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1. Probiotic Profiling of Lactic Acid Bacteria from Local
(Bangladeshi) Pegions’ GIT
B. Sc. Honours Thesis Presentation
11 December, 2018
Lutfee Alom Brishti
Examination Roll No: 140636
Registration No: 1406225
Supervisor
S. M. Khaledur Rahman
Assistant Professor
3. INTRODUCTION
This study is about to isolate Lactic Acid Bacteria from
local pegion’s GIT
A microorganism which, when administered in adequate
amounts, confers a health benefit on the host
Gastrointestinal tract or GI tract begins at the mouth,
includes several important organs, and ends at the cloaca
4. PROBIOTICS
Derived from the Greek language meaning "for life"
Fuller in 1989 redefined probiotics as a live microbial
feed supplement which beneficially affects the host
Most common microorganisms found in probiotics
currently available are lactic acid bacteria especially
Lactobacillus and Bifidobacterium species
These resident microflora in the GIT improving its
intestinal microbial balance
5. AIM AND OBJECTIVES
Isolation and charectarization of Lactic Acid Bacteria from
local pegion’s GIT
Goal of this reserch is to gain scientific and technological
knowledge about Lactic Acid Bacteria from local pegion’s
GIT
6. HEALTH BENEFITS OF PROBIOTICS
Probiotics help balance in digestive system
Probiotics can prevent colorectal cancer
Probiotic supplements improve mental health conditions
Probiotics can help reduce symptoms of certain
digestive disorders
Probiotics may help one’s lose weight and belly fat
7. METHODS & PROCEDURES
Sample collection and sampling
Two local pegions with different age and color collected
from Borobazar,Palbari,Jessore
From two pegions’ GIT six samples (crop, gizzard,
intestine) were collected
Then stored in autoclaved plastic vials at -20⁰C in a
refrigerator
From those 5 samples were selected for further study
8. Media preparation
According to De Man et al, 1960; MRS Agar and Broth
designed to encourage the growth of lactic acid bacteria
MRS formulation was developed by de Man,Rogosa and
Sharpe
Media is selective for lactobacilli
Selectivity can be altered by pH adjustment
9. Distill water
MRS broth powder
Heat and agitate
Sterilize in autoclave
at 1210C at pressure
15 psi for 45 minutes
MRS agar powder
Media
preparation
Liquid
Solid
Cool at room
temperature
Poured into test
tubes/petridish
es
10. Protocol for isolation of lactic acid bacteria
One gram of sample dissolved in 9ml of 0.15% peptone
water solution
Dilluted up to ten logarithmic (10^-10) fold, then
incubated for 48 hours at 37oC into MRS agar plate
Typical LAB characteristics colonies were randomly picked
up and purified by streaking on fresh MRS agar plates
During the tests, culture was kept in MRS agar stabs at
4⁰C temperatures
11. BACTERIAL CHARACTERIZATION (MORPHOLOGICAL TEST)
Gram staining Protocol
According to the protocol of Erkus (2007)
Single
colony
taken onto a
glass slide
Mixed with
distilled
water
Application
of cristal
violet
Application
of iodine
Alcohol
wash
Application of
safranin
Identifying gram
positive/negative
12. Catalase test
Conducted to determine the presence of the enzyme catalase
Catalase enzyme was found in most bacteria
If enzyme present, it would break hydrogen peroxide (H2O2)
and release of free oxygen
13. Protocol
According to ‘Amrita Virtual Lab Collaborative Platform’
A clean glass slide was divided into two sections with lubricant
pencil
One should be labeled as test and the other as control
Colony from
subculture smeared
onto glass slide
Hydrogen
peroxide
Oxygen bubble
produce/catalase
positive
Oxygen bubble
omit/catalase
negative
14. BIOCHEMICAL TEST
NaCl tolerance test protocol
According to Hoque et al., 2010 determination of NaCl tolerance of
isolated LAB
7 test tubes containing MRS broth adjusted with different concentrations
(5-10%) of NaCl
After sterilization, each test tube inoculated with 1% fresh overnight
culture of LABs and
Incubated at 37°C for 24 hours
After incubation, growth were determined by observing turbidity
15. pH tolerance test Protocol
Following Bates, Roger G. Determination of pH: theory and
practice. Wiley (1973) to test pH tolerance
Broth having pH 2.2 and pH 6.6 inoculated with 1% bacterial
suspension at 370C for 24 hours
Then samples were taken at 0, 10, 12, 14,16, 18 and 20 hours and
after 36 hours for cell resistance detection
Non-inoculated broth medium were served as control at pH 2.2
and pH 6.6
16. Bile salt tolerance test protocol
This test using protocol by Graciela and Maria (2001) with some
modifications, Zinedine and Faid (2007)
MRS broths with different concentration (0.15% and 0.3%) of bile Oxgall
used to determine the tolerance and growth rate of isolated LABs
Final pH of the medium adjusted to 6.5 and autoclaved at 1210C
Then 1% overnight cultured isolates of LABs were inoculated into the
MRSO (MRS-Oxgall) broth medium and Incubated at 370C for 24 hours
Survival rates of the isolates measured by Spectrophotometer
Non-inoculated MRS broth served as control
17. Antimicrobial activity
Antagonistic test are formed by well diffusion methods (Balcazar et al.,
2008; Cappuccino and Sherman, 2002)
Vibrio cholera, Escherichia coli, Klebsiella Pneumonia pathogens used
Pathogenic bacteria and isolates culture individually in nutrient broth
and incubated at 370C for 24 hour.
Nutrient
agar
Distilled
water
Media
preparation
Dissolved and
pourd into
petridishes
Pathogens spread
onto petridishes
Gell cutted by
gellcutter and
sample placed
18. Antibiotic resistance test
According to the standard agar diffusion method known as Kirby Bauer (Barry
and Thornsberry, 1985)
Nutrient agar Distilled water
Dissolved then
poured into
petridishes
Media cutted by
gellcutter and
antibiotic disk placed
Isolates spreaded
Media
prepared by
31. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 10 20 30 40 50 60
0.3% Bile salt
Series1 Series2 Series3 Series4 Series5hour
Figure : Growth curve of bile salt tolerance for five isolates
35. DISCUSSION
5 isolates from 6 samples remain at the end of the isolation
Among the 5 strains three are appeared as gram positive, catalase
positive , white;
Two were gram negative, catalase negative, white; Two were rod
and Three were cocci shaped
In this study assume that two rod shaped belongs to Lactobacillus
By analysis of microscopy and biochemical characterization,
it is assumed that another three isolates may be Lactococcus spp
36. CONCLUSION
I completed my study successfully and found a hopeful result
In this study the first step was taken to use the isolates as
cultures
Then I performed some test to determine probiotics
In future, this study can be broaden for probiotic products
production for higher range
This study can be continued to making probiotics more available