Study On Screening And Fermentation Of Fungal Rennet Produing Strain, Purification And Characerization Of The Rennet

Calf rennet was originally derived from the fourth stomach of calves that were not weaning. Since its discovery, it has been widely used in cheese manufacturing. However, due to the development of dairy industry and people's increasing demand for dairy products, slaughters of calves cannot meet the market need for calf rennet anymore. In its substitutes, people pay extensive attention to the production of fungal rennet owing to its advantages of low cost, free from geographic restrictions and convenient extraction with facility.In this research work, various ways of mutagenesis treatment, such as UV,3-alpha,12-alpha-dihydroxy-5-beta-cholan-24-oic aci sodium salt, APPBS, were carried out to study the effectiveness of increasing the productivity of fungal rennet producing strain Mucor meihei and Aspergillus niger. Finally, an effectively rennet-producing strain, A.niger L-2-12 was isolated by screening and second screening. Its producing enzyme activity was 3.39 and 4.28 times of that of the original M.miehei and A.nigre strains respectively.Furthermore, the ingredients of culture media and the cultivation conditions were optimized and the ability of this strain to produce fungal rennet was strengthened. The optimal culture medium has been settled as:9g wheat bran, 1g wheat flour, 0.1g skim milk powder were distributed in 250mL flasks and moistened with 15mL acidic mineral salt solution (g/L MgSO4·7H2O,0.07%; FeSO4·7H2O, 0.09%; 0.2N HCl; pH6.61). After the flasks were inoculated, cultivation was carried out for 98h in a incubator at 37℃. The maximal enzyme activity of fermented product reached 21000SU when strain L-2-12 was incubated under aforementioned conditions. And then, the influence of amino acids in medium were studied. Results show the proline, tryptophan were the most effective.After completion of fermentation, a modified three-stage countercurrent extraction procedure involving solid liquid contact and pressing was used to obtain a relatively high enzyme concentration in the product broth. The solvent used was tap water. The product broth was clarified in a high speed refrigerate, and the supernatant was then vacuum concentrated in a forced circulation evaporator. For preparation of enzyme powder, the crude enzyme was vacuum freeze-drying.The results of characterization showed that the optimal temperature range for milk-clotting was 45-50℃and the optimal pH range was 5.8-6.2, respectively. The enzyme activity was stable under 50℃. It remained activity if it was put at 25℃for 48 hour. The enzyme activity was slightly activity by Ca2+. And the optimal concentration was 0.1%(m/v).