Isolation , characterization and comparative study of lactobacillus sp. using biochemial tests and ftir analysis

This is one of my research work which was carried out to isolate, characterize the Lactobacillus sp. biochemical test and FTIR techniqes

1. Project by:
Vaibhav Kumar Maurya
1.
ISOLATION , CHARACTERIZATION AND
COMPARATIVE STUDY OF LACTOBACILLUS SP. USING
BIOCHEMIAL TESTS AND FTIR ANALYSIS

2. INTRODUCTION:
The Lactobacillus is a gram positive, facultative anaerobic or
microaerophilic, having low GC content, acid tolerant, non sporulating, rod
or cocci .
Lactic acid bacteria comprises Lactobacillus, Leuconostoc,Pediococcus,
Lactococcus , Streptococcus (at core) and Enterococcus, Vagococcus,
Weisella (as peripheral).
Members of Lactic acid bacteria ferment carbohydrates into energy and
lactic acid . Depending upon strain type metabolic pathways differ :
Homofermentative and Heterofermentative.
 FTIR was applied to charaterize Lactobacillus from different samples. In
this Comparitive study, ATCC 7469 was used as reference strain to
characterize and analyse the spectra.
2.

3. OBJECTIVES:
 The aim of this project was to isolate, characterize and do a comparative
study of Lactobacillus sp., from various sources of milk products.
 Isolation of Lactobacillus strains from different samples.
 Characterization by using conventional Gram’s Staining and
Biochemical Tests.
 Determination of best strain by studying the effects of various parameters
like absorbance, pH, free lactic acid formation and colony count.
 FTIR analysis for comparitive study of Lactobacillus strains isolated from
different samples with ATCC 7469 sample .

4. ISOLATION OF LACTOBACILLUS SP.
Conical flask of 100ml MRS broth + 1 conical flask of 100ml MRS agar.
Autoclave
1g bifiliac was poured into each flask of MRS broth & flask were kept on shaker overnight.
15-20ml MRS agar was poured into each autoclaved petri plate.
50µl of broth was spread plated with an L-rod on the MRS agar plates.

Overnight incubation at 37ºC , Gram’s Staining and Biochemical tests
Lactobacillus colonies were subcultured and streaked onto an autoclave plate of MRS
agar.

6.

5. Kulfi sample Bifiliac sample

6. Curd sample Milk sample
10.

7.  BIOCHEMICAL
CHARACTERIZATION
BIOCHEMICAL TESTS:
1.Voges-Proskauer Test (VP test)
Principal : Acetylmethylcarbinol to Diacetyl production .
Procedure :
1. 100ml of Mr VP medium was prepared and autoclaved.
2. 15 ml of medium was poured into 6 test tubes.
3. Each test tubes was inoculated with a single colony of lactobacillus isolated
from Milk, Kulfi, Curd, Bifiliac,Lactobacillus ATCC and left overnight.
Result : Deep rose colour in culture in 15 minutes.
11.

9. 2. Methyl Red Test:
Principal : Production and stabilization of high conc.of acid end products.
Procedure :
•1.100 ml of MR VP medium was prepared and autoclaved.
•2. 15ml of medium was poured into 6 test tubes.
•3. Each test tube was inoculated with a single colony of Lactobacillus,isolated from
Milk,Kulfi, Curd,Bifiliac and Lactobacillus ATCC.
•4. Left for overnight on shaker.
•5. The next day, 1ml methyl red indicator was pippeted and added to each boiling tube
and left at room temp for 15-20 mins
Result : pH 4 – Red colour ( +ve ).

13.

10. 12.
Milk Sample
Kulfi Sample
Curd Sample Bifiliac Sample

11. 3. Catalase test:
Principle : Degrade H
2
O
2
by producing the enzyme Catalase.
Procedure :
1.A single colony was touched with a loop and smear onto a clean slide.
2. 2 drops of hydrogen peroxide was added to smear.
3. Presence or absence of free oxygen gas bubbles was checked .
Result : Bubbles of free oxygen gas is produced .
15.

12. 4. Citrate Utilization test :
Principal : Alkaline medium changes bromothymol blue indicator from green
to deep Prussian blue .
Procedure :
1.200ml of Simmons Citrate Agar was
prepared and autoclaved .
2. 15ml agar each was poured into 6boiling tubes .
3. Each tube is Inoculated with a colony .
4. The boiling tubes were incubated
at 35’C for 48 hours and colour change observed .
Result : No microbial growth at the surfaces of the slants and the medium
remained green thus indicating a negative result .
16.

13. 5. Growth at 15ºC and Growth at 45ºC :
Procedure :
1. Single colonies wer touched with a loop and streaked onto two MRS agar
plates .
2. One MRS agar plate was incubated at 15’C and another was incubated at
45’C overnight .
3. Plates were checked for growth the next day .
Result : No growth of colonies was seen at 15’C. However,growth was seen
at 45’C .
18.

14. •Growth was Observed at 45ºC•Growth was not observed at 15ºC
19.

15. Tests ATCC MILK ICE CREAM LOCAL CURD BIFILIAC
VP test positive Positive Positive Positive Positive
MR test Positive Positive Positive Positive Positive
Citrate utilization testNegative Negative Negative Negative Negative
Catalase test Negative Negative Negative Negative Negative
Growth at 15ºC Negative Negative Negative Negative Negative
Growth at 45ºC Positive Positive Positive positive positive
Summary of Biochemical Tests:

16. EFFECT OF VARIOUS PARAMETERS
ON THE CURDLING OF THE MILK
1.ABSORBANCE : The absorbance of the milk inoculated with
Lactobacillus ATCC 7469 and Lactobacillus isolated from Milk, Vellore
local curd, Ice cream, Bifilac was checked and growth curve plotted.

TIME/SO
URCE
ATCC MILK ICE
CREAM
VELLOR
E CURD
BIFILIAC
1 hour 0.03 0.041 0.034 0.022 0.038
2 hours 0.031 0.042 0.035 0.022 0.038
3 hours 0.039 0.043 0.037 0.023 0.039
4 hours 0.042 0.055 0.043 0.031 0.052
5 hours 0.044 0.067 0.062 0.045 0.069
6 hours 0.052 0.079 0.084 0.069 0.077
7 hours 0.069 0.091 0.099 0.088 0.099
8 hours 0.078 0.109 0.103 0.096 0.12
9 hours 0.084 0.117 0.134 0.108 0.147
10 hours0.11 0.141 0.171 0.134 0.165
11 hours0.112 0.141 0.172 0.156 0.165
12 hours0.112 0.142 0.173 0.156 0.166
Fig. Values of absorbance obtained at 600 nm

17. 0
0.05
0.1
0.15
0.2
0 5 10 15
OD at 600 nm
hour
Comparative Graph
ATCC
MILK
ICE CREAM
VELLORE CURD
BIFILIAC

18. 2. pH: The pH of the milk inoculated with Lactobacillus ATCC 7469 and
Lactobacillus isolated from milk and Vellore local curd. Ice cream. Bifilac
was seen to decrease, indicating the production of Lactic Acid.
Fig. Values of pH obtained
Source/
Time
ATCC MILK VELLOR
E CURD
ICE
CREAM
BIFILIAC
After 1st
hour
6.56 6.4 6.48 6.5 6.4
After 2nd
hour
5.0 5.15 4.85 5 5.38
After 3rd
hour
4.5 4.6 4.63 4.81 4.7
After 4th
hour
4.4 4.32 4.37 4.4 4.15
After 5th
hour
4.15 4.1 4.3 4.33 4.16

20. 3.LACTIC ACID PRODUCTION: Using the ether extraction method, the
amount of free lactic acid produced for 100cc of milk was calculated as per
the formula below:

cc. of 0.2N alkali used
__________________ X 105 = cc of 0.2 NaOH required to neutralize the
lactic acid in 100 cc. of milk
cc of ether decanted
Fig. Values of cc. of 0.2N alkali used to neutralize
the free lactic acid in ether extract for each sample
Source/Tim
e
BifiliacMilk Ice creamVellore
curd
ATCC
1 hour 0.025 0.05 0.05 0.05 0.1
2 hour 0.075 0.06 0.07 0.07 0.15
3 hour 0.08 0.08 0.08 0.08 0.31
4 hour 0.8 0.15 0.32 0.14 0.64
5 hour 1 0.5 0.72 0.9 1.2

21. Fig. Values of cc. of free lactic acid produced per 100cc. of milk
Hence, it was seen that the most amount of lactic acid of 34.12cc. for 100cc. of milk
after 5 hours, was produced by Lactobacillus ATCC 7469 nearest to it Lactobacillus
sp. from Bifilac that produces 26.2 cc for 100 cc of milk.
Bifilac Milk Ice creamVellore local
curd
ATCC 7469
1 hour 0.66 cc 1.34 cc 1.35 cc 1.34 cc 1.62 cc
2 hours 1.93 cc 1.59 cc 1.89 cc 1.88 cc 3.93 cc
3 hours 2.0 cc 2.17 cc 2.11 cc 2.10 cc 8.17 cc
4 hours 21.04 cc 3.98 cc 8.4 cc 3.64 cc 16.8 cc
5 hours 26.5 cc 13.15 cc 18.9 cc 23.69 cc 34.12 cc

22. 4. COLONY COUNT : Samples were collected after every two hours for
every source and spread plated on MRS agar and incubated overnight to
obtain colonies of the desired strain.


After 2
hours
After 4
hours
After 6
hours
After 8
hours
After 10
hours
After 12
hours
Bifiliac54 137 201 369 422 430
ATCC 61 141 250 400 439 444
Milk 72 169 279 391 470 480
Vellore
Curd
65 150 220 319 405 410
Ice Cream40 120 222 336 400 403
Fig. Colony count

23. FTIR ANALYSIS
Preparation of samples for FTIR.
Lyophilization of samples.
 Processing of samples for FTIR.
Spectral analysis of samples.

24. - The process of curdling of milk involves the conversion of lactose to lactic
acid, The prominent groups of lactic acid are the carboxylic group (OH-C=O)
which lies in the range of wave numbers 1760-1670 cm
-1
and the alkyl group
(-CH
3
) which lies in the range of wavenumbers 2960-2850 cm
-1
.
- The detection and increase of these peaks in the spectra obtained by FTIR
indicate the positive production of lactic acid. An increase in peak height is
directly proportional to an increase in quantity.

SPECTRAL ANALYSIS

25. Fig. Comparison of ATCC 7469
sample at 1
st
hour and 7
th
hour

26. Fig. Comparison of Milk
sample at 1
st
hour and 7
th
hour

27. Fig. Comparison of Bifilac
sample at 1
st
hour and 7
th
hour

28. Fig. Comparison of Ice Cream sample at
1
st
hour and 7
th
hour

29. Fig. Comparison of Vellore Local
Curd sample at 1
st
hour and 7
th
hour

30. CONCLUSION
 The aim of this project was to isolate, characterize and do a comparative
study of Lactobacillus sp. from various sources of milk products .
 Characterization was done by using conventional Gram’s Staining and
Biochemical Tests which have shown the presence of Lactobacillus to be
positive.
 After the Gram staining and Biochemical Tests to confirm the presence of
our desired strains we processed our sample by using FTIR.The importance
of this FTIR to characterize the organisms on genus and species level and
determine the best strain, as it states the amount of similarities between the
organisms.
 Effect of various parameters like Absorbance ,pH, amount of free lactic
acid formation and colony count gave the idea of best strain on comparing
with reference ATCC 7469 strain.

31. 22.