Title of Invention

A PROCESS FOR PRODUCTION OF GIBBERELLIC ACID(GA3) IN HIGH YIELD

Abstract

Present invention provides a process of production of gibberellic acid (GA3) from wet or dry biomass of agriculture, fruit and vegetable waste using solid-state fermentation technique. The steps provides preparation of inoculum of Gibberella fujikuroi strain NCIM 1099 or mutants derived there from, incubating the inoculum in sterilized micronutrient rich biomass of solid waste, isolation of gibberellins from fermented solid biomass. The method produces at least 1.2 gm gibberellic acid per kg of waste. The method produces a mixture of gibberellins and other compounds in which gibberellic acid (GA3) is at least 50%.

Full Text

FIELD OF INVENTION The present invention relates to a process for production of Gibberellic acid (GA3) in high yield from fruits, vegetables and agriculture waste. More particularly it relates to a process for production of Gibberellic acid (GA3) in high yield from fruits, vegetables and agriculture waste using fungal strain Gibberella. BACKGROUND AND PRIOR ART OF INVENTION The gibberellins (GAs) are originally identified as secondary metabolites of the fungus Gibberella fujikuroi Saw Wr. (Fusarium moniliforme Sheld.). While these compounds have no apparent role in the fungus, they have been found to elicit a variety of responses in higher plants (seed plants) including shoot elongation, sex expression, fruit growth, and seed germination. The early descriptions of gibberellins -induced elongation, especially those associated with genetic dwarfism, led to the idea that GAs might be naturally-occurring in normal, non dwarf strains of higher plants. The first evidence for the presence of gibberellin-like substances in higher plants came from the fact that semi-purified extracts from such material would mimic a GA-induced growth response when applied to genetic dwarfs. This evidence was soon followed by the isolation and chemical identification of gibberellins (Gas). Gibberellins are well-recognized group of diterpenoids having a typical tetracyclic ring system. More than hundred naturally occurring gibberellins are known. It is a well-established fact that gibberellins, particularly Gibberellic acid (GA3) are a well-known plant growth regulator. Gibberellins are well-recognized group of diterpenoids having a typical tetracyclic ring system. More than hundred naturally occurring gibberellins are known. Many gibberellins are found to occur naturally in all higher plants. Because of potent plant growth regulating property; Gibberellic acid (GA3) is extensively used in agriculture, horticulture, nurseries, greenhouses, viticulture and tea gardens. Gibberellic acid (GA3) at very low concentration ( to extremely low yield, extensive down stream processing and the consequent high capital and operating expenses. Reference may be made by a method described for production of Gibberellic acid by Solid-State Fermentation in Coffee Husk by Machado C. M. M., et al. (Applied Biochemistry and Biotechnology, July 2002,vol.l02, no.1-3, pp.170-192 (14)). Although it uses coffee husk, an agriculture waste as a raw material and produces gibberellic acid using Gibberella fujikuroi LPB 06, yet the yield of gibberellic acid is very low (492.5 mg) as it is well known fact that Gibberellic Acid yield varies according to the use of the selected strain. Also, coffee husk waste used by Machado C. M. M., et al., is region specific. Objects of the invention; The main object of the invention is to provide a process for production of gibberellic acid in high yield (GAa) from fruits, vegetables and agriculture waste. Another object of the present invention is to provide a process for production of gibberellic acid (GAs) in high yield from fruits, vegetables and agriculture waste using fungal strain Gibberella fujikuroi NCIM 1099. Further another object of the present invention is to provide a simple, economical and efficient process for obtaining gibberellins containing high percentage of gibberellic acid (GA3). Summary of the invention; The present invention deals with a process for production of gibberellic acid (GAs) in high yield from fruits, vegetables and agriculture waste using fungal strain Gibberella fujikuroi NCIM 1099 fruits, vegetables and agriculture waste containing dried or wet coarse powder waste of citrus, pineapple, tomato, potato, pea hull, sorghum straw and maize cob etc. It is a simple, economical and efficient process for obtaining gibberellins containing high percentage of gibberellic acid Detailed description of the invention; Accordingly, the present invention provides a process for production of gibberellic acid (GAs) in high yield from fruits, vegetables and agriculture waste using fungal strain Gibberellafujikuroi NCIM 1099, wherein the said process comprising the steps of: a) growing the Gibberella fujikuroi NCIM 1099 on potato dextrose agar (PDA) in slant at low temperarture; b) preparing solid state fermentation media by transferring the biomass obtained from fruits, vegetables and agriculture waste in nutrient medium in sterile condition; c) transferring the Gibberellafujikuroi NCIM 1099 from PDA slant obtained from step (a) in the above said medium obtained from step (b) at 27-31 degree C for at least three days to get the inoculum; d) incubating the inoculum obtained from step (c) with sterilized biomass of waste for 8-10 days at 27-31 degree C to get fermented biomass; e) isolating the gibberellic acid (GAs) from fermented biomass obtained from step (d) by conventional method. In an embodiment of the present invention, the said fungal strain Gibberella fujikuroi NCIM 1099 is obtained from ATCC with accession number 12616. In another embodiment of the present invention, the said fungal strain Gibberella fujikuroi NCIM 1099 has the following characteristics: a) it grows on potato dextrose agar in slant at low temperature; b) colonies of said fungal strain on potato dextrose slants seems the texture like cotton wool. In another embodiment of the present invention, the said fruits, vegetables and agriculture waste are selected from the group consisting of Citrus pomace, pineapple pomace, tomato peel, potato peel, pea pod waste, maize cobs, sorghum straw etc. In further another embodiment of the present invention, the used potato dextrose agar comprises agar, potato infusion form and sucrose/ glucose in the ratio of 3: 40: 4 respectively. In yet another embodiment of the present invention, the said biomass is obtained by drying the fruits, vegetables and agriculture waste followed by pulverizing and passing through mesh aboutl/16". In still another embodiment of the present invention, the used nutrient media comprises glucose, sucrose, or maltose, N^NOs, KH2PO4) MgSO4, 7H2O, ZnSO4, 7H2O, CuSO4, 7H2O, MnSO4, 7H20, Na2MoO4, 2H2O, FeSO4 7H2O in the ratio ranges from 60000: 400: 4500: 1000: 2: 0.1: 0.05: 0.05: 1.5 to 80000: 500: 5500: 1200: 3: 0.3: 0.2: 0.2: 2.5 respectively. In still another embodiment of the present invention, the white-pale-cream colored fungal growth is observed during solid-state fermentation. In still another embodiment of the present invention, the fermented biomass is acidified after observing the white-pale-cream colored fungal growth. In still another embodiment of the present invention, the gibberellic acid is isolated with ethyl acetate using multipurpose solvent extractor. In still another embodiment of the present invention, gibberellic acid is determined by using known method. In still another embodiment of the present invention, gibberellic acid is determined by using high performance liquid chromatography, using reverse flow C -18 column and methanol as mobile phase. In still another embodiment of the present invention, the yield of the gibberellic acid is at least 5-6 gm/kg of waste. Gibberellins are well-recognized group of diterpenoids having a typical tetracyclic ring system. Many gibberellins are found to occur naturally in all higher plants. Gibberellic acid (GAs) is a well-known compound among the group of gibberellins and has been recognized as a potential plant growth regulator. Gibberellic acid (GA3) is extensively used in agriculture, horticulture, nurseries and green houses for regulating cell growth and stimulating the phenomenon of germination. The present invention provides a novel method for utilizing solid waste of agriculture, fruit and vegetable from food processing industries for production of gibberellins, containing high percentage of gibberellic acid. The waste, which is not used for any purpose other than disposal by any way, is used for value added product involving very simple technique. E.g. the method described in Kumar K. P. K. and Lonsane B. K. (1987), used to produce gibberellic acid from wheat bran and rice bran. But these materials are used as an important ingredient in animal and poultry feed formulations. Thus the present invention utilizes solid waste of agriculture, fruit and vegetable for production of gibberellic acid by solid-state fermentation using a fungal strain of Gibberellafujikuroi NCIM 1099. Agriculture waste viz. waste maize cobs, sorghum straws, fruit and vegetable wastes like viz. citrus pomace, pineapple pomace, tomato peel, potato peel, pea hull etc. from fruit and vegetable processing industry has limited utility to make value added products. This invention discloses the process of production of gibberellic acid using the solid waste of fruits and vegetables and agriculture waste which have no utility otherwise and essentially are absolute wastes. The present invention also discloses isolation of gibberellic acid by modified solid-liquid extraction process with high recovery of solvent. The invention provides a novel method utilizing solid waste of fruits and vegetables and agricultural waste as a raw material for producing the value added products. Solid waste of fruits, vegetables and agriculture contains appreciable quantum of nutrients and hence we got more yield of Gibberellic Acid upto 5-6 gm/Kg of waste. This invention describes the production of gibberellic acid by fungal strain Gibberella fujikuroi NCIM 1099 obtained from National collection of Industrial Microorganism, National Chemical Laboratories (NCL), Pune. The strain was grown subsequently on potato dextrose agar in slants, stored at low temperature and used in the experiments. The colonies of the Gibberella fujikuroi on Potato Dextrose agar in slants found the texture like cotton wool. Aqueous nutrient media was prepared which consist of useful source of carbon and nitrogen, low level of inorganic salts like potassium, sodium, magnesium, phosphorus and trace elements like zinc, copper, manganese, molybdenum. 100 ml of aqueous nutrient media was transferred in 500 ml Erlenmeyer flask and closed with surgical cotton plug. The flasks were autoclaved to get sterile biomass. These flasks were inoculated with the culture of Gibberellafujikuroi NCIM 1099 grown on potato dextrose agar at ambient temperature. The inoculated flasks were incubated in a chamber equipped with shaking unit maintained at temperature 29°+2°C. The growth was observed after 3 days. The three days old contents were used as inoculum. The solid waste was mixed with aqueous nutrient media same as used in preparation of inoculum at the level corresponding to the water activity assuring maximum growth of the fungus. The moist solid waste (25g solid waste + 20 ml nutrient media) was charged in 500 ml Erlenmeyer flasks, closed with surgical cotton plug. These flasks were autoclaved to get sterile biomass and cooled to ambient temperature. These flasks were inoculated with 20-25 ml inoculum and incubated in a chamber maintaining at 29°+2°C. The fermentation was allowed for 8 - 10 days. A white-pink fungal mould growth was observed in Erlenmeyer flasks. The flasks were acidified to lower the pH to the extent of pH = 2 to 3.5, to avoid the formation of iso-gibberellic acid. The fermented solid mass was subjected to isolation of crude gibberellic acid using solid-liquid extraction process. The amount of gibberellic acid (GAs) isolated from solid state fermentation media was determined by a known method using high performance liquid chromatography, using reverse flow C -18 column and methanol as mobile phase. The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present invention. Example No.l Composition of Potato Dextrose Agar (PDA) Agar: 15 gm/1 Potato, infusion form: 200 gm/1 Sucrose or glucose: 20 gm/1 The media was prepared using distilled water, gently mixing with glass rod; 3-4 ml was transferred to 25 ml culture tubes. Culture tubes were closed with surgical cotton plug, covered with paper and tied. The tubes were autoclaved at 121°C for 30 minutes and cooled to ambient temperature. These tubes were inoculated with the culture obtained fromNCIM,Pune. Example No. 2 Nutrient Media Preparation Glucose, sucrose, or maltose: 60 - 80 gm/1 NH4NO3 0.4-0.5 gm/1 KH2PO4 4.5-5.5 gm/1 MgSO4,7H20 1-1.2 gm/1 ZnS04, 7H2O 2-3 mg/1 CuSO4, 7H2O 0.1-0.3 mg/1 MnSO4, 7H2O 0.05-0.2 mg/1 Na2MoO4,2H20 0.05-0.2 mg/1 FeS047H20 1.5-2.5 mg/1 Accordingly, the nutrient media was prepared using double distilled water. 100 ml double distilled water was transferred in 500 ml Erlenmeyer flask, closed with surgical cotton plug, covered with paper and tied. The flasks were autoclaved at 121°C for 30 minutes and cooled to ambient temperature. These flasks were inoculated with the culture grown in culture tubes. After a period of 3-4 days, inoculated flasks were used as inoculum for solid- state fermentation. Example No. 3 Solid State Fermentation Media Citrus pomace, pineapple pomace, tomato peel, potato peel, pea pod waste, maize cobs, sorghum straw were used separately as a media for solid state fermentation. These waste were dried at 55-60°C separately. When the materials attained the moisture content below 10%, the materials were pulverized and passed through mesh 1/16". The biomass obtained from each waste was packed and sealed separately in polythene bags and used in the experiments. 25 gm of each type of biomass was transferred in 500 ml Erlenmeyer flask. Then 20 ml nutrient media was added and shaken thoroughly. The inlet of the flasks was packed with surgical cotton plug and covered with paper and tied using thread. The flasks were autoclaved at 121°C for 30 minutes and cooled. Example No. 4 Inoculum preparation 500 ml nutrient media was transferred in 2 lit. Erlenmeyer flask and the inlet of the flasks was packed with surgical cotton plug, covered with paper and tied with thread. The flask was autoclaved at 121°C for 30 minutes and cooled to ambient temperature. Gibberella fujikuroi NCIM 1099 was transferred from PDA slant and incubated for 3-4 days. This preparation was used as inoculum for solid state fermentation. Example No. 5 Solid State fermentation Autoclaved erlenmeyer flasks containing solid waste of fruits or vegetables or agriculture solid waste were inoculated with 20 ml inoculum. The fermentation was allowed to continue for 8 to 10 days. White-pale-cream colored fungal growth was observed. The flasks were acidified using dilute hydrochloric acid to maintain the pH = 2.5. Example No. 6 Isolation of Gibberellins Gibberellins were isolated with ethyl acetate using multipurpose solvent extractor. Example No. 7 Identification and quantification process Identification and quantification was done using HPLC Conditions for HPLC: Gibberellic Acid standard - Merck-Germany 8.14464.001 Column: C-18 Reverse phase Water Mobile phase: HPLC grade Methanol Flow rate: 1 ml/min Wavelength: 212 nm Peak was obtained within a period of 2.7 to 3.5 min. (Table Removed) Advantages; The main advantages of the present invention are: • The yield of Gibberellic Acid (GA3) is about 2 to 8 times higher than the process of submerged fermentation depending upon the type of waste. • Other Gibberellins such as GA4 and GA7 are also produced in the process, which are also known as plant growth regulators. • The process is more economical. • The process is developed for value added use of solid waste of fruit and vegetable processing industries. • The process envisages the recycling, reuse and recovery of valuable product and has positive impact on the process of solid waste management programs of the urban area. We Claim: 1) A process for production of Gibberellic acid using solid state fermentation from plant waste material using fungal strain Gibberella fujikuroi NCIM 1099, wherein the said process comprising the steps of: (a) growing the Gibber ella fujikuroi NCIM 1099 on potato dextrose agar (PDA) in slant preserved below -4°C. (b) preparing solid state fermentation media by transferring the biomass obtained from plant waste material in nutrient medium in sterile condition; (c) transferring the Gibberella fujikuroi NCIM 1099 from potato dextrose agar slant obtained from step (a) in the above said medium obtained from step (b) at 27-31°C for at least three days to get the inoculum; (d) incubating the inoculum obtained from step (c) with sterilized biomass of waste for 8-10days at 27-31°Cto get the fermented biomass; (e) isolating the Gibberellic acid from fermented biomass obtained from step (d) by conventional method. 2) The process as claimed in claim 1, wherein the said fungal strain Gibberella fujikuroi NCIM1099 is obtained from ATCC with accession numberl2616.3. 3) The process as claimed in claim 1, wherein the said fungal strain Gibberella fujikuroi NCIM 1099 has the following characteristics: a. it grows on potato dextrose agar in slant at low temperature; b. colonies of said fungal strain on potato dextrose slants seems like cotton wool. 4) The process as claimed in claim 1, wherein the said plant waste material is selected from the group consisting of citrus pomace, pineapple pomace, tomato peel, potato peel, pea pod waste, maize cobs, sorghum straw etc. 5) The process as claimed in claim 1, wherein the used potato dextrose agar comprises agar, potato infusion form and sucrose/ glucose in the ratio of 3:40: 4 respectively. 6) The process as claimed in claim 1 , wherein the said biomass is obtained by drying the fruits, vegetables and agriculture waste followed by pulverizing and passing through 1/16" mesh size. 7) The process as claimed in claim 1, wherein the used nutrient media comprises glucose, sucrose, or maltose, NH4N03, KH2P04, MgS04.7H20, ZnS04.7H20, CuS04.7H20, MnS04.7H20, Na2Mo04.2H20, FeS047H20 in the ratio ranges from 60000: 400: 4500: 1000: 2: 0.1: 0.05: 0.05: 1.5 to 80000: 500: 5500: 1200: 3: 0.3:0.2:0.2:2.5 respectively. 8) The process as claimed in claim 1, wherein the Gibberellic acid is isolated with ethyl acetate using multipurpose solvent extractor. 9) The process as claimed in claim 1, wherein the yield of the Gibberellic acid is at least 5- 6gm/kg of waste. 10) The process for production of Gibberellic acid in high yield substantially as herein described with reference to the examples accompanying this specification.

Documents:

649-DEL-2006-Abstract-(23-02-2012).pdf

649-del-2006-abstract.pdf

649-DEL-2006-Claims-(23-02-2012).pdf

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649-DEL-2006-Correspondence Others-(23-02-2012)..pdf

649-DEL-2006-Correspondence Others-(23-02-2012).pdf

649-del-2006-correspondence-others-1.pdf

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649-DEL-2006-Description (Complete)-(23-02-2012).pdf

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649-del-2006-form-1.pdf

649-del-2006-form-18.pdf

649-DEL-2006-Form-2-(23-02-2012).pdf

649-del-2006-form-2.pdf

649-DEL-2006-Form-3-(23-02-2012)..pdf

649-DEL-2006-Form-3-(23-02-2012).pdf

649-del-2006-form-3.pdf

649-del-2006-form-5.pdf

649-DEL-2006-Petitiion-137-(23-02-2012).pdf