Original article Milk-clotting enzymes produced by Aspergillus flavo furcatis strains on Amazonic fruit waste Mircella M. Alecrim, 1 Rosana A. Palheta, 2 Maria Francisca S. Teixeira 1 * & Ila Maria de A. Oliveira 3 1 Culture Collection DPUA, Federal University of Amazonas, Manaus 69077-000, Brazil 2 Federal Institute of Amazonas, East Zone Campus, Manaus 69083-000, Brazil 3 Pharmaceutical Science College, Federal University of Amazonas, Manaus 69010-300, Brazil (Received 24 April 2014; Accepted in revised form 2 September 2014) Summary Six strains of Aspergillus flavo furcatis were screened to investigate milk-clotting enzyme production by fermentation in natural liquid medium. The growth media comprised extracts of cupuac ßu exocarp+rice bran [10% or 20% (v/v) CE+RB] and ac ßai waste+rice bran [10% or 20% (v/v) AW+RB] with or without supplementation of 0.1% (w/v) yeast extract and 0.5% (w/v) gelatin. Significant values of milk-clotting activity were determined by A. flavo furcatis DPUA 1461 and DPUA 1608, in the standard and natural media, respectively. According to criteria of clot and whey formations, 8.3% of the samples tested were classified as strong coagulation, 41.70% showed weak coagulation and in 50% was not observed milk coagulation. The enzyme optimal action of DPUA 1608, the selected strain, was at 40 °C and pH 7.0. Milk-clotting proteases were inhibited by pepstatin (94.72%) and moderately inhibited by the others metal ions tested. Keywords Aspergillus flavo furcatis, milk-clotting enzymes, submerged fermentation, waste. Introduction The use of cheese as ingredient in prepared foods for imparting taste, texture and nutritional qualities are valorised. However, there are currently ethical con- cerns that refer negatively the consumption of animal rennet. Also, the shortage of rennet has increased the interest in milk-clotting enzymes to replace that extracted from the stomach of calves. Many studies show that enzymes from plants and micro-organisms are already used as coagulants (Egito et al., 2007; Ah- med et al., 2009; Hashim et al., 2011; Grozdanovic et al., 2013; Tita et al., 2013). Coagulant proteases are synthesised by plants, ani- mals and micro-organisms. However, microbial sources are preferred due to their wide diversity bio- chemical and susceptibility to genetic manipulation. In addition, micro-organisms have a better acceptance by people whose eating habits and religious beliefs are against the use of enzymes from animal origin (Sand- hya et al., 2005; Merheb-Dini et al., 2010). Among microbial sources, enzymes produced by fungi have many advantages because they are normally GRAS (Generally Regarded as Safe) and their extra- cellular enzymes are easily recovered in fermentation process (Sandhya et al., 2005). Milk-clotting enzymes from Rhizomucor miehei, Rhizomucor pusilus, Aspergil- lus oryzae and Endothia parasitica are already used commercially (Shieh et al., 2009). Aspergillus flavo furcatis strains are emerging sources of enzymes to industrial application (Teixeira et al., 2012). According to the morphological characteristics, on Czapek’s solution agar, its colonies spreading rap- idly, attaining a diameter of 6.0–7.0 cm in 10–12 days at room temperature (24–26 °C), dark olive-buff through brownish olive when young, becoming sepia to mummy brown (Raper & Fennell, 1977). Submerged fermentation (SmF) can be used as a method to produce milk-clotting proteases from micro-organisms. In this process, different types of substrates are used including agro-industrial waste with supplementation of carbon and nitrogen sources that can promote diverse proteolytic activities. More- over, in submerged fermentation, the conditions are monitored with greater accuracy when compared to solid state fermentation (Sumantha et al., 2006; Bon et al., 2008; Singhania et al., 2010). The aim of this study was to investigate the produc- tion of milk-clotting proteases by Aspergillus flavo fur- catis strains using extracts of cupuac ßu exocarp *Correspondent: E-mail: mteixeira@ufam.edu.br International Journal of Food Science and Technology 2014 doi:10.1111/ijfs.12677 © 2014 Institute of Food Science and Technology 1