Morphology and growth on selective media
Four Lactobacillus spp.were isolated from the four types offermented yoghurt drinks. Lactobacillus spp. produced creamy whitish colonies on an MRS agar plate (Figure 1) but E. coli showed no growth. The isolates were labeled as A, B, C, and D.
Biochemical Characterization
All of the isolates were characterized according to Bergey's manual of systematic Bacteriology(Buchanan & Gibbons, 1984)(Bergey, 1994).Creamy whitish colonies were further identified for biochemical analysis. All isolates were gram positive, catalase negative, citrate positive, able to coagulate milk, produces no gas from glucose, showed positive results in starch hydrolysis test and negative in bile esculin test (Figure 2, Figure 3). Different morphological and biochemical charateristics are shown in Table 1.
Table 1: Morphological and biochemical characterization of the isolates
Morphological and Biochemical Characteristics
|
Isolate A
|
Isolate B
|
Isolate C
|
Isolate D
|
Morphological Characteristics
|
Cell shape
|
Cocci
|
Cocci
|
Rod
|
Rod
|
Size
|
Small
|
Small
|
Small
|
Small
|
Form
|
Round
|
Round
|
Round
|
Round
|
Surface
|
Shiny
|
Shiny
|
Smooth
|
Smooth
|
Color
|
White creamy
|
White creamy
|
White creamy
|
White creamy
|
Opacity
|
Opaque
|
White creamy
|
White creamy
|
White creamy
|
Gram staining
|
+
|
+
|
+
|
+
|
Biochemical Characteristics
|
MIU
|
-
|
-
|
-
|
-
|
Catalase test
|
-
|
-
|
-
|
-
|
Citrate test
|
+
|
+
|
+
|
+
|
TSI test
|
-
|
-
|
-
|
-
|
Gas from glucose
|
-
|
-
|
-
|
-
|
Milk coagulation test
|
+
|
+
|
+
|
+
|
Starch hydrolysis test
|
+
|
+
|
+
|
+
|
Bile esculin test
|
-
|
-
|
-
|
-
|
+ indicate Positive growth, - indicate negative growth
Screeing for probiotics properties
pH tolerance test
A pH range of 2 to 8 was used in this investigation to observe growth. Based on the results of the experiment, it was found that isolated Lactobacillus spp. from fermented yoghurt beverages can survive in both acidic pH (5 to 6) and basic pH environments (pH 7.5 to 8).Isolates tolarence activity at different pH are shown in Table 2.
Table 2: pH tolerance test
Isolates
|
Optical Density at 620 nm
|
pH 2
|
pH 3
|
pH 4
|
pH 5
|
pH 6
|
pH 7
|
pH 8
|
A
|
0.482±0.0053
|
0.202±0.0065
|
0.454±0.0055
|
1.484±0.0058
|
1.742±0.0085
|
1.856±0.0042
|
1.857±0.0044
|
B
|
0.379±0.0026
|
0.254±0.0057
|
0.277±0.031
|
1.372±0.0038
|
1.77±0.036
|
1.759±0.027
|
1.86±0.019
|
C
|
0.181±0.0055
|
0.166±0.008
|
0.141±0.004
|
1.388±0.027
|
1.57±0.054
|
1.846±0.056
|
1.86±0.065
|
D
|
0.179±0.0056
|
0.173±0.0055
|
0.149±0.0076
|
1.267±0.0049
|
1.464±0.014
|
1.88±0.01
|
1.60±0.012
|
Values are the mean±standard deviation of at least 3 determination
Phenol tolerance test
Four isolates showed a higher growth rate at the concentration of 0.1%. All of the isolates showed the lowest growth rate at 0.3% concentrations. Survival rate at 0.1 and 0.2% concentrations of isolate A and isolate B was higher than Isolate C and Isolate D (Figure 4). Isolate D showed higher growth at 0.4% phenolic concentration..
NaCl tolerance test
At 2%NaCl, the four isolates showed higher growth than the other two concentrations. Isolate A, B and D showed moderate growth at 4% NaCl concentration (Figure 5). At 8%, isolate C showed no growth as the absorbance was lower than the control. This bacterial species was sensitive to higher concentrations of NaCl. But the other three species showed a little growth at 8% NaCl concentrations.
Determination of optimum temperature
Isolate A was able to survive at temperature 25°C, 30°C, 35°C and 40°C and showed growth in MRS agar plate. Wheras isolate B, C, Dshowed growth in MRS agar plate at temperature 25°C, 30°C, 35°C but not in 40°C. Image of representative isolates were shown in figure 6, 7.
Safety analysis
Hemolytic activity
When screened for hemolytic activity, every isolate showed a negative result (Figure 8), indicating that lactic acid bacteria are safe for use by humans.
Antibiotic Sensitivity Test
The disc diffusion method was used to analyze the antibiotic sensitivity pattern of some isolates in order to see the inhibitory effect of any antibiotics against them.Isolates A, B showed resistance against kanamycin (30 µg) whereas isolates C, D showed resistance agaistvancomycin(30 µg)(Table 3).Some images of antibiotic sensitivity pattern of isolates on MHA (Mueller Hinton Agar)were shown in Figure 9.
Table 3: Assay of antibiotic sensitivity pattern
Name of Antibiotic
|
Zone of inhibition (mm)
|
Isolate A
|
Isolate B
|
Isolate C
|
Isolate D
|
Vancomycin (30µg)
|
S
|
S
|
R
|
R
|
Erythromycin (15 µg)
|
I
|
I
|
S
|
S
|
Gentamicin (10 µg)
|
I
|
I
|
S
|
S
|
Tetracycline (30 µg)
|
S
|
S
|
S
|
S
|
Cefoxitin (30 µg)
|
S
|
S
|
I
|
S
|
Amikacin (30 µg)
|
I
|
I
|
S
|
S
|
Ciprofloxacin (5 µg)
|
S
|
S
|
S
|
S
|
Kanamycin (30 µg)
|
R
|
R
|
S
|
S
|
S=Sensitive, R=Resistant, I=Intermediate
Evaluation of antagonistic activity
The isolates were tested for antimicrobial activity against some common pathogenic microorganisms. Isolates A, B, C, D showed maximum zone of inhibition against E. coli and S. aureus whereas all isolates showed resistance against S.typhi and Pseudomonas spp. Each isolates antimicrobial activitywereshown in table 4.
Table 4: Antagonistic activity of isolates against common pathogenic microorganisms
Isolates name
|
Test organisms with zone of diameter (mm)
|
S aureus
|
Enterococcus spp
|
Bacillus spp
|
E. coli
|
S. typhi
|
Pseudomonas spp
|
Klebsiellaspp
|
Serratiaspp
|
Candida albicans
|
Aspergillusspp
|
Trichodermaspp
|
Isolate A
|
++
|
+
|
+
|
++
|
-
|
-
|
-
|
+
|
++
|
+
|
+
|
Isolate B
|
++
|
+
|
+
|
++
|
-
|
-
|
+
|
+
|
+
|
+
|
-
|
Isolate C
|
++
|
+
|
+
|
++
|
-
|
-
|
+
|
-
|
+
|
++
|
+
|
Isolate D
|
++
|
+
|
+
|
++
|
-
|
-
|
+
|
-
|
+
|
+
|
+
|
Zone diameter: - indicates o mm; + indicates (1-5 mm); ++ indicates 6-12mm
Growth rate study
After a certain period, isolate A, isolate B and isolate C showed stationary growth rates but isolate D was different (Figure 10). It showed an increase and decrease from time to time. In the maximum case, the growth of the bacterial species was slowed down after four or five hours. Then their growth level was indifferent.