Title of Invention

"A PROCESS FOR THE PREPARATION OF PHYTASE FROM JACKFRUIT SEED POWDER USING ASPERGILLUS FICCUM."

Abstract A process for the preparation of phytase from Jackfruit seed powder using Aspergillus flccum relates to the production of phytase enzyme using solid state fermentation and using jackfruit seed powder as cabon and nitrogen source suitably supplemented with inorganic salts preferably a ammonium nitrate, magnesium sulphate and Sodium chloride preferably at a concentration in the range of 0.01-3.0% in the said solid medium . The aqueous extract obtained as supernatant from processing has a phytase activity in the range of 82—101 IU/gds within a period of 82-144 hours. Phytase is used as a supplemant to pig and poultry feed.
Full Text Field of the invention
This invention relates to a process for the preparation of phytase from jackfruit seed powder using Aspergillus ficcum by solid -state fermentation.This is a cost effective invention for the efficient production of phytase enzyme under solid-state fermentation using the dried Jackfruit seed particles (coarse and fine) moistened with salt solution and supplemented with a nitrogen source.
BACKGROUND OF THE INVENTION
Recent market trends have clearly shown that enzymes have emerged as big feed supplements. Feed enzymes (protease, xylanase, phytase, and amylase) are the newest segment of animal nutrition market, which is increasing fast. Presently only about 6% of manufactured animal feeds contain enzymes; against 80-90% vitamins which is considered as the largest animal nutrition category [Anons (1998), Enzymes emerge as big feed supplement. Chemical Engineering News, May 4, p.29-302]. Several major animal nutrition companies are getting involved in the area of making dietary enzymes. The current commercial feed supplement is a recombinant Aspergillus niger (formerly called as A.ficuum) phytase (Wodzinski, RJ and UUah AHJ (1996) Advances in applied microbiology. Vol 42, p 263-302, Academic press, Inc, Newyork, NY) or phytase produced by Aspergillus Oryzae (ie Phytase Novo, Novo Nordisk A/S (1995) Bio times 10: 8-9). BASF, which has marketing agreement with dutch enzyme poducer company Gist-Brocades, is the leading marketer of phytase world wide. The trade name given to the feed enzyme containing phytase was ' Natuphos'. BASF has been coUaboratin since early 1990 s with Royal Gist-brocads, a leader feed enzyme technology in Europe, to market feed enzymes worldwide. Since then, Natuphos has been introduced in Europe, Asia, Canada and recently in United States. The enzyme potentially increases phosphorus availability in the feed by as much as 30 %, allowing producers to reduce inorganic phosphorus supplimentation by up to 17 % (BASF Animal nutrition Partner (1996) Vol 7, July) 'AUzyme' is the trade name given by the company AUtech, which as established a manufacturing facility in Mexico for the production of phytase (Anons (1998) Chemical engineering News, 4 may, 29-302; Pandey, A, Szakacs, G., Soccol CR, Rodriguez-Leon JA and Soccol VA. Bores. Technol., 77(3), 203-214 (2001)].). Similarly 'Cenzyme ' is a

product from Cenzone, which is a unique blend of concentrated digestive enzymes including phytase from a fungal source (Cenzone, 1999 http:/www.ras.intemational.com/cenzone/cenzyme.html.). It has application in animal feed. However, presently only about 6 % of manufactured animal feeds contain the feed enzymes, against 80-90% for vitamins, which is considered as the largest animal nutrition category (Anons 1998 Chemical engineering News, 4 may, 29-302). Similarly ' Ronozyme ' is the result of a joint development between F.Hoffmann-La Roch Ltd, Switzerland and Novozymes A/S (previously known as Novo Nordisk), Denmark. Roche provides a full complement of enzyme products for both pig and poultry diets under the brand names Ronozyme and Roxazyme. Gist-Brocades manufactures feed enzyme containing phytase under the trade name of 'Natu-phos'. The largest market share (-40%) in feed enzyme is held by Finnfeeds International, which has marketing agreement with Dutch enzyme produces company Gist-Brocades, is the leading marketers of phytase the world over. 'NOVO industry' too is marketing phytase in Europe [Anons (1998), Enzymes emerge as big feed supplement. Chemical Engineering News, May 4, p.29-302].
Phytase (myo-inositol hexakisphosphate phosphohydrolase (EC.3.1.3.8) catalyses the release of phosphate from phytate (myo-inositol hexakisphosphate), which is the main form of phosphorus predominantly occurring in cereal grains, legumes and oilseeds. Hydrolysis of phytic acid (phytate) to myo-inositol and phosphoric acid is considered an important metabolic process in several bio-systems. Increasingly demanding recent regulations the world over on controlling the agricultural pollution, particularly on phosphorus pollution that limit the phosphorus content in manure, have intensified the phytase research. Environmental pollution due to the high-phosphate manure has resulted in the accumulation of phosphorus at various locations, especially in water bodies. Recent research has mainly been focused on its production and use as a means of reducing inorganic phosphorus supplementation in feed and consequent reductions in fecal phosphorus excretion.
Phytase improves the bioavailability of phosphorus in plant foods for humans [Martinez C, Ros G, Periago MJ, Lopez G, Ortuno J, Rincon (1996), Phytic acid in human nutrition Food Science and Technology International, 2: (4) 201-209]. Pigs and
poultry lack the enzyme needed to digest phytate thereby excreting large amounts of phosphorus in to the environment resulting in pollution. However, for their proper skeletal growth, these animals need phosphorus at suitable concentration. Both these conditions can be tackled effectively by the supplementation of phytase to feed. One alternative approach to decrease the phytic acid content of the agricultural products could be the use of chemical methods, but are generally expensive and affect negatively nutritional quality of the product [Liu, BL, Rafiq, A, Tzeng YM and Rob A (1997), The induction and characterization of phytase and beyond. Enzyme and Microbial Technology, 22,415-24].
Several screening programmes have been carried out aiming at the isolation of different groups of bacteria, yeast and fungi having extra-cellular phytase activity. Lissitskaya et al. [Lissitskaya TB, Shmeleva VG, Vardoian GS, Yakovlev VI (1999), Screening of microorganisms producing phytase, Mikologiya I Fitopatologiya, 33: (6) 402-405] screened micro-organisms producing phytase using museum and soil samples. It was found that moulds metabolized phosphorus more effectively than bacteria. Chen (1998) [Chen JC, (1998), Novel screening method for extra-cellular phytase-producing micro-organisms. Biotechnology Techniques, 12: (10) 759-761] developed a bioassay method for the screening of extra-cellular phytase producing micro-organisms. Gargova et al. (1997) [Gargova S, Roshkova Z, Vancheva G (1997), Screening of fungi for phytase production. Biotechnology Techniques, 11: (4) 221-224] used a two-step procedure to screen some 200 fungi for phytase production.
Schwanniomyces castellii CBS 2863 showed the highest phytase activity in the presence of phytate out of the 21 yeast strains [Lambrechts C, Boze H, Moulin G, Galzy P (1992), Utilization of phytate by some yeasts. Biotechnology Letters, 14: (1) 61-66]). The microbial phytase can be determined in a simple method, by determining the inorganic orthophosphate released on hydrolysis of sodium phytate (Engelen AJ, Vanderheeft FC, Randsdorp PHG, Smit ELC (1994), Simple and rapid-determination of phytase activity. Journal of AOAC International, 77: (3) 760-764].
Phytases can be produced from a host of micro-organisms including bacteria, yeast and fungi. During the past 40-50 years, the use of filamentous fungi for the production of industrial enzymes has rapidly increased. Submerged fermentation (SmF)
has largely been employed as the production technology. However in recent years solid-state fermentation (SSF) has gained much interest for the production of primary and secondary metabolites [Pandey A (1991), Aspects of design of fermenter in solid state fermentation, Process Biochemistry, 26, 355-361; Pandey A (1992), Recent developments in solid state fermentation, Process Biochemistry, 27, 109-117]. SSF systems, which during the previous two decades were termed as 'low technology' system, appear to be promising one for the production of value added 'low volume-high cost' products such as biopharmaceuticals. Also the use of agro-industrial residues offers potential advantage in SSF processes Pandey A, Soccol CR (1998), Bioconversion of biomass: A case study of ligno-cellulosics bioconversions in solid state fermentation, Brazilian Archives of Biology and Technology, 41, 379-390].
Techniques of SmF as well as SSF have been employed for the production of phytases. Types of strains, culture conditions, nature of the substrate and availability of the nutrients are critical factors affecting the yield. A filamentous fungus in SmF is exposed to hydrodynamic forces but in SSF the surface of the solid particles acts as the matrix for the culture.
Several bacterial strains (wild or genetically modified) such as Lactobacillus amylovorus, E. coli, B. subtilis, B. amyloliquefaciens, Klebsiella sp. etc have been employed for phytase synthesis. Sreeramulu et al. (1996) [Sreeramulu G, Srinivasa DS, Nand K, Joseph R (1996), Lactobacillus amylovorus as a phytase producer in submerged culture. Letters In Applied Microbiology, 23: (6) 385-388] evaluated 19 strains of lactic acid producing bacteria of the genera Lactobacillus and Streptococcus for the production of extra-cellular phytase. Lactobacillus amylovorus B4552 produced the maximum amount of phytase, ranging from 125-146 units/ml in SmF using glucose and inorganic phosphate.
Phytase production using yeast culture has generally been carried out in SmF systems. The strains used include Schwanniomyces castellii, S. occidentalis, Hansenula polymorpha, Arxula adeninivoranse, Rhodotorula gracilis etc. Mayer et al. (1999) [Mayer AF, Hellmuth K, Schlieker H, Lopez-Ulibarri R, Oertel S, Dahlems U, Strasser AWM, Van Loon APGM (1999), An expression system matures: A highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula
polymorpha, Biotechnology and Bioengineering, 63: (3) 373-381] developed an efficient process for the low-cost production of phytase using Hansenula polymorpha. Glucose or glucose syrups were used as the main carbon source during fermentation. Compared with the process using glycerol, the use of glucose led to the reduction of more than 80% in the raw material cost. In addition, exceptionally high concentration of active enzyme was obtained in the medium, with phytase representing over 97% of the total accumulated protein.
Several fungal cultures have also been employed for the production of phytase Ahmad et al. (2000) [Ahmad T, Rasool S, Sarwar M, Haq AU, Hasan ZU (2000), Effect of microbial phytase produced from a fungus Aspergillus niger on bioavailability of phosphorus and calcium in broiler chickens. Animal Feed Science and Technology,83: (2) 103-114 used maize starch based medium for the production of phytase in SmF using A. niger. Activity of the enzyme was found to be 1.075 phytase units per minute per ml of the crude culture, pH at 5.5 and temperature at 400C (10 days). Extra-cellular phytase produced by Aspergillus sp. 5990 showed a fivefold higher activity when compared with A. ficuum NRRL 3135. The phytase had a higher optimum temperature for its activity than the commercial enzyme, Natuphos from A. ficuum NRRL 3135 [Kim YO, Lee JK, Kim HK, Yu JH, Oh TK (1998), Cloning of the thermostable phytase gene (phy) from Bacillus sp. DSll and its over-expression in Escherichia coli, FEMS Microbiology Letters, 162: (1) 185-191]. Using a strain of A. carbonarius on canola meal, Alasheh and Duvnjak (1994, 1995a,b) [Alasheh S, Duvnjak Z (1994), Characteristics of phytase produced hy Aspergillus carbonarius Nrc-401121 in canola-meal. Acta Biotechnologica, 14: (3) 223-233; Alasheh S, Duvnjak Z (1995a), Effect of phosphate concentration on phytase productions and the reduction of the phytic acid content in canola-meal by Aspergillus carbonarius during a solid-state fermentation process. Applied Microbiology and Biotechnology, 43, 25-30; Alasheh S, Duvnjak Z (1995b), Phytase production and decrease of phytic acid content in canola-meal by Aspergillus-Carbonarius in solid-state fermentation. World Journal of Microbiology & Biotechnology,ll: (2) 228-231] found 53-60% moisture as the optimum. Addition of glucose at lower concentration (6g) and surfactants such as Na-oleate as Tween-80 in the medium increased biomass growth and enzyme synthesis.
From the literature, it is evident that in spite of the growing significance of this important feed enzyme, (also used to control P pollution) in the international market, there is a lack of suitable process involving a good combination of a suitable microorganism and substrate. Solid-state fermentation offers tremendous potential in this regard. Enzyme production using liquid fermentation is generally expensive due to low yields and high cost in-puts in up- and down stream processing. In SSF higher enzyme yields are attainable and in-put costs in up- and down-stream are relatively much less. Agro-industrial residues could be important in this regard.
The technique of solid-state fermentation has been reported significant for the production of industrially important enzymes and other products and has been elaborated in our laboratory for the production of various enzymes. (Some latest references from our laboratory- Febe Francis, A Sabu, KM Nampoothiri, Sumitra Ramachandran, Sanjoy Ghosh, George Szakacs and Ashok Pandey, Biochemical Engineering Journal, 15, 107-115 (2003); Barbara Bogar, George Szakacs, Ashok Pandey, A Sabu, James C Linden and Robert P Tengerdy, Biotechnology Progress, 19, 312-319 (2003); B Bogar, George Szakacs, R P Tengerdy, J C Linden and Ashok Pandey, Applied Biochemistry and Biotechnology, 102-103, 453-463 (2002); Febe Francis, A Sabu, K Madhavan Nampoothiri, G Szakacs and Ashok Pandey, Journal of Basic Microbiology, 42 (3), 322-326 (2002); Pandey, A. and Radhakrishnan, S: Enzyme and Microbial Technology, 14, 486-88, 1992; Pandey, A. and Radhakrishnan, S: Process Biochemistry, 28, 309 - 12, 1993; Pandey, A. Selvakumar, P. and Ashakumary, L.: World Journal of Microbiology and Biotechnology, 10, 348-49, 1994; Pandey, A., Selvakumar, P., Nigam, P., Soccol, C.R., Current Science, 77, 149-162. 1999; Pandey, A., Selvakumar, P., Soccol, C.R., Soccol, V.T., Krieger, N., Fontana, J.D. Applied Biochemistry and Biotechnology, 81, 35-52. 1999; Pandey, Soccol, C.R., Benjamin, S., Krieger, N., Soccol, V.T. Biotechnology and Applied Biochemistry, 29,119-132. 1999; Pandey, A., Szakacs, G., Soccol, C.R, Rodriguez, A., Soccol, V.T., Bioresource Technology, 77,203-214. 2001).
Selection of Jackfruit seed as substrate for SSF
Jackfruit is believed indigenous to the rain forests of the Western Ghats. It is cultivated at low elevation throughout India, Burma, Cylon, China, Malaysia and East Indies. It is common in Philippines, both cultivated and naturalized in Africa, it is often planted in Kenya, Ugenda and former Zazibar. It is also found in tropical America, West Indies and Northern Brazil. In South India, the jackfruit is a popular food ranking next to the mango and banana in total annual production.
The jackfruit, Artocarpus heterophyllus belongs to the family moraceae. The trees are handsome and stately, 30 to 70 ft tall, with evergreen, alternate, glossy, somewhat leathery leaves to 9 in long, oval on mature wood. Largest of all tree-borne fruits, the jack fruit may be 3 to 8 inches in length (20-90 cm) and 6 to 20 inches in (15-50 cm) wide and weight ranges 10 to 60 or even much as 110 lbs (4.5 - 20 or 50 kg). The "rind" or exterior of the compoimd or aggregate fruit t is green or yellow when ripe and composed of numerous hard, core like points attached to thick and rubbery, pale yellow or whitish walls. The interior consists of large bulbs of yellow, banana-flavored flesh. Each bulb encloses a smooth, oval, light-brow seed covered by a thin white membrane. The seed is 3/4 to 1-1/2 inch (2-4 cm) long and 1/2 to 3/4 inch in thick and is white and crisp within. There may be 100 or up to 500 seeds in a single fruit
Jackfruit seeds are discarded as waste from different agro-industries and higher percentage of fruits and seeds are being wasted as fallen fruits. The seeds are rich in carbohydrate, protein and trace elements. Table below shows the Nutritional value per 100 g of jackfruit seed (Morton, 1987, Jack Fruit, p 58-64, In: Fruits of Warm climate, Juila F.Morton, F.L. (Ed.)
Nutritional value per 100 g of jackfruit seed

(Table Removed)
In general, fresh seeds are considered to be high in starch, low in calcium and iron, good source of vitamins Bl and B2. So this agro industrial residue can be used as substrate for SSF, which provides the essential carbohydrate in the form starch to growth and a solid support for anchorage of cells.
The main objective of this invention was to develop an improved process for the production of phytase enzyme employing solid-state fermentation technology. Such an objective was attainable by utilizing a potential fungal culture, which could utilize Jackfruit seed effectively in SSF for growth and its activity.
Novelty of the invention lies in the use of the dried Jackfruit seed (coarse and fine) powder moistened with salt solution and supplemented with a nitrogen source for production of phytase enzyme under solid-state fermentation.
Accordingly, the present invention provides a process for the preparation of phytase from iackfi^lit seed powder using Aspergillus ficcum comprising:
(a) preparing a solid-state fermentation medium(SSFM) containing jack fiiiit seed powder as carbon source and the components of the said medium comprising 5-20 g jackfruit seed powder moistened with mineral salts from group of ammoniimi nitrate , magnesium sulphate and sodium chloride in the range of 0.01 -3.0 g%) and mixing of said jack fruit seed powder with mineral salts and water being in the ratio of about 5:4:1 and the moisture content of the medium being kept in the range of 40 -75% prior to autoclaving.
(b) inoculating the culture of Aspergillus ficcum (TUBF-1165) to the SSFM (20 %v/w) as prepared in step( a) with spore suspension of about 2xl0^of the said culture and growing the culture in stationary condition for a period ranging 84-144 hours at a temperature ranging 27-32°C imder aseptic condition;
c) diluting the culture as obtained in step b) with 0.05 to 0.1 v/v Tween- 80 distilled water in a ratio of about 1:1;
d) mixing and dislodging of spore and mycelia of fungal culture as obtained in step (c) by shaking on rotary shaker at 150 rpm for a period ranging 30- 40 minutes;
e) filtering the mixture as in step (d) through filter paper to remove fungal culture;
(f) centrifuging the filtrate as obtained in step (e) at about 12000 rpm for a period ranging 15-20 minutes at a temperature of about 4°C in a refi-igerated centrifuge to obtain the supernatant aqueous extract containing phytase enzyme
In an embodiment of the present invention Aspergillus ficuum TUBF -1165 (NRRL 3135) strain used is obtained from the "TUB" Culture Collection of the Technical University of Budapest, Hungary.
In yet another embodiment of the present invention the jack fruit {Artocarpus heterophyllus) seed powder used in medium comprises
(Table Removed)
In another embodiment of the present invention the high yield of enzyme is obtained in the range of about 82- 101 lU /gds with the use of ammonium nitrate concentration in the range of 0.75-2.5 in the solid medium.
In yet another embodiment of the present invention the aqueous liquid obtained as supematant has a phytase activity in the range of 82-101 lU/gds within a period of 84-144 hours of fermentation period
DETAILED DESCRIPTION OF THE INVENTION
Experimental procedure adopted and results obtained are broadly described here:
Micro-organism: Culture of Aspergillus ficuum TUB F-1165 (NRRL 3135) obtained from the "TUB" Culture Collection of the Technical University of Budapest, Hungary was used in the process development studies. It was grown on potato - dextrose - agar medium (Hi- Media, Bombay); preserved at 4° C and sub-cultured once in every three weeks.
Preparation of inoculum: Freeze-dried culture of A. ficuum was grown on potato dextrose agar (PDA) slants. To fully sporulated (7-10 days old) agar slope culture, 10 ml of sterile salt solution containing 0.1% Tween-80 was added. Then the spores were scrapped under strict aseptic conditions. The spore suspension obtained was used as the inoculum. Viable spores in the spores suspension were determined by plate-count (colony count) technique, using a colony counter. In this method, the spore suspension was serially diluted, plated on PDA medium and after incubation at 30°C for 24 hours the spores were counted.
Solid-state fermentation: A known weight of the solid substrate was taken in Erlenmeyer flasks and was moistened with adequate concentration of distilled water containing mineral salts such as NH4 NO3, MgS04.7H20 and NaCl at suitable concentrations between 0.01-3.0 %. SSF resulted in comparatively high titres of enzyme production. This can be well explained with the following examples. Three independent sets of experiments were carried out for each example and the average values are represented here. The standard deviation in each case was less than +3%.
Enzyme recovery: From the fermented solid substrates, enzyme extraction was carried out using distilled water. It was done in two steps by adding equal amount of distilled water containing 0.1% Tween-80 in to the fermented matter .The flask was kept on a rotary shaker at 150 rpm for 30 minutes. The total extraction volume was 100 ml. After this, the solids were separated from the solution by filtering through cheesecloth. This step was repeated again and the solution was centrifuged at 12,000 rpm for 20 min at 40 in a refrigerated centrifuge. The supernatant was collected and used for enzyme assay. Extraction was done at room temperature.
Enzyme assay: Phytase was assayed according to Harland and Harland (1980) [Harland B.F. and Harland J. (1980). Fermentative reduction of phytate in rye, white and whole wheat breads. Cereal Chem., 257,226-229].
The following specifications particularly describe and ascertain the nature of this invention and the manner in which it is to be performed. "High yield of extra-cellular phytase enzyme can be produced under solid-state fermentation employing Aspergillus ficuum in relatively shorter period of time using a cheaply available tropical agro-industrial residue such as Jackfruit seed powder suitably supplemented with a nitrogen source (preferably an ammonium salt) at appropriate concentration which favour the fungal strain's growth and activity". The process offers scope for scale-up for industrial production of this important feed enzyme from a relatively cheap agro residue.
The following examples are given by way of illustration in actual practice and therefore should not be construed to limit the scope of the invention.
Example 1: The solid substrate, Jackfruit seed powder containing coarse and fine particles was taken at a known quantity (5-20g, dry wt basis) in Erlenmeyer flasks and was moistened with mineral salt solution and distilled water in such a way so as to obtain a final moisture content between 40-75 % in the substrate. After thorough mixing, the wet substrate was autoclaved at 121°C for 20 minutes and cooled to room temperature. It was inoculated with the spore suspension containing 2x106 -10 10 spores per millilitre of A ficuum ((20%, v/w inoculum size) and were incubated at 30°C for desired length of period, (24-120 h). Enzyme yields as high as 6.26 lU/gds (gram dry fermented substrate) were achieved in a fixed period of time between 72-168 h with A. ficuum (Table 1) Table 1: Production of phytase enzyme by A. ficuum using Jackfhiit seed powder

(Table Removed)
Example II: Solid-state fermentation was carried out under conditions as employed above under different initial moisture level of the substrate (46- 68%). An enhanced enzyme production from 6.26 lU/gds (Table 1) to 12. 1 lU/gds. was obtained by using the substrate having initial moisture of 48.5 -56.5 %.
Example III: Solid-state fermentation was carried out under conditions as described in examples I and II with further modification by supplementing it with few organic as well as inorganic nitrogen sources independently at a fixed concentrations between 0.5-3.5 %. Addition of inorganic N source .in particular of ammonium salts resulted in noticeable increase in the enzyme production from 12.1 to 34 lU/gds (Table 2) in a fixed period of time between 72-168 h fermentation using A.ficuum
Table 2 Effect of supplementation of various nitrogen sources to jack fruit seed powder based medium for the production of phytase by Solid-state fermentation using A. ficuum

(Table Removed)
NS- Ammonium salts, sodium nitrate, tryptone, yeast extract and peptone
Example IV: Solid state fermentation was carried out under conditions as described in examples 1,11 and 111 with further modification by supplementing it with ammoniacal nitrogen source at different concentrations between 0.5-3.5 %. Supplementation at a suitable concentration between 0.75-2.5%N source resulted in significant increase in the enzyme production from 34 lU/gds to 82 lU/gds in a fixed period of time between 72-168 h of fermentation using A.flccum.
Example V; A time course study was conducted under the above optimum conditions and the study revealed that a mximum of 101 lU/gds phytase was obtained after suitable period of time between 84-144 hours (Table 3)

(Table Removed)
ADVANTAGES
Jackfhiit seeds are discarded as waste from different agro-industries and higher percentage of fruits and seeds are being wasted as fallen fruits. The seeds are rich in carbohydrate, protein and trace elements and it has been used as a substrate for the phytase enzyme. The process of preparation of enzyme is simple and cheap.






We claim:
1. A process for the preparation of phytase from jackfruit seed powder using Aspergillus ficcum comprising:
a. Preparing a solid-state fermentation medium(SSFM) containing jack fruit
seed powder as carbon source and the components of the said medium
comprising 5-20 g jackfruit seed powder moistened with mineral salts from
group of ammonium nitrate , magnesium sulphate and sodium chloride in
the range of 0.01 -3.0 g% and mixing of said jack fruit seed powder with
mineral salts and water being in the ratio of about 5:4:1 and the moisture
content of the medium being kept in the range of 40 -75% prior to
autoclaving.
b. Inoculating the culture of Aspergillus ficcum (TUBF-1165) to the
SSFM(20 %v/w) as prepared in step( a) with spore suspension of 2xl06 of
the said culture and growing the culture in stationary condition for a period
ranging 84- 144 hours at a temperature ranging 27-32°C under aseptic
condition;
c. diluting the culture as obtained in step b) with 0.05 to 0.1 v/vTween-80
distilled water in a ratio of 1:1;
d. mixing and dislodging of spore and mycelia of fungal culture as obtained
in step (c) by shaking on rotary shaker at 150 rpm for a period ranging 30-
40 minutes;
e. filtering the mixture as in step (d) through filter paper to remove fungal
culture;
f. centrifuging the filtrate as obtained in step (e) at 12000 rpm for a period
ranging 15-20 minutes at a temperature of 4°C in a refrigerated centrifuge
to obtain the supernatant aqueous extract containing phytase enzyme

2 A process as claimed in claim 1-3 wherein the jack fruit {Artocarpus
heterophyllus) seed powder used in medium comprises
%w/w
carbohydrate 38.4
Protein 6.6
Fibre 1.5
Ash 2.96
Calcium 0.13
Phosphorus 0.54
Iron 0.005
3 A process as claimed in claim 1, wherein the high yield of enzyme is obtained in the range of about 82- 101 1U /gds with the use of ammonium nitrate concentration in the range of 0.75-2.5 in the solid medium.
4 A process as claimed in Claim 1, wherein the aqueous extract obtained as supernatant has a phytase activity in the range of 82-101 lU/gds within a period of 84-144 hours of fermentation period.
5 A process for the preparation of phytase from jackfruit seed powder using
Aspergillus ficcum as substantially as herein described with reference to
examples accompanying the specification.


Documents:

1891-DEL-2004-Abstract-(03-03-2011).pdf

1891-del-2004-abstract.pdf

1891-DEL-2004-Claims-(25-04-2011).pdf

1891-del-2004-claims.pdf

1891-DEL-2004-Correspondence-Others-(03-03-2011).pdf

1891-DEL-2004-Correspondence-Others-(25-04-2011).pdf

1891-del-2004-correspondence-others.pdf

1891-del-2004-description (complete).pdf

1891-DEL-2004-Form-1-(03-03-2011).pdf

1891-del-2004-form-1.pdf

1891-del-2004-form-18.pdf

1891-del-2004-form-2.pdf

1891-DEL-2004-Form-3-(03-03-2011).pdf

1891-del-2004-form-3.pdf

1891-del-2004-form-5.pdf


Patent Number 248793
Indian Patent Application Number 1891/DEL/2004
PG Journal Number 34/2011
Publication Date 26-Aug-2011
Grant Date 25-Aug-2011
Date of Filing 30-Sep-2004
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110 001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 ASHOK PANDEY REGIONAL RESEARCH LABORATORY CSIR, TRIVANDRYM, INDIA.
2 KESAVAN MADHAVAN NAMPOOTHIRI REGIONAL RESEARCH LABORATORY CSIR, TRIVANDRYM, INDIA.
PCT International Classification Number C12N 9/16
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA