1. Introduction

Protease is one of the most important commercial enzymes used in food processing, cheese making, detergents, dairy industry and leather making ^[12]. The protease constitute two third of total enzymes used in various industries ^[8]. Protease occurs widely in plant and animals, but commercial proteases are produced exclusively from microorganisms. Molds of the genera Aspergillus, Penicillium, Rhizopus and Fusarium oxysporum are especially used for production of protease ^[15]. Protease is extracellular enzyme that can be produced by both submerged and solid-state fermentation. Solid-state fermentation is especially suited for the growth of fungi and it is simple, low cost, and provides high yields of appropriate enzymes ^[16]. Solid-state fermentation can use inexpensive and widely available agricultural residues as substrates. Agroindustrial wastes constitute spent grain, cocoa husk, rice bran, wheat bran, chickpea bran, sawdust and other agricultural waste Among various solid substrates reported, commercial wheat bran has been found to be a suitable substrate for producing mold proteases by solid-state fermentation but wheat bran is not easily available in many regions. In many Asian countries rice bran is less expensive than wheat bran ^[15]. Rice bran is a byproduct of the milling of rice and is a good source of proteins. Utilization of agroindustrial wastes clean environment and increase the economical value of agroindustrial waste. The present study was undertaken to produce protease on rice bran by using F. oxysporum in solid-state fermentation and to determine the effect of moisture, pH, temperature and incubation period on protease production.

2. Materials and Methods

The organism used in present study was F.oxysporum isolated from diseased seed of groundnut by standard blotter method ^[4]. The sample of groundnut seeds were placed on 3 layered moistened blotter discs in 9 cm glass petriplate @ four seeds per plate and incubated for 7 days at 22 ± °C temperature in incubator. Cultural characters were assessed by eye slight and by microscopic examination. Colony morphology was recorded from cultures grown on potato dextrose agar. The morphology of macro and microconidia was studied by using the criteria of Alexopoulos ^[3] and Gerlach and Nirenberg ^[7]. The inoculum was prepared by dispersing the spores from a week-old fungal slant culture in 0.1% Tween -80 solution with a sterile inoculation loop under aseptic condition.

The rice bran was obtained from rice mill of Parbhani (India). Rice bran was cleaned, and washed and dried. It was milled with an electric wearing blender and sieved through 20-40mm mesh sieve. The powder with 0.42 and 0.85mm mesh particle size were collected and used as solid substrate for solid-state fermentation.

Ten gram of rice bran powder with 0.42 and 0.85mm mesh particle size was supplemented with 0.166g K₂HPO₄ and 0.032g MgSO4 in 250mL Erlenmeyer flasks and moistened with 5ml of distilled water. After through mixing with glass rod the moist media was autoclaved for 20min at 121°C.

After cooling to room temperature the moist fermentation media was inoculated with 2ml of spore suspension (10¹² spore/ml) and mixed carefully with sterile glass rod under strictly aseptic conditions to achieve a uniform distribution of inoculums throughout the solid medium. The inoculated flask having initial moisture content of 50% (w/w) was incubated at 30°C for 7 days of incubation. After every 12h of interval 1 g substrate was harvested from flask and transferred to a test tube containing 5ml phosphate buffer (pH 7.4). The content was homogenized and centrifuges at 2000rpm for 30min to remove all particulate matter. The cultural filtrate was filtered through Whatman filter No. 1 paper and used for enzyme assay.

The activity of protease was assayed as suggested by Agrawal et al. ^[1]. To 1ml of supernatant 5ml solution of 2% casein dissolved in 0.5mL^-1 carbonate buffer (pH 10) were mixed. The obtained solution was incubated at 40°C on a rotator shaker at 300rpm for 30min. A 0.5ml of the reaction was withdrawn and the reaction was terminated by adding 1.5ml of pre-chilled trichloroacetic acid (10%). The mixture tube was then immersed in an ice bath for five minute to precipitate all the protein. Precipitate was recovered by centrifugation of mixture at 10,000rpm for 10min. Tyrosine liberated during casein hydrolysis was measured in the supernatant using the method of Lowry et al. ^[11]. A unit of protease activity was defined as the amount of enzyme liberating 1μg tyrosine min⁻¹ at the incubation temperature of 40°C. The protease activity is reported per g of dry solids used in initial extraction.

The effect of incubation period on protease was determined by incubating fermentation medium for different incubation periods in hrs. i.e. 24,48,72,96, 120, 144 and 168h at 35°C with initial substrate pH 7.0.

Initial moisture of substrate plays important role in mold growth and enzyme production in solid-state fermentation. To determine the effect of moisture, substrate was moistened at 40, 45, 50, 55, 60, and 65% with distilled water. The fermentation was carried out for 120h at 35°C.

The inoculated substrate was incubated at different temperatures i.e. 25, 30, 35, 40, 45, and 50°C to observe the effect of temperature, by keeping all other conditions at their optimum level.

In general practice, the pH of solid-state fermentation is almost never controlled during fermentation, only the initial pH of the substrate was adjusted before inoculation. To evaluate production of protease initial pH of substrate was adjusted at 5, 5.5, 6, 6.5, 7 and 7.5 pH with 1N HCl or 1N NaOH. The fermentation was carried out at 35°C to study their effect on enzyme production.

3. Result and Discussion

Protease production by F. oxysporum in solid-state fermentation was studied over a 7- day of incubation period. It is notable that in other studies, enzyme production was studied over an incubation time of 48h for bacteria and 8-9 days for fungi ^[10]. The study of protease production versus incubation time showed that protease production increased with incubation time. Maximum enzyme production was observed at 72h of incubation (70.5U/g) shown in Figure 1. Similar finding were reported by Paranthaman et al. ^[14]. A gradual decrease in enzyme units was observed with increasing incubation period clearly suggesting the enzyme’s role as a primary metabolite, being produced in log phase of the growth of the fungi for utilization of nutrients i.e. protein present in the solid substrate^[2]. The subsequent decrease in the enzyme unit could probably be due to inactivation of the enzyme by synthesis of other metabolites in the medium. These results are in accordance with observation made by Yeoman and Edwards ^[17].

Initial moisture content of solid substrate is important parameter for mold growth and enzyme production in solid-state fermentation ^[5]. Water play important role in physico-chemical properties of the substrate, which affect enzyme production ^[13]. Increase in moisture level is believed to reduce the porosity of the rice bran, thus limiting oxygen transfer. While lower moisture content causes reduction in the solubility of nutrients of the substrate and lower degree of swelling. Therefore the amount of protease production varies as per the initial moisture content of substrate. The effect of initial moisture on protease production is shown in Figure 1 was maximum (70.4U/g) at initial moisture level of 50% (w/w). Similar finding were reported by Jarun et al. ^[9] in the case of Aspergillus oryzae. Substrate moistened at 50% (w/w) level shown maximum protease activity while moisture level above 50% decreased enzyme production as the substrate become less pores and adversely affect on oxygen supply to the mold ^{[5, 15]}.

The effect of different incubation temperature on enzyme production is shown in Figure 1. The optimum temperature for maximum protease production was found at 35°C which was also optimum temperature for A. niger ^[14]. A decrease or increase in incubation temperature reduced the productivity of enzymes. Temperature is an essential factor affecting solid-state fermentation performance because of its importance in microorganisms’ growth and metabolite production ^[13].

Figure 1. Effect of incubation time, moisture, temperature on protease production by F.oxysporum on rice bran in SSF

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The pH in solid-state fermentation is almost never controlled during fermentation; only the initial pH of the substrate is adjusted before incubation. Protease production by microorganism depends on the extracellular pH because culture pH strongly influences many enzymatic processes and transport of various components across the cell membranes, which in turn support the cell growth and product production ^[6]. Optimum pH for protease production by F. oxysporum was recorded at 7.0 as shown in Figure 2. Similar finding were also reported by Paranthaman et al. ^[14].

Figure 2. The effect of Initial pH on protease production by F. oxysporum on rice bran in SSF

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4. Conclusion

The utilization of agroindustrial waste is important factor for cleaning environment and reducing environmental pollution. There are several reports on use of agroindustrial wastes for energy production, biofuel and renewable organic matter. The purpose of present study was to increase the protease production and reduce the cost of enzyme production process. Considering the economic value of other process and substrate, utilization of rice bran as a substrate proved economical. The results of above study revealed that rice bran can be successfully utilized as substrate for protease production by Fusarium oxysporum. Maximum protease production can be achieved at 35°C on incubation period of 72h at initial pH 7.0 of substrate.

References

[1]	Agrawal, D., Partidar, P., Banerjee, T., and Patil, S., “Alkaline protease production by a soil isolate of Beauveria feline under SSF condition: parameter optimization and application to soy protein hydrolysis,” Process Biochem, 40, 1131-1136. 2005.
	In article		CrossRef
[2]	Alagarsamy, S., Paul, D., Chandran, S., George, S., Carlos, R., “Rice bran as a substrate for proteolytic enzyme production,” Brazilian Archives of Biology and technology, 49, 843-851. 2006.
	In article		CrossRef
[3]	Alexopoulos, C.J., “Introductory Mycology” IInd edition. John Wiley and sons, New York. 1962.
	In article
[4]	Anonymous, “International Rules of Seed Testing,” Proc. Int. Seed Test. Assoc., 4, 3-49. 1976.
	In article
[5]	Chisti, Y., “Solid substrate fermentations, enzyme production, food enrichment. In Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation,” Vol 5, ed. by Flickinger MC and Drew SW. Wiley, New York, 2446-2462. 1999.
	In article
[6]	Ellaiah, P., Srinivasulu, B., Adinarayana, K., “A review on microbial alkaline proteases,” J. Sci Ind Res, 61, 690-704. 2002.
	In article
[7]	Gerlach, W., Nirenberg, H., “The genus Fusarium: a pictorial atlas.” Mitteilungen aus der Biologischen Bundesanstalt für Land-und Forstwirtschaft, Berlin-Dahlem, Germany, 1982.
	In article
[8]	Gupta, R., Beg, Q.K., Lorenz, P., “Bacterial Alkaline Protease: Molecular approaches and industrial application,” Appl Microbial Biotechnol, 59, 15-32. 2002.
	In article		CrossRef PubMed
[9]	Jarun, C., Sinsupha, C., Yusuf, C., and Penjit, S., “Protease Production by Aspergillus oryzae in solid-state fermentation using agroindustrial substrate,” J. Chem Tech Biotechnology, 83, 1012-1018. 2008.
	In article		CrossRef
[10]	Kumar, A., Sachdev, A., Balasubramanyam, S.D., Saxena, A.K., and Lata., “Optimization of conditions for production of neutral and alkaline protease from species of Bacillus and Pseudomonas,” Ind. J. Microbiol. 42, 233-236. 2002.
	In article
[11]	Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., “Protein measurement with the Folin phenol reagent,” J. Biol Che, 193, 265-275. 1951.
	In article		PubMed
[12]	Negi, S. and Benerjee, R., “Optimization of amylase and protease production from Aspergillus awamori in single bioreactor through EVOP factorial design technique,” Food Technol Biotechnol. 44, 257-261. 2006.
	In article
[13]	Pandey, A., Sevalkumar, P., Soccol, C.R. and Nigam, P., “Solid state fermentation for the production of industrial enzymes,” Curr Sci, 77, 149-162. 1999.
	In article
[14]	Paranthaman, R., Alagusundaram, K. and Indhumathi, J., “Production of protease from rice mill wastes by Aspergillus niger in solid state fermentation,” World J. Agri Sci, 5, 308-312. 2009.
	In article
[15]	Sandhya, C., Sumantha, A., Szakacs, G. and Pandey, A., “Comparative evolution of Neutral proteases production by Aspergillus oryzae in submerged and slid state-fermentation,” Process Biochem., 40, 2689-2694. 2005.
	In article		CrossRef
[16]	Wang, R., Law, R.C.S. and Webb, C., “Protease Production and Condition by Aspergillus oryzae in Flour fermentation,” Process Biochem, 40, 217-227. 2005.
	In article		CrossRef
[17]	Yeoman, K.H., and Edwards, C., “Protease production by Streptomyces thermovulgaris grown on rapemeal-derived media,” J. Appl Bacteriol, 77, 264-270. 1994.
	In article		CrossRef PubMed