Int.J.Curr.Microbiol.App.
Sci (2017) 6(7): 3975-3980
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 6 Number 7 (2017) pp. 3975-3980
Journal homepage: http://www.ijcmas.com
Original Research Article https://doi.org/10.20546/ijcmas.2017.607.411
Isolation and Screening of Lactic Acid Bacteria for
Acidic pH and Bile Tolerance
A.S. Noor Nawaz1*, K.S. Jagadeesh1 and P.U. Krishnaraj2
1
Department of Agricultural Microbiology, Agriculture College,
Dharwad-580 005, Karnataka, India
2
Department of Agricultural Microbiology, Agriculture College, Vijayapura-586 101,
Karnataka, India
*Corresponding author
ABSTRACT
Keywords As many as 25 lactic acid bacterial strains were isolated from various
sources. These isolates were investigated for their tolerance to low pH (3.0,
Lactic acid
bacteria, Isolates, 4.0 and 5.0) and different bile concentrations (0.3 %, 0.4 %, 0.75 % and 1.0
Probiotics, %). Among 25 isolates, strain 42, 45 and 48 showed very high tolerance to
Acid tolerance, acidic pH and the same isolates also tolerated high level of bile salt
Bile tolerance.
concentration up to 1.0 per cent. Thus, high resistance to low pH values and
Article Info to higher concentrations of bile salts enables these strains to survive in the
Accepted: stomach and intestinal environment and to colonize the gut of the host,
29 June 2017 possess a promising probiotic potential. Hence, these isolates hold potential
Available Online:
10 July 2017 to be used as probiotics.
Introduction
Lactic acid bacteria (LAB) are Gram positive, neutraceuticals has attracted new research
non-spore forming, Catalase negative, acid arena for food scientists and health
tolerant, fastidious, non-motile and facultative professionals (Mozzi et al., 2010; De Vuyst et
anaerobic friendly gut bacteria. They al., 2004).
constitute heterogeneous group of industrially
important bacteria. In food industries, they Traditionally, the fermented foods serve as
used as preservative, acidulant and flavouring good sources of new potential sources of
agents by the virtue of their probiotic traits. LAB. Therefore use of strains of such origin
They are also in use as starter culture in food would be of great importance, consumers who
fermentations such as beverages, yoghurt, suffer from lactose intolerance. In order to
vegetables, cereals, meat, cocoa beans etc. exert its beneficial effect on the host, a
(Doyle and Beauchat, 2007). They are also probiotic strain must be able to survive the
utilized in the development of functional gut passage of humans to reach to the action
foods and more specifically their application site in viable state and should be in sufficient
as vaccines, pro and prebiotics, population (normally 107 cells per ml). Thus,
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Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 3975-3980
in order to survive in and colonize the gut, the Test for bile salt tolerance
bacteria should express high tolerance to low
(acidic) pH and high tolerance to higher Similarly, in order to determine the bile
concentrations of bile (Kirjavainen et al., tolerance potential of LAB isolates two ml of
1998). They are supplemented as probiotics the overnight grown cultures of the test strains
that are live microbial food supplements were centrifuged (10 min at 8000 rpm) using
which beneficially affect the host by centrifuge and the cellular pellet was
improving the intestinal microbial balance. resuspended in two ml of MRS broth
Hence, as many as 25 isolates of lactic acid previously adjusted with bile salts (Oxgall
bacteria were obtained from various sources Himedia) at different levels such as 0.3 %, 0.4
such as traditional artisan curds, fermented %, 0.75 % and 1.0 %.
vegetables, dosa and idli batter, etc. The
present work was aimed to evaluate the The inoculated cultures were incubated at 370
probiotic potential of these LAB isolates in C for 3h. The initial bacterial concentration
terms of acid and bile tolerance properties. and after incubation up to 3 h was recorded
using optical density spectrophotometer at
Materials and Methods 650 nm, the counts were tabulated as CFU per
ml (Table 4). The control treatment was also
Isolation of LAB strains, media and culture maintained and incubated in MRS broth
conditions without any bile salts.
As many as 25 LAB strains were isolated Enumeration of viable LAB
from various sources such as traditional
fermented arisan curds of Karnataka such as Viable cells were enumerated by plating 10-
Kohlar, Banashankari, Almatti areas, fold dilution on MRS agar medium. The
fermented vegetables etc. by the method of plates were incubated at 370C in an incubator
plate count method. The 48 h grown culture for 3 h.
was further purified and maintained on de
Mann Rogosa Sharpe broth (MRS) (De Man The population was enumerated at initial
et al., 1960) at -800 C by DMSO (immediately after inoculation) and final
(cryoprotectant) method. counts at 90th minute by serial dilution and
plate count method at 105 dilution. The values
Test for Acid tolerance obtained were expressed as colony forming
units per mililitre (CFU/ml).
Two ml of overnight grown cultures of the
test strains were centrifuged (10 min at 8000 Results and Discussion
rpm) using centrifuge (HERMLE Z32HK)
and the cellular pellet was resuspended in two The evaluation of probiotic potential of the 25
ml of MRS broth previously adjusted with isolates was carried out in vitro by stimulating
HCl to different pH levels of 3.0, 4.0 and 5.0. the environment of the gut, namely by
The initial bacterial population soon after subjecting the isolates to low pH values and
inoculation and after 3 h of after incubation by exposure to bile salts. The typical transit
was recorded using serial dilution and plate time of the food in the stomach is between 20
count method on MRS agar. The cultures minutes to 3 hours (Hyronimus et al., 2000;
were incubated at 370C in an incubator. The Goldin et al., 1992) and therefore the
viable counts (CFU/ml) are tabulated. treatment was kept until 3 h time span.
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Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 3975-3980
Acid tolerance at pH 5.0. The isolates 42, 45 and 48 showed
considerably higher at low pH of 3 and 4
Before reaching the intestinal tract, probiotic compared to pH 5. (Table 1-3). This study
bacteria must first survive the harsh acidic confirms that the resistance to the low pH is
condition of the stomach. Three pH levels of strain dependent. These results are in
the growth medium were used where confirmation with Mishera et al., (2005),
previously adjusted with HCl ranging from three of their seven isolates Lactobacillus sp.
3.0, 4.0 and 5.0 considering the stomach tolerated pH 3.0. Although the acid tolerance
acidity varies from individual to individual in lactobacilli is highly strain specific,
(Dunne et al., 2001). The results indicated members of the genus Lactobacillus are
that among the 25 isolates studied, all showed acidophilic in nature and are able to grow at
good tolerance to acid but most of the LAB pH 4.0 in foods containing a fermentable
isolates showed poor growth and population carbohydrate (Alakomi et al., 2001).
Table.1 Growth of lactic acid bacterial isolates at pH 3.0
Code No. of the LAB *Mean Population (cfu X 105 per ml)
Sl. No.
isolate Initial After 3 h of incubation
1 2 16.5 116.0
2 3 16.0 125.5
3 4 18.0 118.5
4 6 16.5 164.0
5 35 16.0 117.0
6 39 15.0 108.0
7 40 14.0 96.5
8 42 77.5 258.5
9 45 97.5 292.0
10 48 82.5 266.0
11 52 9.0 107.5
12 53 10.5 87.0
13 54 56.0 170.5
14 57 15.0 67.0
15 58 11.0 69.5
16 59 26.0 164.0
17 60 15.0 60.5
18 61 71.5 192.5
19 63 12.5 51.0
20 65 12.0 102.0
21 66 14.0 102.5
22 69 68.0 186.0
23 72 65.5 175.5
24 73 11.5 101.0
25 75 35.0 173.5
*mean of two replications
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Table.2 Growth of lactic acid bacterial isolates at pH 4.0
Population (cfu X 105 per ml)
Sl. No. Code No. of the LAB isolate After 3 h of
Initial
incubation
1 2 6.5 99.0
2 3 8.0 70.0
3 4 6.5 102.0
4 6 22.0 56.8
5 35 12.5 83.0
6 39 12.5 85.0
7 40 6.5 82.0
8 42 85.5 253.0
9 45 72.0 244.0
10 48 74.5 245.0
11 52 10.5 66.5
12 53 8.0 87.0
13 54 34.5 159.0
14 57 13.5 94.0
15 58 11.0 83.5
16 59 32.0 146.0
17 60 14.0 126.0
18 61 62.0 219.0
19 63 16.0 78.5
20 65 7.5 68.5
21 66 13.5 67.5
22 69 50.5 170.5
23 72 50.0 162.0
24 73 11.0 71.0
25 75 33.0 156.0
Table.3 Growth of lactic acid bacterial isolates at pH 5.0
Population (cfu X 105 per ml)
Sl No. Code No. of the LAB isolate
Initial After 3 h of incubation
1 2 5.0 45.0
2 3 5.5 46.0
3 4 6.0 25.5
4 6 12.5 51.0
5 35 7.0 33.5
6 39 7.0 47.0
7 40 4.0 39.0
8 42 57.0 222.5
9 45 44.0 187.5
10 48 53.5 199.0
11 52 6.5 33.5
12 53 5.0 25.0
13 54 19.5 130.0
14 57 4.0 41.5
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15 58 5.5 41.0
16 59 17.0 99.0
17 60 6.0 35.0
18 61 42.0 176.5
19 63 5.5 41.0
20 65 6.5 42.0
21 66 6.5 45.0
22 69 34.0 156.0
23 72 22.5 147.0
24 73 6.0 36.0
25 75 22.5 121.5
Table.4 Growth of lactic acid bacterial isolates at different bile concentrations (OD values)
LAB 0.3 % 0.4 % 0.75 % 1.0 %
ISOLATE 0:0 h 03:00 h 0:0 h 03:00 h 0:0 h 03:00 h 0:0 h 03:00 h
2 0.120 0.225 0.117 0.680 0.123 0.225 0.122 0.256
3 0.085 0.215 0.090 0.796 0.075 0.081 0.085 0.092
4 0.071 0.265 0.128 0.450 0.114 0.116 0.131 0.203
6 0.053 0.033 0.069 0.066 0.090 0.590 0.090 0.619
35 0.130 0.189 0.112 0.769 0.108 0.239 0.123 0.274
39 0.095 0.505 0.074 0.236 0.084 0.110 0.094 0.121
40 0.076 0.109 0.065 0.749 0.063 0.084 0.080 0.100
42 0.097 1.115 0.120 1.093 0.133 1.099 0.135 1.041
45 0.083 0.070 0.111 0.080 0.120 0.139 0.119 0.933
48 0.110 0.122 0.125 1.175 0.135 1.195 0.190 1.273
52 0.078 0.103 0.053 0.840 0.053 0.080 0.048 0.086
53 0.080 0.097 0.082 1.060 0.086 0.165 0.090 0.199
54 0.037 0.031 0.069 0.069 0.091 0.332 0.092 0.600
57 0.054 0.079 0.022 0.841 0.053 0.091 0.058 0.116
58 0.059 0.066 0.058 0.871 0.056 0.075 0.056 0.073
59 0.051 0.053 0.071 0.071 0.090 0.631 0.091 0.713
60 0.090 0.145 0.109 0.603 0.109 0.178 0.071 0.093
61 0.063 0.055 0.082 0.090 0.100 0.617 0.120 0.831
63 0.150 0.171 0.110 0.756 0.061 0.090 0.062 0.078
65 0.100 0.121 0.069 0.862 0.058 0.127 0.049 0.119
66 0.098 0.251 0.073 0.838 0.071 0.108 0.074 0.125
69 0.081 1.093 0.120 1.095 0.130 0.139 0.120 0.947
72 0.080 0.062 0.090 0.059 0.115 0.851 0.131 0.854
73 0.098 0.237 0.110 0.719 0.098 0.148 0.095 0.149
75 0.050 0.055 0.077 0.081 0.092 0.615 0.120 0.750
The probiotic effect of LAB may partly be Bile tolerance
based on the production of relevant
concentrations of lactic acid in the Bile salts belong to the factors that may
microenvironment, which in combination with significantly affect the viability of LAB in the
bile salts, inhibits the growths of Gram negative GIT, influencing the health of the host.
pathogenic bacteria (Stiles and Holzapfel, Tolerance to bile salts is a prerequisite for
1997). colonization and metabolic activity of the
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bacteria in the small intestine of the host De Vuyst, L., Avonts, L., and Makras, E. 2004.
(Havenaar et al., 1992). This will help LAB to Probiotics, prebiotics, and gut health. In C.
reach the small intestine and colon and Remacle & B. Reusens (Eds.), Functional
contribute in balancing the intestinal microflora Foods, Ageing and Degenerative Disease
(Tambekar and Bhutada, 2010). Cambridge, UK: Woodhead Publishing,
416-482.
After 3 h of incubation in MRS medium Doyle, M.P. and Beuchat, L.R. 2007. Food
supplemented with 0.3% bile all isolates Microbiology: Fundamentals and Frontiers.
showed variable rates of growth (Table 3). All (eds.) Washington, DC: ASM Press.
Goldin, B., Gorbach, S., Saxelin, M., Barakat, S.,
the isolates showed good survivability at lower
Gualtieri, L. and Salminen, S. 1992.
concentrations (0.3 %, 0.4 %). However, few
Survival of Lactobacillus species (Strain
isolates such as 42, 45 and 48 survived even to
GG) in human gastrointestinal tract.
the higher bile concentrations (0.75 % and 1.0 Digestive Dis. Sci., 37: 121-128.
% oxgall). Thus, these results indicate that these Havenaar, R. and Huis in’t Velt, J. 1992.
isolate could be used as potential candidates as Probiotics: A general view. In wood ed.
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disease. London Elsevier Appl. Sci., 209-
In conclusion, the LAB isolates used in the 224.
study showed a high resistance to low pH levels Hyronimus, B., LeMarrec, C., Haji Sassi, A. and
and to higher bile salt concentrations. These Deschamps, A. 2000. Acid and bile
traits may enable them to survive in the stomach tolerance of spore-forming lactic acid
and intestine or even to compete with other bacteria. Int. J. Food Microbiol., 61: 193-
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colonize the gut of the host. The results Mishera, V. and Prasad, D.N. 2005. Application
indicated that isolates 42, 45 and 48 showed of in vitro methods for selection of
very high tolerance to acidic pH as well as to Lactobacillus casei strains as potential
higher bile salt concentrations. Hence, the probiotics. Int. J. Food Microbiol., 103:
results indicating that their potential to be 109-115.
explored as probiotic strains in future. Mozzi, F., Raya, R.R. and Vignolo, G.M. 2010.
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How to cite this article:
Noor Nawaz, A.S., K.S. Jagadeesh and Krishnaraj, P.U. 2017. Isolation and Screening of Lactic Acid
Bacteria for Acidic pH and Bile Tolerance. Int.J.Curr.Microbiol.App.Sci. 6(7): 3975-3980. doi:
https://doi.org/10.20546/ijcmas.2017.607.411
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