Title of Invention

A PROCESS FOR PRODUCTION OF 2-HYDROXY-4-METHOXY BENZALDEHYDE FROM TUBERS OF DECALEPIS HAMILTONII WIGHT & ARN.

Abstract

The present invention relates to a process for production of 2-hydroxy 4 methoxy benzaldehyde from tubers of Decalepis hamiltonii Wight & Arn. The novelty of the present invention is, it provides for the first time an efficient method for microbial elicitation of flavor compound 2-hydroxy-4-methoxy benzaldehyde in tubers of D. hamiltonii . In the present invention Bacterial ( Bacillus cereus, B. subtilis, Psuedomonas sp. Streptococcus, Staphylococcus sp. , Escherichia coli and Agrobacterium tumefaciens LBA 4404 ) , fungal cultures (Aspergillus niger, A.flavus, Penicillium notatum, Rhizopus oligosporus, Fusarium sp. ) and yeast {Saccharomyces cerevisiae, Rhodotorula rubrum ) were used for elicitation of flavour compound 2-hydroxy-4-methoxybenzaldehde in tubers of D. hamiltonii. The treated tubers were incubated and later the flavour compound was isolated by steam distillation method from the tubers .

Full Text

The present invention relates to a process for production of 2-hydroxy 4 methoxy benzaldehyde f rom tubers of Decalepis hamiltonii Wight & Arn. The invention is aimed at development a process for production of 2-hydroxy 4 methoxy benzaldehyde from tubers of Decalepis hamiltonii Wight & Arn/ Decalepis hamiltonii Wight & Arn., (swallow root) belonging to Asclepidaceae is a monogeneric climbing shrub native of the Deccan peninsula and forest areas of Western Ghats of India. It finds use as a culinar/ spice due to its high priced aromatic roots. The roots are markedly fleshy , cylindrical (1-6 cm diameter ) are characterized by a sarasaparilla like taste accompanied by a tingling sensation on the tongue as described in Wealth of India 1952 (Wealth of India 1952, A dicţionar/ of raw materials , CSIR, New Delhi 3: 24). The roots of D.hamiltonii are used as a flavouring principie (Wealth of India, 1990), appetizer (Murthi, P.B.R., and Seshadri.T.R. Proc. Ind.Acad.Sci. 1947; 13A, 221), blood purifier (Jacob.K.C. Madras Agric. Journal. An unrecorded economic product Decalepis hamiltcnii W & Arn., Family Asclepidaceae 1937; 25; 176), and preservative (Phadke, N.Y., Gholap A.S., Ramakrishnan K, Subbulakshmi G. , J.Food Sci.Technol. 1994; 31, 472). Similarly the roots of this taxon as described by Nayar et al. (1978) (Nayar RC, Shetty JKP, Mary Z and Yoganrasimhan 1978 Pharmacological studies of root of Decalepis hamiltonii W & Arn and comparison with Hemidesmus indicus (L.) R.Br. Proc. Indian Acad. Sciences 87 (B) : 37-48) are considered as "Sar/Va Bheda" in Ayurveda where finds use as an alternative to roots of Hemidesmus indicus in the preparation of severa! herbal drugs like Amrutamalaka taila, Drakshadi churna, shatavari rasayana and yeshtimadhu taila. The roots contain 92% fleshy matter and 8% woody core. Of late the highly aromatic roots have been subjected to over exploitation by destructive harvesting that nas endangered the survival of this plant. In the earlier reports by George et al. (George ,J. PeriraJ., Divakar.S., Udayasankar.K and Ravishankar.G.A. Current Science ,1999; 77, 501-502) it was observed that the aromatic roots of D.hamiltonii proved to be a potent bioinsecticide on storage pests at lethal and sub-lethal levels (Indian Patent No. 1301/Del/98). The supercritical extracts of these roots proved to be potent antimicrobial agents (George ,J., Udayasankar, K., Keshava.N and Ravishankar, G.A. Fitoterapia 1999; 70, 172-174). George,J., Bais.H.P. and Ravishankar.G.A. (Current Science ,2000; 79:894-898) were able to regenerate plantlets of D.hamiltonii W&A from leaf callus. Similarly a method for rooting of Decalepis hamiltonii for field transfer was reported earlier (Bais HP, Sudha G, Suresh B &. Ravishankar GA , Curr. Sci, 2000, 79: 408-410; Obul Reddy, B ., Giridhar, P and Ravishankar G.A, Current Science 81(11), 2001,1479-1482). Plant cell culture îs an alternative technology for the production of high value phytochemicals. Root cuitures offer additional advantages such as high growth rate, genetic stability and high biosynthetic capacity. 2-hydroxy-40-mthoxy benzaldehyde is found in roots of D.hamiltonii the single major constituent. Elicitation of secondary metabolites by using fungal , bacteria! and yeast origin have been known (Dicosmo and Misawa 1985 Elicitation of secondary metabolism in plant cultures Trend in Biotechnology 3, 318-322). Ravishankar G.A. and Venkatraman L.V . Elicitation of capsaicin production in freely suspended cells and immobilized cell cultures of Capsicum frutescens mill. Food Biotehcnol. 5: 197-205). There are no prior reports on elicitation of flavour metabolites in Decalepis hamiltonii. So there is a need to develop a method to improve the fiavour content 2-hydroxy -4-methoxy benzaldehyde in tubers of Decalepis hamiltonii in view of its wide range of applications. The main object of the present invention is to provide a process for production of 2-hydroxy 4 methoxy benzaldehyde from tubers of Decalepis hamiltonii Wight & Arn. which obviates the drawback as detailed above. Accordingly a process for production of 2-hydroxy 4 methoxy benzaldehyde from tubers of Decalepis hamiltonii Wight & Arn which comprises; a) cleaning and washing of tubers of Decalepis hamiltonii by known methods, b) soaking the tubers in 200 ml surface cleaning agent such as herein described solution for 5-8 minutes followed by washing with sterile water thrice, c) sterilizing tubers by soaking in the solution of 1-2% NaOCI for 5-7 minutes followed by washing with sterile water for atleast three times. d) soaking of the above obtained tubers in microbial inoculum selected from microbial inoculum Bacillus cereus B. subtilis Escherichia coli Staphylococcuc sp. Streptococcus sp. Pseudomonas sp. Agnobacetrium tumefaciens LBA 4404 Saccharomyces cerevisiae Rhodotorula rubrum Aspergillus niger Fusarium sp. Penicillium notatum Rhizopus oligosporus Optical Density 0.50 to 1.86 0.08 to 0.350 0.55 to 1.65 0.08-0.750 0.09- 0.627 0.150-1.58 0.18-1.08 0.14 to 1.526 0.124-1.86 -1.5X106 to 2.5 X106 -1.5X106 to 2.5 X106 -1.5X106 to 2.5 X106 -1.5X106 to 2.5 X106, e) Slicing the incubated tubers mechanically, f) steam distilling the above said tubers for 5-6 hrs, g) extracting the steam condensate with dichloromethane by known methods, h) concentrating the extract in a flash evaporator, i) dissolving in ethanol to obtain 2-hydorxy-4-methoxy-benzaldehyde, The novelty of the present invention is, it provides for the first time an efficient method for microbial elicitation of flavor compound 2-hydroxy-4-methoxy benzaldehyde in tubers of D. hamiltonii. In the present invention : Bacterial ( Bacillus cereus, B. subtilis, Psuedomonas sp. Streptococcus, Staphylococcus sp. , Escherichia coli and Agrobacterium tumefaciens LBA 4404 ) , fungal cultures (Aspergillus niger, A.flavus, Penicillium notatum, Rhizopus oligosporus, Fusahum sp. ) and yeast (Saccharomyces cerevisiae, Rhodotorula rubrum ) were used for elicitation of flavour compound 2-hydroxy-4-methoxybenzaldehde in tubers of D. hamiltonii. The treated tubers were incubated and later the flavour compound was isolated by steam distillation method from the tubers . The qualitative analysis of the compound was performed by TLC, and quantitative analysis was done by GC(FID). The following examples are given by way of iliustration of the present invention Example 1 Fresh tubers of Decalepis hamiltonii were collected from local market and sorted out to a required size ( approximately 10.0 ± 1.0 cm length and 2.0 ± 0.5 cm diameter). The tubers were cleaned off extraneous matter and soil with 1-2 liters of cold water (15 ±2 °C) / 500 gm tubers. Later they were surface sterilized first with 200 ml of 70% alcohol for 5 seconds followed by washing with sterile water thrice and then with 200 ml solution of Tween 20 (v/v) (3-4 drops in 100 ml water) for 5 minutes to remove the adhering soil particles followed by sterilized distilled water thrice. The tubers were then further subjected to surface sterilization with 200 ml 1% sodium hypochlorite (NaOCI) for 5 minutes followed by water wash thrice under aseptic conditions. The surface of the tubers was blotted with sterilized blotting paper. The fungal stock cultures Aspergillus flavus, Aspergillus niger, Pencillium notatum, Rhizopus oligosporus , Fusarium sp. were used for this study. Fresh culture were made on PDA slants and incubated for 7 days. Then the spores of the respective fungi were used to prepare spore suspension or inoculum in 0.1% sodium lauryl sulphate and diluted ultimately to get a spore density of ~ 2.5 X 106 spore / ml). Bacterial cultures Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Pseudomonas sp., Streptococcus sp. , Staphylococcus sp, E.coli, and Agrobacterium tumefaciens LBA 4404 were used for this study. Bacterial inoculum was prepared by inoculating overnight grown bacterial suspension into nutrient broth medium ( peptone 5.0 gm ; beef extract 3.0 gm; NaCI 5 gm ; water 1000 liter). After incubating at 30°C for 18 hrs the O.D. values of respective bacterial cultures were recorded (Bacillus cereus 1.176 , B.subtilis 0.165 , E. coli 1.172, Staphylococcus sp. 0.127, Streptococcus sp. 0154, Pseudomonas sp. 0.849, Agrobacetrium tumefaciens LBA 4404 0.428 ,). Similarly the yeast Saccharomyces cerevisiae, Rhodotoruia sp. were also used for this study and their inoculums were made as in case of bacteria (S. cerevisiae 0.826, R. rubrum 0.556). The surface sterilized tubers were soaked in bacterial / fungal / suspension ( 50 g tubers / 200 ml inoculum) for 30 minutes after removing peel in one set of experiment and peel was retained in another set. Later the treated tubers were transferred to sterilized polythene bags containing sterilized cotton wetted with water to maintain humidity in side. The polythene bags were stapled and incubated at optimum conditions (28± 1°C for bacteria except E.coli 30°C ) for 72 hrs. The fungal cultures were incubated in dark at room temperature ( 28 ±2°C)for 10 days. Later the tubers were washed in sterilized water 3-4 times to remove the surface growth of respective organisms. Then the washed tubers were mechanically dissected into small pieces of 0.5-1.0 cm diameter. and subjected to steam distillation for 5 hours. The steam condense was extracted with dichloromethane (50 ml x 4). The combined extracts were passed through a funnel containing anhydrous sodium sulphate to remove the water content, concentrated in a flash evaporator and dissolved in 1ml ethanol and stored in closed vials. Quantification of the flavour compound was determined by gas chromatographic analysis (GC) using flame ionization detection (FID). Analysis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) was done by spotting the root extracts on TLC plate along with standard (Fluka Chemicals, Switzeriand) and run in a solvent system comprising of Hexane: Benzene (1:1). Rf of spot coinciding with that of standard (2H4MB) (0.47) was eluted in solvent and UV spectrum was measured on a Perken-Elmer UV-Vis recording spectrophotometer UV-160. Maximum absorption was obtained at 278nm. Quantitative detection was done by GC. The constituent was identified by matching the mass spectra with GC-MS library user generated mass spectral libraries, and also confirmed by comparison with GC retention time of standard sample. The concentrated volatiles were separated by GC, flame ionization detector (FID) with capillary column and GC-MS analysis using a Shimadzu, GC-14B coupled with QP 5000 MS system under the following conditions SPB-1 column (Supelco, USA, 30 m x 0.32 mm, 0.25 |iM film thickness); oven temperature programme, 60° C for 2 min, rising at 2°C/min to 250° C, held for 5 min; injection port temperature 225° C; detector temperature, 250° C; carrier gas helium, flow rate 1ml min"1. The amount of solution injected was 1 ml for analysis . The GC(FID) profiles indicated that there was a six fold increase of flavour compound (2-hydroxy-4-methopxy benzaldehyde )in tubers without peel elicited with B.cereus, four tirnes increase in case of B. subtilis, 2-3 folds increase in case of E.coli, Pseudomonas sp. respectively. Similarly the yeast cultures Saccharomyces cerevisiae showed five times increase of flavour compound (2-hydroxy-4-methopxy benzaldehyde) followed by doubling of flavour compound in case of R.rubrum. Out of the four fungi tried Aspergillus niger only showed marked elicitation (doubled) of flavour compound 2-hydroxy-4-methopxy benzaldehyde in tubers without peel. There was no significant increase of flavour compound in tubers with peel, irrespective of type of microbial elicitation . An average of three samples were shown in Table. 1. Table. 1 Elicitation of flavour compound in tubers of Decalepis hamiltonii by using some biotic elicitors (Table Removed) The values are an average of three samples Example 2 Fresh tubers of Decalepis hamiltonii were col'ected from local market and sorted out to a required size ( approximately 10.0 .: O cm length and 2.0 ± 0.5 cm diameter). The tubers were cleaned off extraneous matter and soil with 1-2 liters of cold water (15 ±2 °C) / 500 gm tubers. Later they were surface sterilized first with 200 ml of 75% alcohol for 10 seconds followed by washing with sterile water thrice and then with 200 ml solution of Tween 20 (v/v) (3-4 drops in 100 ml water) for 8 minutes to remove the adhering soil particles followed by sterilized distilled water thrice. The tubers were then further subjected to surface sterilization with 200 m! 1% sodium hypochlorite (NaOCI) for 7 minutes followed by water wash thrice under aseptic conditions. The surface of the tubers was blotted with sterilized blotting paper. The fungal stock cultures Aspergillus flavus, Aspergillus niger, Pencillium notatum, Rhizopus oligosporus , Fusarium sp. were used for this study. Fresh culture were made on PDA slants and incubated for 7 days. Then the spores of the respective fungi were used to prepare spore suspension or inoculum in 0.1% sodium lauryl sulphate and diluted ultimately to get a spore density of - 2.5 X 106 spore / ml). Bacterial cultures Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Pseudomonas sp., Streptococcus sp. , Staphylococcus sp, E.coli, and Agrobacterium tumefaciens LBA 4404 were used for this study. Bacterial inoculum was prepared by moculating overnight grown bacterial suspension into nutrient broth medium ( peptone 5.0 gm ; beef extract 3.0 gm; NaCI 5 gm ; water 1000 liter). After incubating at 30°C for 18 hrs the O.D. values of respective bacterial cultures were recorded (Bacillus cereus 0.500 , B.subtilis 0.08 , E. coli 0.55, Staphylococcus sp. 0.08, Streptococcus 0.09, Pseudomonas sp. 0.150, Agrobacetrium tumefaciens LBA 4404 0.18). Similarly the yeast Saccharomyces cerevisiae, Rhodotorula sp. were also used for this study and their inoculum were made as in case of bacteria, (S. cerevisiae 0.14 R. rubrum 0.124). The surface sterilized tubers were soaked in bacterial / fungal / suspension ( 50 g tubers / 200 ml inoculum) for 30 minutes after removing peel in one set of experiment and peel was retained in another set. Later the treated tubers were transferred to sterilized polythene bags containing sterilized cotton wetted with water to mamtain humidity in side. The polythene bags were stapled and incubated at optimum conditions (28± 1°C for bacteria except E.coli 30°C ) for 72 hrs. The fungal cultures were incubated in dark at room temperature ( 28 ±2°C)for 10 days. Later the tubers were washed in sterilized water 3-4 times to remove the surface growth of respective organisms. Then the washed tubers were mechanically dissected into small pieces of 0.5-1.0 cm diameter. and subjected to steam distillation for 5 hours. The steam condense was extracted with dichioromethane (50 ml x 4). The combined extracts were passed through a funnel containing anhydrous sodium sulphate to remove the water content, concentrated in a flash evaporator and dissolved in 1 ml ethanol and stored in closed vials. Quantification of the flavour compound was determined by gas chromatographic analysis (GC) using flame ionization detection (FID). Analysis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) was done by spotting the root extracts on TLC plate along with standard (Fluka Chemicals, Switzerland) and run in a solvent system comprising of Hexane: Benzene (1:1). Rf of spot coinciding with that of standard (2H4MB) (0.47) was eluted in solvent and UV spectrum was measured on a Perken-Elmer UV-Vis recording spectrophotometer UV-160. Maximum absorption was obtained at 278nm. Quantitative detection was done by GC. The constituent was identified by matching the mass spectra with GC-MS library user generated mass spectral libraries, and also confirmed by comparison with GC retention time of standard sample. The concentrated volatiles were separated by GC, flame ionization detector (FID) with capillary column and GC-MS analysis using a Shimadzu, GC-14B coupled with QP 5000 MS system under the following conditions SPB-1 column (Supelco, USA, 30 m x 0.32 mm, 0.25 nM film thickness); oven temperature programme, 60° C for 2 min, rising at 2°C/min to 250° C, held for 5 min; injection port temperature 225° C; detector temperature, 250° C; carrier gas helium, flow rate 1ml min"1. The amount of solution injected was 1 ml for analysis . The GC(FID) profiles indicated that there was a six fold increase of flavour compound (2-hydroxy-4-methopxy benzaldehyde )in tubers without peel elicited with B.cereus, two times increase in case of B. subtilis, 1-2 foids increase in case of E.coli, Pseudomonas sp. respectively. Similarly the yeast cultures Saccharomyces cerevisiae showed three times increase of flavour compound (2-hydroxy-4-methopxy benzaldehyde) followed by doubling of flavour compound in case of R.rubrum. Out of the four fungi tried Aspergillus niger only showed marked elicitation (one) of flavour compound 2-hydroxy-4-methopxy benzaldehyde in tubers without peel. There was no significant increase of flavour compound in tubers with peel, irrespective of type of microbial elicitation. An average of three samples were shown in Table.2 Table. 2 Elicitation of flavour compound in tubers of Deca/ep/s hamiltonii by using some biotic elicitors (Table Removed) The values are an average of three samples Example 3 Fresh tubers of Decalepis hamiltonii were collected from local market and sorted out to a required size ( approximately 10.0 ± 1.0 cm length and 2.0 ± 0.5 cm diameter). The tubers were cleaned off extraneous matter and soil with 1-2 liters of cold water (15 ±2 °C) / 500 gm tubers. Later they were surface sterilized first with 200 ml of 70% alcohol for 5 seconds followed by washing with sterile water thrice and then with 200 ml solution of Tween 20 (v/v) (3-4 drops in 100 rnl water) for 5 minutes to remove the adhering soil particles followed by sterilized distilled water thrice. The tubers were then further subjected to surface sterilization with 200 ml 1% sodium hypochlorite (NaOCI) for 5 minutes followed by water wash thrice under aseptic conditions. The surface of the tubers was blotted with sterilized blotting paper. The fungal stock cultures Aspergillus flavus, Aspergillus niger, Pencillium notatum, Rhizopus oligosporus , Fusarium sp. were used for this study. Fresh culture were made on PDA slants and incubated for 7 days. Then the spores of the respective fungi were used to prepare spore suspension or inoculum in 0.1% sodium lauryl sulphate and diluted ultimately to get a spore density of - 2.5 X 106 spore / ml). Bacterial cultures Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Pseudomonas sp., Streptococcus sp, , Staphylococcus sp, E.coli, and Agrobacterium tumefaciens LBA 4404 were used for this study. Bacterial inoculum was prepared by inoculating overnight grown bacterial suspension into nutrient broth medium ( peptone 5.0 gm ; beef extract 3.0 gm; NaCI 5 gm ; water 1000 liter). After incubating at 30°C for 18 hrs the O.D. values of respective bacterial cultures were recorded (Bacillus cereus 1.86 , B.subtilis 0.350 , E. coli 1.65, Staphylococcus sp. 0.750, Streptococcus sp. 0.627, Pseudomonas sp. 1.58, Agrobacetrium tumefaciens LBA 4404 1.08). Similarly the yeast Saccharomyces cerevisiae, Rhodotorula sp. were also used for this study and their inoculums ( S. cerevisiae 1.526, R. rubrum 1.86) were made as in case of bacteria. The surface sterilized tubers were soaken m bacterial / fungal / suspension ( 50 g tubers / 200 ml inoculum) for 30 minutes atter removing peel in one set 'of experiment and peel was retained in another set. Later the treated tubers were transferred to sterilized polythene bags containing sterilized cotlon wetted with water to maintain humidity in side. The polythene bags were stapled and incubated at optimum conditions (28± 1°C for bacteria except E.coli 30°C ) for 72 hrs. The fungal cultures were incubated in dark at room temperature ( 28 ±2°C)for 10 days. Later the tubers were washed in sterilized water 3-4 times to remove the surface growth of respective organism Then the washed tubers were mechanically dissected into small pieces of 0.5-1.0 cm diameter. and subjected to sleam distillation for 5 hours. The steam condense was extracted with dichioromethane (50 ml x 4). The combined extracls were passed through a funnel containing anhydrous sodium sulphate to remove the water content, concentrated in a flash evaporator and dissolved in 1ml ethanol and stored in closed vials. Quantification of the flavour compound was determined by gas chromatographic analysis (GC) using flame ionization detection (FID). Analysis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) was done by spotting the root extracts on TLC plate along with standard (Fluka Chemicals, Switzerland) and run in a solvent system comprising of Hexane: Benzene (1:1). Rf of spot coinciding with that of standard (2H4MB) (0.47) was eluted in solvent and UV spectrum was measured on a Perken-Elmer UV-Vis recording spectrophotomeler UV-160. Maximum absorption was obtained at 278nm. Quantitative detection was done by GC(FID). The constituent was identified by matching the mass spectra with GC-MS library user generated mass spectral libraries, and also confirmed by comparison with GC retenlion time of standard sample. The concentrated volatiles were separated by GC, flame ionization detector (FID) with capillary column and GC-MS analysis using a Shimadzu, GC-14B coupled with QP 5000 MS system under the following conditions SPB-1 column (Supelco, USA, 30 m x 0.32 mm, 0,25 (iM film thickness); oven temperature programme, 60° C for 2 min, rising at 2°C/min to 250° C, held for 5 min; injection port lemperalure 225° C; delector temperature, 250° C; carrier gas helium, flow râie 1ml min"1. The amount of solution injected was 1 ml for analysis . The GC(FID) profiles indicated that there was a six fold increase of flavour compound (2-hydroxy-4-methopxy benzaldehyde )in tubers without peel elicited with B.cereus, 3.8 times increase in case of B. subtilis, 2-3 folds increase in case of E.coli, Pseudomonas sp. respectively. Similarly the yeast cultures Saccharomyces cerevisiae showed 4.8 times increase of flavour compound (2-hydroxy-4-methopxy benzaldehyde) followed by doubling of flavour compound in case of R.rubrum. Out of the four fungi tried Aspergillus niger only showed marked elicitation (1.2 fold) of flavour compound 2-hydroxy-4-methopxy benzaldehyde in tubers without peel. There was no significant increase of flavour compound in tubers with peel, irrespective of type of microbial elicitation . An average three samples were presented in Table.3 Table. 3 Elicitation of flavour compound in tubers of Decalepis hamiltonii by using some biotic elicitors (Table Removed) *The values are an average of three samples The main advantages of the present invention are: 1. The development of efficient method for enhancement of flavor metabolite 2- hydroxy -4-methoxy benzaldehyde in tubers of D. hamiltonii. 2. Elicitation of flavour metabolite by microbial cultures is achieved after the harvest, and this process can be adopted prior to extraction of the flavour compound. 3. Elicitation of flavour metabolite also reduces the requirement of biomass by over 3 folds , hence enhancing the productivity of flavour metabolite yield. We claim: 1. A process for production of 2-hydroxy 4 methoxy benzaldehyde from tubers of Decalepis hamiltonii Wight & Arn which comprises; a) cleaning and washing of tubers of Decalepis hamiltonii by known methods, b) soaking the tubers in 200 ml surface cleaning agent such as herein described solution for 5-8 minutes followed by washing with sterile water thrice, c) sterilizing tubers by soaking in the solution of 1-2% NaOCI for 5-7 minutes followed by washing with sterile water for atleast three times. d) soaking of the above obtained tubers in microbial inoculum selected from microbial inoculum Optical Density Bacillus cereus 0.50 to 1.86 B. subtilis 0.08 to 0.350 Escherichia coli 0.55 to 1.65 Staphylococcuc sp. 0.08 - 0.750 Streptococcus sp. 0.09-0.627 Pseudomonas sp. 0.150-1.58 Agrobacetrium tumefaciens LBA 4404 0.18 - 1.08 Saccharomyces cerevisiae 0.14 to 1.526 Rhodotorula rubrum 0.124 - 1.86 Aspergillus niger ~ 1.5 X 106 to 2.5 X 106 Fusarium sp. ~ 1.5 X 106 to 2.5 X 106 Penicillium notatum ~ 1.5 X 106 to 2.5 X 106 Rhizopus oligosporus ~ 1.5 X 106 to 2.5 X 106, e) Slicing the incubated tubers mechanically, f) steam distilling the above said tubers for 5-6 hrs, g) extracting the steam condensate with dichloromethane by known methods, h) concentrating the extract in a flash evaporator, i) dissolving in ethanol to obtain 2-hydorxy-4-methoxy-benzaldehyde, 2. A process for production of 2-hydroxy 4 methoxy benzaldehyde from tubers of Decalepis hamiltonii Wight & Arn as claimed in claim 1, which is here in described with reference to examples accompanying specifications.

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439-DEL-2003-Abstract-(15-12-2008).pdf

439-del-2003-abstract.pdf

439-DEL-2003-Claims-(15-12-2008).pdf

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439-DEL-2003-Correspondence-Others-(15-12-2008).pdf

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439-DEL-2003-Description (Complete)-(15-12-2008).pdf

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