Cellulase and β-glucosidase activity of crude enzyme extracted from fresh and expired soybean tempeh
Abstract
Background and Objectives: Enzymes are protein biomolecules that act as catalysts, including cellulase and β-glucosidase with extensive applications. Thus, this work aimed to contrast the activity of both enzymes from tempeh fermented by Rhizopus microsporus at 2 and 7 days of incubation.
Materials and Methods: Incubated tempeh was tested for quality evaluation. The crude extracts of cellulase and β-glucosidase were obtained by extracting tempeh with a cold phosphate buffer solution. The presence of the R. microsporus was examined through microscopic identification, and molecular identification using PCR amplification. Cellulase activity was determined using 3.5-dinitrosalicylic acid (DNS) reagent, whereas β-glucosidase activity was evaluated by measuring the release of p-nitrophenol from p-nitrophenyl-β-D-glucopyranoside (p-NPG).
Results: The protein content and water content increase with the length of fermentation time. Microscopic identification and molecular identification confirmed the presence of R. microsporus. The highest cellulase activity was found in fresh tempeh (2-day incubation) at 0.0911 U/mL at pH 7, while the highest β-glucosidase activity was found in expired tempeh (7-day incubation) at 0.0042 U/mL at pH 5.
Conclusion: These findings indicate that the standard quality contributed to the differences in the enzymatic profile of tempeh incubated for 2 and 7 days.
1. Uzuner S, Cekmecelioglu D. Enzymes in the beverage industry. In: Enzymes in Food Biotechnology: Production, Applications and Future Prospects. Elsevier; 2019. pp. 29-43.
2. Naher L, Fatin SN, Sheikh MAH, Azeez LA, Siddiquee S, Zain NM, et al. Cellulase enzyme production from filamentous fungi Trichoderma reesei and Aspergillus awamori in submerged fermentation with rice straw. J Fungi (Basel) 2021; 7: 868.
3. Demirkan E, Kut D, Karakaya E, Yıldırım I, Liaqat F, Khazi MI. Sustainable bio-finishing of denim fabric using a novel thermostable cellulase from mutant Bacillus subtilis IE3 for reduced environmental impact. Int J Biol Macromol 2026; 353: 151223.
4. Ejaz U, Sohail M, Ghanemi A. Cellulases: From bioactivity to a variety of industrial applications. Biomimetics (Basel) 2021; 6: 44.
5. Palugan L, Filippin I, Cirilli M, Moutaharrik S, Zema L, Cerea M, et al. Cellulase as an “active” excipient in prolonged-release HPMC matrices: A novel strategy towards zero-order release kinetics. Int J Pharm 2021; 607: 121005.
6. Muradova M, Proskura A, Canon F, Aleksandrova I, Schwartz M, Heydel JM, et al. Unlocking Flavor Potential Using Microbial β-Glucosidases in Food Processing. Foods 2023; 12: 4484.
7. de Morais Souto B, Barbosa MF, Sales RM, Moura SC, Araújo AD, Quirino BF. The potential of β-glucosidases for aroma and flavor improvement in the food industry. The Microbe 2023; 1: 100004.
8. Ouyang B, Wang G, Zhang N, Zuo J, Huang Y, Zhao X. Recent advances in β-Glucosidase sequence and structure engineering: a brief review. Molecules 2023;28: 4990.
9. Nurmilah S, Frediansyah A, Cahyana Y, Utama GL. Biotransformation and health potential of isoflavones by microorganisms in Indonesian traditional fermented soy products: A review. J Agric Food Res 2024;18: 101365.
10. Subali D, Christos RE, Givianty VT, Ranti AV, Kartawidjajaputra F, Antono L, et al. Soy-Based Tempeh Rich in Paraprobiotics Properties as Functional Sports Food: More Than a Protein Source. Nutrients 2023;15: 2599.
11. Hariyanto I, Hsieh CW, Hsu YH, Chen LG, Chu CS, Weng BBC. In vitro and in vivo assessments of anti-hyperglycemic properties of soybean residue fermented with Rhizopus oligosporus and Lactiplantibacillus plantarum. Life (basel) 2022; 12: 1716.
12. Martín-Miguélez JM, Bross J, Prado D, Merino E, Perisé Moré R, Otero J, et al. Review: Rhizopus sp. beyond tempeh - an Occidental approach to mold-based fermentations. Int J Gastron Food Sci 2025; 39: 101090.
13. Wiesel I, Rehm HJ, Bisping B. Improvement of tempe fermentations by application of mixed cultures consisting of Rhizopus sp. and bacterial strains. Appl Microbiol Biotechnol 1997; 47:218-225.
14. Pamekas TTL, Fibri DLN, Supriyadi S. Volatile compounds and aroma descriptions in overfermented tempe (tempe bosok) with different packaging and blanching treatment. Int J Gastron Food Sci 2025; 39: 101105.
15. Badan Standardisasi Nasional (BSN). SNI 3144-2015: Tempe kedelai. In Jakarta; 2015. p. 2. Available from: https://akses-sni.bsn.go.id/viewsni/baca/6165
16. Badan Standardisasi Nasional (BSN). SNI 3144 - 2009: Tempe kedelai. In Jakarta: BSN; 2009. Available from: https://akses-sni.bsn.go.id/viewsni/baca/3823
17. Michail M, Vasiliadou M, Zotos A. Partial purification and comparison of precipitation techniques of proteolytic enzymes from trout (Salmo gairdnerii) heads. Food Chem 2006; 97: 50-55.
18. Huang YC, Wu BH, Chu YL, Chang WC, Wu MC. Effects of tempeh fermentation with lactobacillus plantarum and Rhizopus oligosporus on streptozotocin-induced type II diabetes mellitus in rats. Nutrients 2018; 10:1143.
19. Romulo A, Surya R. Tempe: A traditional fermented food of Indonesia and its health benefits. Int J Gastron Food Sci 2021; 26: 100413.
20. Boukid F, Hassoun A, Zouari A, Tülbek MÇ, Mefleh M, Aït-Kaddour A, et al. Fermentation for Designing Innovative Plant-Based Meat and Dairy Alternatives. Foods 2023; 12: 1005.
21. Yarlina VP, Djali M, Andoyo R, Nurmilah S, Lani MN. Metagenomic insights into enhancing protein content and digestibility in Jack Bean (Canavalia ensiformis) tempeh: Unraveling microbial dynamics during fermentation. Appl Food Res 2024; 4: 100588.
22. Lopez MJ, Mohiuddin SS. Biochemistry, Essential Amino Acids. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 [cited 2026 May 17]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557845/
23. Pasini E, Corsetti G, Aquilani R, Romano C, Picca A, Calvani R, et al. Protein-amino acid metabolism disarrangements: The hidden enemy of chronic age-related conditions. Nutrients 2018;10: 391.
24. Prativi MBN, Astuti DI, Putri SP, Laviña WA, Fukusaki E, Aditiawati P. Metabolite Changes in Indonesian Tempe Production from Raw Soybeans to Over-Fermented Tempe. Metabolites 2023; 13: 300.
25. Witono Y, Widjanarko SB, Mujianto, Rachmawati DT. Amino acids identification of over fermented tempeh, the hydrolysate and the seasoning product hydrolysed by calotropin from crown flower (Calotropis gigantea). Int J Adv Sci Eng Inf Technol 2015; 5: 103.
26. Nkhata SG, Ayua E, Kamau EH, Shingiro JB. Fermentation and germination improve nutritional value of cereals and legumes through activation of endogenous enzymes. Food Sci Nutr 2018; 6: 2446-2458.
27. Kadar AD, Astawan M, Putri SP, Fukusaki E. Metabolomics based study of the effect of raw materials to the end product of tempe—an indonesian fermented soybean. Metabolites 2020; 10: 367.
28. Hartanti AT, Rahayu G, Hidayat I. Rhizopus Species from Fresh Tempeh Collected from Several Regions in Indonesia. HAYATI J Biosci 2015; 22: 136-142.
29. Sjamsuridzal W, Khasanah M, Febriani R, Vebliza Y, Oetari A, Santoso I, et al. The Effect of The use of Commercial Tempeh Starter on The Diversity of Rhizopus Tempeh in Indonesia. Sci Rep [Internet] 2021; 11: 23932.
30. Beye M, Fahsi N, Raoult D, Fournier PE. Careful use of 16S rRNA gene sequence similarity values for the identification of Mycobacterium species. New Microbes New Infect 2018; 22: 24-29.
31. Aggarwal NK, Goyal V, Saini A, Yadav A, Gupta R. Enzymatic saccharification of pretreated rice straw by cellulases from Aspergillus niger BK01. 3 Biotech 2017; 7: 158.
32. Pandey AK, Negi S. Enhanced cellulase recovery in SSF from Rhizopus oryzae SN5 and immobilization for multi-batch saccharification of carboxymethylcellulose. Biocatal Agric Biotechnol 2020; 26: 101656.
33. Khadka S, Khadka D, Poudel RC, Bhandari M, Baidya P, Sijapati J, et al. Production optimization and biochemical characterization of cellulase from Geobacillus sp. KP43 isolated from hot spring water of Nepal. Biomed Res Int 2022; 2022: 6840409.
34. Ye M, Sun L, Yang R, Wang Z, Qi K. The optimization of fermentation conditions for producing cellulase of bacillus amyloliquefaciens and its application to goose feed. R Soc Open Sci 2017; 4: 171012.
35. Prasad P, Singh T, Bedi S. Characterization of the cellulolytic enzyme produced by Streptomyces griseorubens (Accession No. AB184139) isolated from Indian soil. J King Saud Univ Sci 2013; 25: 245-250.
36. Kupski L, Pagnussatt FA, Buffon JG, Furlong EB. Endoglucanase and total cellulase from newly isolated Rhizopus oryzae and Trichoderma reesei: Production, characterization, and thermal stability. Appl Biochem Biotechnol 2014; 172: 458-468.
37. Fernández-López MG, Batista-García RA, Aréchiga-Carvajal ET. Alkaliphilic/alkali tolerant fungi: molecular , biochemical , and biotechnological aspects. J Fungi (Basel) 2023; 9: 652.
38. Mustafa HK, Anwer SS, Zrary TJ. Influence of pH, agitation speed, and temperature on growth of fungi isolated from Koya, Iraq. Kuwait J Sci 2023; 50: 657-664.
39. Michlmayr H, Schümann C, Barreira Braz Da Silva NM, Kulbe KD, Del Hierro AM. Isolation and basic characterization of a β-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture. J Appl Microbiol 2010; 108: 550-559.
40. Sparringa RA, Kendall M, Westby A, Owens JD. Effects of temperature, pH, water activity and CO2 concentration on growth of Rhizopus oligosporus NRRL 2710. J Appl Microbiol 2002; 92: 329-337.
41. Decker CH, Visser J, Schreier P. β-Glucosidases from five black Aspergillus species: Study of their physico-chemical and biocatalytic properties. J Agric Food Chem 2000; 48: 4929-4936.
2. Naher L, Fatin SN, Sheikh MAH, Azeez LA, Siddiquee S, Zain NM, et al. Cellulase enzyme production from filamentous fungi Trichoderma reesei and Aspergillus awamori in submerged fermentation with rice straw. J Fungi (Basel) 2021; 7: 868.
3. Demirkan E, Kut D, Karakaya E, Yıldırım I, Liaqat F, Khazi MI. Sustainable bio-finishing of denim fabric using a novel thermostable cellulase from mutant Bacillus subtilis IE3 for reduced environmental impact. Int J Biol Macromol 2026; 353: 151223.
4. Ejaz U, Sohail M, Ghanemi A. Cellulases: From bioactivity to a variety of industrial applications. Biomimetics (Basel) 2021; 6: 44.
5. Palugan L, Filippin I, Cirilli M, Moutaharrik S, Zema L, Cerea M, et al. Cellulase as an “active” excipient in prolonged-release HPMC matrices: A novel strategy towards zero-order release kinetics. Int J Pharm 2021; 607: 121005.
6. Muradova M, Proskura A, Canon F, Aleksandrova I, Schwartz M, Heydel JM, et al. Unlocking Flavor Potential Using Microbial β-Glucosidases in Food Processing. Foods 2023; 12: 4484.
7. de Morais Souto B, Barbosa MF, Sales RM, Moura SC, Araújo AD, Quirino BF. The potential of β-glucosidases for aroma and flavor improvement in the food industry. The Microbe 2023; 1: 100004.
8. Ouyang B, Wang G, Zhang N, Zuo J, Huang Y, Zhao X. Recent advances in β-Glucosidase sequence and structure engineering: a brief review. Molecules 2023;28: 4990.
9. Nurmilah S, Frediansyah A, Cahyana Y, Utama GL. Biotransformation and health potential of isoflavones by microorganisms in Indonesian traditional fermented soy products: A review. J Agric Food Res 2024;18: 101365.
10. Subali D, Christos RE, Givianty VT, Ranti AV, Kartawidjajaputra F, Antono L, et al. Soy-Based Tempeh Rich in Paraprobiotics Properties as Functional Sports Food: More Than a Protein Source. Nutrients 2023;15: 2599.
11. Hariyanto I, Hsieh CW, Hsu YH, Chen LG, Chu CS, Weng BBC. In vitro and in vivo assessments of anti-hyperglycemic properties of soybean residue fermented with Rhizopus oligosporus and Lactiplantibacillus plantarum. Life (basel) 2022; 12: 1716.
12. Martín-Miguélez JM, Bross J, Prado D, Merino E, Perisé Moré R, Otero J, et al. Review: Rhizopus sp. beyond tempeh - an Occidental approach to mold-based fermentations. Int J Gastron Food Sci 2025; 39: 101090.
13. Wiesel I, Rehm HJ, Bisping B. Improvement of tempe fermentations by application of mixed cultures consisting of Rhizopus sp. and bacterial strains. Appl Microbiol Biotechnol 1997; 47:218-225.
14. Pamekas TTL, Fibri DLN, Supriyadi S. Volatile compounds and aroma descriptions in overfermented tempe (tempe bosok) with different packaging and blanching treatment. Int J Gastron Food Sci 2025; 39: 101105.
15. Badan Standardisasi Nasional (BSN). SNI 3144-2015: Tempe kedelai. In Jakarta; 2015. p. 2. Available from: https://akses-sni.bsn.go.id/viewsni/baca/6165
16. Badan Standardisasi Nasional (BSN). SNI 3144 - 2009: Tempe kedelai. In Jakarta: BSN; 2009. Available from: https://akses-sni.bsn.go.id/viewsni/baca/3823
17. Michail M, Vasiliadou M, Zotos A. Partial purification and comparison of precipitation techniques of proteolytic enzymes from trout (Salmo gairdnerii) heads. Food Chem 2006; 97: 50-55.
18. Huang YC, Wu BH, Chu YL, Chang WC, Wu MC. Effects of tempeh fermentation with lactobacillus plantarum and Rhizopus oligosporus on streptozotocin-induced type II diabetes mellitus in rats. Nutrients 2018; 10:1143.
19. Romulo A, Surya R. Tempe: A traditional fermented food of Indonesia and its health benefits. Int J Gastron Food Sci 2021; 26: 100413.
20. Boukid F, Hassoun A, Zouari A, Tülbek MÇ, Mefleh M, Aït-Kaddour A, et al. Fermentation for Designing Innovative Plant-Based Meat and Dairy Alternatives. Foods 2023; 12: 1005.
21. Yarlina VP, Djali M, Andoyo R, Nurmilah S, Lani MN. Metagenomic insights into enhancing protein content and digestibility in Jack Bean (Canavalia ensiformis) tempeh: Unraveling microbial dynamics during fermentation. Appl Food Res 2024; 4: 100588.
22. Lopez MJ, Mohiuddin SS. Biochemistry, Essential Amino Acids. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 [cited 2026 May 17]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557845/
23. Pasini E, Corsetti G, Aquilani R, Romano C, Picca A, Calvani R, et al. Protein-amino acid metabolism disarrangements: The hidden enemy of chronic age-related conditions. Nutrients 2018;10: 391.
24. Prativi MBN, Astuti DI, Putri SP, Laviña WA, Fukusaki E, Aditiawati P. Metabolite Changes in Indonesian Tempe Production from Raw Soybeans to Over-Fermented Tempe. Metabolites 2023; 13: 300.
25. Witono Y, Widjanarko SB, Mujianto, Rachmawati DT. Amino acids identification of over fermented tempeh, the hydrolysate and the seasoning product hydrolysed by calotropin from crown flower (Calotropis gigantea). Int J Adv Sci Eng Inf Technol 2015; 5: 103.
26. Nkhata SG, Ayua E, Kamau EH, Shingiro JB. Fermentation and germination improve nutritional value of cereals and legumes through activation of endogenous enzymes. Food Sci Nutr 2018; 6: 2446-2458.
27. Kadar AD, Astawan M, Putri SP, Fukusaki E. Metabolomics based study of the effect of raw materials to the end product of tempe—an indonesian fermented soybean. Metabolites 2020; 10: 367.
28. Hartanti AT, Rahayu G, Hidayat I. Rhizopus Species from Fresh Tempeh Collected from Several Regions in Indonesia. HAYATI J Biosci 2015; 22: 136-142.
29. Sjamsuridzal W, Khasanah M, Febriani R, Vebliza Y, Oetari A, Santoso I, et al. The Effect of The use of Commercial Tempeh Starter on The Diversity of Rhizopus Tempeh in Indonesia. Sci Rep [Internet] 2021; 11: 23932.
30. Beye M, Fahsi N, Raoult D, Fournier PE. Careful use of 16S rRNA gene sequence similarity values for the identification of Mycobacterium species. New Microbes New Infect 2018; 22: 24-29.
31. Aggarwal NK, Goyal V, Saini A, Yadav A, Gupta R. Enzymatic saccharification of pretreated rice straw by cellulases from Aspergillus niger BK01. 3 Biotech 2017; 7: 158.
32. Pandey AK, Negi S. Enhanced cellulase recovery in SSF from Rhizopus oryzae SN5 and immobilization for multi-batch saccharification of carboxymethylcellulose. Biocatal Agric Biotechnol 2020; 26: 101656.
33. Khadka S, Khadka D, Poudel RC, Bhandari M, Baidya P, Sijapati J, et al. Production optimization and biochemical characterization of cellulase from Geobacillus sp. KP43 isolated from hot spring water of Nepal. Biomed Res Int 2022; 2022: 6840409.
34. Ye M, Sun L, Yang R, Wang Z, Qi K. The optimization of fermentation conditions for producing cellulase of bacillus amyloliquefaciens and its application to goose feed. R Soc Open Sci 2017; 4: 171012.
35. Prasad P, Singh T, Bedi S. Characterization of the cellulolytic enzyme produced by Streptomyces griseorubens (Accession No. AB184139) isolated from Indian soil. J King Saud Univ Sci 2013; 25: 245-250.
36. Kupski L, Pagnussatt FA, Buffon JG, Furlong EB. Endoglucanase and total cellulase from newly isolated Rhizopus oryzae and Trichoderma reesei: Production, characterization, and thermal stability. Appl Biochem Biotechnol 2014; 172: 458-468.
37. Fernández-López MG, Batista-García RA, Aréchiga-Carvajal ET. Alkaliphilic/alkali tolerant fungi: molecular , biochemical , and biotechnological aspects. J Fungi (Basel) 2023; 9: 652.
38. Mustafa HK, Anwer SS, Zrary TJ. Influence of pH, agitation speed, and temperature on growth of fungi isolated from Koya, Iraq. Kuwait J Sci 2023; 50: 657-664.
39. Michlmayr H, Schümann C, Barreira Braz Da Silva NM, Kulbe KD, Del Hierro AM. Isolation and basic characterization of a β-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture. J Appl Microbiol 2010; 108: 550-559.
40. Sparringa RA, Kendall M, Westby A, Owens JD. Effects of temperature, pH, water activity and CO2 concentration on growth of Rhizopus oligosporus NRRL 2710. J Appl Microbiol 2002; 92: 329-337.
41. Decker CH, Visser J, Schreier P. β-Glucosidases from five black Aspergillus species: Study of their physico-chemical and biocatalytic properties. J Agric Food Chem 2000; 48: 4929-4936.
| Files | ||
| Issue | Vol 18 No 3 (2026) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v18i3.21671 | |
| Keywords | ||
| Cellulase β-glucosidase Rhizopus microsporus Tempeh | ||
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How to Cite
1.
Karlina Y, Rizaldy D, Riani C, Faramayuda F, Sukrasno S. Cellulase and β-glucosidase activity of crude enzyme extracted from fresh and expired soybean tempeh. Iran J Microbiol. 2026;18(3):439-447.
