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 4ºC.
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.
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
Cupp-Enyard,
C. (2008). Sigma non-specific protease activity assay: Casein as substrate.
Journal of Visualized Experiments, 19, 82-99. https://doi.org/10.3791/899
DoctorMurry.com.
(2015). Healing power of protease. Retrieved from
http://www.doctormurry.com/healing-power-of-protease
Dhaliwal,
K. M., Patil, R. N., & Sambare, S. (2018). Production of proteases using
solid-state fermentation. International Journal of Scientific Research in
Science, Engineering and Technology, 5(4), 2394-4099.
Muthulakshmi,
C., Gomathi, D., Kumar, D. G., Ravikumar, G., Kalaiselvi, M., & Uma, C.
(2011). Production, purification and characterization of protease by
Aspergillus flavus under solid state fermentation. Jordan Journal of Biological
Sciences, 4(3), 137-148.
Kademi,
F., Abachi, S., Mortazavi, A., Ehsani, M. A., Tabatabaei, M. R., &
Malekzadeh, F. A. (2013). Optimization of fungal rennet production by local
isolate of Rhizomucor nainifalensis under solid-substrate fermentation system.
Kunitz,
M. (1947). Crystalline soybean trypsin inhibitor II: General properties.
Journal of General Physiology, 30, 291-310.
Ramachandran,
N., & Arutselvi, R. (2013). Partial purification and characterization of
protease enzyme from Nomuraea rileyi. International Journal of Pharmaceutical
Sciences and Research, 12(6), 1-11.
Rodarte,
M. P., Dias, D. R., Vilela, D. M., & Schwan, R. F. (2011). Proteolytic
activities of bacteria, yeasts, and filamentous fungi isolated from coffee
fruit (Coffea arabica L.). Acta Scientiarum Agronomy, 33(3), 457-464.
Sadh,
P. K., Duhan, S., & Duhan, J. S. (2018). Agro-industrial wastes and their
utilization using solid state fermentation: A review. Bioresources and
Bioprocessing, 5(1), 1-15.
Sirinividya,
S. (2012). Production and characterization of an acid protease from a local
Aspergillus spp. by solid-state fermentation. Archives of Applied Science
Research, 4(1), 188-199.
Syed,
S. A., & Vidhale, W. W. (2013). Protease production by Fusarium oxysporum
in solid-state fermentation using rice bran. American Journal of Microbiology
Research, 1(3), 45-47. https://doi.org/10.12691/ajmr-1-3-2
Piyawan,
C., Gunjana, T., Savitree, L., Hirohide, T., Osa, O., & Kazunobu, M.
(2003). Quinoprotein alcohol dehydrogenase is involved in catabolic acetate
production while NAD-dependent alcohol dehydrogenase in ethanol assimilation in
Acetobacter pasteurianus SKU1108. Journal of Bioscience and Bioengineering,
96(6), 564-571. https://doi.org/10.1016/s1389-1723(04)70150-4
Xin,
Z., Yao, S., & Ha, T., Chuiquin. (2021). Novel method for analysis of
protease activity: The casein plate method and its application. ACS Omega,
6(5), 367-3675. https://doi.org/10.1021/acsomega
