Title of Invention	"A PROCESS FOR THE PREPARATION OF A MOSQUITO LARVICIDAL FORMULATION OF BACILLUS THURINGIENSIS VAR. ISRAELENSIS"
Abstract	A process for the preparation of a mosquito larvicidal formulation from bacillus thuringiensis var. israelensis comprising preparing an inoculum from • seed of bacillus thuringiensis var. iaraelensis in atleast three stages, subjecting said inoculum to the step of fermentation, harvesting the cell mass obtained from said fermentation, spray drying and formulating said cell mass, said formulation/mixture being Subjection to the step of extrusion, curing and drying to obtain bacillus thuringiensis var. israelensis having mosquito larvicidal properties.

Title of Invention

"A PROCESS FOR THE PREPARATION OF A MOSQUITO LARVICIDAL FORMULATION OF BACILLUS THURINGIENSIS VAR. ISRAELENSIS"

Abstract

A process for the preparation of a mosquito larvicidal formulation from bacillus thuringiensis var. israelensis comprising preparing an inoculum from • seed of bacillus thuringiensis var. iaraelensis in atleast three stages, subjecting said inoculum to the step of fermentation, harvesting the cell mass obtained from said fermentation, spray drying and formulating said cell mass, said formulation/mixture being Subjection to the step of extrusion, curing and drying to obtain bacillus thuringiensis var. israelensis having mosquito larvicidal properties.

Full Text	This invention relates to a process for the preparation of a mosquito larvicidal -formulation of bacillus thuringiensia var. israelensis. Bacillus thuringi ensis var. israel ensis is a known bacterial strain and processes for the preparation thereof are known in the art. However, such known processes produced the aforesaid bacterial strain which * did not possess suitable level of activity against mosqui to 1 arvae. An ob:iect of this invention is tD propose an improved process far the preparation of a mosquito larvicidal formulation of bacillus thuringi ensis var, Israel ensis having suitable level of larvicidal activity- A further ofoiect of this invention is to propose a process for preparing a slow-release floating formulation of bacillus thuringiensis var. israelensis to serve as a moire efficient agent for destroying mosquito larvae. At the outset of the description follows, it is to be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only an exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching o-f the invention and not intended to be taken restrictivety. According to this invention, there is provided a process -for the preparation of a mosquito larvicidal formulation from bacillus thuringiensis ver israelensis comprising preparing an inoculum from a seed of bacillus thuringiensis ver. israelensis in at leant three stages, subjecting said inoculum to the step of fermentation, harvesting the cell mass obtained from said fermentation, spray drying and formulating said cell mass, said formulation/mixture being subjection to the step of extrusion, curing and drying to obtain bacillus thuringiensis ver . israelensis having mosquito larvicidal properties. In accordance with this invention comprises in subjecting a seed of bacillus thuringiensis ver. israelensis bacterial strain to the step o-f population growth. Preferably but without implying any limitations thereto, the step o-f population growth is effected in three stages. In the first stage the seed of -said bacterial strain is suspended in a medium at a pH 7.0-7.5 for a period of 6-8 hour to obtain a culture. fhe culture is transferred to fresh nutrient media having constituents similar to the medium of the first stage in atleast a further two stages for each a period of 6—8 hour to obtain an inoculum. The inoculum so obtained is subjected to the step of fermentation in a nutrient medium containing soyaflour, peanut oil and sterile water at a pH of 7.0—7.2 and for a period so as to obtain a cell mass, The said cell mass is harvested by precipitating with hydrochloric acid. The supernatant is discarded and the precipitated cell mass is then spray-dried and formulated. The step of formulation consists in a first step of mixing cell mass with sodium alginate, said mixture is diluted with water, extruded as granules into calcium chloride solution and said granules allowed to cure. The granules are then dried and packed in perforated sachets with polystyrene beads. ft process for the preparation of a mosquito larvicidal formulation of bacillus thuringiensis var. israelensis according to a ' preferred embodiment is herein described and illustrated in the accompanying drawings whereins — Fig.l shows the flow chart of the process. The process proposed by the present invention and as shown in the flow of chart of fig.l comprises transferring the seed/spores of the strain, obtained from the lyophiiized seed lot, into a test tuba having 8-12ml of seed medium and sterile water maintained at a pH between 7.0-7,5 to allow the growth at a temperature of 29 to 32°C for 6-8 hr. at the shaking speed of 225-275 r,p.m. to obtain a culture. The seed medium comprises 8-8—1.27. peptone, 0.8-1.27. yeast extract, 0,3-0.77. glucose, 0.8-1.27. salt Solution and sterile water having pH 7-7.5. The salt solution comprises 1.37. calcium chloride, 17. magnesium chloride, 0.287. manganese sulfate, 0.5% zinc sulfate, 0.01% ferrous sulfate, 0.01% copper sulfate, 0.037. sodium chloride in 0.1 molar hydrochloric acid. The grown culture so obtained is transferred into a conical flask having 80-I20mi preferably 100ml of seed medium as herein described above and sterile water at a pH 7.0—7.5 to grow the culture at 28~32°C for 6-8 hour and at a stiring/shaking speed of 225-275 r.p.m. preferably 250 r.p.m. Again the grown culture so obtained is transferred into another conical flask having 1Q00— 2200ml preferably 2080ml of seed medium as mentioned hereinabove and sterile water at a pH 7-7.5 so as to all the growth of the culture at a temperature 28-32°C -for 6-8 hr. at 225-275 r.p.m. stiring/shaking speed. The grown culture so obtained is then transferred into a tomenter/bioreactor having. 55-65 litre o-f production medium containing, 2.5% soyaflour or 1.57. Jaggery, 0.57. yeast extract, 0.01% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.3% sodium chloride 0.01% ferrous sulfate, 0.5% peptone or 0.5% corn steep liquor and 1% salt solution and 0.1% peanut oil and sterile water at a pH of 7—7.5 to allow the culture growth at a temperature 28-32°C for 24-30 hr. at 200-250 r.p.m- and with 30-50% dissolved oxygen. The growth up ceilmass so obtained is then harvested from the above culture either by continuous centrifugation at 25000-30000 r.p.m. or by precipitation with 5 N hydrochloric acid or 0.8% potassium aluminium sulfate. The cellmass is resuapended with sterile water at the ratio of 2s 100 and is then spray dried with inlet temperature at 170-200°C and outlet temperature 70-80°C. The dried cellmass is mixed with 3~8% solution of sodium alginate in water in the ratio of l:l weight/volume. The mixture is then diluted three times with sterile water. the pH is adjusted to 6-5-7. Ihe mixture ia then extruded into granules in 0.2 M calcium chloride solution and is subjected to the step of curing for 3—6 hr. The cured granules are dried at a temperature 30— 40°C for 24-28 hr and the dried granules so obtained are then packed in perforated sachets aiongwith polystyrene bends at the ratio of 100:1. WE CLAIMS:- 1. A process for the preparation of a mosquito larvicidal formulation from bacillus thuringiensis var. israelensis comprising preparing an inoculum from a seed of bacillus thuringiensis var. israelensis in atleast three stages, subjecting said inoculum to the step of fermentation, harvesting the cell mass obtained from said fermentation, spray drying and formulating said cell mass, said formulation/mixture being subjection to the step of extrusion, curing and drying to obtain bacillus thuringiensis var. israelensis having mosquito larvicidal properties. 2. A process as claimed in claim 1 wherein said inoculum is prepared by first growing the seed o+ bacillus thuringiensis var. israelensis are first grown up in a test tube having 0-12ml seed medium for 6-6 hr. at a temperature 28-32°C and shaking/stiring speed 225-275 r.p.m- 3. A process as claimed in claim 2, wherein said grown up seed culture is further grown up in a conical flask having 88J-100ml of said seed medium for 6-8 hr. at 2B-32°C and 225-275 r.p.m. 4. A process as claimed in claim 3 wherien said grown up culture is further grown up into a conical flask having 1800~2200ml seed medium for 6-8 br at the temperature 28-32°C and at a shaking speed of 225-275 r.p.m. 5. A process as claimed in any of the claims 2,3 and 4 wherein said seed medium comprises 0.8-1.2% peptone 0.8-1.27. yeast extract, 0.3-0.7% glucose and 0.8-1.2% salt solution and sterile water maintained at a pH between 7.0-7.5. 6. A process as claimed in claim 5, wherein said salt solution comprises 1.37. calcium chloride, 1% magnesium chloride, 0.283, manganese sulfate, 0.05% line sulfate, 0.01% ferrous sulfate, 0.01% copper sulfate, 0.3% sodium chloride in 0,1 molar hydrochloric acid. 7. A process as claimed in claim 1 wherein the step of fermentation comprises in transferring the grown up culture medium into a fomenter having 55-65 litre production medium, allowing growing of said culture, for 24-30 hr at a temperature 28-32°C and at a shaking speed 200-250 r .p.m. in the presence of 30-50% dissolved oxygen. 8. A process as claimed in claim 7 wherein said production medium comprises 2.5% soyaflour or a mixture of 1.5% Jaggery, 8.5% yeast extract, 0.01% potassium dihydrogen phosphate, 0.052. magnesium sulfate, 0.3% sodium chloride, 0.01% ferrous sulfate and 0.57. peptone or . a mixture of 5.07. corn steep and 1% said salt •solution and 0.1% peanut oil and sterile water at a pH of 7.0-7,. 5. 9. A process as claimed in claims 1 and 7 wherein said grown—up cellmass is harvested by continuous centrifugation at 25000-30000 r.p.m. 10. A process as claimed in claim 9 wherein said grown up cellmass is harvested by precipitation with 5 N hydrochloric acid or 0.0% potassium aluminium sulfate. 11. A process as claimed in claim 10 wherein the harvested cellmass ore resuspended with sterile water at a ratio of 2:100 and are subjected to the step of spray drying with inlet temperature of 70-80°C. 12. A process as claimed in claim 1 wherein said step of formulation comprises in mixing the dried cell mass with a 3-8% solution of sodium alginate in water at the ratio of lsl weight/volume, diluting said formulation mixture three times with sterile water and adjusting the pH between 6.5-7. 13. A process as claimed in claim 1 wherein the step of extrusion comprises in extruding the said formulation mixture in the form of granules into 0.2m calcium chloride solution and subjecting said granules to the sttep of curing for 3—6 hr. and then drying said granules to the step of curing 3-6 hr. and then drying said granules at a temperature 30-40°C for 24—20 hr. to obtain the mosquito larvicidai formulation of bacillus thuringiensis ver. israelensis. 14,. A process for the preparation of a mosquito larvicidai formulation of bacillus thuringienais var. israelensis substantially as herein described and illustrated.

Full Text

This invention relates to a process for the preparation of a mosquito larvicidal -formulation of bacillus thuringiensia var. israelensis.
Bacillus thuringi ensis var. israel ensis is a known bacterial strain and processes for the preparation thereof are known in the art. However, such known processes produced the aforesaid bacterial strain which
*
did not possess suitable level of activity against mosqui to 1 arvae.
An ob:iect of this invention is tD propose an improved process far the preparation of a mosquito larvicidal formulation of bacillus thuringi ensis var, Israel ensis having suitable level of larvicidal
activity-
A further ofoiect of this invention is to propose a process for preparing a slow-release floating formulation of bacillus thuringiensis var. israelensis to serve as a moire efficient agent for destroying mosquito larvae.
At the outset of the description follows, it is to be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only an exemplary
embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching o-f the invention and not intended to be taken restrictivety.
According to this invention, there is provided a process -for the preparation of a mosquito larvicidal formulation from bacillus thuringiensis ver israelensis comprising preparing an inoculum from a seed of bacillus thuringiensis ver. israelensis in at leant three stages, subjecting said inoculum to the step of fermentation, harvesting the cell mass obtained from said fermentation, spray drying and formulating said cell mass, said formulation/mixture being subjection to the step of extrusion, curing and drying to obtain bacillus thuringiensis ver . israelensis having mosquito larvicidal properties.
In accordance with this invention comprises in subjecting a seed of bacillus thuringiensis ver. israelensis bacterial strain to the step o-f population growth. Preferably but without implying any limitations thereto, the step o-f population growth is effected in three stages. In the first stage the seed of -said

bacterial strain is suspended in a medium at a pH 7.0-7.5 for a period of 6-8 hour to obtain a culture. fhe culture is transferred to fresh nutrient media having constituents similar to the medium of the first stage in atleast a further two stages for each a period of 6—8 hour to obtain an inoculum. The inoculum so obtained is subjected to the step of fermentation in a nutrient medium containing soyaflour, peanut oil and sterile water at a pH of 7.0—7.2 and for a period so as to obtain a cell mass, The said cell mass is harvested by precipitating with hydrochloric acid. The supernatant is discarded and the precipitated cell mass is then spray-dried and formulated.
The step of formulation consists in a first step of mixing cell mass with sodium alginate, said mixture is diluted with water, extruded as granules into calcium chloride solution and said granules allowed to cure. The granules are then dried and packed in perforated sachets with polystyrene beads.
ft process for the preparation of a mosquito larvicidal formulation of bacillus thuringiensis var. israelensis according to a ' preferred embodiment is herein described and illustrated in the accompanying drawings whereins —
Fig.l shows the flow chart of the process.
The process proposed by the present invention and as shown in the flow of chart of fig.l comprises transferring the seed/spores of the strain, obtained from the lyophiiized seed lot, into a test tuba having 8-12ml of seed medium and sterile water maintained at a pH between 7.0-7,5 to allow the growth at a temperature of 29 to 32°C for 6-8 hr. at the shaking speed of 225-275 r,p.m. to obtain a culture.
The seed medium comprises 8-8—1.27. peptone, 0.8-1.27. yeast extract, 0,3-0.77. glucose, 0.8-1.27. salt Solution and sterile water having pH 7-7.5. The salt solution comprises 1.37. calcium chloride, 17. magnesium chloride, 0.287. manganese sulfate, 0.5% zinc sulfate, 0.01% ferrous sulfate, 0.01% copper sulfate, 0.037. sodium chloride in 0.1 molar hydrochloric acid.
The grown culture so obtained is transferred into a conical flask having 80-I20mi preferably 100ml of seed medium as herein described above and sterile water at a pH 7.0—7.5 to grow the culture at 28~32°C for 6-8 hour and at a stiring/shaking speed of 225-275 r.p.m. preferably 250 r.p.m.
Again the grown culture so obtained is transferred into another conical flask having 1Q00—

2200ml preferably 2080ml of seed medium as mentioned hereinabove and sterile water at a pH 7-7.5 so as to all the growth of the culture at a temperature 28-32°C -for 6-8 hr. at 225-275 r.p.m. stiring/shaking speed.
The grown culture so obtained is then transferred into a tomenter/bioreactor having. 55-65 litre o-f production medium containing, 2.5% soyaflour or 1.57. Jaggery, 0.57. yeast extract, 0.01% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.3% sodium chloride 0.01% ferrous sulfate, 0.5% peptone or 0.5% corn steep liquor and 1% salt solution and 0.1% peanut oil and sterile water at a pH of 7—7.5 to allow the culture growth at a temperature 28-32°C for 24-30 hr. at 200-250 r.p.m- and with 30-50% dissolved oxygen.
The growth up ceilmass so obtained is then harvested from the above culture either by continuous centrifugation at 25000-30000 r.p.m. or by precipitation with 5 N hydrochloric acid or 0.8% potassium aluminium sulfate. The cellmass is resuapended with sterile water at the ratio of 2s 100 and is then spray dried with inlet temperature at 170-200°C and outlet temperature 70-80°C. The dried cellmass is mixed with 3~8% solution of sodium alginate in water in the ratio of l:l weight/volume. The mixture is then diluted three times with sterile
water. the pH is adjusted to 6-5-7. Ihe mixture ia
then extruded into granules in 0.2 M calcium chloride solution and is subjected to the step of curing for 3—6 hr. The cured granules are dried at a temperature 30— 40°C for 24-28 hr and the dried granules so obtained are then packed in perforated sachets aiongwith polystyrene bends at the ratio of 100:1.

WE CLAIMS:-
1. A process for the preparation of a mosquito
larvicidal formulation from bacillus thuringiensis var.
israelensis comprising preparing an inoculum from a
seed of bacillus thuringiensis var. israelensis in
atleast three stages, subjecting said inoculum to the
step of fermentation, harvesting the cell mass obtained
from said fermentation, spray drying and formulating
said cell mass, said formulation/mixture being
subjection to the step of extrusion, curing and drying
to obtain bacillus thuringiensis var. israelensis
having mosquito larvicidal properties.
2. A process as claimed in claim 1 wherein said inoculum is prepared by first growing the seed o+ bacillus thuringiensis var. israelensis are first grown up in a test tube having 0-12ml seed medium for 6-6 hr. at a temperature 28-32°C and shaking/stiring speed 225-275 r.p.m-
3. A process as claimed in claim 2, wherein said grown up seed culture is further grown up in a conical flask having 88J-100ml of said seed medium for 6-8 hr. at 2B-32°C and 225-275 r.p.m.
4. A process as claimed in claim 3 wherien said grown
up culture is further grown up into a conical flask
having 1800~2200ml seed medium for 6-8 br at the temperature 28-32°C and at a shaking speed of 225-275 r.p.m.
5. A process as claimed in any of the claims 2,3 and
4 wherein said seed medium comprises 0.8-1.2% peptone
0.8-1.27. yeast extract, 0.3-0.7% glucose and 0.8-1.2%
salt solution and sterile water maintained at a pH
between 7.0-7.5.
6. A process as claimed in claim 5, wherein said salt solution comprises 1.37. calcium chloride, 1% magnesium chloride, 0.283, manganese sulfate, 0.05% line sulfate, 0.01% ferrous sulfate, 0.01% copper sulfate, 0.3% sodium chloride in 0,1 molar hydrochloric acid.
7. A process as claimed in claim 1 wherein the step of fermentation comprises in transferring the grown up culture medium into a fomenter having 55-65 litre production medium, allowing growing of said culture, for 24-30 hr at a temperature 28-32°C and at a shaking speed 200-250 r .p.m. in the presence of 30-50% dissolved oxygen.
8. A process as claimed in claim 7 wherein said production medium comprises 2.5% soyaflour or a mixture of 1.5% Jaggery, 8.5% yeast extract, 0.01% potassium
dihydrogen phosphate, 0.052. magnesium sulfate, 0.3% sodium chloride, 0.01% ferrous sulfate and 0.57. peptone or . a mixture of 5.07. corn steep and 1% said salt •solution and 0.1% peanut oil and sterile water at a pH of 7.0-7,. 5.
9. A process as claimed in claims 1 and 7 wherein
said grown—up cellmass is harvested by continuous
centrifugation at 25000-30000 r.p.m.
10. A process as claimed in claim 9 wherein said grown
up cellmass is harvested by precipitation with 5 N
hydrochloric acid or 0.0% potassium aluminium sulfate.
11. A process as claimed in claim 10 wherein the
harvested cellmass ore resuspended with sterile water at
a ratio of 2:100 and are subjected to the step of spray
drying with inlet temperature of 70-80°C.
12. A process as claimed in claim 1 wherein said step
of formulation comprises in mixing the dried cell mass
with a 3-8% solution of sodium alginate in water at the
ratio of lsl weight/volume, diluting said formulation
mixture three times with sterile water and adjusting the
pH between 6.5-7.
13. A process as claimed in claim 1 wherein the step
of extrusion comprises in extruding the said formulation
mixture in the form of granules into 0.2m calcium
chloride solution and subjecting said granules to the sttep of curing for 3—6 hr. and then drying said granules to the step of curing 3-6 hr. and then drying said granules at a temperature 30-40°C for 24—20 hr. to obtain the mosquito larvicidai formulation of bacillus thuringiensis ver. israelensis.
14,. A process for the preparation of a mosquito larvicidai formulation of bacillus thuringienais var. israelensis substantially as herein described and illustrated.

Documents:

219-del-1999-abstract.pdf

219-del-1999-claims.pdf

219-del-1999-correspondence-others.pdf

219-del-1999-correspondence-po.pdf

219-del-1999-description (complete).pdf

219-del-1999-drawings.pdf

219-del-1999-form-1.pdf

219-del-1999-form-2.pdf

219-del-1999-form-3.pdf

« Previous Patent

Next Patent »

Patent Number

233543

Indian Patent Application Number

219/DEL/1999

PG Journal Number

14/2009

Publication Date

27-Mar-2009

Grant Date

30-Mar-2009

Date of Filing

10-Feb-1999

Name of Patentee

INDIAN COUNCIL OF MEDICAL RESEARCH

Applicant Address

ANSARI NAGAR, NEW DELHI- 110 029, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	KOTHANDAPANI BALARAMAN	ANSARI NAGAR, NEW DELHI - 100 029, INDIA
2	A.M. MANONMANI	ANSARI NAGAR, NEW DELHI - 100 029, INDIA
3	S. L. HOTI	ANSARI NAGAR, NEW DELHI - 100 029, INDIA

PCT International Classification Number

A61K

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA