pediococcus acidilactici and 
Lactobacillus paracasei. The biochemical and API CHL50 Kit compared to 16S 
rRNA, the result of this study using molecular methods very important for typing 
newly isolated microorganisms. 
49 
3.5 CONCLUSION 
Dairy industry seeks for novel enzyme sources, and microbial coagulants have several 
advantages over animal and plant source. Lactic acid bacteria are used in the 
production of a wide range of dairy products and have influences on flavor 
development and ripening. In this study a total of 135 LABs isolates were isolated 
from different Malaysian fermented foods were screened for produce extracellular 
enzyme based on skim milk agar. Two LBAs isolated from belacan, and pekasam 
were identified by (API 50CHL) and confirmed by the sequence analysis of 16S 
rDNA gene as Pediococcus acidilactici SH, and Lactobacillus paracasei CFI. The 
results show the properties of the extracellular enzyme produce by (SH) and (CF 1) 
isolates that indicate the possibility of the use of these extracellular enzymes in the 
dairy manufacture.