Title of Invention	"BIOCIDAL COMPOSITION AND THE PREPARATION THEREOF"
Abstract	The present invention relates to a biocidal composition, and the process of preparation thereof. The composition can be used to attract gravid mosquitoes to lay their eggs and subsequently kill the larvae emerged from said eggs and thereby controlling the vector mosquito propagation.

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BACKGROUND AND PRIOR ART Recent invention in the development of biopesticides offer opportunities for the worldwide exploitation of biological control agents as substitutes for more hazardous and environmentally unacceptable synthetic insecticides for inclusion in Integrated Pest Management programs. The latest advancement and high value obtained through development, exploitation and use of biopesticides in developing countries is enormous. The production, marketing and use of biopesticides in a developing nation can establish indigenous employment opportunities; reduce health risks and costs due to chemical poisoning and environmental damage besides improving export earnings by reducing chemical residue levels on export commodities. In addition to this, there are the benefits obtained through the extra control achieved by preserving natural enemies and indigenous biodiversity. Mosquitoes transmit some of the world's most life threatening and debilitating parasitic and viral diseases, including malaria, filariasis, Japanese encephalitis and dengue fever. Alarmingly, these diseases are on the rise in many tropical and sub-tropical areas. Approaches to reduce the incidence of these diseases have focussed largely on controlling mosquito populations with chemical insecticides and by physical barrier methods or by using drugs to prevent infection caused by disease spreading parasites. Every year about 500 million people are estimated to be affected by malaria, transmitted by Anopheles species of mosquitoes with about 3 million deaths. The world burden of lymphatic filariasis is estimated to be 250 million people infected primarily by filariasis parasites (Wuchereria bancrofti, Brugia malayi and Brugia timori). About 100 million people are infected every year by dengue viruses transmitted by Aedes species of mosquitoes with about 24,000 deaths. Therefore, mosquito control programs to overcome communicable diseases in human society have received greater attention. Several mosquito control strategies have been adopted to control these diseases, but perils of epidemics still loom large in most States in the country. The last decade has witnessed an increased interest in biological mosquito control agents. Currently, use of microorganisms as a source of biological control agents for mosquito control has started after the discovery of several strains of bacteria. Since the discovery of B. sphaericus isolates, more than 300 strains have been found to be highly pathogenic to mosquito larvae including the most studied strains (Bs 1593, 2362, 2297, IAB 59). Bs has 49 serotypes and out of that six serotypes are having mosquitocidal effect. The most toxic strain belongs to serotypes H5, H6 and H25. A process of producing bacterial bio-pesticides (Bs and Bti) in mosquito control program is developed by the Applicant of the present invention and has filed a patent application bearing Indian Patent Application No. 319/Del/2005. In continuation of this invention, further intensive research was conducted to explore the potential use of remaining culture filtrates from bird feathers (i.e. left out filtrate used in the above-said patent application) for completely unrelated industrial application. The Applicant has carried out further research work to utilize the culture filtrates for altogether different utilization which includes attracting the gravid female mosquitoes for laying the eggs and thereafter leading to complete death of larvae on the surface of medium as vital part in mosquito control program. Thus, the novelty of this invention lies with culture filtrates derived from bird feathers. This biological material was reported earlier by the inventor as an excellent source for efficient production of bio-pesticides at a cost effective manner to control mosquito larvae by harvesting the bacterial cells and crystal toxins. Database search from all over the world reveals that no report has been published to date in this important area of research utilizing this inexpensive bio-waste material for attracting disease transmitting female mosquitoes. It was observed that using this material is relatively easy to follow by any one, extremely inexpensive, produce effective toxins to kill mosquito larvae. Further, the raw material is rich in nitrogenous source (structural proteins) in the form of keratin. Bird feathers have been produced in huge quantities as unused waste materials from poultry processing industries and broiler shops. Several approaches have been implemented for disposing feather waste including burning, treatment for animal feed and degradation of feathers with bacteria. Feathers normally accumulate with proteins and keratins are major class of structural proteins that are highly resistant to biological degradation. Common enzymes, which do not break down protein, such as trypsin, do not affect keratin. However, the enzyme keratinase produced by a bacterial strain (Bacillus licheniformis) degrade the keratins which are ultimately useful for animal feed and fertilizer. The total protein content was analysed and found that the feather-extract contain high content of proteins, which enables rapid utilization of this nitrogenous source by bacteria for growth and production of spores and crystals. Keratins, like other proteins, are made up of long string of various amino acids, which fold into a final 3-dimensional form. Alpha helix and beta sheet are common parts of such a 3-dimensional form. Long, thin biological structures often contain alpha helixes, while flat structures are often built from beta sheets. Some keratins are rich in alpha helix structures, while others are mostly beta sheet. These two major types of keratin are known as alpha-keratin and beta-keratin. Bird feathers are largely composed of beta-keratin. Based on the high content of total protein on the medium, it is assumed that the protein present in the liquid growth medium plays vital role by being degraded with bacteria (Bs and Bti) for the synthesis of toxins to exhibit toxic effects on mosquito larvae. Bird feathers are produced in larger quantities as disposal waste by the poultry resources (industries, shops, markets etc) in both developed and developing countries. Up to date, several kinds of approaches have been used for disposing these feather wastes, such as land filling, burning, natural gas production, fertilizer and cleaning of certain fabrics. Therefore, to consider this opportune, the present invention is the first one to utilize completely the bird feathers as the cheapest source of bio-insecticide and larvicides. OBJECT OF THE PRESENT INVENTION The primary object of the present invention is to produce a low cost lethal mosquito attractant. Another object of the present invention is to develop a bacterial culture filtrates by an economical process to prepare the lethal mosquito attractant. Sill in another aspect of the present invention is to optimize the strength of the attractant to attract disease transmitting gravid mosquitoes to lay eggs on the surface of medium. Yet in another object of the present invention is to achieve complete mortality of larvae emerged from egg rafts and thereby controlling the mosquito emergence. Yet in another object of the present invention is to extract and purify extra cellular toxins from culture filtrates. Yet in another object of the present invention is to screen the extra cellular toxins produced by Bs and Bti from the culture filtrates. Still in another aspect of the present invention is to identify the active principles act as toxins present in the bacterial culture nitrate. Further, in one another embodiment of the present invention is to study the toxicity of extra cellular proteins against mosquito larvae for understanding the higher toxic value of Bs and Bti'-based bio-pesticides. Yet in another object of the present invention is to characterize extra cellular toxin individually for mosquitocidal activity. BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES TABLE 1: Nomenclature of toxic polypeptides obtained from culture filtrates from Bs TABLE 2: Nomenclature of toxic polypeptides obtained from culture filtrates from Bti TABLE 3: LC so and LC 90 values of Culex quinquefasciatus treated with new bacterial toxins produced from culture filtrates BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES Figure 1: Toxins from bacterial culture filtrate [(a)=Bs and (b)=Bti] Figure 2: Sedimentation of extra cellular proteins from Bacillus sphaericus (Bs) and Bacillus thuringiensis serovar israelensis (Bti) cultured filtrates figure 3: Photomicrograph of extra-cellular toxins from bacteria produced from culture filtrates [(a) =Bs and (b) =Bti] Figure 4: Mosquito oviposition attractancy of culture filtrate of Bs culture filtrate Figure 5: Mosquito oviposition attractancy of culture filtrate of Bti culture filtrate Figure 6: Percentage of egg raft laid by Culex on the surface of culture filtrate (Bs) and P-cresol Figure 7: Percentage of egg raft laid by Culex on the surface of culture filtrate (Bti) and P-cresol Figure 8: Oviposition attractancy by culture filtrates (Bs) and conventional medium Figure 9: Oviposition attractancy by culture filtrates (Bti) and conventional medium Figure 10: Dead larvae aggregated on the surface of culture filtrate without further progress on metamorphosis. Figure 11: Extra cellular toxins from Bti by NATIVE PAGE. Figure 12: Extra cellular toxins from B. sphaericus by NATIVE PAGE Figure 13: Characterization of Bs-extracellular toxins by Thin Layer Chromatography Figure 14: Characterization of Bft'-extracellular toxins by Thin Layer Chromatography Figure 15: Fast Performance Liquid Chromatography (FPLC) peak of Bs culture filtrate toxins Figure 16: Fast Performance Liquid Chromatography (FPLC) peak of Bti culture filtrate toxins DETAILED DESCRIPTION OF THE INVENTION Accordingly, the present invention describes a new bacterial culture filtrates (bacteria cell-free medium) comprising aqueous extract of bird feathers. In the first embodiment of the present invention a biocidal composition comprising at least one or in plurality of the following polypeptides in the range of 6 to 60 % by wt, exhibiting FPLC peaks at retention times and absorbance as mentioned below along with carriers such as herein described: (Table removed) Still in another embodiment of the present invention, wherein the carrier is selected from a group comprising phosphate buffer and water. Yet in another embodiment of the present invention, wherein polypeptides A to C are obtained from culture filtrate medium such as herein described by using Bacillus sphaericus Neide (Bs) and polypeptides D to F by using Bacillus thuringiensis serovar israelensis de Barjac (Bti). Further in another embodiment of the present invention, wherein biocidal composition is insecticidal composition. Yet in another embodiment of the present invention, wherein insecticidal composition is larvicidal composition. In one more another embodiment of the present invention, wherein the larvicidal composition is mosquito larvicidal composition. Still in another embodiment of the present invention is a method for inhibiting propagation of mosquito, said method comprising steps of attracting gravid mosquito onto composition of the first embodiment and immobilizing larvae produced thereafter to inhibit the propagation of mosquito. Yet in another embodiment of the present invention, wherein the said composition is having a concentration in the range of 0.1 to 0.001 mg/litre. One more embodiment of the present invention relates to a process of obtaining consortium of polypeptides toxic to insect larvae, exhibiting FPLC peaks at retention times as mentioned below: (Table removed) said process comprising the steps: boiling 6-12 wt%/vol. air-dried bird feathers with water to obtain bird feather extract, raising the pH of the above said extract in the range of 7.5 to 8.0, culturing Bacillus sphaericus Neide (Bs) and Bacillus thuringiensis serovar israelensis de Barjac (Bti) in the extract of step (b) for a period of at least 48 hours, and separating bacterial colonies from the extract of step (c) to obtain the said consortium of polypeptides, optionally purifying individual polypeptides by conventional methods. MATERIALS AND METHODS Bacterial strains Two wild strains of bacteria {Bacillus sphaericus IAB 59 and B. thuringiensis serovar israelensis H-14) are used in the present invention. Nutrient Yeast Extracts Mineral Salt (NYSM) Bacterial culture filtrates produced from standard conventional medium (NYSM) was used as basis, (gram per litre): glucose 5, peptone 5, NaCl 5, beef extract 3, yeast extract 0.5, MgCh 0.203, CaCl2 0.102, MnCh 0.01, pH adjusted to 7.5. Bird Feather Extract (BFE) Bird feathers discarded as wastes from poultry processing industries were brought to laboratory, wet dried and stored at room temperature (28-30°C). The dried feathers were weighed, mixed up with ordinary tap water (100 gram/litre) and boiled. After cooling, the bird feather extracts (BFE) were filtered through muslin cloth and pH was adjusted to 7.5. Bird feather extract also includes the aqueous extract of bird feather powder. The cultures of both NYSM and BFE were dispensed separately into flasks and sterilized. A known volume of pre-cultures of Bs and Bti (50 ul) were inoculated into culture media and allowed to grow on a shaker at the agitation speed of 200 rpm at 30°C. Bacterial spores/crystals were harvested from culture medium by continuous centrifugation process (13,000 rpm, 30 min) and the bacterial spores/crystals free culture filtrates were removed carefully. Biochemical techniques The extra cellular proteins from culture filtrates were precipitated by ammonium sulphate precipitation technique (60 %saturation) and after 24 hours from cooled condition they were centrifuged (at 13,000 rpm, 30 min) and pellets were removed. The filtrate samples were appropriately dissolved in distilled water and dialysis was carried out against Tris-HCl, l0mM (pH 6.8) by over night at -4 °C. Lyophilization was completed and thereafter the toxic proteins were qualitatively screened by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Mosquito attraction by culture filtrates Different concentrations of Bs and Bti culture filtrates (concentration range: 5 ppm to 35 ppm) and a known oviposition attractant (p-cresol, a volatile pentane) (at 15 ppm) were prepared in wax coated paper cups containing tap water. The cups containing culture filtrate solutions were placed on the floor of a mosquito cage (size: 2.5ft x 2.5ft x 2.5ft). Exactly one hundred numbers of fully gravid female mosquitoes were exposed into the mosquito cage. The paper cups containing respective concentrations were carefully kept in the inner side corners of the cage. Total egg rafts laid in each cup were carefully removed and counted. Percentage of egg rafts collected in each concentration was calculated from the total number of egg rafts collected in both new culture medium and control medium. Oviposition active index (OAI) of new culture filtrate medium and the known medium (p-cresol) was calculated using the following derivations: (Table removed) Where, Nt = number of egg rafts laid in experimental medium; Ns = egg rafts laid in control medium. Mosquito larval bioassays Mosquitocidal toxins of Bs and Bti extracted from culture filtrate of bird feathers were bioassayed against mosquito larvae of Culex quinquefasciatus, (early third stage). Different concentrations of culture filtrates were appropriately diluted with tap water (from 5 ppm to 35 ppm) in wax coated cups. Bioassay experiments were replicated (three replicates) and repeated for two times. The larval mortality was recorded after 24 and 48 hours for Bti and Bs culture filtrate toxins respectively and dosage mortality regressions were analysed. Poly Acrylamide Gel Electrophoresis (SDS-PAGE) A total of 5 ug proteins from culture filtrates of Bs and Bti were incubated with an equal volume of protein sample buffer and boiled for 5 min and separated on SDS-PAGE. The protein bands were stained with Coommasie Brilliant Blue R-250 and visualized. For NATIVE-PAGE, Sodium Dodecyl Sulphate was not used and samples along with loading buffer were not boiled. Thin Layer Chromatography (TLC) The active principles from culture filtrates of Bs and Bti were isolated by TLC. They were lyophilised (tempt: -50° C; vacuum pressure: 0.1m bar) and the lyophilised culture filtrate was extracted with methanol, subjected to ascend on the surface of silica plates using chloroform-methanol (70:30) as solvent. The principle toxins were visualized by iodine vapour. The relative frequency values were measured by comparing solvent front. Fast Performance Liquid Chromatography (FPLC) The active proteins present in the culture filtrates were purified and characterized by FPLC analysis. The proteins in the filtrates were fractionated by gel-filtration using sephacryl S-300. The protein fractions were eluted using Tris-HCl (0.05M), containing sodium chloride buffer (0.1M pH 7.5) and monitored at 280 run. Fractions (2 ml each) collected were bio assayed for larvicidal activity. The present invention is explained further in the following specific examples, which are not to be construed as limiting the scope of the invention. EXAMPLE 1 New Toxins from Bacterial culture filtrates In the present invention, the bacterial strains i.e. B.sphaericus (Bs) and B.thuringiensis serovar israelensis (Bti) were grown in experimental culture medium (bird feathers) and conventional medium (NYSM) and spores/crystals released from respective medium were separated by centrifugation process. The remaining culture filtrates were added with ammonium sulphate (60%) and after precipitation; the precipitates were removed, dialysed and lyophilised. The toxins released from filtrates were shown in Figures 1 A (Bs) and B (Bti). Visually the colour of the samples is entirely different from the known crystal toxin (Figure 2). Photo micrographic observation reveals that the size and structure of toxins from culture nitrates are different from crystals (Figure 3). EXAMPLE 2 Mosquito oviposition attraction stimulated by bacterial culture filtrates Mosquito oviposition attractancy was examined for culture filtrates and known mosquito attractant. As shown in Figures 4 and 5, oviposition attraction was significantly very high at the dosage level of 15 ppm in all culture media examined, whereas very low oviposition attraction was observed against a known oviposition attractant (p-cresol) at 25 ppm dosage level. The percentage of egg rafts laid by females of Culex quinquefasciatus on the surface of culture media, as well as the known oviposition attractant (p-cresol) has also shown in Figures 6 and 7. The findings depicted clearly that the Bs and Bti culture media and the plain media produced from NYSM and BFE exhibited a highest level of oviposition attraction (>70%). Oviposition Active Index (OAI) as calculated by attraction of mosquitoes from experimental and control medium was found to be at the highest rate (>0.9) in all bacterial culture media tested. The oviposition attractancy of new culture medium (BFE) was also compared with standard culture medium (NYSM) as shown in Figures 8 and 9. Here also, it was observed that high level of attraction was seen in new culture medium than the standard medium. EXAMPLE 3 Mosquito larval killing effect by the induction of bacterial culture filtrates It was invented further from these findings that the young larvae (1st instar) hatched from egg rafts on the surface of Bs/Bti culture filtrates died immediately without further progress on metamorphosis. The dead larvae aggregated in one area on the surface of medium and in latter stage the grouped larvae immersed down (Figure 10). This feature does not exist in plain culture media even though they attract mosquitoes considerably. This invention has great impact on the novelty of new compounds present in the culture filtrates. EXAMPLE 4 Mosquitocidal toxins from culture filtrates Extra cellular toxic proteins were analysed by NATIVE PAGE in the present invention. A new array of protein profiles from Bs and Bti were visualized. The culture filtrate of Bs contains polypeptides with molecular masses of 300, 250, 205,150,132,100, 80, 70, 60 and 15 kDa proteins. Similarly, culture filtrate of Bti contains polypeptides with molecular masses 210, 200, 90, 75, 60, 45 and 35 kDa proteins. All these proteins are new group of toxins, which exhibit similar kinds of mosquitocidal action as in crystal proteins, but they are entirely different from crystal toxins. The invention suggests that though the toxins are entirely different but they evolved from a common organism of a bacterial strain (Bs or Bti) (Fig. 11 and 12). EXAMPLE 5 Active principles from bacterial culture filtrates In the present invention, the active principles synthesized from Bs and Bti during sporulation process in the culture media (NYSM and BFE) was analyzed by Thin Layer Chromatography (TLC) and the results are given in Figures 13 to 14. As depicted in the figures, there were five active principles of secondary metabolites (Rf values 0.87, 0.72, 0.50, 0.41 and 0.15) which were isolated during bacterial growth period (i.e. from 36 to 72 hours) and these results were comparable between two culture media. These active compounds attracted the gravid female mosquitoes for laying eggs. So, the utility of these compounds is very important as they could be used for trapping the mosquitoes for identification, surveillance and control. The present invention shows that the microbial metabolites released during growth of bacteria are very attractive to gravid females of Culex quinquefasciatus and hence the new compounds identified could be useful in integrated mosquito control programs. EXAMPLE 7 Characterization of new toxins from culture filtrates The extra cellular origin of active principles invented in the culture filtrates of Bs and Bti were purified and characterized further for screening the mosquitocidal action. Three peaks were characterized through Fast Performance Liquid Chromatography (FPLC), which were found to be mosquito larvicidal in nature (Figure 15 and 16) . The retention time and absorbance values of Bs and Bti are given in Table 1 and 2. This invention proved further by characterizing the newly identified exotoxins for mosquitocidal activity. EXAMPLE 8 Mosquitocidal activity of toxins produced from culture filtrates Mosquito larval toxicity with B.sphaericus and Bti produced from culture filtrates from conventional (NYSM) and experimental medium (bird feathers) were claimed. Bioassays were conducted with mosquito species of Culex quinquefasciatus and lethal concentrations were expressed in milligram toxin per litre. As shown in Table 3, the LC50 and LC90 values for B.sphaericus produced from bird feathers were 0.015 and 0.14 mg/lit respectively. Similarly the LC50 and LC90 values for Bti were 0.23 and 0.25 mg/lit respectively. These toxicity values didn't deviate statistically from conventional medium indicating the potential values of culture filtrate produced from bird feathers. ADVANTAGES OF THE INVENTION Identification of novel mosquitocidal toxins that differ in structure and mode of action from those produced by B.sphaericus (Bs) and B.thuringiensis sub sp. israelensis (Bti) may be of great interest. The current invention is the first one that the extra cellular insecticidal toxins (exotoxins) of Bs and Bti produced from bird feather culture filtrates excellently attracting the disease causing female mosquitoes for laying their eggs. Further it is new to report that the freshly emerged mosquito larvae on the surface of water medium died immediately and thus controlling the mosquito larvae to emerge further to the environment. Thus, by using oviposition attractants, vector mosquitoes could be attracted to chosen sites for laying eggs and thereafter killing young mosquito larvae. This novel invention invites the people at various levels in the society especially, the executives from pesticide companies, pesticide policy makers, scientists, health workers from all over the world to follow this new technology on the integrated vector management programs. Because, oviposition attractants are very important as they could be used in mosquito trapping for identification, surveillance and vector control. (Table removed) We Claim: 1. A biocidal composition comprising the following polypeptides in the range of 6 to 60% by wt, exhibiting FPLC peaks at retention times and absorbance as mentioned below along with carriers such as herein described: (Table removed) 2. A biocidal composition as claimed in claim 1, wherein the carrier is selected from a group comprising phosphate buffer and water. 3. A method for inhibiting propagation of mosquito, said method comprising steps of attracting gravid mosquito onto composition of claim 1 and immobilizing larvae produced thereafter to inhibit the propagation of mosquito wherein the said composition is having a concentration in the range of 0.1 to 0.001 mg/litre. 4. A process for obtaining consortium of polypeptides toxic to insect larvae, exhibiting FPLC peaks at retention times as mentioned below: (Table removed) said process comprising the steps: a) boiling 6-12 wt%vol. air-dried bird feathers with water to obtain bird feather extract, b) raising the pH of the above said extract in the range of 7.5 to 8.0, c) culturing Bacillus sphaericus Neide (Bs) and Bacillus thuringiensis serovar israelensis de Barjac (Bti) in the extract of step (b) for a period of at least 48 hours, and d) separating bacterial colonies from the extract of step (c) to obtain the said consortium of polypeptides, e) optionally purifying individual polypeptides by conventional methods.

Documents:

358-del-2006-abstract.pdf

358-del-2006-claims.pdf

358-del-2006-Correspondence Others-(11-04-2012).pdf

358-DEL-2006-Correspondence Others-(12-03-2012).pdf

358-del-2006-correspondence-others.pdf

358-del-2006-correspondence-po.pdf

358-del-2006-description (complete).pdf

358-del-2006-drawings.pdf

358-del-2006-form-1.pdf

358-del-2006-form-18.pdf

358-del-2006-form-2.pdf

358-del-2006-form-26.pdf

358-del-2006-form-3.pdf

358-del-2006-form-5.pdf

358-del-2006-GPA-(11-04-2012).pdf