RESUMO
In a conventional microorganism-mediated biological process for degradation of keratinous waste material the production of keratin-specific proteases (i.e., keratinases) and the hydrolysis of keratin-rich residual biomass both take place during the same stage of the bioprocess and, as a consequence, occur simultaneously under suboptimal conditions. In the present study the keratinolytic actinomycete Amycolatopsis keratiniphila D2 was successfully employed to biodegrade thermally pretreated porcine bristles at high solids loading (16% w/v) via a novel cultivation methodology. Indeed, the two-stage submerged fermentation process developed in this work enabled to efficiently recover, in a single unit operation, about 73% of the protein material contained in the keratinous biowaste structure, resulting in an overall accumulation of 89.3â¯g·L-1 protein-rich hydrolysate and a productivity of 427â¯mg crude soluble proteins per litre per hour. The obtained protein hydrolysate powder displayed a 2.2-fold increase in its in vitro pepsin digestibility (95%) with respect to the non-hydrolysed pretreated substrate (43%). In addition, the chromatogram obtained by size-exclusion chromatography analysis of the final product indicated that, among the identified fractions, those consisting of small peptides and free amino acids were the most abundantly present inside the analysed sample. Given these facts it is possible to conclude that the soluble proteins, peptides and free amino acids recovered through the newly designed two-stage bioextraction process could represent a viable alternative source of protein in animal feed formulation.
Assuntos
Queratinas , Hidrolisados de Proteína , Animais , Bactérias Aeróbias , Fermentação , Hidrólise , Peptídeo Hidrolases , SuínosRESUMO
Due to its sensitivity and speed of execution, detection of ATP by luciferin-luciferase reaction is a widely spread system to highlight cell viability. The paper describes the methodology followed to successfully run the assay in the presence of yeast cells of two strains of the yeast Saccharomyces cerevisiae, BY4741 and CEN.PK2-1C and emphasizes the importance of correctly determining the contact time between the lysing agent and the yeast cells. Once this was established, luciferin-luciferase reaction was exploited to determine the maximum specific rate of growth, as well as cell viability in a series of routine tests. The results obtained in this preliminary study highlighted that using luciferin-luciferase can imply an over-estimation of maximum specific growth rate with respect to that determined by optical density and/or viable count. On the contrary, the bioluminescence assay gave the possibility to highlight, if employed together with viable count, physiological changes occurring in yeast cells as response to stressful environmental conditions such as those deriving from exposure of yeast cells to high temperature or those depending on the operative conditions applied during fed-batch operations.