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Comparison of Protease Activity between Two Fungal Strains and Commercial Rennet on Different Substrates

Sambo S¹; Adamu SA²; Haruna IA³

¹Department of Biology, Shehu Shagari College of Education, Sokoto, Nigeria.

²Department of Microbiology, Sokoto State University, Sokoto, Nigeria.

³Department of Entrepreneurship Umaru Ali Shinkafi Polytechnic, Sokoto.

INTRODUCTION

Microbial enzymes, particularly proteases, play essential roles across diverse industries, including food processing, pharmaceuticals, leather production, industrial waste treatment, and detergent formulation. Among them, acid proteases are especially valued for their protein-coagulating abilities. Proteolytic enzymes, or proteases, encompass a range of enzymes that break down proteins into smaller units; these include pancreatic protease, chymosin, trypsin, bromelain (from pineapple), papain (from papaya), fungal proteases, and serrapeptase (from soil bacteria) (Doctor Murray.com, 2015). Protease activity is commonly measured in units, with one unit defined as the amount of enzyme that catalyzes the formation or consumption of 1 µmol of reaction product or substrate per minute (Piyawan et al., 2003).

The rapid growth in population and food processing has led to a significant increase in agro-industrial waste, which contains various sugars, minerals, and proteins. Microorganisms have the potential to use these waste products as raw materials through fermentation processes (Sadh et al., 2018). This study examines the protease activity of two fungal strains, Aspergillus niger and Fusarium oxysporum, along with commercial rennet, using cost-effective agro-industrial substrates such as wheat bran (WB), rice bran (RB), millet bran (MB), soybean husk (SBH), banana peel powder (BPP), and casein.

Protease activities are typically measured by tracking either the reduction in substrate protein concentration or the increase in free amino acids or polypeptide concentration following enzymatic hydrolysis (Xin et al., 2021). This research will provides insights into the potential of these fungal strains to produce proteases economically by utilizing readily available waste substrates.

MATERIALS AND METHODS

Solid-State Fermentation for the Screening of Substrate for Protease production from the Isolated Fungi

Six types of media (which included casein, wheat bran, millet bran, rice bran, banana peel powder and soya bean meal) were screened as media for the production of protease from the isolated fungi using Solid State Fermentation (SSF) method. Crude enzyme was exacted after the fermentation (using the procedure described in section 3.7.1) and assayed for the acid protease activity. For the SSF, 5.0g of each substrate was taken in a 250ml Erlenmeyer flask separately, each was moistened with salt solutions; composition (% W/V) as follows: sodium nitrate 0.2, potassium dihydrogen phosphate 0.1, magnesium sulphate 0.05, potassium dihydrogen phosphate 0.1, magnesium sulphate 0.05, potassium chloride 0.05, ferrous sulphate concentration, and zinc sulphate concentration at pH of 7.0 were used to achieve the desired moisture content. The mixture was sterilized at 121 ^ºC at 15 min, cooled and inoculated with 1 ml of fungal spore suspension (10⁶ spores/ml) and incubated at 30 ^ºC for 72 hrs (Sirividya, 2012). The same procedure was used for the remaining substrates and commercial rennet.

Enzymes Extraction (EE)

After 72 hrs of fermentation 5.0 g of the fermented material was mixed with 30 ml of 0.1ml phosphate buffer and homogenised by shaking for 30 min and filtered through cheese cloth. Cell free supernatant was obtained by centrifuging the extract at 10,000 rpm for 30 min. The centrifuged extract was filtered through Whatman No.1 filter paper. The volume of filtrate containing the crude enzyme was measured, recorded and used for activity and protease assay (Sirividya, 2012).

Purification of Extracted Enzymes

The crude enzyme extracted from the samples was purified by subjecting to ammonium sulphate precipitation. The filtrate was taken and 70% fraction of ammonium sulphate was added slowly to the supernatant. While adding the ammonium sulphate, the culture was kept in ice blocks, then, the mixture was incubated overnight in refrigerator at 4^ºC. On the next day the mixture was centrifuged at 12,000 rpm for 10 mins. The pellet was collected and dissolved in 1M tris HCL (Ramachandran and Arutselvi, 2013) adopted by Kademi et al., (2013). A1m of enzyme solution was added to 5 ml of substrate solution containing 1.2% of casein solution in 0.05M phosphate buffer (pH 6.0). The mixture was incubated at 35 ^ºC for 10 min. Five milliliters (5.0 ml) of 0.44Mole Tri- chloro acetic acid was added to inhibit the reaction and the mixture was allowed to stand for 15 min before centrifuging at 10,000 rpm for 10 min at 4 ^ºC to reduce the resulting precipitate. Two milliliters (2ml) of the supernatant was added to 5 ml of NaOH solution (0.28N). Later, 1.5ml of Folin’s phenol reagent was added to the mixture at 35 ^ºC for 10 min and measured at 660nm. Enzyme activity was expressed in units per ml (U/ML) (Kademi et al., 2013). To have a comparing standard, tyrosine standard solution were prepared and concentrations of 0, 4, 8, 12, 16 and 20mg/l and their absorbances were measured at 660nm using spectrophotometer. A standard curve was generated by plotting the change in absorbance in the standard on Y axis versus the amount (in micro moles) for each of the standard concentration on X axis and formula below was used to calculate values of both the standard and test samples and the result was expressed in unit per milliliters (Cupp-Enyard, 2008).

Units per ml enzyme = Umole tyrosine equivalent release X total volume of Assay/ Vol. of enzyme (in mls) of assay X time of assay in minutes X Vol. in ml of enzymes used Umole tyrosine equivalent = Absorbances for samples and standard

Total volume of assay = 11ml

Volume of enzymes = 1ml

Time of assay = 10 minutes

Volume of enzymes used = 2ml

RESULTS

Protease activity values of supernatant enzymes of various substrate treated with A. niger are presented in Figure 1, where CS had the highest PA of 0.733±0.012U/ML and comparison among the values of PA obtained revealed that there was significant differences among all the values except for WB and BPP, SBH and MB; with SBH having the lowest PA of 0.026±0.06.

On treatment of the substrates with F. oxysporum, the highest PA was from supernatant enzymes of CS with 1.130±0.065, followed by 0.363±0.067 from supernatant of SBH. There was significant difference among the various PA values (Figure 2).

Analysis of the PA values obtained from treatment of the substrate with Commercial Rennet, Figure 3 showed that CS had the highest PA, which was 0.519±0.028, followed by RB with 0.246±0.010. However, there was no significant difference between PA obtained for RB and that obtained in SBH (0.210±0.017). There was significant difference for all other values.

Figure 1: Effects of Various Substrates on Protease Activity of Supernatant Enzyme Produced by A. niger

Figure 2: Effects of Various Substrates on Protease Activity of Supernatant Enzyme Produced by F. oxysporum

Figure 3: Effects of Various Substrates on Protease Activity of Supernatant Enzyme Produced by Commercial Rennet.

DISCUSSION

A one-way ANOVA followed by Bonferroni’s multiple comparison test was used to analyze protease activity values, showing significant protease activity for both fungal strains and commercial rennet (CR) on casein, with F. oxysporum achieving the highest activity at 1.13 ± 0.06 U/mL. This aligns with findings by Rodate et al. (2011), who isolated 144 microorganisms from coffee fruit and observed proteolytic activity on casein agar, noting that 2.6% of yeast and 50% of saprophytic fungi—including F. moniliforme, F. solani, A. dimorphicus, A. ochraceus, P. fellutans, and P. waksmanii demonstrated strong protease activity.

Wheat bran proved particularly effective for cultivating protease enzymes with both fungal strains and CR, yielding over 0.01 U/mL. This result for A. niger supports findings by Dhaliwal et al. (2018), who reported a protease yield of 1.785 U/mL from A. niger ATCC 16404 grown on wheat bran at pH 6.0 and 28 ± 2ºC under solid-state fermentation (SSF), surpassing the 1.487 U/mL obtained from rice bran. The higher protein content in wheat bran (14-16%) compared to rice bran (7-8%) likely contributes to this difference. High protease activity from A. niger on wheat bran was also reported by Muthulakshmi et al. (2011) and Mukhtar and Ikram-ul-Haq (2013).

CR maintained steady protease activity across all substrates except millet bran (MB) and banana peel powder (BPP). A. niger showed stronger enzyme activity with BPP than with soybean husk (SBH), MB, or rice bran (RB), while F. oxysporum exhibited higher protease activity on RB than A. niger, corroborating the findings of Syed and Vidhale (2013), who observed 70.5 U/g of protease activity from F. oxysporum on rice bran after 72 hours of incubation.

Overall, F. oxysporum achieved protease activity above 0.01 U/mL across all substrates, outperforming both A. niger and CR. These results indicate that F. oxysporum could be effectively utilized for protease production on various low-cost substrates, presenting potential for optimization and scale-up for commercial applications.

CONCLUSION

This research concludes that Fusarium oxysporum and Aspergillus niger exhibit significant potential as sources of protease enzymes when cultivated on agro-industrial substrates, with F. oxysporum consistently demonstrating higher protease activity across all substrates compared to A. niger and commercial rennet. Among the substrates tested, wheat bran proved to be the most effective medium for enzyme production, likely due to its higher protein content. Furthermore, F. oxysporum displayed strong protease activity on rice bran, underscoring its adaptability and efficacy in utilizing diverse, low-cost substrates. These findings suggest that F. oxysporum could be optimized for large-scale protease production using inexpensive agro-industrial wastes, offering a cost-effective, sustainable enzyme source with potential applications in food processing, pharmaceuticals, and other industries. This study highlights the feasibility of using fungal strains to produce protease enzymes, adding value to agro-industrial byproducts and contributing to waste reduction.

ACKNOWLEDGEMENT

We wish to express our sincere gratitude to Shehu Shagari College of Education, Sokoto and Tertiary Education Trust Fund (TETFUND, Abuja) for research sponsorship.

CONFLICT OF INTEREST

The authors have declared that no competing interests exist.

REFERENCES

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