High Pressure Homogenization Of Porcine Pepsin Protease: Effects On Enzyme Activity, Stability, Milk Coagulation Profile And Gel Development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G' value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.105Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [2012/13509-6, 2010/02540-1

Effect of High-Pressure Technologies on Enzymes Applied in Food Processing

High isostatic pressure (HIP) and high-pressure homogenization (HPH) are considered important physical technologies that able to induce changes on enzymes. HIP and HPH are emerging food processing technologies that involve the use of ultra high pressures (up to 1200 MPa for HIP and up to 400 MPa for HPH), where the first process is based on the principle that the maintenance of a product inside vessels at high pressures induces changes in the molecules conformation and, consequently, in the functionality of polysaccharides, proteins and enzymes. To the contrary, for HPH process, the high shear and sudden pressure drop are the responsible phenomena for the changes on the processed product. This chapter aims to evaluate comparatively the effects of HIP and HPH on the activity of enzymes currently applied in food industry and to identify the main structural changes induced by each process. The overall evaluation of the results shows that mild conditions of both processes were recently highlighted as able to improve the activity and the stability of several enzymes, whereas extreme process conditions (pressure, time and temperature) induce enzyme denaturation with consequent reduction of biological activity. Considering the complexity and diversity involved in the enzyme structure and its ability to react, it is not possible to determine specific conditions that each process is able to promote increase or reduction of enzyme activity, being necessary to evaluate HIP and HPH for each enzyme. Finally, in terms of molecular structure, the effect of HIP and HPH on enzymes can be explained by the alterations in the quaternary, tertiary and secondary structures of enzymes, which directly affects its active site configuration

Influence of high pressure homogenization on commercial protease from Rhizomucor miehei: Effects on proteolytic and milk-clotting activities

AbstractThis work studied the influence of high pressure homogenization (HPH) on a commercial fungal protease. The enzyme solutions (2 and 20 g/100 mL) were processed up to 190 MPa and the proteolytic activity (PA), milk-clotting activity (MCA) and the rheological behavior of the milk coagulation phase were evaluated. The effects of multi-pass (three cycles) HPH at 25 and 190 MPa was evaluated for enzyme processed at concentration of 2 g/100 mL. No differences in PA and MCA were observed for the samples of 2 g/100 mL of enzyme concentration processed by HPH. On the other hand, increase in PA (∼3%) and MCA (∼10%) were observed for the enzymes processed at 190 MPa at high concentration, which consequent faster clotting and higher consistency of the milk gel. The multi-pass increased PA (≤6%) but did not alter MCA nor improved the milk coagulation phase. The results highlight that the energy supplied from HPH to enzyme at low concentration is not enough to promote positive changes in the enzyme coagulant profile; however, the HPH of solution with high enzyme concentration showed a positive effect, indicating that the collisions between enzymes during the process was important to reach the observed changes

Avaliação da imuno-expressão das proteínas P53, E-caderina,Cox-2 e EGFR no prognóstico do carcinoma de vesícula biliar ressecado

BACKGROUND: Gallbladder carcinoma presents a dismal prognosis. Choice treatment is surgical resection that is associated a high levels of both morbidity and mortality. Best knowledgement of prognostic factors may result a better selection of patient either for surgical or multimodal treatment. AIM: To evaluate tecidual immunoexpression of P53, E-cadherin, Cox-2, and EGFR proteins and to correlate these findings with resected gallbladder adenocarcinoma survival. METHODS: Clinical, laboratorial, surgical, and anatomopathological reports of a series of gallbladder adenocarcinoma patients were collected by individualized questionary. Total sample was 42 patients. Median of age was 72 years (35-87). There were seven men and 35 women. Lesion distribuition in according TNM state was the following: T1 (n=2), T2 (n=5), T3 (n=31), T4 (n=4). Twenty-three patients underwent radical resection (R0), while 19 palliative surgery (R1-R2). A block of tissue microarray with neoplasic tissue of each patient was confected. It was performed evaluation of P53, E-Caderine, COX-2, and EGFR proteins imunoexpression. These findings were correlated with overall survival. RESULTS: Five-year survival was 28%. The median of global survival was eight months. Only immunoexpression of EGFR protein was considered independent variable at multivariated analysis. CONCLUSION: Final prognosis was influenced by over-expression of EGFR protein in tumoral tissue.RACIONAL: O carcinoma de vesícula biliar apresenta mau prognóstico. O tratamento de escolha é a ressecção cirúrgica que está associado à alta morbimortalidade. O melhor conhecimento de fatores prognósticos pode resultar em melhor seleção dos doente para o tratamento cirúrgico e multimodal. OBJETIVOS: Avaliar a imunoexpressão tecidual das proteínas P53, E-caderina, Cox-2 e EGFR e correlacionar com a sobrevida do adenocarcinoma de vesícula biliar ressecado. MÉTODO: Os dados clínicos, laboratoriais, cirúrgicos e anatomopatológicos de uma série de doentes operados por adenocarcinoma de vesicula biliar foram coletados. A casuística total foi de 42 doentes. A mediana de idade foi de 72 anos (35-87). Foram sete homens e 35 mulheres. A distribuição da lesão de acordo com TNM foi a seguinte: T1 (n=2), T2 (n=5), T3 (n=31), T4 (n=4). Vinte três doentes realizaram ressecção radical (R0) enquanto 19 operação paliativa (R1-R2). Um bloco de tissue microarray foi confeccionado com tecido neoplásico de cada doente. para avaliação da imunoexpressão das proteínas P53, E-Caderina, COX-2 e EGFR. Esses achados foram correlacionados com prognóstico final dos doentes. RESULTADOS: A sobrevida estimada em cinco anos foi de 28%. A mediana de sobrevida global foi de oito meses. Apenas a imunoexpressão da proteína EGFR foi considerada variável independente no prognóstico dos doentes. CONCLUSÃO: Pior prognóstico teve relação com a imunoexpressão aumentada da proteína EGFR no tecido tumoral.Hospital do Servidor Público Estadual de São Paulo Francisco Morato de OliveiraUniversidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Avaliação do estadiamento, prognóstico e mortalidade do câncer colorretal mediante marcadores tumorais: receptor erbB-2 e caderinas

PURPOSE: To evaluate the prognostic significance and correlation with staging and degree of cell differentiation of the tumoral expression of the proteins c-erbB-2 and E-cadherin, in patients with colorectal adenocarcinoma. METHODS: The study included 117 patients with an average age of 63.1 years and an average follow-up duration of 28.1 months. The disease-free interval, survival, incidence of recurrence and specific mortality were evaluated. c-erbB-2 anti-oncoprotein antibodies (Dako) were utilized via the streptavidin-biotin technique. Samples were considered to be positive for c-erbB-2 if 10% or more of the tumor cell membranes were stained.The anti-E-cadherin antibodies (Dako), evaluated this protein and is considered positive, if 50% or more of the cell membranes were stained. Statistical analysis was performed using Pearson's chi-squared test, Fisher's exact test, Kaplan-Meier's estimator, the log-rank test and Wilcoxon's test (Breslow version), setting the level of statistical significance at 5% (p<0.05). RESULTS: 52 of 108 patients studied for c-erbB-2 were positive (48,1%), 47 of 93 patients studied for E-cadherin were negative (50,5%). These data do not express any correlation with TNM (tumor, node and metastasis) staging and the degree of cell differentiation or with the tumor recurrence rate. The disease-free interval among patients who were positive for c-erbB-2 and negative for E-cadherin was 68.0 months and did not differ from those with c-erbB-2 negative and E-cadherin positive ( 55.0 months - p = 0.5510). The average survival among patients positive for c-erbB-2 and negative for E-cadherin was 75 months without statistical significance difference with the other group ( 61 months - p = 0.5256). Specific mortality occurred in 20.0% of the cases and did not correlate with the expression of c-erbB-2 (p=0,446), E-cadherin (p=0,883). CONCLUSION: The tumoral expression of c-erbB-2 and E-cadherin did not demonstrate a correlation with the staging and degree of cell differentiation, and it did not present prognostic value regarding disease recurrence, disease-free interval, survival and specific mortality among patients with colorectal adenocarcinoma.OBJETIVO: Avaliação da expressão tumoral das proteínas c-erbB-2 e E-caderina e sua relação com o prognóstico, estadiamento e grau de diferenciação celular, em doentes com câncer colo-retal . MÉTODOS: O estudo incluiu 117 doentes com média de idade de 63.1 anos e com acompanhamento médio de 28.1 meses. O intervalo livre de doença, sobrevida, índice de recidiva e mortalidade específica foram os parâmetros avaliados. Anticorpos anti-oncoproteína c-erbB-2 (Dako) foram utilizados pela técnica da estreptavidiva-biotina. Considerou-se como positiva a presença desta proteína quando mais de 10% das células tumorais estivessem coradas. A proteína E-caderina foi estudada pelo anticorpo anti-E-caderina (Dako), sendo computada como positiva a amostra que apresentasse 50% ou mais das células coradas. A análise estatística utilizou o teste do qui-quadrado de Pearson, o teste exato de Fischer, a curva de Kaplan-Meier, o teste de log-rank e o teste de Wilcoxon ( variante de Breslow),sendo estabelecido nível de significância de 5%( p<0,05). RESULTADOS: 52 de 108 doentes estudados para c-erbB-2 foram positivos (48,1%), 47 de 93 doentes estudados para E-caderina foram negativos (50,5%). Estes dados não mostraram relação com estadiamento TNM (tumor, nódulo e metástase), com o grau de diferenciação celular e índice de recidiva tumoral. O intervalo livre de doença para os doentes positivos para c-erbB-2 e negativos para E-caderina foi de 68.0 meses e não diferiu daqueles que foram negativos para c-erbB-2 e positivos para E-caderina ( 55.0 meses - p = 0.5510). A sobrevida média para os doentes positivos para c-erbB-2 e negativos para E-caderina foi 75 meses sem diferença estatisticamente significante com o outro grupo de comparação( 61 meses - p = 0.5256). A mortalidade específica foi de 20.0% dos casos e não se correlacionou com a expressão do c-erbB-2 (p=0,446) ou da E-caderina(p=0,883). CONCLUSÃO: A expressão das proteínas c-erbB-2 e E-caderina em doentes portadores de adenocarcinoma colo-retal não apresentou correlação com o estadiamento e grau de diferenciação celular. Não houve da mesma forma relação com o prognóstico, no que diz respeito ao índice de recidiva da doença, intervalo livre de doença, sobrevida e mortalidade específica.University Center Uni-FOAUNIFESP Division of Gastrointestinal SurgeryUNIFESP Pathology DepartmentUNIFESP, Division of Gastrointestinal SurgeryUNIFESP, Pathology DepartmentSciEL

Análise da correlação entre a expressão da p53 e do bcl-2 com o estadiamento e o prognóstico do adenocarcinoma colorretal

PURPOSE: To analyze the correlation between p53 and bcl-2 expression and colorectal adenocarcinoma staging and prognosis. METHODS: This was a retrospective series of 125 colorectal adenocarcinoma patients (67 women and 58 men; ages 30-87 years) who underwent surgery with curative intent. The mean follow-up was 28.5 months (range: 2-96 months). TNM staging, tumor recurrence, survival and cancer-related mortality were analyzed. Immunoreactivity was evaluated using DO7 (Dako) for p53 and K492 (Dako) for bcl-2. Tumors with accumulation of staining for cytoplasmic bcl-2 or nuclear p53 in more than 10% of cells were considered positive. Statistical analysis utilized Pearson chi-squared, log-rank and Wilcoxon tests, and Kaplan-Meier survival estimation (significance level: p<0.05). RESULTS: p53+ was found in 11.8% (14/118), bcl-2+ in 50% (58/116) and associated p53+/bcl-2+ in 6.4% (7/109) of the tumors. There was no significant correlation between expression of these biomarkers and TNM I, II, III and IV staging (p=0.385 for p53; p=0.461 for bcl-2). For tumor recurrence, p53+ was found in 9.5% (2/21), bcl-2+ in 50% (11/22), and associated p53+/bcl-2+ in 5.2% (1/19) of the tumors (p=0.714, p=1.000 and p=0.960, respectively). For survival analysis, p53+: 57 months (45.0-68.0), bcl-2+: 78 (37.0-89.0), and p53+/bcl-2+: 62 (56.0-68.0) (p=0.319). For cancer-related mortality, p53+: 8.3% (3/36), bcl-2+: 47.2% (17/36), and p53+/bcl-2+: 5.9% (2/36) of the patients (p=0.432, p=0.688 and p=0.907, respectively). CONCLUSION: No correlation was found between tumor expression of p53 and bcl-2 and the TNM staging, recurrence, survival and cancer-related mortality in colorectal adenocarcinoma.OBJETIVO: Analisar a correlação entre a expressão da p53 e do bcl-2 com o estadiamento e prognóstico do adenocarcinoma colorretal. MÉTODOS: Foi realizado o estudo de uma série retrospectiva de 125 doentes com adenocarcinoma colorretal (67 mulheres e 58 homens; 30 a 87 anos de idade), que se submeteram ao tratamento cirúrgico com intenção curativa. O tempo médio de seguimento foi de 28,5 meses (variação de 2 a 96 meses). O estadiamento TNM, a recidiva tumoral, a sobrevida e a mortalidade relacionada com o câncer foram analisados. A reação imunohistoquímica utilizada foi o DO& (Dako) para o p53 e o K492 (Dako) para o bcl-2. Tumores com intensidade de coloração citoplásmica para o bcl-2 e nuclear para o p53, acima de 10% de células foram considerados positivos. A análise estatística utilizada foi o teste qui-quadrado de Pearson, log-rank, Wilcoxon e estimativa de sobrevida de Kaplan-Meier (nível de significância : p<0,05). RESULTADOS: p53+ foi encontrado em 11.8% (14/118), bcl-2+ em 50% (58/116) e associados p53+/bcl-2+ em 6.4% (7/109) dos tumores. Não foi encontrado correlação significante entre a expressão tumoral destes marcadores e o estadiamento TNM I, II, III e IV (p=0.385 para a p53; p=0.461 para o bcl-2). Na recidiva tumoral, p53+ foi encontrado em 9.5% (2/21), bcl-2+ em 50% (11/22), e p53+/bcl-2+ associados em 5.2% (1/19) dos tumores (p=0.714, p=1.000 e p=0.960, respectivamente). Na análise de sobrevida, p53+: 57 meses (45.0-68.0), bcl-2+: 78 (37.0-89.0), e p53+/bcl-2+: 62 (56.0-68.0) (p=0.319). Para mortalidade relacionada com câncer, p53+: 8.3% (3/36), bcl-2+: 47.2% (17/36), e p53+/bcl-2+: 5.9% (2/36) dos pacientes (p=0.432, p=0.688 and p=0.907, respectivamente). CONCLUSÃO: Nenhuma correlação significante foi encontrada entre a expressão tumoral da p53 e do bcl-2 com o estadiamento TNM, recidiva, sobrevida e mortalidade relacionada com câncer.Centro Universitário de Volta RedondaUNIFESP-EPM Pathology DepartmentUNIFESP-EPM Surgical Gastroenterology DepartmentUNIFESP, EPM, Pathology DepartmentUNIFESP, EPM Surgical Gastroenterology DepartmentSciEL