Microbial Growth Promotion by Using a Digestive Ayurvedic Formulation.

S. Mondal*¹, D. Saha², D. Mridha¹, M. Jana¹ and S. Kayal³

¹Dept. of Pharmacy, Bharat Technology, Banitabla, Uluberia-711316, W. B.

²School of Pharmacy, Chouksey Engineering College, Bilaspur- 495004, C. G.

³Dept. of Pharmacy, Kanak Manjari Institute of Pharmaceutical Sciences, Rourkela-769015, Orissa.

ABSTRACT:

A number of medicinal plants are available which helps both in growth promotion and inhibition of different micro-organisms. Micro-organisms having capability to produce several diseases and also by secreting different enzymes which help our body to develop.

Different extracts obtained against each process like cold infusion, hot infusion, decoction and maceration were suitably diluted with doubled distilled water. Several different samples were prepared and marked as Papain extract, Combined extract, Combined extract with Papain, Mixed Extract and Mixed Extract with Papain. Different cell cultures of four different species of micro-organisms were used based on different dilution and type of extraction. They are applied to observe the growth promotion of those beneficial micro-organisms. The maximum growth was observed in case of those extracts obtained from hot infusion and decoction process.

KEYWORDS: Microbial, Digestive, Ayurvedic.

INTRODUCTION:

The principle of microbiological study by using plant materials plays an important role in the both cases of growth exhibition and inhibition of different microorganisms. Micro-organisms having the capability to produce several diseases and also by secreting different enzymes which help our body to develop. Beneficial microorganisms also serve our body by balancing the immunity or resistance level.

The use of medicinal plants and their extracts for the cure of localized and specific human infection as well as cattle infection is an age old practice. Different extracts of medicinal plants helps both in growth promotion and inhibition of different microorganisms. ^{1, 2, 4}

MATERIAL AND METHODS:

1. Preparation of the extracts :

Preparation of the samples :^{1, 2}

Five different extracts obtained against each process like Cold infusion, Hot infusion, Decoction and Maceration were suitably diluted with doubled distilled water to get desired effects.

Sample 1: Papain extract

Sample 2: Combined extract

Sample 3: Combined extract with Papain

Sample 4: Mixed extract

Sample 5: Mixed extract with Papain

Sterilization of the extracts:¹ Suitably diluted extracts were sterilized by autoclaving at a temperature of 121 C and pressure of 15lb.

2. Collection and maintenance of Microbial strains:³

Collection of different microbial strains used as test organisms had been done from Central Drugs Laboratory, Kolkata. One day after collection of the strains of Escherichia coli (ATCC – 10536), Saccharomyces cerevisiae (ATCC - 2601), Lactobacillus plantarum or arabinosus (ATCC - 8014) and Lactobacillus leichmannii (ATCC - 7830); two subcultures for each microorganisms were made and kept in incubator fixed at a temperature of 37⁰C.

For E. Coli, S. Cerevisaie Slant cultures were made where as stab cultures were made for L. Plantarum or L. Arabinosus and L. Leichmannii.

As a medium of growth nutrient agar media was prepared for E. Coli and S. Cerevisiae whereas for Lactobacillus species MRS Agar media was prepared.

3. Washing of glass materials:¹

Required amount of glass materials were firstly soaked in soap water for 30 minutes. Then these were washed by tap water followed by distilled water. After that washed again by acetone, allowed to dry for 30 minutes and finally rinsed with alcohol.

4. Dry heat sterilization of the glass materials:¹

All the dried glass apparatus were subjected to sterilize in for dry heat sterilization in hot air oven. Firstly the temperature was fixed at 50 C for 20 minutes and then temperature was allowed to rise in 100 C for 30 minutes. After that again the temperature was fixed at 140 C for 45 minutes and finally it was adjusted to 160 C for 1hr.

5. Preparation of sterile water and normal saline:^{2, 3}

a) In a 250 ml clean and dry conical flask 250 ml of distilled water was taken and cotton plugged followed by wrapping and tiding it with brown paper and thread.

b) 225 mg. of sodium chloride weighed in a 250 ml. conical flask and 250 ml. of distilled water was added to this. It was also cotton plugged followed by wrapping and tiding it with brown paper and thread. Both of this was sterilized in autoclave at 121⁰ C temperature and 15 lb pressure for 20 minutes.

5.

6. Preparation of microbial suspension and centrifugation:³

Subcultures of different microorganisms were taken and 5 ml. of normal saline was added to the each test tube. Shake each one for 2 mins. Then each microbial suspension was transferred aseptically in different sterile centrifuge tube. Then it was allowed for centrifugation at a rate of 3000 rpm for 10 minutes.

Then took another 4 sterile test tubes and suspended microbial cells were transferred aseptically by the help of sterile spatula. 10 ml. of sterile normal saline was added to each test tube and suitable dilutions were made aseptically in case of E. coli and S. cerevisiae.

In case of L. plantarum or arabinosus and L. leichmannii after collection of suspended microbial cells 5 ml. each of MRS broth and sterile normal saline was added in 4 different test tubes and suitable dilutions were made.

7. Preparation of nutrient agar media : ¹

Nutrient agar media was prepared individually for E. coli and S. cerevisiae.

Composition of the media

Agar – 5 gm.

Beef Extract –2. 5 gm.

Yeast Extract –2. 5 gm.

Sodium Chloride –1. 5 gm.

Distilled Water –250 ml.

Firstly in balance no. 1 sodium chloride was weighed in prescribed amount in a 250 ml. conical flask. Then in balance no. 2 required amount of beef extract and yeast extract was weighed. 250 ml. of distilled water was added to this. Then the weighed materials were subjected to warm in water bath until the beef extract and yeast extract was completely dissolved. Then pH was adjusted in between 7. 4 by using NaOH solution. Finally required amount of agar was weighed in balance no. 2 and added to the conical flask with continuous stirring by the help of a clean and dried glass rod.

The straw like yellow coloured solution was divided equally in 25 different clean and dried test tubes at an amount of 10 ml. and cotton plugged. All the test tubes were taken in a 1000 ml beaker and wrapped with brown paper and tied with a thread. Finally it was autoclaved at a temperature of 121 C and 15 lb pressure.

8. Preparation of MRS broth media : ²

MRS broth media was prepared individually for L. leichmannii and L. plantarum or arabinosus.

Composition of the media

Proteose peptone – 10 gms. /lit.

Beef extract – 10 gms. /lit.

Yeast extract – 5 gms. /lit.

Dextrose – 20 gms. /lit.

Polysorbate 80 – 1 gm. /lit.

Ammonium citrate – 2 gms. /lit.

Sodium acetate – 5 gms. /lit.

Magnesium Sulphate – 0. 10 gm. /lit

Manganese Sulphate – 0. 05 gm. /lit.

Dipotassium phosphate – 2 gms. /lit.

13. 78 gms. of MRS broth media was weighed in a 250 ml. of clean and dry conical flask in balance no. 1 and 250 ml. of distilled water was added to this. pH was adjusted to 6. 5 by using NaOH solution.

The total light yellow coloured solution was divided equally at an amount of 10 ml. in 25 test tubes. After cotton plugging of the test tubes all are taken in a 1000 ml. beaker and wrapped by brown paper followed by tieing up with a thread. Finally it was subjected to autoclaving at a temperature of 121⁰C and 15 lb pressure.

9. Inoculums of the microorganisms and preparation of plates and test tubes:^{3, 5}

For E. coli and S. cerevisiae sterile nutrient agar media was poured in 21 different sterile petridishes. Just after that 0. 5 ml. of suitably diluted microbial suspensions were added in every plate. Then the plates were rounded clockwise very carefully. For next 10 minutes the all the plates were allowed to solidify.

The first plate left as control and other 20 plates were marked for individual extracts along with their extraction procedure.

After formation of solid bed 1 ml. of individual extracts were given in specified plates except the control one. Then the plates were rounded as before clockwise for uniform spreading of the extracts.

After preparation of the plates they were allowed to keep in the incubator fixed at 37 C for 48 hours.

For L. plantarum or arabinosus and L. leichmannii sterile nutrient MRS broth media was poured in 21 different sterile test tubes. Just after that 0. 5 ml. of suitably diluted microbial suspensions were added in every test tube. Then the test tubes were shacked with hands very carefully. For next 5 minutes the all the test tubes were allowed to stand.

The first test tube left as the control and another 20 test tubes were marked for individual extracts along with their extraction procedure.

After that 1 ml of the plant extracts obtained by different processes were given in individual test tube except the control. Then test tubes were shacked with hands. After preparation of the test tubes, they were allowed to keep in the incubator fixed at 37 C for 48 hours.

Table no. 1 and 2 regarding the observation of E. Coli and S. Cerevisiae in different test tubes in order to observe the turbidity occurred. The observations had been made in respect to the control. In the observation charts ++ stands same as control the, +++ stands for more growth observed than control. [C. I. stands for cold infusion, H. I. stands for hot infusion, D stands for decoction, M stands for maceration]

RESULTS:

From the observation table we can come to the point that mixed extracts obtained from the processes of cold infusion, hot infusion and decoction with or without papain enhance the growth of E. coli.

In case of S. cerevisiae different extracts containing papain enhance more growth.

Table no. 3 and 4 regarding the observation of Lactobacillus species in different test tubes in order to observe the turbidity occurred. The test tubes are compared with the control one. ++ stands for same as control and +++ stands for more turbid than control test tube.

For L. plantarum or arabinosus combined and mixed extracts enhance the growth to some extent especially in case of all the extraction procedures.

STAB CULTURE OF LACTOBACILLUS PLANTARUM OR ARABINOSUS

STAB CULTURE OF LACTOBACILLUS LEICHMANNII

PREPARATION OF PLATES

Table no. 1: Growth Chart of E. coli

Extraction procedure	Type of extracts
	Papain	Combined	Combined with papain	Mixed	Mixed with papain
C. I.	++	++	++	+++	+++
H. I.	++	++	++	+++	+++
D	++	++	++	+++	++
M	++	++	++	++	++

Table no. 2: Growth Chart of S. cerevisiae

Extraction procedure	Type of extracts
	Papain	Combined	Combined with papain	Mixed	Mixed with papain
C. I.	+++	++	+++	++	+++
H. I.	+++	++	+++	+++	++
D	+++	++	+++	+++	+++
M	+++	++	+++	++	+++

Table no. 3: Growth Chart of Lactobacillus plantarum or arabinosus

Extraction procedure	Type of extracts
	Papain	Combined	Combined with papain	Mixed	Mixed with papain
C. I.	++	+++	+++	+++	+++
H. I.	++	+++	+++	+++	+++
D	++	+++	+++	+++	+++
M	++	+++	++	+++	++

Table no. 4: Growth Chart of Lactobacillus leichmannii

Extraction procedure	Type of extracts
	Papain	Combined	Combined with papain	Mixed	Mixed with papain
C. I.	++	++	+++	+++	+++
H. I.	++	+++	+++	+++	+++
D	++	+++	+++	+++	+++
M	++	++	++	+++	++

INOCULUM OF MICROBIAL SUSPENSION

DISCUSSION:

Before moving to the microbiological study papain is added in different extracts (combined and mixed) in its powder form. Four different micro-organisms were used, two from lactobacillus species such as Lactobacillus plantarum or arabinosus and Lactobacillus leichmannii; rest two are E. coli (beneficial strain) and Saccharomyces cerevesiae.

By applying those extracts, growth profile of the above commencular micro-organisms which help in our digestion, were studied and favorable growth was observed.

In all aspects it is clear that growth promotion of different commencular micro-organisms, used in the present study, gave favorable growth in samples obtained from hot infusion and Decoction process.

It is expected that those beneficial micro-organisms will give favorable growth if the extracts used in the formulation, obtained by Hot infusion or Decoction process.

Considering the above study, the designed formulation will be capable to bring a new dimension, after proper standardization and clinical trials, to overcome day to day problems regarding indigestion.

REFERENCES:

1. Board, R. G. & Lovelock, D. W., Some Methods for Microbiologal Assay. Society of Applied Bacteriology, Technical Series, 1975, 8.

2. Kavanagh, F., Analytical Microbiology New York & London: Academy Press., 1972, 2.

3. Fisher, Bacterial Culture Media, 2^nd Edition Fisher Scientific Company, USA, 1996.

4. Shivranjan, V. V., and Balachandran, I., Ayurvedic Drugs and their Plant sources, Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi, 1940, 91.

5. Washington, J. A, Diagn, Microbial. Dis., 1988, 9^th edition, 135-138.

Received on 26. 10. 2010

Accepted on 03. 12. 2010

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 3(1): Jan. - Feb. 2011, 26-29