Optimization of Phytase Production from Rhodotorula mucilaginosa RG-PK20 Using Agricultural Waste

Seprianto(1) , C. C. Y. Utama(2) , V. Melani(3) , P. Handayani(4) , Sukarman(5)
(1) Department of Biotechnology, Faculty of Health Sciences, Universitas Esa Unggul,
(2) Department of Biotechnology, Faculty of Health Sciences, Universitas Esa Unggul,
(3) Department of Nutrition, Faculty of Health Sciences, Universitas Esa Unggul,
(4) Department of Public Health, Faculty of Health Sciences, Universitas Esa Unggul,
(5) National Research and Innovation Agency

Abstract

Some ingredients in poultry feed contain phytic acid, which prevents the absorption of nutrients. Microbial phytase enzymes can help with this problem. However, the phytase-producing gene of the novel yeast Rhodotorula mucilaginosa RG-PK20 has been constrained by the high cost of traditional substrates such as sodium phytate. The aim of this study is to evaluate the production of phytase from R. mucilaginosa RG-PK20 using a phytic acid source (substrate) from agricultural waste, with in vitro testing in poultry feed. The fermentation process was conducted utilizing a substrate-to-medium-to-yeast culture ratio of 1:1:1 v/v/w over a period of four days at various temperatures (25, 28, and 30 °C) and pH levels (3, 4, 5, and 6, with 7 as a control). Glucose and urea supplements were given when the optimal conditions were established by measuring the phytase content and activity. The molecular weight of the phytase was confirmed by SDS-PAGE analysis, and the ability of the enzyme to hydrolyze phytic acid was evaluated in vitro. Corn cobs generated the highest amount of phytase, with a concentration of 25.29 mg/mL and activity of 4.46 U/mL. The in vitro tests revealed an 81% reduction in phytic acid levels in poultry feed. These results demonstrate the potential of phytase derived from R. mucilaginosa RG-PK20 to reduce phytic acid in poultry feed ingredients.

Full text article

Generated from XML file

References

Adebo, J. A., Njobeh, P. B., Gbashi, S., Oyedeji, A. B., Ogundele, O. M., Oyeyinka, S. A., & Adebo, O. A. (2022). Fermentation of cereals and legumes: Impact on nutritional constituents and nutrient bioavailability. Fermentation, 8(2), p.63. https://doi.org/10.3390/fermentation8020063

Casey, A., &d Walsh, G. (2004). Identification and characterization of a phytase of potential commercial interest. Journal of biotechnology, 110(3), pp.313-322. https://doi.org/10.1016/j.jbiotec.2004.03.001

Chen, H., Li, D., Zhao, J., Xiao, K., & Wang, K. (2018). Effects of nitrogen addition on activities of soil nitrogen acquisition enzymes: A meta-analysis. Agriculture, Ecosystems & Environment, 252, 126-131. https://doi.org/10.1016/j.agee.2017.09.032

Chen, W., & Xu, D. (2023). Phytic acid and its interactions in food components, health benefits, and applications: A comprehensive review. Trends in Food Science & Technology, 141, 104201. https://doi.org/10.1016/j.tifs.2023.104201

Chen, T., Shi, Y., Peng, C., Tang, L., Chen, Y., Wang, T., Wang, Z., Wang, S., & Li, Z. (2022). Transcriptome analysis on key metabolic pathways in Rhodotorula mucilaginosa Under Pb(II) Stress. Applied and Environmental Microbiology, 88(7), 1–13. https://doi.org/10.1128/aem.02215-21

Chondrou, T., Adamidi, N., Lygouras, D., Hirota, S. A., Androutsos, O., & Svolos, V. (2024). Dietary phytic acid, dephytinization, and phytase supplementation alter trace element bioavailability—a narrative review of human interventions. Nutrients, 16(23), 0–14. https://doi.org/10.3390/nu16234069

Classen, H. L., Apajalahti, J., Svihus, B., & Choct, M. (2016). The role of the crop in poultry production. World’s Poultry Science Journal, 72(3), 459–472. https://doi.org/10.1017/S004393391600026X

Coban, H. B., & Demirci, A. (2014). Enhanced submerged Aspergillus ficuum phytase production by implementation of fed-batch fermentation. Bioprocess and Biosystems Engineering, 37(12), 2579–2586. https://doi.org/10.1007/s00449-014-1236-z

Da Cunha, M. M. L., Dos Santos, L. P. B., Dornelas-Ribeiro, M., Vermelho, A. B., & Rozental, S. (2009). Identification, antifungal susceptibility and scanning electron microscopy of a keratinolytic strain of Rhodotorula mucilaginosa: A primary causative agent of onychomycosis. FEMS Immunology and Medical Microbiology, 55(3), 396–403. https://doi.org/10.1111/j.1574-695X.2009.00534.x

Dailin, D. J., Hanapi, S. Z., Elsayed, E. A., Sukmawati, D., Azelee, N. I. W., Eyahmalay, J., Siwapiragam, V., & El Enshasy, H. (2019). Fungal phytases: biotechnological applications in food and feed industries. Recent Advancement in White Biotechnology Through Fungi: Volume 2: Perspective for Value-Added Products and Environments, pp.65-99. https://doi.org/10.1007/978-3-030-14846-1_2

Deak, N. A., & Johnson, L. A. (2007). Fate of phytic acid in producing soy protein ingredients. Journal of the American Oil Chemists’ Society, 84(4), 369–376. https://doi.org/10.1007/s11746-007-1050-8

El-Hack, M. E. A., Alagawany, M., Arif, M., Emam, M., Saeed, M., Arain, M. A., Siyal, F. A., Patra, A., Elnesr, S. S., & Khan, R. U. (2018). The uses of microbial phytase as a feed additive in poultry nutrition - A review. Annals of Animal Science, 18(3), 639–658. https://doi.org/10.2478/aoas-2018-0009

El-Ziney, M. G., Zaid, E. A. A., & El-Naggar, M. Y. (2018). Characterization of carotenogenic Rhodotorula strains isolated from delta region, Egypt and their potential for carotenoids production. Journal of Pure and Applied Microbiology, 12(2), 587–599. https://doi.org/10.22207/JPAM.12.2.17

Feizollahi, E., Mirmahdi, R. S., Zoghi, A., Zijlstra, R. T., Roopesh, M. S., & Vasanthan, T. (2021). Review of the beneficial and anti-nutritional qualities of phytic acid, and procedures for removing it from food products. Food Research International, 143, 110284. https://doi.org/10.1016/j.foodres.2021.110284

Fukuji, T. S., Ferreira, D. L., Soares, A. L., Prete, C. E. C., & Ida, E. I. (2008). Ácido Fítico De Híbridos De Milho E Alguns Produtos Industrializados. Acta Scientiarum - Agronomy, 30(1), 31–35. https://doi.org/10.4025/actasciagron.v30i1.1125

Gattlen, J., Zinn, M., Guimond, S., Körner, E., Amberg, C., & Mauclaire, L. (2011). Biofilm formation by the yeast Rhodotorula mucilaginosa: process, repeatability and cell attachment in a continuous biofilm reactor. Biofouling, 27(9), 979–991. https://doi.org/10.1080/08927014.2011.619657

Gocheva, Y., Engibarov, S., Lazarkevich, I., & Eneva, R. (2023). Phytases - types, sources, and factors affecting their activity. Acta Microbiologica Bulgarica, 39(3), 249–263. https://doi.org/10.59393/amb23390305

Gowthami, G. A., & Gunashree, B. S. (2023). Production of metabolites from mutant strains of Rhodotorula minuta upon varied temperature. Research Square, 1(1), 1–12. https://doi.org/10.21203/rs.3.rs-3379006/v1

Heydari-Majd, M., Milani, H., Monjazeb Marvdashti, L., & Kamali, M. (2024). Phytase-producing fungi from processed cheese: isolation, screening, and optimization of production parameters. Journal of Microbiota, 1(1), 1–8. https://doi.org/10.5812/jmb-148356

Hue, L. A., Nhan, N. T. T., & Chon, N. M. (2023). Isolation and identification of carotenoid-producing Rhodotorula sp. from soils collected in the coastal environment of Kien Giang and Tra Vinh provinces. Vietnam Journal of Science Technology and Engenering, 65(2), 71–78. https://doi.org/10.31276/VJSTE.65(2).71-78

Jain, J., Sapna, & Singh, B. (2016). Characteristics and biotechnological applications of bacterial phytases. Process Biochemistry, 51(2), 159–169. https://doi.org/10.1016/j.procbio.2015.12.004

Jain, J., & Singh, B. (2017). Phytase production and development of an ideal dephytinization process for amelioration of food nutrition using microbial phytases. Applied Biochemistry and Biotechnology, 181(4), 1485–1495. https://doi.org/10.1007/s12010-016-2297-z

Jlali, M., Hincelin, C., Francesch, M., Rougier, T., Cozannet, P., Ozbek, S., Ceccantini, M., Yavuz, B., Preynat, A., & Devillard, E. (2023). A novel bacterial 6-phytase improves productive performance, precaecal digestibility of phosphorus, and bone mineralization in laying hens fed a corn-soybean meal diet low in calcium and available phosphorus. Journal of Poultry Science, 60(2). https://doi.org/10.2141/jpsa.2023019

Joshi, S., & Satyanarayana, T. (2017). Characteristics and multifarious potential applications of HAP phytase of the unconventional yeast Pichia anomala BT - developments in fungal biology and applied mycology. In T. Satyanarayana, S. K. Deshmukh, & B. N. Johri (Eds.), Developments in Fungal Biology and Applied Mycology (pp. 265–278). Springer Singapore. https://doi.org/10.1007/978-981-10-4768-8_14

Joudaki, H., Aria, N., Moravej, R., Rezaei Yazdi, M., Emami-Karvani, Z., & Hamblin, M. R. (2023). Microbial phytases: properties and applications in the food industry. Current Microbiology, 80(12), 1–13. https://doi.org/10.1007/s00284-023-03471-1

Kanti, A., 2017. Potensi kapang, Aspergillus niger, Rhizopus oryzae dan Neurospora sitophila sebagai penghasil ezim fitase dan amilase pada substrate ampas tahu. Buletin Peternakan, 41(1). https://doi.org/10.21059/buletinpeternak.v41i1.13337

Khan, N., Zaman, R., & Elahi, M. (1991). Effect of heat treatments on the phytic acid content of maize products. Journal of the Science of Food and Agriculture, 54(1), 153–156. https://doi.org/10.1002/jsfa.2740540117

Koni, T. N. I., Paga, A., & Asrul. (2024). Calcium, phosphorus, and phytic acid of fermented rice bran. IOP Conference Series: Earth and Environmental Science, 1360(1). https://doi.org/10.1088/1755-1315/1360/1/012010

Kumar, A., Lal, M. K., Kar, S. S., Nayak, L., Ngangkham, U., Samantaray, S., & Sharma, S. G. (2017). Bioavailability of iron and zinc as affected by phytic acid content in rice grain. Journal of Food Biochemistry, 41(6), 1–9. https://doi.org/10.1111/jfbc.12413

Li, G. E., Wu, X. Q., Ye, J. R., Hou, L., Zhou, A. D., & Zhao, L. (2013). Isolation and identification of phytate-degrading rhizobacteria with activity of improving growth of poplar and masson pine. World Journal of Microbiology and Biotechnology, 29(11), 2181–2193. https://doi.org/10.1007/s11274-013-1384-3

Mohammadi-Kouchesfahani, M., Hamidi-Esfahani, Z., & Azizi, M. H. (2019). Isolation and identification of lactic acid bacteria with phytase activity from sourdough. Food Science and Nutrition, 7(11), 3700–3708. https://doi.org/10.1002/fsn3.1229

Muniroh, A., Suja’i, M., Wibowo, A., Saputra, K. H., Yunita, E., & Herwanto, S. (2021). Perubahan kandungan asam fitat dan asam amino esensial bahan-bahan organik pakan yang difermentasi ragi tempe. Jurnal Bioteknologi & Biosains Indonesia, 8(1), 42–56.

Naghavi, F. S., Hanachi, P., & Saboora, A. (2014). Effect of temperature, pH and salinity on carotenoid production in Rodotorula mucilaginosa. Research in Biotechnology, 5(4), 1–04. www.researchinbiotechnology.com

Naves, L. de P., Corrêa, A. D., Bertechini, A. G., Gomide, E. M., & dos Santos, C. D. (2012). Effect of ph and temperature on the activity of phytase products used in broiler nutrition. Revista Brasileira de Ciencia Avicola. Brazilian Journal of Poultry Science, 14(3), 181–185. https://doi.org/10.1590/S1516-635X2012000300004

Nissar, J., Ahad, T., Naik, H. R., & Hussain, S. Z. (2017). A review phytic acid: As antinutrient or nutraceutical. Journal of Pharmacognosy and Phytochemistry, 6(6), 1554–1560. https://www.phytojournal.com/archives/2017.v6.i6.2263/a-review-phytic-acid-as-antinutrient-or-nutraceutical

Ogunremi, O. R., Sanni, A. I., & Agrawal, R. (2015). Probiotic potentials of yeasts isolated from some cereal-based Nigerian traditional fermented food products. Journal of Applied Microbiology, 119(3), 797–808. https://doi.org/10.1111/jam.12875

Pable, A., Gujar, P., & Khire, J. M. (2014). Selection of phytase producing yeast strains for improved mineral mobilization and dephytinization of chickpea flour. Journal of Food Biochemistry, 38(1), 18–27. https://doi.org/10.1111/jfbc.12020

Philippi, H., Sommerfeld, V., Olukosi, O. A., Windisch, W., Monteiro, A., & Rodehutscord, M. (2023). Effect of dietary zinc source, zinc concentration, and exogenous phytase on intestinal phytate degradation products, bone mineralization, and zinc status of broiler chickens. Poultry Science, 102(12), 103160. https://doi.org/10.1016/j.psj.2023.103160

Pires, E. B. E., de Freitas, A. J., Souza, F. F. e., Salgado, R. L., Guimarães, V. M., Pereira, F. A., & Eller, M. R. (2019). Production of fungal phytases from agroindustrial byproducts for pig diets. Scientific Reports, 9(1), 1–9. https://doi.org/10.1038/s41598-019-45720-z

Pragya, Sharma, K. K., & Singh, B. (2023). Phytase from Aspergillus oryzae SBS50: Biocatalytic reduction of anti-nutritional factor and exhibiting vanadium-dependent haloperoxidase activity. Biocatalysis and Agricultural Biotechnology, 52, 102840. https://doi.org/10.1016/j.bcab.2023.102840

Pramitha, J. L., Joel, A. J., Srinivas, S., Sreeja, R., Hossain, F., & Ravikesavan, R. (2020). Enumerating the phytic acid content in maize germplasm and formulation of reference set to enhance the breeding for low phytic acid. Physiology and Molecular Biology of Plants, 26(2), 353–365. https://doi.org/10.1007/s12298-019-00725-w

Priyodip, P., & Balaji, S. (2024). Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126. International Microbiology, 27(1), 37–47. https://doi.org/10.1007/s10123-023-00390-w

Quan, C.-S., Tian, W.-J., Fan, S.-D., & Kikuchi, J.-I. (2004). Purification and properties of a low-molecular-weight phytase from Cladosporium sp. FP-1. Journal of Bioscience and Bioengineering, 97(4), 260–266. https://doi.org/10.1016/S1389-1723(04)70201-7

Qvirist, L., Carlsson, N.-G., & Andlid, T. (2015). Assessing phytase activity–methods, definitions and pitfalls. Journal of Biological Methods, 2(1), 1. https://doi.org/10.14440/jbm.2015.58

Riviere, A., Nothof, T., Greiner, R., Tranchimand, S., Noiret, N., Robert, F. and Mireaux, M., 2021. In vitro assessment of enzymatic phytate dephosphorylation during digestive process of different feeds and feed ingredients. Animal Feed Science and Technology, 281, 115096. https://doi.org/10.1016/j.anifeedsci.2021.115096

Sadh, P. K., Kumar, S., Chawla, P., & Duhan, J.S. (2018). Fermentation: a boon for production of bioactive compounds by processing of food industries wastes (by-products). Molecules, 23(10), p.2560. https://doi.org/10.3390/molecules23102560

Sapna, Singh, B., Singh, D., & Sharma, K. K. (2013). Microbial phytases in skirmishing and management of environmental phosphorus pollution BT - biotechnology for environmental management and resource recovery. In R. C. Kuhad & A. Singh (Eds.), Biotechnology for Environmental Management and Resource Recovery (pp. 239–260). Springer India. https://doi.org/10.1007/978-81-322-0876-1_13

Sasirekha, B., Bedashree, T., & Chamka, K. L. (2012). Optimization and partial purification of extracellular phytase from Pseudomonas aeruginosa p6. European Journal of Experimental Biology, 2 (1), 95–104. www.pelagiaresearchlibrary.com

Scarcella, A. S., Junior, R. B., Bastos, R. G., & Magri, M. M. R. (2017). Temperature, ph and carbon source, affect drastically indole acetic acid production of plant growth promoting yeasts. Brazilian Journal of Chemical Engineering, 34(2), 429–438. https://doi.org/10.1590/0104-6632.20170342s20150541

Seprianto, S., Wahyuni, F. D., Novianti, T., Turnip, O. N., & Saputra, I. K. (2023a). Isolation and identification of yeast from fermented raisins extract as probiotic candidates. AIP Conference Proceedings, 2634(020093), 1–7. https://doi.org/10.1063/5.0111410

Seprianto, Wulansari, W., Wahyuni, F. D., & Novianti, T. (2023b). Optimization of the annealing temperature specific primers for detection of phytase gene from Rhodotorula mucilaginosa RG-PK20. Indonesian Journal of Biotechnology and Biodiversity, 7(2), 72–81.

Singh, Kunze, G., & Satyanarayana, T. (2011). Developments in biochemical aspects and biotechnological applications of microbial phytases. Biotechnology and Molecular Biology Review, 63(3), 69–87. http://www.academicjournals.org/BMBR

Singh, V., Mehra, R., Bisht, S., Shekhar, M., & Kumar, A. (2018). Phytin: a nutritional inhibitor in food and feed - review of strategies and challenges to overcome the menace in maize. International Journal of Current Microbiology and Applied Sciences, 7(06), 3264–3279. https://doi.org/10.20546/ijcmas.2018.706.384

Sumengen, M., Dincer, S., & Kaya, A. (2013). Production and characterization of phytase from Lactobacillus plantarum. Food Biotechnology, 27(2), 105–118. https://doi.org/10.1080/08905436.2013.781507

Sun, J., Li, M., Tang, Z., Zhang, X., Chen, J. and Sun, Z., 2020. Effects of Rhodotorula mucilaginosa fermentation product on the laying performance, egg quality, jejunal mucosal morphology and intestinal microbiota of hens. Journal of applied microbiology, 128(1), pp.54-64. https://doi.org/10.1111/jam.14467

Tariq, M., Nawaz, M., Anjum, A. A., Sana, S., Hafeez, M. A., Nazir, J., Shahzad, W., & Najeeb, M. I. (2017). Production and characterization of phytase from indigenous Aspergillus niger isolates. Pakistan Journal of Agricultural Sciences, 54(04), 799–806. https://doi.org/10.21162/PAKJAS/17.5517

Teigiserova, D. A., Bourgine, J., & Thomsen, M. (2021). Closing the loop of cereal waste and residues with sustainable technologies: An overview of enzyme production via fungal solid-state fermentation. Sustainable Production and Consumption, 27, 845–857. https://doi.org/10.1016/j.spc.2021.02.010

Tian, D., Cheng, X., Wang, L., Hu, J., Zhou, N., Xia, J., Xu, M., Zhang, L., Gao, H., Ye, X., & Zhang, C. (2022). Remediation of lead-contaminated water by red yeast and different types of phosphate. Frontiers in Bioengineering and Biotechnology, 10(March), 1–8. https://doi.org/10.3389/fbioe.2022.775058

Vilanculos, S. L., Svanberg, U., & Andlid, T. (2024). Phytate degradation in composite wheat/cassava/sorghum bread: Effects of phytase-secreting yeasts and addition of yeast extracts. Food Science and Nutrition, 12(1), 216–226. https://doi.org/10.1002/fsn3.3754

Wei, X., Wang, X., Zhou, B., & Zhou, H. (2006). Effect of urea on activity and conformation of a glycoprotein. Tsinghua Science & Technology, 11(4), 400–407. https://doi.org/10.1016/S1007-0214(06)70208-5

Yu, P., Wang, X.-T., & Liu, J.-W. (2015). Purification and characterization of a novel cold-adapted phytase from Rhodotorula mucilaginosa strain JMUY14 isolated from Antarctic. Journal of Basic Microbiology, 55(8), 1029–1039. https://doi.org/10.1002/jobm.201400865

Zohari, M., Akhavan Sepahy, A., & Amini, K. (2021). Molecular cloning and anti-cancer activity of carotenoid pigments isolated from Micrococcus spp. and Rhodotorula spp. Journal of Sciences, Islamic Republic of Iran, 32(1), 29–37.

Authors

Seprianto
seprianto@esaunggul.ac.id (Primary Contact)
C. C. Y. Utama
V. Melani
P. Handayani
Sukarman
Seprianto, Utama, C. C. Y., Melani, V., Handayani, P., & Sukarman. (2025). Optimization of Phytase Production from Rhodotorula mucilaginosa RG-PK20 Using Agricultural Waste. Tropical Animal Science Journal, 48(4), 328-337. https://doi.org/10.5398/tasj.2025.48.4.328

Article Details

How to Cite

Seprianto, Utama, C. C. Y., Melani, V., Handayani, P., & Sukarman. (2025). Optimization of Phytase Production from Rhodotorula mucilaginosa RG-PK20 Using Agricultural Waste. Tropical Animal Science Journal, 48(4), 328-337. https://doi.org/10.5398/tasj.2025.48.4.328