Orientador : Yong Kun Park / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos e Agricola / Made available in DSpace on 2018-07-14T18:34:17Z (GMT). No. of bitstreams: 1 Ferreira_JoaoCarlosDias_M.pdf: 5016372 bytes, checksum: af920e6ebcf3027ffa745ac39b043f84 (MD5) Previous issue date: 1983 / Resumo: De urna amostra de quatro abacaxis '(Ananas comosus (L.) merril) cv Pérola, foram extraldos, por homogenização da polpa, sem adição de água, 2 800 ml de suco. Ao mesmo foi adicionado (NH4}2 504 a uma concentração de 40% de saturação. Após um perIodo de 16 horas de repouso a 40C a amostra foi centrifugada e o precipitado descartado. Ao sobrenadante foi adicionado (NH~}2 504 até 80% de saturação. Após 2lhoras de repouso a 4 C o precipitado foi separado por centrif~ gaçao e em seguida dialisado contra H20 durante 48 horas a 40C e contra tampao fosfato 0,05 M pH 6,0 durante outras 48 horas a 4 C. Após fracionamento com sulfato de amônio a peroxidase foi purificada em colunas de Dietilaminoetil celulose (DEAE-celulose) e carboximetil celulose (CM-celulose) sucessivarnen te. Na cromatografia em DEAE-celulose foram obtidas 5 frações distintas, eluidas com gradiente de NaCl. Em seguida cada uma das cinco frações foi submetida a cromatografia em c~ luna de CM-celulose. Na cromatografia da primeira fração ob tida da coluna de DEAE-celulose (em coluna de 'eM-celulose) foram obtidas duas frações denominadas fração V e fração VI. Na crornatografia em coluna de CM-celulose de cada uma das outras quatro frações obtidas na cromatografia em coluna de DEAE-celulose, foi obtida uma só fração em cada caso, sendo denominadas frações I, II, III e IV. As 6 frações contendo atividade de peroxidase foram dialisadas e liofilizadas para os estudos posteriores. Para a determinação do pH ótimo de atividade das vã rias isoenzimas da peroxidase do abacaxi foram utilizados os tampões acetato e fosfato. As frações I, II e III apresenta ram um pH ótimo de 5,2; a fração IV apresentou um ótimo de atividade a pH 5,4 e as frações V e VI apresentaram pH ótimo de 5,0. O estudo da estabilidade térmica foi conduzido submetendo-se as diversas frações a tratamentos a 75°, 80°, 85° e 90 C por tempos de 30",1',2' e 3'; sendo logo a seguir d~ terminadas as atividades residuais. Utilizando-se a equação de Arrhenius determinaram-se as energias de inativação que foram da ordem de 23 a 37 kcal/mol para as várias frações / Abstract: From a sample of four pineapples . {Ananas comosus (L. ) merril) Perola breed, 2 800 ml of juice were extracted, by homogenization of the pulp, without water addition. Anm:>nÍUm sulfate at a concentration of 40% of saturation was added to the jui ce. After a period of resting, the sample was centrifugated and the precipitate was discarded. Enough (NH4)2 504 was ad ded to he supernatant in order to reach a concentration of 80% of saturation. A new resting period was followed by cen trifugation. The resulting supernatant was discarded, and the precipitate was dialised. The purification of the peroxidase started afterwards, with the use of the Ionic cromatography technique. First of alI the dialised was applied to a previously prepared Die thyl-aminoethylcellulose (DEAE-celulose) oolumn and eluted from it with the use of increasing concentrations of NaCI. Five distinct fractions were obtained in this phase. Only the first fraction was shown not to link i tself to DEAE~e'el lulose and was therefore called "the unlinked fraction". The four other anionic fractions were identified by the numbers I through IV. Each one of the five fractions was dialised and added to a previously prepared carboxymethylcellulose(CM celulose) column and eluted from it with increasing concen trations of Na CI. When submitted to CM-cellulose c hroma to graphy the unlinked fraction generated two other fractions, (V and VI) one of them classificated as no-ionic because it didn't linked itself to the CM-cellulose too, and the last one classificated as cationic because it linked itself to CM-cellulose. Fractions I through IV, when submitted to an identical process generated each one a single final fraction. The anionic character of fractions I through IV was shown by the fact that none of their final fractions linked itself to CM-eellulose. The six fraetions were dialised and liofilised for storage in order to allow later experiments. The optimum pH determination for eaeh one of the seve ral isoenzimes of pineapple peroxidase were made using sodium aeetate bUffer solutions for the aeidie pH and phosphate buffer solutions for the basie pH The following values we re obtained: 5,2 for three of the anionie isoenzimes; 5,4 for the other and 5,0 for the ~wo other ones. The study of the thermal stability of the was eondueted submitig the several fraetions to at 75°, 80°, 85° and 900C for periods of 30", 1', 2' and 3'; followed by imediate determination of the remainding peroxi dase aetivities. With the utilization of the Arrhenius equ~ tion, the inativation energies were determined and were to range from 23 to 37 keal/mol for the several fraetions / Mestrado / Mestre em Tecnologia de Alimentos

An enzyme-linked immunosorbent assay (ELISA) for pantothenate has been developed. Antibodies induced in rabbits against bovine serum albumin-pantothenate conjugate were specifically purified by affinity chromatography. This process served to reduce the amount of endogenous pantothenate attached to the antibody, as well as to purify the antibody. The purified antibodies were covalently linked to alkaline phosphatase (Sigma type VII) with glutaraldehyde (0.05% aqueous solution). An immobilized pantothenate substrate was first obtained by attaching human serum albumin-pantothenate conjugate to the surface of polystyrene culture tubes by passive adsorption. The binding of the enzyme labelled antibody (E-AB} to this substrate is proportionately inhibited by free pantothenate as standards or as samples for analysis. The inhibition of E-AB immobilization was quantitated at 405 nm by the hydrolysis of p-nitrophenyl phosphate as indicated by the formation of p-nitrophenol. A standard curve was plotted on log logit paper, and was linear in the range of 2 through 1000 ng pantothenate. Initial experiments show that the ELISA will be useful in assessing pantothenate in deproteinized blood samples and in food extracts.

Thesis (M.S.)--University of Michigan, 1989.

Thesis (M.S.)--University of Michigan, 1983.

Thesis (M.S.)--University of Michigan, 1989.

Thesis (M.S.)--University of Michigan, 1983.

Thesis (M.S.)--University of Michigan, 1989.

Thesis (M.S.)--University of Michigan, 1989.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.