production of dextran from dairy sources

2. PRODUCTION AND CHARACTERISATION
OF
BY LACTOBACILLUS PARACASEI FROM DAIRY
SAMPLES

3. • Dextran is group of high molecular mass polysaccharides.
• Composed of complex branched Glucan chains of different
lengths(3to2000kd).
• It is used to medicinally as an Antithrombotic (antiplatelet) to
reduce blood viscosity.
Introduction Of Dextran

4. • Dextran was first discovered by Louis Pasteur as a microbial
product in wine.
• Scheilberin (1874) confirmed that this microbial
polysaccharide with the empirical formula (C6H10O6) n and
therefore named as “dextran”.
• Dextran is produced by species of Leuconostoc, Lactobacillus
, Streptococcus and Acetobacter.
• It finds various other industrial applications .

5. Objectives
 Isolation of bacterial stain from dairy sources.
 Biochemical Characterization of the isolated bacterial stain and
identification.
 Confirmation of the identified bacterial stain using Sanger’s sequencing
method.
 Production of Dextran by the bacterial stain isolated from dairy and
vegetable sources.
 Characterization and confirmation of Dextran using FTIR analysis.

6. D
E
X
T
R
A
N
Isolation of colonies
Characterization of
Bacterial strain
DNA sequencing
c
c
v
Production of
DEXTRAN
Characterization of
DEXTRAN
Application Of
DEXTRAN
Experimental design

7. • The Lactobacillus sp. were isolated from available sources :
Fermented vegetables :
 cabbage
 cauliflower
 pumpkins
 carrot and tomato
Dairy products :
 Yoghurt
 Milk
Materials and Methods
 Sources

8. MSE agar was prepared
and then pour into the petridish
and allowed to solidify.
Sample (milk) was
collected and was
diluted
Transfer 0.1ml of
Serial diluted sample
onto agar plates
Spread the inoculum on the
surface of the agar by L rod
method
The plate was
Incubated at
25ºC at 24 hrs
Colourless, mucous
colonies
were developed

9. 
• Gram staining
• Indole test
• Oxidase test
• Catalase production test
The isolated bacterial strain was were confirmed the
Morphological and biochemical test.
Morphological and Biochemical characterization
of bacterial isolate:

10.  Conformation by sanger’s sequencing
 The sample was sent for analysis of 16s r RNA Analysis
in the laboratory of Yazzah genomics, Chennai. To analysis
 DNA isolation
 PCR
 Sequencing
NCBI – BLAST
 Phylogenetic tree construction

11. DEXTRAN PRODUCTION

12. Specific Dextran production Broth
A loopful of culture of colonies was inoculated in
10 ml of Dextran production broth
This Inoculum mainly used for Dextran production
Preparation of inoculum

13. Culture was incubated at 26˚.C
for 18 hrs.
40 ml of Sterile Dextran production
Medium was prepared and then added to
60 ml of inoculum medium
pH was adjusted at 5.5 to make
more it viscous
Production of Dextran
dextran
production
medium

14. After 18 hours of incubation, the culture medium was
precipitated by using chilled ethanol.
Precipitation of Dextran
Equal amount of
ethanol was added and
stirred well and
centrifuged at 4000
rpm at 30 minutes.
The supernatant was
discarded
Chilled ethanol
was added with constant
stirring and precipitates
of dextran was obtained
It was allowed to stand
for 5–10 minutes and
supernatant was again
decanted.

15. • precipitated Dextran was washed with distilled water
and centrifuged thrice to get purified Dextran .
• Sterile distilled water will be added step wise to make
a paste of dextran in water.
Purification Of Dextran

16.  RESULTS and DISCUSSIONS

17. Identification organism
The Bacterial strain were initially confirmed by growing it on
the selective MSE agar. Then they were confirmed by
phenotypic and biochemical characterization.
Figure 1: Isolation bacterial sp by spread plate method- The plate shows individual
colonies of bacterial isolate

18. Biochemical Test Results
Gram staining Positive
Motility Non-Motile
Oxidase Negative
Catalase Negative
Indole Negative
Morphological and biochemical characterization o f bacterial
strain

19. Gram staining - positive
Indole test - Negative
Oxidase - Negative

20. Conformation of bacterial strain by sanger’s sequencing

23. Figure 10: Sanger sequencing

24. sequence
>KIR_800R.ab1 821
CCGGTCTTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCG
CCTTCG
CCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATG
GAGT
TCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGC
TTCCT
CGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTG
CGCTCG
CTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTAC
CGCG
GCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGT
CACGCC
GACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTT
TACGA
CCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTT
GCGTCC
ATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGC
CGTGTC
TCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATC

25.  NCBI - BLAST
Figure 11: The figures shows that Nucleotide sequence analysis of BLAST

26. Figure 12: Description Query coverage analysis of BLAST

27. Figure 15: Multiple Sequence alignment and Similarity studies

28.  Phylogenetic tree construction
Figure 16: Phylogenetic tree construction – The figure shows the
relationship of the Lactobacillus paracasei sp with its related
bacteria

29. Dextran production
 Preparation of inoculum
Medium pH: ± 6. 9
Temperature: 25 °C
Incubation time: 24hrs
Figure17: Preparation of inoculum

30.  Dextran production
Figure18: Production of Dextran in MSE Dextran preparation media
Medium pH: ± 5.7
Temperature: 25 °C
Incubation time: 18hrs

31.  Precipitation and Purification of Dextran
Centrifuge speed: 4000 rpm
Minutes: 10mins

32.  conformation of Dextran by FTIR analysis

33.  Application of Dextran
 Protein stabilization
 Vaccines
 Antiplaetlet
 food

34. • Sarwat, F., Qader, S. A. U., Aman, A., & Ahmed, N. (2008).
Production & characterization of a unique dextran from an
indigenous Leuconostoc mesenteroides CMG713.Int. J. Biol. Sci,
4(6), 379-386.
• Üretimi, D. (2005). Production of dextran by newly isolated
strains of Leuconostoc mesenteroides PCSIR-4 and PCSIR-
9.TürkBiyokimyaDergisi [Turkish Journal of Biochemistry-Turk J
Biochem], 31(1), 21-26.
 References