ISSN print edition: 0366-6352
ISSN electronic edition: 1336-9075
Registr. No.: MK SR 9/7

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Fungal strain selection for protease production by solid-state fermentation using agro-industrial waste as substrates

M. A. Lizardi-Jiménez, J. Ricardo-Díaz, T. A. Quiñones-Muñoz, F. Hernández-Rosas, and R. Hernández-Martínez

CONACYT, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México



Abstract: Screening of the fungal strains, Aspergillus niger, Trichoderma harzianum, and Rhizopus microsporus var. chinensis for protease production was realised by monitoring the presence of clear zones on skimmed milk agar plates. Additionally, screening of the radial growth on vanilla waste, cane bagasse, and pineapple crown plates was performed. Radial growth data of the fungal strains on skimmed milk agar and agro-industrial waste plates were adjusted to the Gompertz model. The results indicate that R. microsporus var. chinensis and pineapple crown were adequate for the production of milk-clotting enzymes by solid-state fermentation. Crude enzyme extract was produced via solid-state fermentation using two particle sizes [2–2.6 mm (SSF1) and a mixture of less than 1 mm with 2–2.6 mm, at a ratio of 1:1(SSF2)]. The highest milk-clotting and proteolytic activities were shown within 12 h and 48 h of fermentation for SSF1 and SSF2, respectively. The optimum milk-clotting activity presented by the crude enzyme extract was at pH 6.5, 40 °C, and 0.04 M CaCl2. The ratio of milk-clotting activity to proteolytic activity (9.7) presented by the crude enzyme extract indicates potential for use as a calf rennet substitute.

Keywords: Milk-clotting enzyme ; Protease ; Solid-state fermentation ; Pineapple waste ; Agro-industrial wastes ; Fungal strains 

Full paper is available at

DOI: 10.1007/s11696-019-00814-w


Chemical Papers 73 (10) 2603–2610 (2019)

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