Extração e purificação de proteínas estruturadoras de gelo obtidas de folhas de trigo

• Autor(a) principal: Campelo, Flávia Assunção
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2946
• topic: Trigo - Folhas; Gelo - Extração; Gelo - Purificação; Gelo - Estrutura - Proteína; Wheat - Leaves; Ice - Extraction; Ice - Purification; Ice - Structure - Protein; CNPQ::CIENCIAS AGRARIAS::CIENCIA E TECNOLOGIA DE ALIMENTOS::CIENCIA DE ALIMENTOS

When certain organisms are subjected to low temperatures, they tend to develop survival mechanisms, such as the production of specific proteins, known as ice structuring proteins (ISP). These proteins belong to a class of polypeptides capable of inhibiting the growth of ice crystals during the nucleation, thereby reducing the damage to the cell structure of these organisms caused by the formation of crystals. In processed food subjected to freezing, the control of ice crystals growth is critical in preventing damage to the final product quality, and, therefore, it is an area of interest to industry researchers. In spite of ISP having already been extracted from wheat and other cereals, a methodology for appropriate and efficient extraction and purification has not been defined, which is the purpose of this study. Winter cultivars (Ok Bullet and Endurance) and spring (Pioneer) were sown in plastic pots. Three replications of each cultivar were grown in controlled conditions, in a growth chamber with cooling system, with temperatures between 4 - 7 ° (cold acclimated plants). Another three replicates C were kept under optimal growth conditions, at temperatures of 23 25 ° One pot per C. replicate was withdrawn from the chamber every seven days, and their leaves were collected for study. This process was repeated over a period of 49 days of growth, with and without acclimatization. The lyophilized leaves of each cultivar were divided into two groups and each passed through an extraction process, E1 or E2. In the E1 group, leaves were cut, ground with Tris -HCl (pH=7.4), centrifuged, and filtered to obtain a crude extract containing the proteins. E2 leaves were ground with ascorbic acid: calcium chloride (pH=3.0) and passed through an impregnation process in a vacuum for the extraction of proteins. The extracts obtained by E1 and E2 were subjected to three purification processes, P1, P2 and P3. In the P1 methodology, samples were treated with acetone, P2 with ethyl ether: petroleum ether (1:1), and in the P3 methodology chloroform was used. In the P1 methodology, extracts were stirred in vortex, centrifuged and the precipitate resuspended for obtaining purified extract. In P2 they were stirred in vortex and decanted. In P3 the extracts were stirred in vortex and centrifuged. The crude extracts were purified and analyzed by electrophoresis in SDS - PAGE. The solids content ranged from 1446.00 to 1543.00 mg/100g for E1, and for E2 the values ranged from 850.00 to 980.00 mg/100g. In the E1 group, the Pioneiro cultivar showed protein content of 201,84 mg/100 g, Ok Bullet and Endurance 217.71 and 270.49 mg/100g respectively. In the E2 group these values were 118,45, 357.96 and 395.33 mg/100g. The extracts obtained from the Pioneiro cultivar for E1 and E2 showed ash content of 2.0% and 1.98% respectively, Ok Bullet (2.11% and 2.07 %) and Endurance (2.13 % and 2.10 %). In extracts of Pioneiro wheat acclimated to cold, it was obtained by extraction methodology E1, one protein in the range from 45.0 to 66.0 kDa, and another in the range 30.0 to 45.0 kDa. For Ok Bullet and Endurance, as well as the above conditions, a third band of 20.1 30.0 kDa was observed, as well as two others, one in the range 66.0 97.0 kDa and the other in the range 30.0 45.0 kDa. For the E2 extraction methodology, only one protein range was observed from 20.1 to 30.0 kDa for the three cultivars. In gels of both extractions (E1 and E2), the presence of ISP in range from 20.1 to 30.0 kDa was observed. Compared to the control extracts from E1 and E2, the P1 purification methodology produced a significant effect only for the Pioneiro cultivar, resulting in a lower content of crude protein after purification. The P3 procedure produced similar effect in the Ok Bullet and Endurance cultivars, compared to both controls. The P2 purification method reduced the protein content for all varieties in the E1 and E2 extracts, so it is not recommended for any variety. After the process of purification for Pioneiro cultivar, a protein was identified in the range of 20.1 to 30.0 kDa and another in the range of 45.0 to 66.0 kDa. For the Ok Bullet and Endurance cultivars, in addition to the two found in the Pioneiro, the presence of two more proteins were identified, one in the range of 66.0 to 97.0 kDa and the other 45.0 to 66.0 kDa, the same ranges that were observed in the extracts electrophoretic profiles obtained from the E1 group. For the gels of the purified extracts, the same proteins observed in the crude extracts of the E2 group, with molecular mass from 20.1 to 30.0 kDa were observed. The E1 extraction methodology was the most efficient for the Pioneiro cultivar while E2 was best for the Ok Bullet and Endurance cultivars. Purification with chloroform (P3) was more effective for the Pioneiro cultivar while acetone (P1) was better for the OK Bullet and Endurance cultivars. This study indicates that research results undertaken abroad are not necessarily suited to wheat cultivars in Brazil. Brazilian wheat cultivars are essentially spring-harvested, and require specific research, whereas they demonstrate to be potential sources for obtaining ISP with different characteristics from those produced by winter-harvested wheat.