On behalf of the Organizing Committees for the “Assam Botany Congress” and the “ International Conference on Plant Science”

1. Comparative Studies on Phytochemical
Components and Curd Probiotics against
Escherichia coli and Klebsiella sp. in In
vitro Analysis
On behalf of the Organizing Committees for the “Assam Botany
Congress” and the “ International Conference on Plant Science”
Avra Pratim Chowdhury*1(a), Bhanu Preya Sharma1(b), Bhriganka Bharadwaj1(c), DR. Abul Manchur2,
1.a. Ph.D. research scholar, Department of Botany, Gauhati University, India.
1. b. M. Sc. Research, Assam down town University, Assam. India
1.c. Assistant Professor, , Assam down town University, Assam. India
2. Professor, Department of Microbiology, University of Chittagong, Bangladesh.

2. Introduction
 Medicinal plant besides therapeutic agents are also a big source of information for a
wide variety of chemical constituents which could be developed as drugs with precise
selectivity. Nyctanthes arbor-tristis is small ornamental tree known across the country
for its fragment white flowers.
 It has been reported to possess hepatoprotective, anti-leishmanial, antiviral and anti-
fungal activities and ulcerogenic activities. The plant also possess anti-allergic, anti-
malarial, antihelminthic and antioxidant activities.
 Taxonomical Classification
kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Lamiales
Family: Oleaceae
Genus: Nyctanthes
Species: Nyctanthes arbor-tristis
So the beneficial aspect of using phytochemicals and probiotic, have already been
subjected in research purposes. But pharmaceutically processed combination of
different species of probiotics and plant medicines used against intestinal
pathogen, for this antagonistic action is required for in vitro analysis.

3. PURPOSE OF THE STUDY:
Comparative analysis stood for studying out
comes of medicinal and phytochemical effect of
individual gut health.
To find out the basic differentiation between curd
probiotics and phytochemical components
against isolated pathogens in public health
significance.
To assure the significant activities of antagonistic
actions against gram negative characterized
organisms and their functions as selective
isolates.

4. Objectives :
 To carry out phytochemical analysis of leaf extract of plant
Nyctanthes arbor-tristis.
 To determine antimicrobial activity of leaf extract of Nyctanthes
arbor-tristis against Escherichia coli.
 Screening of probiotic from curd according to their isolation
and identifications.
 Comparative analytical study on characteristics of
phytochemicals and curd probiotics against the implemented
and isolated pathogens.
Aims:
Assessment of visible and non visible findings of antagonistic
action of Phytochemical Components and Curd Probio against
isolated infectious pathogens.

5. Collection of the plant sample Extraction processes (Aqueous , methanol and
ether) Phytochemical analysis of these extracts Study of microscopic
characteristics of bacteria Biochemical characteristics of bacteria
Antimicrobial activity by well diffusion method using MHA (Muller Hinton Agar)
media
Phytochemical Analysis
A B C
D E F
B. Positive result for carbohydrate
C. Positive result for phenol
D. Positive result for protein
E. Positive result for Flavonoid
F. Positive result for steroid
A. Positive test for Alkaloids
Saponin and Tanin Negative, absent in extrac
Aqueous
Extract
Methanol
Extract
Petroleum
Ether
Extract

6. Screening of Curd
Probiotics
 Samples: Home made Curd, Curd from Beverage.
 Dilutions: 5 fold and 10 fold, Total diluted Samples: 20
 Methodology:
Spread plate Mix culture Pure culture TSI slant Colony
characterization Isolation Screening Biochemical test
Staining Identification
Identification 2 : Cultural, morphological and biochemical characteristics
of the isolate SC15 was compared with the standard description given in
Bergey‘s Manual of Determinative Bacteriology - 8th ed. (Buchanan and
Gibbons, 1974) and 9th ed. (Holt et al., 1984) and it was found closely
related with the
genus Streptococcus described by Rosenbach (1884). The isolate was
provisionally identified as Streptococcus lactis (Lister) LÖhnis (1909).
Identification 1: The morphological, cultural, Biochemical characteristics of
isolates SC 10 and SC17 were
compared with standard description of ‘Bergey’s Manual of Determinative
Bacteriology’-8th Ed. (Buchanan and Gibbons, 1974) and found closely
related with the genus Streptococcus, and Lactobacillus sp.
Fig. Streptococcus sp.
Fig. Sub
terminal spores
in Lactobacillus
sp.

7. Identification of Isolated
Pathogens
Sample
Total 20
MacConkey
agar
CLED agar Indole test MR
test
Citrate
utilization
Urease
test
Urine
3, 15, 17,
20
Pinkish translucent
colonies
Pale yellow
colonies
- - + +
Sample
Total 20
Indole Methyl red Catalase Carbohydrate fermentation
Glucose Lactose Sucrose
Urine
5, 10, 12
+ve +ve +ve +ve +ve +ve
Identification: Cultural, morphological and biochemical characteristics of the isolate A17c was
compared with the standard description given in Bergey’s Manual of Determinative Bacteriology - 8th
ed. (Buchanan and Gibbons, 1974) and 9th ed. (Holt et al., 1984) and it was found closely related with
the genus Klebsiella (described by Schroeter 1886). The isolate was provisionally identified as
Klebsiella pneumoni. (Trevisan 1887).
Identification: The morphological, cultural and biochemical characteristics of isolate A10c was
compared with standard description of ‘Bergey’s Manual of Determinative Bacteriology’-8th Ed.
(Buchanan and Gibbons, 1974) and found closely related with the genus Escherichia . The isolate
was provisionally identified as Escherichia coli. Escherichia coli described by Escherich et al., in
1885.

8. In vitro Analysis and
Comparative Studies
Fig: In Muller-Hinton agar the effect of
Nyctanthes arbor-tristis aquous extract
( by well diffusion method) against E.
coli.
Fig: In Muller-Hinton agar the effect of
Nyctanthes arbor-tristis methanol and
petroleum ether extract ( by well diffusion
method)against E. coli.
Samples
(autoclaved three
times and mix with
MacConkey medium.)
Control
(MacConkey
Agar plate)
Centralized
MacConkey agar
Peripheral
Nutrient agar
Inference
(Heat labile toxin of
probiotics
antagonists itself)
E. coli - + + No
Curd - - + Yes
Klebsiella sp. - + + No
A. Control, no growth
of Probiotics.
B. Curd Probiotics acts
Antagonistic growth on toxins
itself.
Phytochemical Extract
Showed better Antagonistic Effect
Than Curd Probiotics against Pathogen.

9. Significance of Research
Test
microorganism
Zone of inhibition
(mm)
Escherichia coli Aqueous 15mm
Methanol 25mm
Petroleum ether
3mm
Phytochemical Extract
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6
Probio+ Klebsiella
colonies
Probio colonies
◊ Probiotics toxin antagonistic against itself,
but k toxin of Klebsiella sp. non
antagonist by itself to it. So it is
impossible to develop killed vaccine
from this pathogen.
◊ Gram-positive spore forming probiotic has
no antagonistic effect on Gram negative
pathogen.
Fig. Anti microbial susceptibility test
of curd and contaminated
Klebsiella sp. samples. (Graphical view)
Draw Back
1. Non experimented by Molecular application
2. Study based on Applied Microbiology
3. Non implemented for high automated machine
reading
4. Phytochemical studies based on manual protocols.

10. Conclusions
• The plant extracts were assayed for phytochemical screening and
antibacterial activity against Escherichia coli.
• All the extracts showed effect against the isolated bacteria of all the
extracts. Methanol extracts was most effective for its antibacterial
activity against E. coli compared to aqueous and petroleum ether
extracts where as Klebsiella sp. non antagonist and non resist against
food probiotics and its toxins.
• Here researcher on his conclusive view, stands for excessive doses of
probiotics along with antibiotics causes death of probio and produced
toxin itself against normal gut flora resulting antagonistic action.
Respective doctors and Practitioner carry out their treatment against
urinary tract pathogen along with probiotics supplements. Here
researcher conclusion is for comparative analyzed between
phytochemicals and food probiotics, to asses for medicinal residues by
in vitro studies.

11. REFERENCE
1. Bansal S, Bharatiand AJ, and Bansal YK. (2012) Efficient In-vitro Regeneration of a Medicinal
Plant Harsinghar (Nyctanthes arbor-tristis Linn.). Plant Tissue Cult. & Biotech. 22(2):137-142.
2. Bhadouria RS, Bhargava S, and Pancholi SS. (2012) Isolation and characterization of two
alkaloids from the ethanolic extract of nyctanthes arbor-tristis linn. Leaves. Advance. Res. Pharma. &
Biolog; 2(4):342-344.
3. Champa Shandhar HK, and Kaur M. (2015) An update on Nyctanthes arbor-tristis Linn. Journal
of Internationale Pharmaceutica Sciencia, P (1) :77-86.
4. Ogura Y, et al. (2001) experimented frame shift mutation in NOD2 associated with susceptibility to
Crohn’s disease. Nature; P (411) :603–6.
5. Schultz M, Linde HJ, Lehn N, et al. (2003) studied on immune stimulatory consequences of oral
administration of Lactobacillus rhamnosus strain (GG) in healthy volunteers. J Dairy Res; P (70)
:165–73.
6. Saikali et al. (2004) described preliminary observations on effect of Lactobacillus sporogenes on
serum lipid levels in hyper cholesterol patients. Indian J Med Res; P (92):431-432.

12. Poster And Articles:

13. Sincerest Acknowledge to:
DR. Diganta Narzary, Associate Professor, Gauhati University, India.
DR. Niraj Agarwala, Assistant Professor, Gauhati University, India.
Dr. P. C. Sarma, Principal, Allied Health Sciences. Assam down town University
Dr. Moni. P. Bhuiyan, Medical Microbiologist, SRL Diagnostic, Assam, India.
The End
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