Produção de transglutaminase microbiana e aplicação em pão sem-glúten

Abstract

The transglutaminases (EC 2.3.2.13) are transferase enzymes capable of catalyzing reactions such as acyl transfer between γ-carboxamide groups of glutamine residues and amino groups. Microbial transglutaminases (MTGases) have a great importance in food industry, because they polymerize foods rich in protein, leading to physical changes in their structure and consequently increasing their value considerably. The aim of this work was to produce MTGase from selected microbial strain using agro-industrial residues by Submerged Fermentation (SF) and by Solid State Fermentation (SSF). Additionally, the crude enzyme was concentrated, characterized and used in gluten-free bread preparation. 116 strains were tested. The strain Streptosporangium roseum V02 was selected because it produced higher enzyme activities. Furthermore, the influence of growth medium components and process conditions, such as soluble starch, peptone, yeast extract, rice flour, corn steep liquor (CSL), pH and inoculum concentration by SF, and inoculum concentration, water/wheat bran, CSL, KH2PO4 and MgSO4 by SSF was evaluated. for the SF experiments, the proposed medium (pH 7.0), in g·L-1, consisted of 40.0 rice flour, 30.0 peptone, 2.0 K2HPO4, 2.0 KH2PO4 and 1.0 MgSO4, and inoculum concentration of 107 CFU/mL-1. The MTGase highest production was 0.2 U/mL on 4 days of fermentation by SF. For the SSF experiments, the proposed medium composition (pH 7.0) consisted of 80 mL/100 g water/wheat bran, 0.1 % KH2PO4, 0.1 % MgSO4 and micronutrients, and an inoculum concentration of 106 CFU·g-1. The highest enzyme production was 0.8 U/gms on 8 days of fermentation. The crude enzyme produced by SSF was lyophilized using sucrose (5% w/w) as a lyoprotectant. The optimum temperature and pH of the enzyme were 45 °C and 6.6, respectively. The enzyme was stable at 35 °C, although it has shown mild stability at 50 °C, temperature, which the enzyme had lost 50 % of its activity in a period of 30 min. At 60 and 70 °C, the enzyme lost its activity almost completely after a period of 30 and 10 min, respectively. The addition of MTGase in the gluten-free bread dough increased specific volume and hardness. It was observed that 0.2 U/100 g (MTGase S. roseum V02 activity on flour basis) increased the specific volume and hardness in 13 % and 62 %, respectively. Increasing the concentration of the enzyme MTGase S. roseum V02 to 2.0 U/100 g (flour basis), the specific volume and hardness increased 6 % and 70 %, respectively, when compared to the control sample. A commercial enzyme was used in a concentration of 0.2 U/100 g (flour basis) and it was not observed a significant increase on the specific volume and hardness of the dough, when compared to the control. On the other hand, a higher concentration resulted in a significant increase on the specific volume and hardness to 11 % and 46 %, respectively. The application of 0.2 U/100g MTGase S. roseum V02 in a flour basis showed better results comparing to the commercial enzyme in the gluten-free bread dough preparation