Title of Invention

"PROCESS FOR MANUFACTURE OF INDIGO FROM INDIGO PLANTS"

Abstract

This invention relates to a process for manufacture of indigo from plants using acid extraction and subsequent neutralization for use as a dye and as a pigment. In this process extracts of Indigofera plant material are prepared with acids at a pH that reversibly inhibits the endogenous indimulsin enzyme. The indigo is precipitated from the clarified extracts by neutralizing it with alkali and collected by known processes. The process is eco-friendly, simple, rapid, produces better quality indigo and does away with the cumbersome requirements of maintaining anoxic conditions of traditional processes. By this method indigo can be prepared in an hour as compared to 10-15 hours required for the prior art processes.

Full Text

Field of Invention: This invention relates to a process for manufacture of indigo from plants using acid extraction and subsequent neutralization and oxidation for use as a dye and as a pigment. This invention is particularly related to a process wherein the indican containing plant material is extracted at very a low pH and avoids the traditional fermentation step. Background and Prior Art: Indigo has been prepared from time immemorial by a fermentation process. Plants of the Indigofera, Isatis and Polygonum genera containing the glucoside - indican - are fermented to enable the endogenous indimulsin break down the glucoside under anaerobic conditions. The indoxyls formed due to the enzymatic action join together in pairs to form leuco-indigotin that is water soluble in the prevalent reduced environment. This leuco-indigotin diffuses out of the plant material into the fermentation liquor. The anoxic fermented liquor is separated from the plant material and oxidised by air to obtain indigo blue that is insoluble in water. The conventional processes for extraction of indigo from Indigofera have been reviewed in the Wealth of India [The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products. Raw Materials, Part V, (1997) pp 173-174. New Delhi: CSIR]. According to the review the wet process is universally adopted in the extraction of indigo. In this process, freshly harvested plants are steeped in water in specially constructed vat; the water is warmed if necessary to 32°C. Fermentation sets in and the glucoside breaks down by the action of indimulsin, naturally present in the plant, to glucose and indoxyl. The fermentation is completed in 10-15 hours. The yellow liquor is run into beating (oxidising) vats equipped with paddle wheels; when the wheels are rotated, the liquor is thrown up into the air as a fine spray and the indoxyl gets oxidised into indigo, which settles down as a fine blue mud at the bottom of the vat. The supernatant water (seeth water) is then drawn off. Better yields are obtained by fermenting the leaves under acid condition and effecting oxidation by blowing ammonia, steam and air simultaneously into the fermented liquor by means of an injector. Addition of sodium nitrate or Dhak gum (from Butea monosperma) facilitates the settling of the indigo mud. The oxidised product is stirred with boiling water containing a little dilute sulphuric acid, allowed to settle and the clear liquor decanted: it is then washed with boiling water filtered through cloth and pressed. The product is cut into 3-inch cubes and air-dried to about 6% moisture. The cubes are brushed to remove surface mould growth, if any, polished and packed in wooden chests. The average yield is 25% of the total extractable colouring matter. In Europe, indigo has been extracted from Isatis (woad) as described by Hurry [Hurry, Jamieson B (1930) The Woad Plant and its Dye. (3rd ed. 1973) Clifton, N.J.: Augustus M. Kelly, pp 24-27]. The ancient process to make indigo from Isatis (woad) is as follows: The leaves are crushed, drained, partly dried and kneaded into balls. The balls are dried slowly and powdered. The powder is sprinkled with water, fermented for 9 weeks at 125°F, after which it emits an odour of ammonia. The resultant clay-like material is thoroughly dried and packed for use by dyers. Recently a new process for preparing indigo from woad has been developed and described by Garfield [Garfield, Simon (2000) Mauve. London: Faber and Faber, p 143]. This method involves making of an extract of leaves with hot water to burst the cells and liberate the precursors into the extraction liquid. The extract is then cooled, made alkaline by adding lime and aerated. The ancient process of drying and fermentation is then followed. The indigo produced by the prior art processes described above is of low quality. It contains many impurities such as indigo red, indigo brown, indigo gluten, etc., in appreciable amounts. The prior art process is time consuming. Process variables such as pH, temperature and redox potential levels affect the quality of the final product. These were never controlled in the prior art process leading to substantial variation in the quality (hue) of the product. If the leuco-indigotin comes in contact with air within or outside the plant material, it would get oxidised and precipitate therein, thereby lowering yields of indigo blue. The Coventry lime process was used, where slaked lime was added to the fermenting liquor to neutralize the effects of the acidity and improve the quality of indigo [Rawson C (1899) Report on the cultivation and manufacture of indigo in Bengal, p 25. For The Indigo Defence Association, Ltd.]. In a European patent applicatio "EP0754734 (publication 1997) a process is described to prepare natural dyes from plant matter including indigo. In this method natural dyes are prepared by drying, cleaning and pulverizing plant material and subjecting to the step of extraction consisting of a first soaking in an aqueous solution of pH 4-10 followed by a second step of heating. The solution is filtered and concentrated. In this prior art method to make natural dyes, the drying and extraction is done in the presence of air. Under these conditions any indigotin formed will precipitate in the plant matter. This indigo is not soluble at the pH (4-10) of the extracting solution. Hence, whatever indigo is formed will be lost in the plant matter. In an US patent US5042989 (1991) a-method to prepare lustrous natural dyes from plant matter of which indigo is an example, is performed by drying collected (dead) plant material at temperature at or above 70°C; pulverizing the dried plant material; extracting it with water; adding sufficient acid or base to the extract such that its pH is similar to the pH of the plant material in the living state. In this prior art process the plant material is dried at temperatures above 70°C. At this temperature the indimulsin of lndigofera looses its activity rapidly, and at 80°C and above it is quickly and irreversibly inactivated. The pH of the extraction medium is also adjusted to the pH of the fresh plant tissue, which in the case oflndigofera is about pH 8. At this pH any residual plant enzyme would be active and since the extraction is done in the presence of air, indigotin formed would precipitate in the plant matter and be lost. Hence, both the prior art processes described in EP0754734 and US5042989 to make natural dyes cannot be used to make indigo. All prior art process used to make indigo try to keep oxygen out of the extraction medium. This is difficult to achieve in practice, is time consuming and expensive. Object: The principle object of the present invention is to develop a process to produce indigo of high quality in a quick manner and with a better recovery. Summary of the invention: Accordingly the present invention describes a process for manufacture of indigo from indigo plants comprising following steps: (i) placing the young twigs or cut plants of Indigofera, Isatis, Polygonum, Wrightia genera or any other plant that synthesises indican in a steeping tank, in such a manner as they will remain submerged in the acidic water with which the tank will be filled in the next step; (ii) filling the said tank containing plants, with highly acidic warm water at pH between 1-3 at 2-10 litres per 1 kg of plant material, in such a manner as to avoid any air getting entrapped in the system; (iii) steeping the plants in the said acidic water for about 15 minutes to 4 hours at 40-70°C with or without addition of exogenous indimulsin like enzymes; (iv) separating and clarifying the acidic acid extract from the plants and its suspended matter; (v) rapidly raising the pH of the said separated clarified acidic liquor to about 5.5-10 by addition of basic substances excluding those which form insoluble salt in this process step to give neutralised extract, and then oxidising it with oxidising agents including air; (vi) allowing the neutralised extract to precipitate as oxidation proceeds and settle indigo, with or without addition of flocculating agents; (vii) separating said precipitated indigo from said neutralised extract, and purifying it if required. Description of the invention: The mesophyll cells in the leaf of Indigofera tinctoria (Indigofera sumatrana) and Indigofera arrecta contain the ß-glucoside 'indican' which is the precursor of indigo [Leake HM (1905) The Localization of the Indigo-Producing Substance in Indigo-yielding Plants. Annals of Botany XIX no. LXXIV: 298-31]. Indimulsin (the ß- glucosidase enzyme) that cleaves the indican into glucose and indoxyl is also present in the leaves [Bergtheil, Cyril (1904) The Fermentation of the Indigo-plant. JCS.T 85, vol. II: 870-892; and Perkin AG, Bloxam WP (1907) Indican Part I. JCS.T, 91, 1715]. In the plant Polygonum tinctorium, the ß-glucosidase is present in the stroma of the chloroplast and the indican is present in vesicles of the same cells [Yoshiko, M., et al (1997) Intracellular localization of beta-glucosidase and indican in Indigo plant. Plant Biology '97, Abstract No. 679]. In the present process of manufacture of indigo plants of any of the genera Indigofera, Isatis, Polygonum, Wrightia and/or other plants containing indican may be used. Preferably the species are Amorpha fruticosa, Asclepias tingens, Baptisia tinctoria, Galega tinctoria, Gymnema tingens, Indigofera arrecta, Indigofera articulata (I. argentea; I. caerulea), Indigofera disperma, Indigofera glabra, Indigofera hirsuta (I. glauca), Indigofera leptostycha, Indigofera longeracemosa, Indigofera pseudo-tinctoria, Indigofera suffruticosa (I. anil), Indigofera tinctoria (I. sumatrana), Isatis alpina, Isatis indigotica, Isatis leptostycha, Isatis tinctoria, Justicia tinctoria, Marsdenia tinctoria, Polygonum tinctorium, Ruellia indigotica, Saphora tinctoria, Scabosa succisa, Spilanthes tinctoria, Tephrosia apollinea, Tephrosia purpurea, Tephrosia tinctoria, Wrightia tinctoria (including plants with synonymic names). In the present invention the bundles of the plant material are arranged with all branches facing in a single direction and transported to the steeping tanks. Alternatively young twigs of the above plants comprising of four expanded leaves and a bud can be plucked and transported to the steeping tanks. The steeping tank is made of, or lined with, an acid resistant material comprising of either epoxy, fibre-glass, teflon, polyethylene, polypropylene, etc. The presence of indican in the plants is ascertained by subjecting the leaves to the action of membrane disrupting agents (such as chloroform, diethyl ether or ice-salt mixture) or physical conditions (such as freeze-thawing) that allow the indican and ß-glucosidase that are normally apart to interact and produce indigo in situ in the leaves. For this purpose the young leaves are immersed for 5-10 seconds in membrane-solubilizing organic solvents such as chloroform or diethyl ether. The leaves are immediately rinsed in water to remove the organic solvent and incubated in a moist atmosphere. Alternatively the leaves are kept at below -15°C (in freezing solutions such as ice-common salt mixture) for 15 min. The leaves are then thawed and washed to remove traces of the freezing solution and incubated in a moist atmosphere. The leaves are repeatedly treated with 1 N caustic soda to remove coloured soluble substances such as carotenoids and the blue colouration of the leaf is ascertained using a colour chart. The depth of colour of the pinna is proportional to the indigo content of the leaf. The bundles are arranged in a vertical manner (with the tips of the branches directed upwards) in a tank that may have a drain valve fitted at the bottom. The tank is fitted with an arrangement of pipes to enable introduction of water at multiple sites from the bottom to maintain temperature uniformity within a small range. The tank may also be fitted with an airtight cover having an outlet that can be connected to a vacuum pump. In order to keep the plants submerged, the bundles are fastened in place or weighed down. Acidic warm water at 40-70oC, preferably between 45-55°C, and free of heavy metals (such as mercury, copper, zinc, cadmium and organic amines at concentrations that affect indimulsin or ß-glucosidase activity) to which is added about 0.1-0.4% v/v of a strong mineral acid such as sulphuric acid, muriatic acid, phosphoric acid, nitric acid but not arsenic acid; or about 1-5% v/v of a weak organic acid such as acetic acid, lactic acid or propionic acid but not tartaric acid; or about 1-10% w/v of salts of strong acids and weak bases chosen from a group of salts such as aluminium chloride, aluminium sulphate, aluminium potassium sulphate, ammonium chloride, ammonium sulphate, etc., or mixtures thereof, is filled into the tank from the bottom so as to submerge the plant material. Salts of aluminium besides acidifying the steeping water, will complex with the endogenous tannins and proteins and yield a clearer extract and the indigo that is produced later will be of a better quality. Aluminium salts remaining behind in the extract will aid precipitation of the indigo in the settling step. About 100-500 g of fresh plant material is used per litre of acidic solution and the pH of the aqueous extract should remain between 1 and 3, preferably between 1.8 and 2.6, so as to reversibly inhibit the action of endogenous indimulsin. The temperature of the water should not fall below 50°C. This may be done insulating the tank and heating the extract by radiative heaters, immersion heaters, passing oxygen free steam through the steeping water, or by drawing the water from under the surface, circulating the water through heat exchangers and reintroducing it at the bottom. The pH of the circulating water may also be maintained within the limits by addition of acid or alkali. Some acids irreversibly inhibit the activity of ß-glucosidase (such as arsenic acid), while others adversely affect the formation of indigo blue from indoxyl (such as tartaric acid). Such acids or their salts should not be chosen to acidulate the steeping water or to adjust its pH. During the filling process, a low pressure may be applied to the air outlet attached to the cover. This will cause the bubbles adhering to the leaf hairs to get detached and will increase the area of contact between the liquid and leaf. In the above steps, the low pH of the extract will inhibit the indimulsin, the elevated temperatures and the increased area of contact between liquid and leaf will hasten the diffusion of indican and indimulsin out of the plant tissue. The plants are steeped in this acidic solution for 15 minutes-4 hours, preferably for 60 min. Besides seepage; the plant constituents can also be extracted into the acidic solution by pulverization, grinding, mincing, or homogenization. The liquor is drained out and the suspended matter is separated from the dissolved matter by filtration, flocculation or centrifugation. The residual soluble substances can be re-extracted from the separated solid matter. The indigo can be produced from the extracts by adding exogenous indimulsin-like enzymes (ß-glucosidases or enzyme complexes containing ß-glucosidase) which will work at this low pH or by rapidly neutralizing the acid. The pH of the clarified extracts is rapidly raised by the addition of a basic substance to a value between 5.5-10, the best pH being in the range of 7-9. The basic substances can be an organic or inorganic base or a salt of a weak acid with a strong base. The basic substance can be chosen from a group of substances such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonia, ammonium hydroxide, extract of plant ash that are alkaline, stale urine (prepared by keeping the urine of dairy or draught animals or humans for 2-3 days at room temperature), organic amines, etc. The basic substance chosen should not form an insoluble salt with any component of the extract. The rapid neutralization will prevent the inactivation of the indimulsin at the intermediate pH values of 3-5 and reactivate the enzyme at pH at 5.5-10. The active indimulsin will cleave the indican into indoxyl and glucose. Two indoxyl molecules will unite to form indigo blue in an oxidising environment. In this process there is no microbial activity involved and hence it cannot be classified as a fermentative process. Indigo is formed by enzymatic cleavage either with endogenous indimulsin or added ß-glucosidase. Any soluble leuco-indigotin may be completely oxidised. Oxidising agents used for this purpose are selected from a group of substances such as air, oxygen, hydrogen peroxide, ammonium persulphate, sodium peroxide, sodium perborate, etc. Aeration is done by beating the extract, using a rotating paddle, an arrangement of aerators, sparging compressed air through it, ejecting the liquid into the air as a fountain or pumping the liquid as a spray back into the tank. To aid settling of the indigo blue, flocculating agents, at a concentration of around 0.5-2% w/v, are added to the extract. The flocculating agents are selected from a group of substances such as natural gums such as dhak gum, xanthan gum, guar gum, gum acacia, gum ghatti, tragacanth gum and/or polymers such as polyethylene glycol, polyvinylpyrollidone and/or settling salts such as sodium nitrate, aluminium chloride, aluminium sulphate, aluminium potassium sulphate. After the addition of flocculating agents the extract may be heated up to 70°C to aid flocculation. The use of flocculating agents will be influenced by the end use of the product. For example the presence of aluminium in the indigo will be toxic to the organisms used to reduce indigo in the dye application step. The indigo precipitate is separated from the seet liquor. The separation is brought about by conventional separation methods such as settling and decantation (draining), filtration and/or centrifugation. The settled indigo is collected and transferred to a vessel where it is purified by heating/boiling with dilute sodium or potassium hydroxide, followed by washing with water, then boiling in dilute muriatic or sulphuric acid and washing with water and processing in a desired form such as powder, paste or cake. Examples: The invention will now be illustrated with the help of examples. The examples are by way of illustration only and in no way restrict the scope of the invention. Drawings: Figure 1 - shows activity of indimulsin over pH range 1-10 and Figure 2 - shows absorbance spectra of the sample of the product of example 2 and that of synthetic indigo sample. Example 1 Note: All chemicals used unless indicated otherwise were of AR grade. The water used was distilled water. In this experiment the stability of indimulsin enzyme from fresh and tender leaves of Indigofera tinctoria was determined. a) Fresh and tender leaves Indigofera tinctoria are plucked and 10 g were weighed and transferred to a mortar. b) 10 mL of distilled water was added to the mortar and the leaves were ground at ambient temperature. c) The macerated leaf tissue was transferred to centrifuge tubes using another 40 mL of distilled water. d) The tubes were centrifuged at 12,000 rpm in a tabletop centrifuge for 30 min at ambient temperature. e) The supernatant was collected in a glass test tube and kept on ice. It was the source of the indimulsin used in the experiment. f) Buffers at a final concentration of 50 mM were prepared to span the pH range of 1-10 at a resolution of 1 pH unit. Enzyme assay buffer was prepared at pH 5.8. A buffer was prepared by taking 100 mL of the acid component at 100 mM concentration in a 250 mL glass beaker, a teflon coated spin-bar was immersed in it, and the beaker kept on a magnetic stirrer. A combined pH electrode was standardised and inserted into the liquid. The spin bar was rotated and the alkali (IN KOH or NaOH) was added slowly until the desired pH value was reached. All the contents were transferred to a 250 mL measuring cylinder and the volume made up to 200 mL with distilled water. The 50 mM buffers were mixed and stored in glass-stoppered bottles. For pH 1 and 2, the pH of HC1 was adjusted to the desired value with KOH. For pH 3, 4, 5 and 6, the pH of acetic acid was adjusted to the desired value with NaOH. For pH 7 and 8, the pH of sodium dihydrogen phosphate was adjusted to the desired value with NaOH. For pH 9 and 10, the pH of ortho boric acid was adjusted to the desired value with NaOH. The enzyme assay buffer was prepared by adjusting the pH of citric acid to 5.8 with NaOH. gi) To microcentrifuge tube marked A 100 µL of buffer at pH 1 was added; gii) To microcentrifuge tube marked B 100 µL of buffer at pH 2 was added; giii) To microcentrifuge tube marked C 100 µL of buffer at pH 3 was added; giv) To microcentrifuge tube marked D 100 µL of buffer at pH 4 was added; gv) To microcentrifuge tube marked E 100 µL of buffer at pH 5 was added; gvi) To microcentrifuge tube marked F 100 µL of buffer at pH 6 was added; gvii) To microcentrifuge tube marked G 100 µL of buffer at pH 7 was added; gviii) To microcentrifuge tube marked H 100 µL of buffer at pH 8 was added; gix) To microcentrifuge tube marked 1100 µL of buffer at pH 9 was added; gx) To microcentrifuge tube marked J 100 µL of buffer at pH 10 was added. h) The substrate buffer used to assay indimulsin was p-nitrophenyl ß-glucoside (PNPG). It was dissolved in 50 mM sodium citrate buffer at pH 5.8 at a final concentration of 1 mM. i) To tube marked A, 100 µL of indimulsin prepared above was added, the contents mixed and tube incubated at 50°C for 30 min. j) The test was terminated by transferring (at the end of the incubation period) 40 uL of the enzyme from tube A to glass tube marked A1 containing 960 uL of enzyme assay buffer (50 mM sodium citrate at pH 5.8). The contents were mixed. k) 1 mL of substrate buffer (1 mM PNPG in 50 mM sodium citrate at pH 5.8) was added to tube A', the contents mixed and the mixture incubated at 40°C for 20 min. 1) The reaction was stopped by adding 2 mL of 1 M sodium carbonate to tube A". The contents were mixed. The product formed due to the activity of the enzyme was determined by measuring the absorbance of the solution in tube A1 at 400 nm measured against a blank containing buffers, substrate and boiled inactivated enzyme. m) For the positive control 20 µL of buffer added to tube A was taken in a glass tube marked A". 980 µL of enzyme assay buffer was added and contents mixed. 20 uL of indimulsin prepared above was added and the contents mixed. 1 mL of substrate buffer was added and the contents mixed. The mixture incubated at 40°C for 20 min. The reaction was stopped and the enzyme activity determined as described in step 1. n) The process (i) - (m) was repeated for the tubes B, C, D, E, F, G, H, I and J. Result: The activity of the enzyme as a percentage of the control is plotted in Fig. 1. The enzyme showed good thermal stability with 100% activity at pH values of 7, 8, 9 and 10, while at pH 6, it showed approximately 65% activity. The enzyme was completely inactive when pre-incubated at pH 1, 3, 4 and 5. However, rather unexpectedly, the enzyme showed approximately 70% activity at pH 2. This moderate stability at a specific and low pH value was used to develop a process that could generate high-quality indigo rapidly. Example 2 Note: All chemicals used were of LR grade. Well water was used for the extraction. In this process indigo was prepared from fresh leaves of lndigofera tinctoria. a) 50 L of water was warmed to 70°C in a stainless steel tank (SS 316) placed on a gas stove and 100 mL of ortho-phosphoric acid (cone.) was added to the warm water. A blanket was wrapped around the tank to prevent heat loss. b) 5 kg fresh leaves, in lots of 500 g was added to the warm dilute acid. The leaves were immersed and stirred with a wooden pole. c) The pH was measured and was brought to 1.8-2.3, by addition of phosphoric acid. d) The mixture was incubated at 70°C for 30 min with intermittent stirring. e) The mouth of the tank was covered with a cloth and the tank was inverted to collect the drained extract. The leaves were discarded; the tank was washed and refilled with the extract. f) A pH electrode was inserted into the extract. 10 N NaOH was prepared in a beaker and rapidly poured into the tank. The extract was stirred during the addition. The addition of NaOH was stopped when the pH of the extract reached 8. g) 500 g polyethylene glycol (PEG 6000) was added to the neutralized extract and dissolved. The tank was heated on a stove till the temperature of the extract reached 70°C. The extract was left to stand for 30 minutes. h) The supernatant was drained out and the volume of the precipitate was measured to be 200 mL. i) Two litres of hot 0.1% w/v NaOH at 70-80°C was added to the blue precipitate and the mixture stirred for 10-15 minutes. The mixture was cooled and the precipitate collected by centrifugation. j) Two litres of hot 0.1% v/v sulphuric acid at 70-80°C was added to the blue precipitate and the mixture stirred for 10-15 minutes. The mixture was cooled and the precipitate was allowed to settle and the supernatant collected. (The acidic supernatant was neutralized with the alkaline supernatant collected in the above step and then discarded), k) The settled precipitate was washed with 1 L of 0.1% w/v sodium carbonate followed by a wash with water. 1) The precipitate was dried away from sunlight in a stream of hot air. Yield was 24.3 g. Examination of the product: 10 mg of precipitate was dissolved in 1 mL concentrated sulphuric acid and diluted with 99 mL of glacial acetic acid. The absorbance was measured at 600 nm. 10 mg of synthetic indigo procured from BASF was used as the standard. This shows about 90% purity. The spectral absorbance of the synthetic indigo and the extracted natural indigo obtained in the example 2 is depicted in Fig.2. 10 mg of indigo was dissolved in 1 mL concentrated sulphuric acid and diluted with 99 mL of glacial acetic acid and analyzed. The graph in Fig.2 indicates that the absorbance of natural indigo is about 90% that of the synthetic. The absorbance curve of the natural indigo is broader than that of synthetic indigo in the violet region indicating that natural indigo contains some impurities of orange-red colouration. Advantages of the invention: The process described above is simple, quick and does away with the cumbersome procedures required to maintain anoxic conditions during the fermentation. Indigo can be obtained in 60 minutes as compared to 10-15 hours in the conventional process. The purity of indigotin obtained is better than what is presently available. The sect water serves as a complete fertilizer when phosphoric acid is used for the acidification, potassium hydroxide for the rapid neutralization and with or without gums as flocculating agents. Organic indigo can also be prepared when organic acetic acid is used for the acidification; plant ash extract that is alkaline or stale urine is employed for rapid neutralization and with or without use of gums as flocculating agents. We claim 1. A process for manufacture of indigo from indigo plants comprising following steps: (i) placing the young twigs or cut plants of Indigofera, Isatis, Polygonum, Wrightia genera or any other plant that synthesises indican in a steeping tank, in such a manner as they will remain submerged in the acidic water with which the tank will be filled in the next step; (ii) filling the said tank containing plants, with highly acidic warm water at pH between 1-3 at 2-10 litres per 1 kg of plant material, in such a manner as to avoid any air getting entrapped in the system; (iii) steeping the plants in the said acidic water for 15 minutes to 4 hours at 40-70°C with or without addition of exogenous indimulsin like enzymes; (iv) separating and clarifying the acidic acid extract from the plants and its suspended matter; (v) rapidly raising the pH of the said separated clarified acidic liquor to about 5.5-10 by addition of basic substances excluding those which form insoluble salt in this process step to give neutralised extract, and then oxidising it with oxidising agents including air; (vi) allowing the neutralised extract to precipitate as oxidation proceeds and settle indigo, with or without addition of flocculating agents; (vii) separating said precipitated indigo from said neutralised extract, and purifying it if required. 2. A process for manufacture of indigo from plants as claimed in claim 1, wherein said plants are of Amorpha fruticosa, Asclepias tingens, Baptisia tinctoria, Galega tinctoria, Gymnema tingens, Indigofera arrecta, Indigofera articulata (I. argentea; I. caerulea), Indigofera disperma, Indigofera glabra, Indigofera hirsuta (I. glauca), Indigofera leptostycha, Indigofera longeracemosa, Indigofera pseudo-tinctoria, Indigofera suffruticosa (I. anil), Indigofera tinctoria (I. sumatrana), Isatis alpina, Isatis indigotica, Isatis leptostycha, Isatis tinctoria, Justicia tinctoria, Marsdenia tinctoria, Polygonum tinctorium, Ruellia indigotica, Saphora tinctoria, Scabosa succisa, Spilanthes tinctoria, Tephrosia apollinea, Tephrosia purpurea, Tephrosia tinctoria, Wrightia tinctoria (including plants with synonymic names). 3. A process for manufacture of indigo from indigo plants as claimed in claim 1- 2, wherein said acidic warm water is maintained at pH 1-3 preferably at 1.8- 2.6. 4. A process for manufacture of indigo from indigo plants as claimed in claims 1 to 3, wherein acids are added to form the said acidic warm water, said acids chosen from sulphuric acid, muriatic acid, phosphoric acid, nitric acid, acetic acid, lactic acid, propionic acid but not arsenic acid or tartaric acid which adversely affect indigo production, and salts of strong acids and weak bases chosen from a group of salts such as aluminium chloride, aluminium sulphate, aluminium potassium sulphate, ammonium chloride, ammonium sulphate, etc., but not salts of arsenates or tartarates and mixtures thereof. 5. A process for manufacture of indigo from indigo plants as claimed in any claim 1-4, wherein the temperature of the acid extract at step (iii) is maintained by using radiative heat, internal heaters, direct oxygen free steam, and/or circulating said liquor through external heat exchangers or the like but without introducing any air into the system. 6. A process for manufacture of indigo from indigo plants as claimed in any claim 1-5, wherein said exogenous indimulsin-like enzymes are selected from those that are active at acidic pH such as a group of enzymes comprising of ß-glucosidase or enzyme complexes containing ß-glucosidase activity. 7. A process for manufacture of indigo from indigo plants as claimed in any claim 1-6, wherein said basic substances added at step (v) are selected from a group of substances such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonia, ammonium hydroxide, extract of plant ash that are alkaline, stale urine, organic amines, etc., but not those forming insoluble salts with any component of the extract. 8. A process for manufacture of indigo from indigo plants as claimed in any claim 1-7, wherein said oxidisinp agents are selected from a group of substances such as air, oxygen, hydrogen peroxide, ammonium persulphate, sodium peroxide, sodium perborate, efc. 9. A process for manufacture of indigo from indigo plants as claimed in any claim 1-8, wherein said oxidation is carried out by aeration using any of the conventional processes such as rotating paddles, arrangement of aerators, sparging compressed air, ejecting liquid in to air as a fountain, pumping said liquid as a spray back into the tank. 10. A process for manufacture of indigo from indigo plants as claimed in any claim 1-9, wherein said separation of indigo from neutralised extracts is brought about by conventional separation methods such as settling and decantation, filtration and/or centrifugation. 11. A process for manufacture of indigo from indigo plants as claimed in any claim 1-10, wherein flocculating agents added at step (vi) are selected from a group of flocculating agents comprising natural gums such as dhak gum, xanthan gum, guar gum, gum acacia, gum ghatti, tragacanth gum and/or polymers such as polyethylene glycol, polyvinylpyrollidone and/or .settling salts such as sodium nitrate, aluminium chloride, aluminium sulphate, aluminium potassium sulphate. 12. An improved process for manufacture of indigo from indigo plants as claimed in any claim 1-11, wherein the precipitated indigo is purified by first heating/boiling in dilute sodium or potassium hydroxide, followed by washing with water, then boiling in dilute muriatic or sulphuric acid and washing with water and processing in a desired form such as powder, paste or cake. 13. A process for manufacture of indigo from indigo plants substantially as herein described in the text, in the examples and in the drawings.

Documents:

908-del-2002-abstract.pdf

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908-del-2002-correspondence-others.pdf

908-del-2002-correspondence-po.pdf

908-del-2002-description (complete).pdf

908-del-2002-drawings.pdf

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908-del-2002-form-2.pdf

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