Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-18T11:49:54Z No. of bitstreams: 1 2014 - Ramon de Sousa Rego.pdf: 1491511 bytes, checksum: 746021e21f53b13713de3e8e94d03121 (MD5) / Made available in DSpace on 2016-10-18T11:49:54Z (GMT). No. of bitstreams: 1 2014 - Ramon de Sousa Rego.pdf: 1491511 bytes, checksum: 746021e21f53b13713de3e8e94d03121 (MD5) Previous issue date: 2014-08-26 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / The protein quality of milk of goats and digestibility of the lipid fraction are important factors that stand out when compared to milk from cows. The ?-lactoglobulin is the higher abundant protein in whey ruminants being produced in the mammary gland during lactation. It may represent up to 12% of the total protein. We seek to present work was to evaluate the gene ?-Lactoglobulin (BLG) with their genetic polymorphisms in the promoter regions 5 'and 3' UTR and associate them the characteristics of milk production in experimental goat herd. For this 150 goats (Capra hircus) of Saanen and Alpine, genotyped for polymorphisms of the BLG gene were used. For genotyping 10 mL of blood per animal were collected. The blood was used for DNA extraction and amplification of two fragments of the BLG gene by means of polymerase chain reaction (PCR). The amplified fragments were submitted to electrophoresis on polyacrylamide gel and evaluated by by restriction fragment length polymorphism (PCR-RFLP). The SmaI and SacII enzymes were used for the promoter (promoter + exon 1 region) region and the region of exon 7 (exon 7 + region 3 '), respectively. The differences in cutting patterns were visualized on a polyacrylamide gel. In the promoter region of the single nucleotide polymorphism (SNP) at position -60 (C/T) was identified and was associated with the percentage of protein in milk. This result suggests a relationship between genotypes of the promoter region of the BLG gene and the protein level of the milk. The presence of two polymorphisms +4641 (I2/I3) and +4601 (A/G) was observed in the region of exon 7. The I2-I3 variation is characterized by a repeat sequence of 10 bp, varying in two and three times, and it being possible to identify by electrophoresis. The I3 variation was often very low, and there was no association between this polymorphism and any production trait. Polymorphism +4601 (G/A) was identified by enzyme SacII digestion. Alleles were identified frequencies close to those reported by other authors and were associated with the percentage of fat in milk goats, and the animals with the S1S2 genotype had higher fat percentage than S1S1 animals and S2S2. The increased production of lipid content may be related to the characteristic of transporting fatty acids ?-lactoglob / A qualidade proteica do leite de cabras e a digestibilidade da fra??o lip?dica s?o importantes fatores que o destacam, quando comparado ao leite de vacas. A ?-Lactoglobulina ? a prote?na de maior abundancia no soro do leite em ruminantes sendo produzida na gl?ndula mam?ria durante o per?odo de lacta??o. Ela pode representar at? 12% do total proteico. Buscamos com o presente trabalho, avaliar o gene da ?-Lactoglobulina (BLG) com seus polimorfismos gen?ticos nas regi?es promotoras 5? e 3? UTR e associ?-los as caracter?sticas de produ??o de leite no rebanho caprino experimental. Para isso foram utilizadas 150 cabras (Capra hircus) das ra?as Saanen e Alpinas, genotipadas para os polimorfismos do gene BLG. Para a genotipagem foram coletados 10 mL de sangue por animal. O sangue foi utilizado para a extra??o de DNA e amplifica??o de 2 fragmentos do gene BLG por interm?dio da rea??o em cadeia da polimerase (PCR). Os fragmentos amplificados foram ent?o submetidos ? eletroforese em gel de poliacrilamida e avaliados pelo polimorfismo no tamanho do fragmento por restri??o (PCR-RFLP). Foram utilizadas as enzimas SmaI e SacII, para a regi?o promotora (regi?o promotora + ?xon 1) e para a regi?o do ?xon 7 (?xon 7 + regi?o 3?), respectivamente. As diferen?as nos padr?es de corte foram visualizadas em gel de poliacrilamida. Na regi?o promotora o polimorfismo de base ?nica (SNP) na posi??o -60 (C/T) foi identificado e apresentou associa??o com a percentagem de prote?nas no leite. Esse resultado sugere uma rela??o entre os gen?tipos da regi?o promotora do gene BLG e o n?vel proteico do leite. Foi observado na regi?o do ?xon 7 a presen?a de dois polimorfismos +4641 (I2/I3) e +4601 (A/G). A varia??o I2-I3 ? caracterizada pela repeti??o de uma sequ?ncia de 10 pb, variando em duas e tr?s vezes, e sendo poss?vel identifica-la por eletroforese. A varia??o I3 teve frequ?ncia muito baixa, e n?o houve associa??o entre esse polimorfismo e nenhuma caracter?stica de produ??o. O polimorfismo +4601 (A/G) foi identificado por meio da digest?o da enzima SacII. Os alelos identificados tiveram frequ?ncias pr?ximas ?s relatadas por outros autores e apresentaram associa??o com a percentagem de gordura no leite cabras, sendo que os animais com o gen?tipo S1S2 apresentaram maior percentagem de gordura que animais S1S1 e S2S2. A maior produ??o de conte?do lip?dico pode estar relacionada com a caracter?stica de transporte de ?cidos graxos da ?-Lactoglobulina

Made available in DSpace on 2014-12-17T15:01:49Z (GMT). No. of bitstreams: 1 JorgeSC_TESE.pdf: 1730447 bytes, checksum: 39fc94239eeb6e994140c5c0f9a2cc23 (MD5) Previous issue date: 2010-06-22 / Expanded Bed Adsorption plays an important role in the downstream processing mainly for reducing costs as well as steps besides could handling cells homogenates or fermentation broth. In this work Expanded Bed Adsorption was used to recover and purify whey proteins from coalho cheese manufacture using Streamline DEAE and Streamline SP both ionic resins as well as a hydrophobic resin Streamline Phenyl. A column of 2.6 cm inner diameter with 30 cm in height was coupled to a peristaltic pump. Hydrodynamics study was carried out with the three resins using Tris-HCl buffer in concentration of 30, 50 and 70 mM, with pH ranging from 7.0 to 8.0. In this case, assays of the expansion degree as well as Residence Time Distribution (RTD) were carried out. For the recovery and purification steps, a whey sample of 200 mL, was submitted to a column with 25mL of resin previously equilibrated with Tris/HCl (50 mM, pH 7.0) using a expanded bed. After washing, elution was carried out according the technique used. For ionic adsorption elution was carried out using 100 mL of Tris/HCl (50 mM, pH 7.0 in 1M NaCl). For Hydrophobyc interaction elution was carried out using Tris/HCl (50 mM, pH 7.0). Adsorption runs were carried out using the three resins as well as theirs combination. Results showed that for hydrodynamics studies a linear fit was observed for the three resins with a correlation coefficient (R2) about 0.9. In this case, Streamline Phenyl showed highest expansion degree reaching an expansion degree (H0/H) of 2.2. Bed porosity was of 0.7 when both resins Streamline DEAE and Streamline SP were used with StremLine Phenyl showing the highest bed porosity about 0.75. The number of theorical plates were 109, 41.5 and 17.8 and the axial dipersion coefficient (Daxial) were 0.5, 1.4 and 3.7 x 10-6 m2/s, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. Whey proteins were adsorved fastly for the three resins with equilibrium reached in 10 minutes. Breakthrough curves showed that most of proteins stays in flowthrough as well as washing steps with 84, 77 and 96%, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. It was observed protein peaks during elution for the three resins used. According to these peaks were identified 6 protein bands that could probably be albumin (69 KDa), lactoferrin (76 KDa), lactoperoxidase (89 KDa), β-lactoglobulin (18,3 KDa) e α-lactoalbumin (14 KDa), as well as the dimer of beta-lactoglobulin. The combined system compound for the elution of Streamline DEAE applied to the Streamline SP showed the best purification of whey proteins, mainly of the α-lactoalbumina / A adsor??o em leito expandido vem se destacando como uma t?cnica promissora dentro do downstream processing por ser de f?cil manuseio, baixo custo, diminuir etapas de processamento e utilizar o material particulado no seu estado natural. Portanto, o presente trabalho teve como objetivo recuperar e purificar prote?nas presentes no soro de queijo tipo coalho, atrav?s da t?cnica de adsor??o em leito expandido, utilizando resinas de troca ani?nica Streamline DEAE e troca cati?nica Streamline SP e intera??o hidrof?bica Streamline Phenyl,. Foi utilizada uma coluna de 2,6 cm de di?metro interno por 30 cm de altura, acoplada a uma bomba perist?ltica. Para o estudo do sistema foram realizados testes de hidrodin?mica e corridas de adsor??o, com as tr?s resinas, na presen?a de tamp?es Tris-HCl nas concentra??es 30, 50 e 70 mM, com pHs ajustados usando HCl para 7,0; 7,5 e 8,0. Para os testes hidrodin?micos foram estudados a expans?o do leito e a Distribui??o do Tempo de Resid?ncia (DTR). Na etapa de recupera??o e purifica??o, uma amostra de solu??o de soro de 200 mL foi aplicada, a temperatura ambiente, a uma coluna contendo resina (25 mL) previamente equilibrada em tamp?o Tris/HCl (50 mM e pH 7,0), ap?s lavagem efetuou-se a elui??o de acordo com o tipo de t?cnica utilizada. Dessa forma, para adsor??o com troca i?nica a elui??o ocorria com adi??o do eluente 100 mL Tris/HCl (50 mM, pH 7,0 em NaCl 1M). No caso de intera??o hidrof?bica, o eluente consistia de Tris/HCl (50 mM e pH 7,0). Os ensaios de adsor??o foram realizados com as resinas Streamline DEAE, Streamline SP e Streamline Phenyl e suas combina??es. Os resultados mostraram que para as condi??es em que foram realizados os ensaios fluidodin?micos e para o tipo de coluna utilizada, houve uma tend?ncia a linearidade, o coeficiente de correla??o (R2) foi da ordem de 0,9 e que a resina Streamline Phenyl obteve um maior grau de expans?o que as outras resinas, chegando a uma rela??o H0/H de 2,2. A porosidade do leito usando as resinas DEAE e SP foi de 0,70 e da resina Phenyl foi um pouco maior, em torno de 0,75. O n?mero de pratos te?ricos foi 109, 41,5 e 17,8 e o coeficiente de dispers?o axial (Daxial) foi de 0,5, 1,4 e 3,7 x 10-6 m2/s, para as resinas Streamline DEAE, Streamline SP e Streamline Phenyl, respectivamente. As prote?nas do soro s?o adsorvidas nas tr?s resinas e a concentra??o de prote?na em solu??o diminui rapidamente nos primeiros instantes do processo de adsor??o, sendo o equil?brio alcan?ado nos primeiros 10 minutos. Ao se aplicar o soro bruto sem tratamento para as tr?s resinas at? a satura??o (ruptura), embora exista adsor??o das prote?nas para essas resinas, perde-se grande parte dessas prote?nas nas etapas de passante e lavagem. Essas perdas somam 84, 77 e 96%, para as resinas Streamline DEAE, Streamline SP e Strealine Phenyl, respectivamente. Entretanto, pode-se recuperar 16, 23 e 4%, respectivamente, para as tr?s resinas. As tr?s resinas estudadas apresentaram picos de prote?nas na elui??o. De acordo com esses picos, foram identificadas 6 bandas de prote?nas. Provavelmente essas prote?nas sejam: albumina (69 KDa), lactoferrina (76 KDa) e lactoperoxidase (89 KDa), β-lactoglobulina (18,3 KDa) e α-lactoalbumina (14 KDa), d?mero da β -lactoglobulina. Portanto, as resinas estudadas s?o compat?veis para serem utilizadas em leito expandido. O sistema formado pela elui??o da Streamline DEAE quando foi aplicada na resina Streamline SP, tende a uma melhor purifica??o das prote?nas do soro, principalmente da α-lactoalbumina