BACKGROUND OF THE INVENTION N-acetyl beta D glucosaminidase, also called NAGase, is an enzyme used in industry for various applications e. g. baking in food industry, cell lysis in process industry and medical applications.

Xylanase, especially endo-xylanase, is another enzyme used widely in industry for various applications e. g. baking in food industry and preparation of fruit juice in beverage industry. Both the enzymes when put together in a composition have synergistic effect in the above mentioned applications. In most of the applications, the ratio of units of NAGase: Xylanase is desired to be approximately 1: 50.

Aspergillussp., particularly A nigeris known to produce these enzymes when cultivated using solid substrate fermentation technique. Because of the unpredictability of the fermentation and downstream processing techniques, the desired ratio of approximately 1 : 50 is not achieved. It is observed that generally the xylanase activity is low. Such a product is highly undesirable, as its application performance is very poor on comparing it with the regular ratio of NAGase: Xylanase desirable, which is approximately 1: 50.

Also it was found that when the organism is cultivated using submerged fermentation technique, it produces NAGase in

insignificant amount. In this case the xylanase activity is much higher and this ratio of activities also is undesirable, as its application performance is very poor on comparing it with the regular ratio of NAGase: Xylanase desirable which is approximately 1 : 50.

The possible solution to the present problem would be to prepare the enzymes independently and mix at the final stage as concentrates or powders. But this would involve independent fermentation and independent purification processes for obtaining the enzyme components. This would involve independent use of the downstream equipment and would be time consuming and costly.

The instant invention disclosed a novel process to overcome the problems mentioned in the background.

SUMMARY OF THE INVENTION The instant invention relates to a novel process for the preparation of an enzyme composition.

The novel process for the preparation of an enzyme composition comprises: a) cultivation of a microorganism using solid substrate fermentation technique, b) cultivation of a microorganism using submerged fermentation technique, c) after fermentation, combination of the koji of the solid substrate fermentation and the broth of the submerged fermentation,

d) extraction of the koji of the solid substrate fermentation with the broth of the submerged fermentation, optionally extracting the koji with aqueous solvent, and affording the combined extract, e) removal of microorganisms and/or particles from the extract by microfiltration, optional dilution of the retentate with an aqueous solvent and re-microfiltration f) purification and concentration of the enzymes using ultrafiltration g) optional addition of stabilizer and/or preservative to the enzyme composition h) optional drying of the concentrate The enzyme in powder form as obtained in step h can be used for application in food industry.

The enzyme concentrate obtained in step f can also be used for application in food industry.

The novel process of the instant invention has following advantages: 1. Desired product quality 2. Economic 3. Time saving 4. High yields 5. No separate downstream processing required for the enzyme components DETAILED DESCRIPTION OF THE INVENTION As mentioned earlier, the instant invention relates to a novel process for the preparation of an enzyme composition.

DEFINITIONS Enzyme composition A mixture of one or more enzymes with or without excipients.

NAGase N acetyl beta D glucosaminidase is an enzyme, which catalyzes hydrolysis of terminal non-reducing N-acetyl-D- hexosamine residues in N-acetyl-beta-D-hexosaminides. The enzyme acts on N-acetyl-glucosides and N-acetyl-galactosides.

NAGase unit One NAGase unit is defined as the amount of the enzyme which will act on one mole of the substrate per minute under the conditions of 37°C for 10 minutes at pH 5.0.

Xylanase Endo-1, 4-beta-xylanase is an enzyme, which catalyzes endohydrolysis of 1, 4-beta-D-xylosidic linkages in xylan.

Xylanase unit One xylanase unit is defined as the increase in reciprocal viscosity per hour per lml of enzyme as used in the assay.

"Solid state fermentation"or"solid state cultivation" The term"solid state fermentation"or"solid state cultivation", sometimes referred to as"semi-solid state fermentation"as used herein, means the process of fermenting microorganisms on a solid medium/matrix that provides anchorage points and/or nutrients for the microorganisms in the absence of any freely flowing substance. The amount of water in the solid medium can vary. For example, the solid medium could be almost dry, or it could be slushy. A person skilled in the art knows that the

terms"solid state fermentation"and"semi-solid state fermentation"is interchangeable.

A solid porous matrix which may be biodegradable or not, but has a large surface area per unit volume for a ready microbial growth on the solid-gas interface. The matrix generally absorbs water amounting one or several times its dry weight with a relatively high water activity on the solid-gas interface in order to allow high rates of biochemical processes. The solid-gas interface should be a good habitat for the fast development of specific cultures of moulds, yeast or bacteria, either in pure or mixed cultures. The energy consumption of the process is low. It does not require high volumes of ingredients and does not involve high cost technology.

Koji The solid substrate used for solid state or solid substrate fermentation inoculated with microorganism.

Submerged fermentation "Submerged fermentation"or"liquid substrate fermentation" means a process of cultivating microorganisms wherein the microorganisms are submerged in the liquid medium. Generally the water content of the medium is generally high so that the media flows or can be agitated using impeller. Most of the media ingredients are soluble in the solvent used. The process parameters like pH, temperature, feeding of nutrients, aeration can be manipulated easily. The process involves high-energy consumption.

High volumes of ingredients and high cost technology are involved in this type of fermentation.

Broth The medium used for submerged fermentation inoculated with microorganism.

Microfiltration A process for separation of microorganisms, cell debris as well as other particles from the broth or koji extract. This process employs microfiltration membranes with a nominal pore size of 0.2 micron.

Ultrafiltration A process for the separation, purification and concentration of molecules based on their molecular weight. This process employs ultrafiltration membranes with a molecular weight cutoff required to separate the desired molecules from undesired molecules.

Drying Drying is a process that causes vaporization of solvents of a system to results in concentration or solidification of the other components of the solution. The drying may be spray drying or freeze-drying. Generally the spray drying is done at a temperature higher than the ambient temperature and the freeze-drying is done at a temperature lower than the ambient temperature.

Baking The different steps involved in baking are as follows : Kneading, which is performed by mixing all the above said ingredients. Temp: 25-28°C Time 7-15 minute

Flour fermentation to allow the enzymes to act and make the environment more condusive for the yeast to ferment.

Molding, wherein the fermented batter is cast in a mould before baking.

Proofing, involves keeping the mould containing the bread batter at 35°C for 40 min in a chamber having ~85% Relative Humidity.

Baking, is performed in an oven at temperature of 220°C for 40 min Cooling and Slicing.

The bread can be prepared by methods known in the literature per se.

Preservatives Substances added to foods to prevent or delay spoilage from fungal or bacterial action or chemical interactions (e. g. , potassium sorbate, a potassium salt of sorbic acid applied in a water spray or baked products).

The novel process for the preparation of an enzyme composition comprises: a) cultivation of a microorganism using solid substrate fermentation technique, b) cultivation of a microorganism using submerged fermentation technique, c) after fermentation, combination of the koji of the solid substrate fermentation and the broth of the submerged fermentation,

d) extraction of the koji of the solid substrate fermentation with the broth of the submerged fermentation, optionally extracting the koji with aqueous solvent, and affording the combined extract, e) removal of microorganisms and/or particles from the extract by microfiltration, optional dilution of the retentate with an aqueous solvent and re-microfiltration f) purification and concentration of the enzymes using ultrafiltration, g) optional addition of stabilizer and/or preservative to the enzyme composition, h) optional drying of the concentrate.

The enzyme in powder form as obtained in step h can be used for application. The enzyme concentrate obtained in step f can also be used for application. Preferably the enzyme composition is used in food industry.

The microorganism is selected from bacteria, yeast or fungi.

The solid substrate for fermentation is selected from wheat bran, wheat rava, oat meal, broken wheat, boiled rice, rice bran, rice rava, beaten rice, maize bran, maize grits, oat bran, bagasse, tapioca residue, soy grits, soy flakes, rice flakes, ceramic beads, glass beads, sponge or a mixture of two or more of these.

The carbon source for fermentation is selected from glucose, sucrose, starch (maize, wheat, tapioca, potato), modified starch, maltose, malto-dextrin, soybean oil, acetate or a mixture of two or more of these.

The nitrogen source for fermentation is selected from ammonium sulfate, dried yeast, ammonium nitrate, sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soy peptone, soy flour, cotton seed flour, corn steep liquor or a mixture of two or more of these.

The media can be supplemented with macro and/or micro nutrients as known in fermentation methods per se.

After the fermentation the koji from the solid substrate fermentation can be mixed with the broth from the submerged fermentation.

The mixture can be allowed to stand to effect extraction of enzymes from the koji into the aqueous phase of the broth. After the extraction, the liquid and solid phases can be separated by filtration, sedimentation or centrifugation. The solid phase can be then re-extracted with water and the extract can be combined with the first extract.

The thus obtained extract can be then subjected to microfiltration to remove microorganisms and/or solid particles of size above the cutoff of the membrane. The retentate of the microfiltration can be diluted with water and again subjected to microfiltration to improve the yields.

All the microfiltration permeates can be combined together and subjected to ultrafiltration using membranes of desired molecular weight cutoff. The molecular weight cutoff of the membrane controls the molecules'passage through the pore.

Ultrafiltration can be used for purification of the desired molecules

from the smaller or larger molecules by careful selection of the low or high molecular weight cutoff membranes.

The molecules of interest can be concentrated as well as purified from smaller molecules if a low molecular weight cutoff membrane is selected.

The thus purified and concentrated enzyme mixture can directly be used for application.

The purified and concentrated enzyme mixture can be mixed with preservatives and/or stabilizers.

The purified and concentrated enzyme mixture without or with preservatives and/or stabilizers can be subjected to drying.

Generally spray drying is employed to afford powder of the enzyme composition.

It was surprisingly found that one microorganism, Aspergillus niger, can be used for the production of both the enzymes. Also it was surprising that the microorganism when cultivated using submerged fermentation technique produces only the xylanase component and when cultivated using solid substrate fermentation technique produces only the NAGase component. Optimization of media ingredients and cultivation conditions was also found to increase the production of both the components.

In particular, the novel process for the preparation of an enzyme composition comprises: a) cultivation of microorganism using solid substrate fermentation technique, b) cultivation of microorganism using submerged fermentation technique,

c) after fermentation, combination of the koji of the solid substrate fermentation and the broth of the submerged fermentation, d) extraction of the koji of the solid substrate fermentation with the broth of the submerged fermentation, optionally extracting the koji with aqueous solvent, and affording the combined extract, e) removal of microorganisms and/or particles from the extract by microfiltration, optional dilution of the retentate with an aqueous solvent and re-microfiltration, f) purification and concentration of the enzymes using ultrafiltration, g) optional addition of stabilizer and/or preservative to the enzyme composition, h) optional drying of the concentrate.

The microorganism selected is fungus. Preferably the microorganism is selected from Aspergillus species. Preferably the microorganism is Aspergillus niger.

The aqueous solvent used for extraction is water or an aqueous buffer.

The extract is separated from the spent koji by filtration, sedimentation or centrifugation. Preferably, the extract is separated from the koji by filtration.

The removal of microorganisms and/or particles from the extract is done by microfiltration. The retentate of the microfiltration is diluted with an aqueous solvent, preferably water

and re-microfiltration to improve yields. The microfiltration is carried out using membranes of 0.2-micron cutoff.

The permeate obtained after microfiltration is subjected to ultrafiltration. Preferably, the ultrafiltration membrane is selected of approx. 5000 Da molecular weight cutoff. This effects purification of the enzyme mixtures from all particles or molecules below 5000 Da molecular weight and also results in concentration as water is removed from the solution.

One or more preservatives are added to the thus obtained concentrate. Preferably, the preservative is selected from benzoate, sorbate, paraben, salts or any other suitable preservative. One or more stabilizers are added to the enzyme concentrate. The stabilizer is selected one or more among sodium chloride, potassium chloride, ammonium sulfate, sugars, polyols or any other suitable stabilizer.

The enzyme in powder form as obtained in step h can be used for application in food industry.

The enzyme concentrate obtained in step f can also be used for application in food industry.

The following examples further illustrate the invention, it being understood that the invention is not intended to be limited by the details disclosed therein.

EXAMPLES Reference example 1 A batch producing NAGASE and xylanase by solid substrate fermentation has in the final product a Nagase activity of 120u/g and xylanase activity of 3000u/g. This gives ratio of NAGase to

xylanase :: 1 : 25. This ratio is not desirable for the application in baking trial.

Reference example 2 In submerged fermentation, in a below mentioned optimized media, the culture produces Xylanase (1000 u/g) and very negligible NAGase (1u/g). This gives a ratio of NAGase: xylanase :: 1: 1000. This ratio is also undesirable for the baking application.

Example 1 A. ENDOXYLANASE BY SUBMERGED FERMENTATION Production of an endo-xylanase by submerged fermentation was evaluated as follows : PREPARATION OF SEED CULTURE a) Storage of the culture Aspergillus niger colonies were selected from MEA plates grown for 5-7 days after sporulation and the spores in the order 10^5 were suspended in 15% Glycerol and stored in-85°C. One spore vial (containing 2 ml) was grown at 30°C in soybean casein digest medium (casein enzymatic hydrolyzate 17g/l, papaic digest of soyabean meal 3. 0g/l, NaCI 5 g/l, di-potassium phosphate 2. 5g/l, dextrose 2. 5g/1). b) Pre-seed culture A vial of spores was thawed, inoculated and grown into 50ml of soybean casein digest medium for 48 hrs at 240 rpm at 30°C. c) Seed culture

The pre-seed culture (10%) was subcultured into seed medium i. e. soybean casein digest medium was incubated for 48 hr at 240 rpm at 30°C.

FERMENTATION The seed medium (7% v/v) was inoculated into a 5-L fermentor. The fermentor contained cellulose fibre and malt extract as a carbon source along with cottonseed flour and yeast extract as nitrogen Source (Production Medium). The fermentor vessel initially contained approximately 3 L of production medium, which contains the ingredients as given in Table 1.

The fermentor was then inoculated with the fungal seed culture, and was maintained at 35°C with 0.66 wm air and agitation ranging from 356 rpm to 1200rpm as a batch run. The DO was maintained between 45-50%. The fermentor broth was harvested at 48 hour when the fermentor attained a pH of 6.0.

B. NAGASE BY SOLID SUBSTRATE FERMENTATION Production of NAGaseby Solid Substrate Fermentation was evaluated as follows : PREPARATION OF THE SEED CULTURE a) Storage of the culture Aspergillus nigercolonies were selected from MEA plates grown for 5-7 days till sporulation and the spores in the order 10^5 were suspended in 15% Glycerol and stored in-85 0 C. One Spore vial (containing 2 ml) was grown at 30°C in malt extract medium (casein enzymatic hydrolyzate 17g/I, papaic digest of soyabean meal 3. 0g/l, NaCl 5 9/l, di-potassium phosphate 2. 5g/l, dextrose 2. 5g/I).

b) Pre-seed culture A vial of spores was thawed, and inoculated and grown into 5 ml of malt extract broth (5ml) for 24 hr at 30°C c) Seed culture The pre-seed culture was subcultured into Seed Medium (10%), Maida (4.14), soy peptone (0.306), MAP (0. 0162), and glucoamylase (0.0054), 48 hr at 240rpm at 30°C.

FERMENTATION The seed medium (10% v/v) was inoculated into solid substrate medium containing wheat bran (4 kg) with water (10: 3).

C. COMBINED DOWNSTREAM OF ENDOXYLANASE PRODUCED BY SUBMERGED FERMENTATION WITH NAGASE PRODUCED BY SOLID SUBSTRATE FERMENTATION SOAKING 4-kg broth from submerged fermentation containing xylanase was harvested at around 48-log hr. The 4 Kg Koji produced by solid substrate fermentation containing NAGase was harvested, mixed and soaked with broth obtained from submerged fermentation. The mixture was held at ambient temperature for 2-3 hour.

EXTRACTION The NAGase was extracted from the koji by the broth containing xylanase. The mixture was separated by filtration. The solid was re-extracted with water. The extracts were combined to a final volume of 24 L.

NAGase u/g= 6. 2 Xylanase u/g= 346

Ratio: 55 MICROFILTRATION The combined extract (24 L) was subjected to microfiltration using 0.2-micron hollow fibre membrane. After collection of the first microfiltration permeate, the retentate was diluted and mixed with equal quantity of water and subjected to microfiltration again.

All microfiltration permeates were combined to obtain 30 L microfiltration permeate.

ULTRAFILTRATION The microfiltration permeate (30 L) was subjected to ultrafiltration using hollow fibre membranes of 5000 Da molecular weight cutoff. The solution was circulated to obtain 1 L concentrate.

NAGase u/g= 120 Xylanase u/g= 6100 Ratio 1 : 51 PRESERVATION AND STABILISATION To the concentrate 0.2% sodium benzoate and 0.1% potassium sorbate were added as preservatives. The concentrate was stabilized by adding 18% KCI.

DRYING The stabilized concentrate was dried in a spray drier. The drying conditions were inlet temp: 170°C and outlet temp: 70°C at a flow rate of 8 ml/min.

NAGase u/g= 475 Xylanase u/g= 24000 Ratio=1 : 51

The enzyme composition powder obtained had moisture content of 0.9%.

At each step samples were assayed for the activity of xylanase, NAGase and protein.

Example 2 A. ENDOXYLANASE BY SUBMERGED FERMENTATION Production of the endo-xylanase by submerged fermentation was evaluated as follows : PREPARATION OF SEED CULTURE Preparation of see culture was done as explained in Example 1.

FERMENTATION The seed medium (10% v/v) was inoculated into a 500-L fermentor. The fermentor contained cellulose fibre and malt extract as a carbon source along with cottonseed flour and yeast extract as nitrogen source (Production Medium). The 500-L fermentor vessel initially contained approximately 300 L of production medium, which contained the ingredients as given in Example 1.

The fermentor was then inoculated with the fungal seed culture, and was maintained at 35°C with 0.66 wm air and agitation ranging from 50 rpm to 500 rpm as a batch run. The DO was maintained between 40-50%. The fermentor broth was harvested at 50th hour when the fermentor attained a pH of 6.2.

B. NAGASE BY SOLID SUBSTRATE FERMENTATION Production of NAGase by solid substrate fermentation was evaluated as follows : PREPARATION OF THE SEED CULTURE

Preparation of see culture was done as explained in Example 1.

FERMENTATION The seed medium (10% v/v) was inoculated in solid substrate medium containing wheat bran (300 kg) and water (10: 3).

C. COMBINED DOWNSTREAM PROCESSINF OF ENDOXYLANASE PROODUCED BY SUBMERGED FERMENTATION WITH NAGASE PRODUCED BY SOLID SUBSTRATE FERMENTATION SOAKING 300-kg broth from submerged fermentation containing xylanase was harvested at around 48-log hr. The 300 Kg Koji produced by solid substrate fermentation containing NAGase was harvested, mixed and soaked with broth obtained from Submerged Fermentation. The mixture was held at ambient temperature for 4 hour.

EXTRACTION The NAGase was extracted from the koji by the broth containing xylanase. The mixture was separated by filtration. The solid was re-extracted with water. The extracts were combined to a final volume of 1800 L.

MICROFILTRATION The combined extract (1800 L) was subjected to microfiltration using 0.2-micron hollow fibre membrane. After collection of the first microfiltration permeate, the retentate was diluted with equal quantity of water and subjected to microfiltration

again. All microfiltration permeates were combined to obtain 2400 L microfiltration permeate.

ULTRAFILTRATION The microfiltration permeate was subjected to ultrafiltration using hollow fibre membranes of 5000 Da molecular weight cutoff.

The solution was circulated to obtain 80 L concentrate.

PRESERVATION AND STABILIZATION To the concentrate 0.2% Sodium benzoate and 0.1% potassium sorbate were added as preservatives. The concentrate was stabilized by adding 18% KCI.

DRYING The stabilized concentrate was dried in a spray drier. The drying conditions were inlet temp: 170°C and outlet temp: 70°C at a flow rate of 50 L/hr.

The enzyme composition powder obtained had moisture content of 0.5%.

At each step samples were assays for the activity of xylanase, xylanase and protein.

The NAGase to Xylanase ratio was 1 : 55 APPLICATION OF THE ENZYME COMPOSITION-BREAD MAKING APPLICATION EXAMPLE 1 Control 1 Ingredients for Bread making: Flour, Sugar, Salt, Yeast, Fat, shortening and Enzyme amylase, NAGase from solid substrate fermentation.

Quality of Bread: The results are disclosed in Table 2 Result : Bread having only NAGase did not show desired characteristics.

APPLICATION EXAMPLE 2 Control 2 Ingredients for Bread making: Flour, Sugar, Salt, Yeast, Fat, shortening and enzyme amylase, xylanase from submerged fermentation.

Quality of Bread: The results are disclosed in Table 2 Result : Bread having only xylanase did not show desired characteristics.

APPLICATION EXAMPLE 3 Test 1 NAGase + xylanase (1: 25) Ingredients for Bread making: Flour, Sugar, Salt, Yeast, Fat, shortening and Enzyme amylase, NAGase from solid substrate fermentation and xylanase from submerged fermentation mixed in powder state.

Quality of Bread: The results are disclosed in Table 2.

Result: Bread having NAGase+Xylanase in a ratio of 1 : 25, did not show desired characteristics.

Quality of Bread: See table 2 APPLICATION EXAMPLE 4 Test 2

NAGase + Xylanse (1 : 50) Ingredients for Bread making: Flour, Sugar, Salt, Yeast, Fat, shortening and Enzyme amylase, NAGase from solid substrate fermentation and xylanase from submerged fermentation obtained by the process of instant invention.

Result : Bread having NAGase + xylanase (1: 50), showed the desired characteristics.

Quality of Bread: The results are disclosed in Table 2.

Table 1 Production medium Sr. No Ingredient % 1 Vitacel 2.5 2 Cotton Seed 1. 5 Flour (CSF) 3 Soy Isolate 0.2 4 Malt Extract 2.0 5 Yeast Extract 0.38 6 Ammonium 0.2 suplhate 7 Magnesium 0.05 sulphate 8 Calcium 0.4 carbonate 9 Antifoam 0. 05 PH = 7. 0 + 0. 2 Table 2 QUALITY OF BREAD Firmness Loaf"texture"Net Moisture Shelf life (g) volume (hole size) appearance (day) (cu. cm) Control 650 1430 Large and Large and open 26 2 1 open Control 600 1440 Large and Large and open 28 2 2 open Test 1 450 1490 Moderate Moderate 36 4 Test 2 250 1560 Fine and Fine and close 42 6 dose