Project by:

Vaibhav Kumar Maurya

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ISOLATION , CHARACTERIZATION AND COMPARATIVE STUDY OF LACTOBACILLUS SP. USING

BIOCHEMIAL TESTS AND FTIR ANALYSIS

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.

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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 .

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.

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Kulfi sample Bifiliac sample

Curd sample Milk sample

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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.

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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 ).

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Milk Sample Kulfi Sample

Curd Sample Bifiliac Sample

3. Catalase test:

Principle : Degrade  H2O2 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 .

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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 .

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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 .

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• Growth was Observed at 45ºC • Growth was not observed at 15ºC

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Tests ATCC MILK ICE CREAM LOCAL CURD BIFILIAC

VP test positive Positive Positive Positive Positive

MR test Positive Positive Positive Positive Positive

Citrate utilization test Negative 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:

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/

SOURCE

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 hours 0.11 0.141 0.171 0.134 0.165

11 hours 0.112 0.141 0.172 0.156 0.165

12 hours 0.112 0.142 0.173 0.156 0.166

Fig. Values of absorbance obtained at 600 nm

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

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

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 milkcc 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/

Time

Bifiliac Milk Ice cream Vellore

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

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 cream Vellore 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

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

Bifiliac 54 137 201 369 422 430

ATCC 61 141 250 400 439 444

Milk 72 169 279 391 470 480

Vellore

Curd

65 150 220 319 405 410

Ice Cream 40 120 222 336 400 403

Fig. Colony count

FTIR ANALYSIS

Preparation of samples for FTIR.

Lyophilization of samples.

Processing of samples for FTIR.

Spectral analysis of samples.

- 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 (-CH3) 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

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

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

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

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

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

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.

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