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I nternational J ournal of Universal Pharmacy and Bio Sciences 3(3): May-J une 2014
INTERNATIONAL JOURNAL OF UNIVERSAL
PHARMACY AND BIO SCIENCES
IMPACT FACTOR 1.89***
ICV 5.13***
Bio Sciences Research Article !!!

ANALYSIS OF PROBIOTIC PROPERTIES OF LACTOBACI LLUS SPP FROM
SELECTIVE REGIONAL CURDS
Mangalanayaki R* And Nandhini C
P.G. and Research Department of Microbiology and A Division of Biotechnology. S.T.E.T.
Womens college, Sundarakkottai, Mannargudi614001, Thiruvarur(Dt), Tamilnadu, India

KEYWORDS:

Regional curds,
Lactobacillus spp., Gastric
juice, Disc diffusssion
method.
For Correspondence:
Mangalanayaki R*
Address: P.G. and
Research Department of
Microbiology and A
Division of
Biotechnology. S.T.E.T.
Womens college,
Sundarakkottai,
Mannargudi 614001,
Thiruvarur (Dt),
Tamilnadu, India.
Email:
[email protected]



ABSTRACT
Curd is a potential source of probiotic Lactobacilli. In the present study
Lactobacillus spp., were isolated from two regional curds in Thiruvarur
District in Tamilnadu which were identified on the basis of their colony
morphologies and some biochemical tests. It was observed that isolated
Lactobacillus spp., were resistance to inhibitors substances like phenol
of 0.4% 1% NaCl (1-10 %) and 0.3% bile acid. The maximum growth
was observed at pH 5.0 for Lactobacilli isiolated from curd at
Mannargudi region . The organic acid was able to produce in skim milk
were determined by titrimetric method. Their susceptibility for to
selective five antibiotics was determined in term of Disc diffussion
method. In the method the Lactobacilli was very sensitive to
clindamycine, antibiotic the zone of inhibition was (24mm) followed by
kanamycine (16mm) and Gentamycine (12mm) to kill the Lactobacilli.
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INTRODUCTION:
The probiotics are living health promoting microorganisms which will in corporate in to various kinds
of food. Lactic acid bacteria widely distributed in the nature and occurring indigenous micro flora in
that play an important role in many food and feed fermentations with increased shelf life. Lactobacilli
have a protective role against vaginal infections. Improper function of the immune system that is
related to decreased tolerance to indigenous microflora bowel disease (1) consumption of the probiotic
curd did not result in significant increase in receptor expression whereas consumption of regular curd
did suggesting that this strain may suppress a curd induced immune inflammatory response (2).
Studies on the effects of culture containing dairy products or probiotic bacteria on cholesterol level
have yielded equivocal results (3). Curds as part of milk that coagulated when the milk sources or is
treated with enzymes. Curd is used to make cheese or a coagulated liquid that resembles milk curd.
Curd is also cheap and easily available sources (4). LAB are most important group of microorganisms
used in food fermentation they contribute to the fast and texture of fermented products and inhibit food
spoilage and pathogenic bacteria by producing antimicrobial substances. Lactobacilli are ubiquitous
and widespread commensal bacteria in the human and animal as adjuvant against gastrointestinal
disorders as dietary supplements and biological food processors based on their fermentative
prosperities (5). The aim of this study was to isolates safe and potential probiotic Lactobacillus spp
from different dairy products.
MATERIALS AND METHODS
Collections of samples
Curd samples were collected from Mannargudi and Needamangalam at Thiruvarur District in
Tamilnadu. Samples were stored aseptically in low temperature (-4 C) refrigerator to protect from
contamination and deterioration.
Culture Media
MRS medium (De Man and Rogosa and Sharpe)
Isolation of Bacteria
Bacteria were isolated from curds by using MRS medium 10g of each sample was dissolved in to
MRS broth they were shaken homogeneously and were incubated at 37 C for 24 h in aerobic condition.
In the presence of 10% Co
2
was created for removal of unwanted bacteria . Finally the single colony
bacteria were observed by some colony morphology and some physiological test (Grams staining and
Catalase reaction) Tab1.
Determination of optimal growth and pH
For the determination of optimal pH of Lactobacillus, 1 % [v/v] fresh over night culture or
Lactobacillus were inoculated into MRS broth with varying pH ranging from 2.5 -8.5. The pH were
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adjusted with concentrated acetic acid [99 %] and 5N NaOH. The inoculated broth was incubated at
anaerobic condition for 24 h at 37 C in the presence of 10% CO
2
. After 24 h of incubation the growth
of the bacteria were measured using a spectrophotometer, at 560 nm against the
]
against the
uninoculated broth as control Tab 2..
Assay of NaCl Tolerance
Ten test tubes containing MRS broth were adjusted with different concentration [1-10%] of NaCl.
After sterilization, each tube was inoculated with 1ml fresh culture of Lactobacillus and incubated at
37 C for 24 h. After 24 h of incubation their growth were determined by observing their turbidity.
Maximum growth were indicated as double positive [++], normal growth as single positive [ + ] and
no growth were indicated as negative sign [ - ] Tab3.
Quantification of Organic Acid and Determination of pH

One percent active culture of Lactobacillus was used to inoculate 10% sterilized skim milk and initial
pH 6.68was determined by a digital electrode meter. The inoculated skim milk was incubated at 37

C
for 72 h, and samples were collected the in every 24 h, 48 h and 72 h and liquids portion was filtered
and then titrated against 0.1 N NaOH Tab 4.
Assay of bile tolerance
Bile tolerances of isolated Lactobacillus from curd were determined using standard procedure (6) .The
bile salt solutions were prepared using oxgall powder. The powder was rehydrated by preparing 10 g
dry powder base in 90 ml distilled water. From this solution final concentrations of 1 %, 2 % were
prepared. Sterile double distilled water without oxgall [pH 6.4] was used as control. All solutions were
autoclaved and stored at room temperature until used. 10ml of each solution was added to each and
incubated at 37 C aerobically control and incubated at 37

C aerobically. One ml of culture was taken
out from each tube after 3, 8 and 12 h time intervals and dilutions were prepared in sterile 9 ml 0.85 %
saline blanks. Appropriate dilutions were plated in MRS agar and incubated at 37

C for 24- 48 h Tab
5.
Assay for Gastric juice Resistance:
Gastric juice resistance of isolated Lactobacillus from curds were assay using the standard procedure
(6).
Antibiotic sensitivity Test
Disc diffusion Method:
In the disc diffusion method to analyze the antibiotic sensitivity of Lactobacillus by using five
different antibiotic disc (8) Tab6.

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RESULT AND DISCUSSION:
In the present study to isolate and identify the effective Lactobacillus the Lactobacillcs from curd
samples of two different region from Thiruvarur district. They to determine the optimal growth pH and
sensitivity against different antibiotic for isolated Lactobacillus spp . The isolated Lactobacilli were
able to survive and multiply 0.05 , 0.15 and 0.3% bile acid . It is also able to survive in gastric
environment at low pH but they were not able to multiply. In the presence of per ml artificial gastric
juice in the MRS medium the colony forming unit at 0 h, 1 h, 2 h, 3 h, 4 h, and 24 h were
1.5x10
4
(CFU/ml), 1.6x10
4
(CFU/ml), 1.5x10
4
(CFU/ml), 1.36x10
4
(CFU/ml), 1.4x10
4
(CFU/ml)and
1.59x10
4
(CFU/ml), respectively. On the other hand, they were able to survive and had partial
multiplication abilities at pH 6.60.
In disc diffusion method the Lactobacillus was very sensitive to clindamicine(24mm) the zone of
inhibition followed by kanamicine and gentamicine (16mm,12mm) . But cefloxin and tetracycline
(10mm,6mm) have low antibacterial activity against Lactobacilli.
In experimented design of bile concentration were 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 (6). Therefore
selection of probiotic bacteria for human concentration it must be endurable to bile
concentration(7).This investigation indicates that there is a minor variation in organic acid production
due to their regional variation the findings has the connection with findings (9).
CONCLUSION:
LAB were successfully isolated from curd. Probiotic activities of both the isolates were determined
both of them resistant to low pH , tolerance against bile salts and antimicrobial activity against
microorganisms. They are able to tolerate inhibitory substances such as 0.05-0.3% bile acid. NaCl also
able to survive in stimulated gastric pH2.2 condition. The isolated Lactobacillus were able to produce
organic acid in milk. Further production of Lactic acid bacteria to higher cell density which could used
as probiotic either in tablet in starter culture was suggested. LAB isolated from traditional dairy
products in these region can be used as potentially probiotic bacteria with promoting host specific
health






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Table-1 Morphological, Physicological and Biochemical Characteristics of Isolated Lactobacilli
Characteristics Isolate -1 Isolate -2 Isolate-3
ColonyMorphology

1.0 mm , whiter
rough and irregular
Small 0.1 0.5 mm rough
dull and brownish
Small circular
white- creamy in
colors
Gram staining + + +
Motility Test Non motile Non motile Non motile
Catalase Test - - -
Endospore Test - - -
0.4% Phenol + + +
Milk Coagulation + + +
Nacl Tolerance + + +
Note: (+) Positive, (-) Negative
Table-2 Optimal Growth and pH of Isolated Lactobacillus Spp from Needmangalam Curd Sample
pH Optical Density
Isolate from
Mannargudi
Curd
Isolate from
Needamangalam
Curd
2.5 0.0 0.01
3 0.2 0.3
3.5 0.1 0.3
4 1.8 0.2
4.5 1.5 1.1
5 1.2 2.0
5.5 1.2 1.8
6 1.0 1.7
6.5 1.1 1.7
7 0.5 1.5
7.5 1.1 1.3
8 0.0 1.0
8.5 0.1 1.0
9 1.7 1.9
Note: Isolate I Mannargudi, Isolate II Needamangal




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Table-3 Tolerance to NaCl Isolated Lactobacilli
Concentration of NaCl
(%)
Lactobacilli isolated
from Mannargudi
Region
Lactobacilli isolated
from Needamangalam
Region
1


++
++
2

+ +
3

+ +
4 + +

5
+ +

6
+ +

7

+ +
8

+ +
9 + +

10
- -
Note: (++) Good Growth; (+) Visible Growth; (-) No Growth
Table-4 Organic Acids (%) and pH

in Skim Milk Produced By Isolated Lactobacillus Spp
Sources of bacteria Name of bacteria
Incubation
time (hour)
Incubation
Temperature
(
o
C)
Organic
Acid (%)
P
H


Mannargudi Curd

Lactobacillus spp
24
48
72
37
o
C
37
o
C
37
o
C
2.289
6.610
6.529
5.08
3.71
6.68

Needamangalam Curd

Lactobacillus spp
24
48
72
37
o
C
37
o
C
37
o
C
2.120
3.919
6.259
5.11
4.48
3.88

Table-5 Bile Tolerance and Gastric juice resistant for Lactobacillus Spp from
Needamangalam Curd Sample
Incubation Time pH 6.2
CFU/ml


pH 6.60
CFU/ml
0 1.5x10
4
1.9x10
4
1.93 x10
4
1 1.6 x10
4
1.83 x10
4
2 1.5 x10
4
1.89 x10
4
3 1.36 x10
4
1.94 x10
4

4 1.4 x10
4
1.94 x10
4

24 1.59 x10
4
1.94 x10
4


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Table-6 Antibiotic Sensitivity Test by Disc Diffusion Method For Lactobacillus From
Needamangalam Curd Sample
Antibiotic Lactobacillus
Gentamicin 12mm
Tetracyclines 6mm
Clindamicines 24mm
Kanamycines 16mm
Cephalexin 10mm

REFERENCES:
1. Merger , M ., and K . Croitoxo 1998. Infections in the immunopathogenesis of chronic
inflammatory bowel disease. Immunol. 10: 69-78.
2. Trapp , C . L. C .C. Chang , G .M. Halpern, C. L. Keen, C. L. and M .e and Gershurn 1993.
The influence of chronic yoghurt consumption on populations of young and adult. Int . J.
Immunotha. 9:53-64
3. Taylor, G .R.T., and C.M. Williams 1998. Effects of probiotics and prebiotics on blood lipids.
Brit, J., Nutr. 80 (suppl-1) S225-S230.
4. Shruthy V.V. pavithra M . Ghose 2011. Probiotic potential among Lactic bacteria isolated from
curd. IJRAP,2(2), pp 602-609.
5. Baesley S. 2004 . Isolation , identification and exploitation of lactic acid bacteria from human
and animal microbiota. PhD thesis Helsinky: University of Helsinki.
6. Graciela , F ., V . De and P .T Maria 2001. Food Microbiology protocols (J . F. T. Spencer
and A . L . R . D Spencer , ED .) , Totowa , New Jersey : Human Press Inc ., chap . 21
(probiotic properities of Lactobacilli ) PP:173 -181.
7. Gilliland , S. E., T . E Stey and L . J. Bush , 1994 Importance of bile tolerance from L .
acidophilus used a dietary J .Dairy Science champaign .67: 30-45
8. Kirby Bauer, 1960 Antibiotic susceptibility of testing by a standard single disc method . A ma.
Journal pathology 36: 492-496.
9. Haddadin, M.S.Y., S.S. Awaisheh and R. K. Roninson , 2004 The production of yoghurt with
probiotic bacteria isolated from infants in Jordan. pakistan J. Nutritional3(5): 290-293.