Coagulante de Aspergillus para elaboração de queijo com biomassa de macrofungo

Abstract

Milk-clotting proteases are important enzymes used in the dairy industry. Due to the increasing demand for cheese production, the increase in the price of animal rennet and cultural issues, the search for alternative coagulating sources, among them microbial, is necessary. The objective of this work was to investigate the production of coagulant proteases by Aspergillus flavo furcatis DPUA 1608 by the fermentation technology for the elaboration of cheese with macrofungo biomass. The culture was reactivated, authenticated and evaluated for the production of aflatoxins in solid medium. The inoculum medium was selected using SAB+GLI, SAB+SAC, BDA+GLI and BDA+SAC. The liquid fermentation medium was selected from a base saline solution (MA01). In the other media, 1% (w/v) glucose (MAGLi) and 1% (w/v) sucrose (MASac), respectively, were added. At the end of the fermentation, the biomass was separated from the crude extract by vacuum filtration. The medium and inoculum in which the best milk coagulant activity was determined were used to optimize the production of coagulant enzymes: carbon and nitrogen sources, inoculum age and size, fermentation time, temperature, agitation and initial pH of the medium. The enzymes of the selected crude extract were characterized as to pH and temperature (optimum activity and stability) and effect of inhibitors. In vitro cytotoxicity testing was also performed with the test on human fibroblasts and commercial sheep blood. At the end of the characterization, a cheese supplemented with mushroom biomass (Pleurotus ostreatoroseus) was elaborated and the physical-chemical and microbiological analyzes of the product were determined. The culture medium of the inoculum and fermentation medium that promoted higher milk coagulant activities were BDA+SAC and MA01, respectively. The milk coagulant enzymes were best produced in submerged fermentation medium according to the following parameters: age (5 days) and inoculum size (106 spores per ml medium), initial pH of the medium (pH 7), fermentation (3 days), fermentation temperature (30°C), stirring (150 rpm), carbon source (0.1% (w/v) starch and nitrogen (peptone 0.1% (w/v)). According to the proteolytic activity, the enzymes presented optimum activity at pH 7 at 50 °C. They were stable up to 40 °C and pH 5 to 9. The Zn2+ and Cu2+ ions promoted the greatest reduction of activity and iodoacetic acid was the inhibitor of higher interference. For the milk coagulant activity, the enzymes were more active at pH 7 and temperature of 45 °C. They remained stable from pH 5 to 9 and temperature from 25 to 55 °C. Zn 2+ and K + promoted increased activity and again iodoacetic acid was the inhibitor of greater interference, characterizing the enzymes as cysteine protease. There was no detection of toxicity according to the tests. The microbiological analisys is in agreement with the vigent legislation. The cheeses presented increasing in the amount of carbohidrates, protein, ashes and humidity according to the increasing of mushroom concentration. However, was also observed that the increasing in mushrrom concentration promoted the decreasing in the amount of lipids and also the total energy Aspergillus flavo furcatis DPUA 1608 has potential as microbial source of milkclotting enzymes and can be used in dairy industry to cheese manufacturing.