Enhanced therapeutic value

1. “ENHANCEMENT OF ANTIMICROBIAL
POTENTIAL OF PHYLLANTHUS NIRURI
USING FERMENTATION”
PRINCIPLE INVESTIGATOR: TEAM MEMBERS:
Dr. M.S. Dinesh Ms. Vaishnavi Venugopalan
Associate professor
Dept. of Biotechnology, PES Institute of Technology

2. INTRODUCTION
FERMENTATION:
 Fermentation is one of the oldest forms of technology in the world. It
can be defined as “slow decomposition process of organic
substances induced by micro-organisms or by complex
heterogeneous substances (enzymes) of plant or animal
origin”(Walker,1988).
 It is a biochemical change that is brought about by the anaerobic or
facultatively anaerobic oxidation of carbohydrates by either
microorganisms or enzymes (Buttock, 1988). It is relatively an
efficient, low energy preservation process, which increases the shelf
life and decreases the need for refrigeration or other of food
preservation technology.

3. HERB:
 Herb is a plant whose stem is not woody and its parts like flowers, seeds
and leaves are used for medicine, perfume and flavor.
 They have high rate of curability, healing power and have less side
effects. Herbs helps to maintain a high role in balancing various
processes in the body, promote general well being and health (Vipul,
1999).
HERB USED FOR THE STUDY:
 Phyllanthus niruri
MICROORGANISM USED FOR FERMENTATION:
 Lactobacillus acidophilus (Std. Isolates & Isolates from herbal surfaces)

4. OBJECTIVES
 Screening the efficiency of indigenous medicinal herb Phyllanthus
niruri extract for production of antimicrobial agents.
 Optimization of the process of fermenting the herb.
 Carrying out fermentation of the herb using Lactobacillus acidophilus
isolates from the herbal surfaces. As a reference, fermentation of
standard media using standard Lactobacillus acidophilus is also
carried out.
 Testing the fermented herbs for antimicrobial activity against
common bacterial pathogens.

5. DESCRIPTION OF THE HERB USED
Phyllanthus niruri
 Commonly known as Amla, the Indian gooseberry or nelli belongs to
Euphorbiaceae family.
 It is an ingredient of almost 175 ayurvedic formulations.
 Fruits are commonly used in the treatment of hemorrhages,
diarrheas, dysentry, jaundice, cough and anaemia.
 The fruit is one of the richest sources of vitamin C. It contains, on an
average 500 to 700mg of ascorbic acid per 100g of pulp.
 The extract of boiled leaves is used for gargling and the tender
leaves are used against indigestions.
 Flowers of Amla give a laxative effect and the fresh roots relive from
jaundice

7. DESCRIPTION OF MICROORGANISM
USED FOR FERMENTATION
 L.acidophilus gets its name from lacto- meaning milk, -bacillus
meaning rod-like in shape, and acidophilus meaning acid-loving.
 Thrives in more acidic environments than most related
microorganisms (pH 4-5 or lower) and grows best at 30 degrees
Celsius.
 Occurs naturally in the human and animal gastrointestinal tract,
mouth and vagina.
 Ferments lactose into lactic acid, like many (but not all) lactic acid
bacteria
 Certain related species (known as heterofermentive) also produce
ethanol, carbon dioxide, and acetic acid this way.
 L. acidophilus itself (a homofermentative microorganism) produces
only lactic acid.

8. METHODOLOGY
ISOLATION OF LACTIC ACID BACTERIA :
 Leaves of Phyllanthus niruri were washed with water to isolate lactic acid
bacteria on Mann Rogosa Sharpe’s agar.
 Reference/ Commercial strains were also maintained on MRS media.
IDENTIFACTION OF ISOLATED STRAINS:
 Colony morphology
 Microscopic observation – Gram staining for Lactic acid bacterial cultures
 Gelatin hydrolysis and catalase activity

9.  Gram staining:
Innoculum of 24hrs old is to be used for gram staining. After making thin
smears of the bacterial cultures on a slide and air drying it, it would be heat
fixed and covered with crystal voilet stain for 30 seconds and then washed
with distilled water. It would then be stained with gram’s iodine for 60 seconds
and then washed in 95% ethyl alcohol. Distilled water washed slides would
then be stained with safrannin for 30 seconds, washed with distilled water,
dried with absorbent paper and observed under the microscope using oil-
immersion objective.
 Gelatin hydrolysis:
Inoculation of bacterial cultures into gelatin agar test tubes using stab culture
and incubation for 48hrs. Liquefaction shows the presence of cultures.
 Catalase activity:
Preparation of thick smears on slides flooded with 3% hydrogen peroxide
which is then observed for bubble formation.

10. PREPARATION OF STARTER
CULTURE
 A loop full inoculums of lactic acid bacterial culture was transferred
to a test tube with 5ml of sterilized MRS broth and kept overnight at
25 degrees for growth.
 This culture was added to 100ml sterile leaf extract in 250ml flask.
 The tubes were kept at 37 degrees for 2days in an incubator with
shaking facility of 100rpm and used at 5%(v/v) for fermentation.

11. FERMENTATION OF MEDICINAL
HERBS
 The medicinal herbs was used for fermentation bacterial isolates
prepared earlier. The pest and disease free leaves were harvested
after eliminating its surface microbial load using running water. 20g
of each medicinal herb was considered for fermentation.
 After crushing the leaves using pestle-mortar, 5%(v/v) of starter
culture was added to it and kept for fermentation. Fermented
samples were collected every 5days for about 15 days (according to
the growth curve of LAB).
 These samples were then used for further analysis and evaluation.

12. FERMENTATION SET UP
FERMENTED SAMPLES

13. PATHOGENS TESTED:
 Bacillus cereus
 Staphylococcus aureus
 Salmonella typhi
 Escherichia coli
 Klebsiella spp.
 Pseudomonas aeruginosa.
 Clostridium spp.

14. SALIENT FINDINGS
 Antimicrobial properties of the herb is enhanced by 40-60% after
fermentation
 The pathogens E.coli and S.aureus are most more sensitive to the
fermented products than the other pathogens that were tested.
 The order of sensitivity is as follows:
E.coli > S.aureus > S.tyhpi > P.aeruginosa > B.cereus > Clostridium
spp. > Klebsiella spp.
 The antimicrobial potential of the fermented herb increases with
fermentation time.