SUMMARY OF REPORT OF WORK DONE Title of Minor Research Project: Study on Solid State Bioprocessing of Rice Mill Byproducts for Food Grade Enzymes Production. File No: 47: F: 1625/10 (WRO), 16 /3/2011 Principal Investigator: Timande S.P. INTRODUCTION In Gondia, thousands of tons of rice milling by-products are produced every year. Presently, as rice bran has nutritive value ,it is used to feed cattle, poultry etc, and rice husk is burnt in furnace to boil water in rice mills. When these by-products and kept unused, they create pollution. For value addition to these products and better economic returns, in this project work bioprocessing of by-products to produce enzymes using microorganisms was carried out. The overall significance of the project is to get low cost enzyme preparation using cheap byproducts available in Gondia City through tools of biotechnology. On the other hand, by using the byproducts of rice mills as substrate for enzyme production, the project aims to convert the byproduct that earlier have caused pollution into useful products for food industry. Thus, low valued byproducts of rice milling industry can effectively be used for better economic returns. The In-House production of enzyme will create more employment and income, which can expedite the new ways of saving foreign exchange reserves. In this project work, an attempt will be made to produce, thermostable-neutral protease and thermostable-acidic amylase from rice-mill byproducts by solid-state fermentation. OBJECTIVES Following are the objectives of this research, To isolate strains of filamentous fungi capable of producing desired enzymes, to screen a set of microbial strains strongly producing the desired enzymes (thermostable acidic amylase and thermostable - neutral protease) from isolated microbes, to study effect of cultural conditions on enzyme production potential of hyper producer by solid state fermentation using rice mill byproduct, to improve- enzyme production capability of selected microbial strains by conventional method, to produce enzymes using rice bran and rice husk under optimized conditions using wild and mutated strains of hyper producer and to study pH and temperature optima of enzyme activities as well as pH and temperature stability of enzymes. METHODOLOGY Production of thermostable acidic amylase and thermostable neutral protease Isolation of Filamentous Fungi Screening for amylase and protease producers

Identification of hyper producer Study on effect of cultural conditions on enzyme production by hyper producer (Optimization of process parameters for amylase and protease production) Enzyme Characterization. Study on improvement of enzyme production of selected strain (hyper producer) by traditional method (UV-irradiation). RESULTS Production of thermostable acidic amylase Isolation of Filamentous Fungi, Screening for amylase producers and Identification of hyper producerA total of 42 mold isolates were obtained from soil and compost samples of district Gondia, M.S. by serial dilution and plating on Potato Dextrose Agar medium with antibiotic streptomycin. The isolated fungi were initially screened for amylase production based on zone of clearance on starch agar plates. Among the 42 isolates, 20 of them had the ability to produce amylase. The three (S5, S6 & C4) highest amylase producing fungi selected on the basis of zone of hydrolysis were further screened for amylase production by secondary screening. The three highest amylase producing strains were further assayed to quantitatively evaluate the amylase production using solid state fermentation technique. It was evident from the finding that mold S6 isolated from crop soil was an effective producer of amylase. The mold s6 isolate from crop soil gave maximum amylase activity. Based on the colony morphology and microscopic observation the S6 mold strain producing maximum amylase was identified as Aspergillus species. Aspergillus species which showed the best enzyme production was chosen and designated as Aspergillus species S6. The production medium was optimized for this strain. Study on effect of cultural conditions on amylase production by hyper producer (Aspergillus species S6) (Optimization of process parameters) Enzyme yield showed increasing trend when various nutritional and environmental parameters were optimized. Optimization of fermentation conditions for amylase production Process Parameters Fermentation period Inoculum volume Ph Temperature Nitrogen sources Sugars

Optimum 5 days 4% v/w 6.0 30°C Yeast extract Glucose

Amylase activity (u/ml) 1.19 1.22 1.45 1.63 1.72 1.78

Kinetics of amylase from Aspergillus species S6 The optimum pH and temperature for maximum activity of Aspergillus species S6 amylase are 6.0 (amylase activity-1.25) and 55°C (amylase activity-1.36 u/ml) (Fig: 8) .Above 55°C, there is decline in enzyme activity. This study revealed that the best pH for amylase activity was 6.0.The enzyme is stable (Amylase activity) for 15 min, at temperature 55°c and 6.0 pH. In order to study the effect of substrate concentration various substrate concentrations ranging from 0.5-5.5 % were used. Reaction rate verses substrate concentration curve was plotted to determine whether the enzyme obeys Michaelis-Menten kinetics, and constants were determined from a Line Weaver-Burk plot. The Vmax and Km values were determined against rice bran as substrate. Amylase of Aspergillus species S6 has Vmax of 120.0 U/min and Km of 1.2 mg/ml. Study on improvement of enzyme production of selected strain (hyper producer) by traditional method. UV - mutation. In acid amylase production by wild and mutant strains of Aspergillus through SSF enhanced acid amylase activity was achieved with physical mutant Aspergillus species S6-UV-10. Extracellular amylase production was performed using the both wild and developed mutant strain (hyper producer on rice bran and rice husk] in SSF. The acid amylase activity of wild strain of Aspergillus species {Aspergillus species S6} and mutant strain of Aspergillus species (Aspergillus species S6 -UV-10) using rice bran were 1.68 and 1.98 u/ml respectively. The acid amylase activity of wild strain of Aspergillus species (Aspergillus species S6) and mutant strain of Aspergillus species (Aspergillus species S6 -UV-10) using rice husk were 1.45 and 1.58 u/ml respectively. Production of thermostable neutral protease Isolation of Filamentous Fungi, Screening for protease producers and Identification of hperproducer A total of 42 mold isolates were obtained from soil and compost samples of district Gondia, M.S. by serial dilution and plating on Potato Dextrose Agar medium with antibiotic streptomycin. Among the 20 amylase producers, 12 had the ability to produce protease. The three (S5, C2 & C3) highest protease producing fungi selected on the basis of zone of hydrolysis were further screened for protease production by secondary screening. The mold C2 isolated from compost gave maximum protease activity. Based on the colony morphology and microscopic observation the C2 mold strain producing maximum protease was identified as Aspergillus species. Aspergillus species which showed the best enzyme production was chosen and designated as Aspergillus species C2. The production medium was optimized for this strain.

Study on effect of cultural conditions on protease production by hyper producer (Aspergillus species C2) (Optimization of process parameters) Enzyme yield showed increasing trend when various parameters were optimized Optimization of fermentation conditions for protease production Process Parameters Fermentation period Inoculum volume pH Temperature Nitrogen sources Sugars

Optimum Values 7 days 3% v/w 6.5 30°C Tryptone sucrose

amylase activity (u/ml) 1.24 1.18 1.38 1.46 1.63 1.67

Kinetics of protease from Aspergillus species C2 This study revealed that the best pH for protease activity was 7.0,(Protease activity-1.38 u/ml).Optimum temperature for maximum protease activity was 50°(Protease activity-1.42u/ml). Above 50°C, there is decline in enzyme activity. The enzyme is stable for 10 min, at temperature 50°C and 7.0 pH (Protease activity- 1.37u/ml). The Vmax and Km values were determined against rice bran as substrate. Amylase of Aspergillus species C2 has Vmax of 42.0 U/min and Km of 1.0 mg/ml. Study on improvement of enzyme production of selected strain (hyper producer) by traditional method. UV - mutation. The neutral protease activity of wild strain of Aspergillus species (Aspergillus species C2) and mutant strain of Aspergillus species (Aspergillus species C2 -UV-10) using rice bran were 1.65 and 1. 87 u/ml respectively. The neutral protease activity of wild strain of Aspergillus species (Aspergillus species C2) and mutant strain of Aspergillus species (Aspergillus species C2 -UV10) using rice husk were 1.25 and 1.48 u/ml respectively CONCLUSION It may be concluded from this work that Aspergillus species has the potential to produce highly thermostable neutral protease and thermostable acid amylase which could have potential application for wide range of industries. In the long run, such studies on the production of amylase and protease by solid state fermentation technique may develop a process for the production of amylase and protease using rice bran and rice husk. The economic utilization of

biomass will help in their control as well as for reducing their adverse impact on environment. Local level employment can be generated once the technology is commercially viable. Further research in this area is required to reduce the cost of production of amylase and protease by solid state fermentation to search for more efficient producer with higher specific activities and greater efficiency.

ACKNOWLEDGEMENTS This minor research project was awarded by University Grants Commission, New Delhi, India. I acknowledge to Commission, for financial assistance. I wish to acknowledge the kind cooperation given to me by the management of Gondia Education Society and Principal, Dhote Bandhu Science College, Gondia, M.S., India.