Desenvolvimento de um modelo preditivo para identificação de perda de estabilidade e ocorrência de proteólise em leite UAT / Development of a predicative model for identification of loss of stability and proteolysis occurrence in UHT milk

Este trabalho se propôs a desenvolver um modelo preditivo para identificação de perda de estabilidade e de sedimentação em leite UAT por determinação da atividade enzimática de aminopeptidase no leite por espectrofotometria. Foram analisadas amostras de leite cru, pasteurizado e UAT após envase durante seis meses, na região Sul do Brasil. Acidez, crioscopia, gordura, extrato seco total, extrato seco desengordurado e densidade foram analisados nos leites cru e pasteurizado. Amostras de leite cru foram ainda submetidas à análise de contagem de psicrotróficos e à atividade de aminopeptidase, e amostras de leite UAT estocadas foram analisadas quanto ao grau de proteólise mediante análises sensoriais e atividade de aminopeptidase. Alterações sensoriais foram observadas em tempos de estocagem menores para amostras originadas de leite cru com contagem de psicrotróficos acima de 107 UFC mL-1. Não houve correlação entre a atividade de aminopeptidase e proteólise e também não foi observada correlação significativa entre os parâmetros físico-químicos e a ocorrência de proteólise no leite estocado. O modelo estudado não foi apto para predizer perda de estabilidade e ocorrência de proteólise no leite UAT.(AU)

The aim of this work was to develop a predictive model for identifying loss of stability and sedimentation in UHT milk by determining the enzymatic activity of aminopeptidasis in milk by spectrophotometry. Samples of raw milk, pasteurized and UHT after filling for 6 months in Southern Brazil were analyzed. Acidity, freezing point, fat, total solids, nonfat solids and density were analyzed in raw and pasteurized milk. Raw milk samples were also subjected to psychrotrophic count analysis and aminopeptidasis activity and UAT samples of stored milk were analyzed for degree of proteolysis through sensory analysis and aminopeptidasis activity. Sensory changes were observed in smaller storage time for samples of raw milk originated with psychrotrophic count above 107 CFU ml-1. There was no correlation between aminopeptidasis activity and proteolysis and there was also no significant correlation between physicochemical parameters and the occurrence of proteolysis in stored milk. The model was unable to predict loss of stability and occurrence of proteolysis in UHT milk.(AU)

Proteínas do soro lácteo de vacas da raça Jersey durante a lactação / Whey proteins from Jersey cows during lactation

Para avaliar as proteínas do soro lácteo durante a lactação, o soro obtido a partir de 48 amostras de leite coletadas de 12 vacas da raça Jersey antes da ordenha foi estudado. Os animais foram distribuídos em três grupos: terço inicial (30-120 dias de lactação), terço médio (121-210 dias de lactação) e terço final da lactação (mais de 211 dias de lactação). O proteinograma consistiu da concentração de proteína total do soro lácteo, determinado pelo método de biureto e da eletroforese em gel de poliacrilamida (SDS-PAGE). A diminuição gradual e significativa de algumas frações do soro de leite foi observada durante a lactação, albumina, lactoferrina, imunoglobulinas, β-lactoglobulina e α-lactoalbumina. Os valores de normalidade obtidos para as proteínas do soro do leite de vacas Jersey foram: proteína total do soro de leite 569,0-713,0mg/dL, lactoferrina 36,0-49,0mg/dL, albumina 24,0-34.0mg/dL, cadeia pesada de imunoglobulina 38,0-51,0 mg/dL; cadeia leve de imunoglobulina 59,0-95,0mg/dL, β-lactoglobulina 207,0-256,0mg/dL, α-lactoalbumina 117,0-157,0mg/dL, proteína com 226 KDa 5,80-12.0mg/dL, e proteína com 118 kDa 2,30-6.80mg/dL.

To evaluate the whey proteins during the lactation, whey obtained from 48 milk samples collected from 12 Jersey cows before milking were studied. Cows were distributed into three groups as follows: early (30-120 days), middle (121-210 days) and end of lactation (more than 211 days). The proteinogram consisted of total proteins concentration determined by biuret method and polyacrymalide gel electrophoresis (SDS-PAGE). A gradual and significant decrease of some fractions of the whey was observed during the lactation cycle albumin, lactoferrin, immunoglobulins, β-lactoglobulin and α -lactoalbumin. The normal ranges obtained for the whey proteins of Jersey cows were: whey total proteins 569.0 to 713.0mg/dL, lactoferrin 36.0 to 49.0mg/dL, albumin 24.0 to 34.0mg/dL, immunoglobulin's heavy chain 38.0 and 51.0mg/dL; immunoglobulin's light chain 59.0 to 95.0mg/dL, β-lactoglobulin 207.0 to 256.0mg/dL, α-lactoalbumin 117.0 to 157.0mg/dL; protein with 226 KDa 5.80 to 12.0mg/dL, and protein with 118 KDa 2.30 to 6.80mg/dL.

Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i) motile ii) rod shaped iii) non spore forming iv) catalase and amylase positive v) able to grow in presence of 10 percent NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7) exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98 percent sequence similarity, 1201 bp). The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 percent activity at 80 0C after 4 h and more than 70 percent activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 percent active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

Whey upgrading by enzyme biocatalysis

Whey is a co-product of processes for the production of cheese and casein that retains most of the lactose content in milk. World production of whey is estimated around 200 million tons per year with an increase rate of about 2 percent/per year. Milk production is seasonal, so surplus whey is unavoidable. Traditionally, whey producers have considered it as a nuisance and strategies of whey handling have been mostly oriented to their more convenient disposal. This vision has been steadily evolving because of the upgrading potential of whey major components (lactose and whey proteins), but also because of more stringent regulations of waste disposal. Only the big cheese manufacturing companies are in the position of implementing technologies for their recovery and upgrading, so there is a major challenge in incorporating medium and small size producers to a platform of whey utilization, conciliating industrial interest with environmental protection within the framework of sustainable development. Within this context, among the many technological options for whey upgrading, transformation of whey components by enzyme biocatalysis appears as prominent. In fact, enzymes are green catalysts that can perform a myriad of transformation reactions under mild conditions and with strict specificity, so reducing production costs and environmental burden. This review pretends to highlight the impact of biocatalysis within a platform of whey upgrading. Technological options are shortly reviewed and then an in-depth and critical appraisal of enzyme technologies for whey upgrading is presented, with a special focus on newly developed enzymatic processes of organic synthesis, where the added value is high, being then a powerful driving force for industrial implementation.

Obtenção de hidrolisados enzimáticos do concentrado proteico do soro de leite com baixo teor de fenilalanina / Preparation of whey protein concentrate hydrolysates with low phenylalanine content

The objective of this work was to obtain enzymatic hydrolysates of wheyprotein concentrate with low phenylalanine content. Seventeen enzymatic hydrolysates were prepared and the effects of some parameters, such as type of enzyme; enzyme: substrate ratio; substrate concentration and time of hydrolysis were evaluated. Activated carbon was used as adsorbent and showed to be efficient for removing phenylalanine, leading to values above 70%. The best results were otained using pancreatin in an enzyme: substrate ratio of 1:100, a substrate concentration of 10% after 5 hours of hydrolysis, obtaining 81.3% of removal and a final phenylalanine content of 394.1 mg/100g of hydrolysate.

El objetivo de la investigación fue la obtención de hidrolizados enzimáticos con bajo contenido de fenilalanina, a partir de concentrado proteico de suero de leche. Se prepararon 17 hidrolizados enzimáticos y se evaluó el efecto de algunos parámetros como: tipo de enzima, relación enzima: sustrato, concentración de la materia prima y tiempo de hidrólisis. Carbón activado fue utilizado como medio adsorbente, y se mostró eficaz en la remoción de fenilalanina, se obteniendo porcentuales sobre 70%. El mejor resultado fué encontrado usando pancreatina en una relación enzima: sustrato de 1:100, con concentración de materia prima de 10% y 5 horas de hidrólisis, consiguiendo 81,3% de remoción y el contenido final de fenilalaninade 394,1 mg/100g de hidrolizado

O presente trabalho teve como objetivo a obtenção de hidrolisados enzimáticos, a partir do concentrado proteico do soro do leite combaixo teor de fenilalanina. Foram preparados 17 hidrolisados enzimáticos, avaliando-se o efeito de alguns parâmetros, como tipo de enzima, relação enzima: substrato, concentração da matéria-prima e tempo de hidrólise. O carvão ativado foi utilizado como meio adsorvente, e mostrou-se eficaz na remoção da fenilalanina, obtendo-se percentuais acima de 70%. O melhor resultado foi encontrado utilizando-se apancreatina na relação enzima: substrato de 1:100, com a concentração da matéria-prima de 10% após 5 horas de hidrólise, atingindo 81,3% de remoção e o teor final de fenilalaninade 394,1mg/100g de hidrolisado.

Isolation of high quality DNA: a protocol combining “rennet” and glass milk

High quality DNA is essential for many molecular biology techniques. However, the reagents used for that purpose usually are expensive and/or cause a high environmental impact. Here, we describe two alternative protocols that use inexpensive reagents and are not hazardous to the environment. The first protocol utilizes the enzyme chymosin, normally used as “rennet” in cheese production and which is easily obtained on the commercial market. The second protocol uses “rennet DNA extraction protocol” combined with the DNA binding capacity of glass powder (glass milk), which can easily be “home made”. The first protocol is used when a high yield of DNA is needed, whereas the second protocol is used for production of a higher quality DNA, being able to work with sparse samples.

Atividade lipolítica do leite com células somáticas ajustadas para diferentes níveis / Lipolytic activity of milk with different somatic cells levels

Estimou-se a origem da atividade lipolítica do leite com contagens de células somáticas (CCS) ajustadas para diferentes níveis. A partir de lotes de leite cru com baixa (100.000 cel/ml) e alta (1.000.000 cel/ml) CCS, formaram-se três tratamentos: A) leite com baixa CCS; B) leite com alta CCS; C) leite originalmente com baixa CCS e com adição de células somáticas. O leite foi submetido à pasteurização e armazenado por 21 dias sob refrigeração a 6ºC. Durante o período de armazenamento, foram coletadas amostras nos dias 1, 7, 14 e 21 para avaliação da concentração de ácidos graxos livres (AGL). Não foi observado efeito de tratamento sobre a concentração de AGL, durante todo o período de armazenamento e nem em cada dia de coleta isoladamente, cujas médias de AGL, após 21 dias, foram de 0,181; 0,159 e 0,153meq/kg, respectivamente, para os tratamentos A, B e C. Observou-se efeito do tempo de armazenamento sobre a concentração de AGL do leite, independentemente dos tratamentos, com teores de AGL de 0,121meq/kg para o dia 1 e de 0,219 para o dia 21. Pode-se concluir que, independentemente da contagem de células somáticas do leite cru, ocorre aumento da lipólise do leite pasteurizado durante o período de armazenamento. A adição de células somáticas ao leite, originalmente com baixa CCS, não aumentou a taxa de lipólise durante o armazenamento refrigerado

The lipolytic activity of milk with different somatic cell counts (SCC) was investigated in samples with low (100,000 cells.ml-1) and high (1,000,000 cells.mlÕ) SCC in order to obtain the following treatments: A) low SCC milk; B) high SCC milk; C) low SCC milk with somatic cells added, taken from high SCC milk. All samples were pasteurized and kept refrigerated at 6ºC for 21 days. Repeated measures during storage time were performed from pasteurized milk at days 1, 7, 14 and 21 to evaluate free fatty acid (FFA) concentrations. No effect of treatments was observed on milk FFA concentration during storage period, nor during each day of sampling, with FFA averaging 0.181; 0.159 and 0.153meq/kg for treatments A, B and C, respectively. FFA concentration in pasteurized milk increased during the storage period, independently of the milk SCC, as the level of FFA was 0.121 meq/kg on day 1 and 0.219 on day 21. Addition of somatic cells to low somatic cell milk did not increase lipolysis rate of milk during refrigerated storage. It can be concluded that pasteurized milk fat breakdown during refrigerated storage is not related to enzymes from somatic cells

Determinación cuantitativa de proteasas de bacterias psicrotróficas aisladas de leche cruda / Quantitative determination of proteases of isolated psychotropic bacteria in raw milk

El uso generalizado de la refrigeración de la leche cruda ha contribuido a mantener la calidad de ésta, pero ha traído como consecuencia la selección de una carga psicotrófica, que durante su desarrollo produce enzimas termoresistentes responsables, en parte, del deterioro de productos de larga vida. Este estudio se diseñó para la cuantificación de la actividad proteolítica de seis microorganismos que corresponden a Pseudomonas fluorescens R12 y R13, Pseudomonas putida R20, Micrococcus luteus R16, Bacillus circulans R5 y Serratia liquefasciens R4, aislados y caracterizados en la leche cruda. Las seis cepas fueron cultivadas en caldo leche al 11/100. Para las curvas de crecimiento, los datos experimentales se ajustaron al modelo de Baranyi. La Pseudomonas putida R20 presentó mejor cinética de crecimiento con máx de 0,1066h-1 y un tiempo de duplicación de 6,5023h. En la determinación de la actividad proteolítica, según Hübner, se estableció que Bacillus circulans R5 a 5ºC produjo en la hora 4 de fermentación la mayor cantidad de proteasas (3,618UP/mL) en comparación con las demás cepas de estudio. Finalmente se estableció que la temperatura donde existe mayor actividad proteolítica fue 5ºC, que al incrementarse provoca una disminución considerable en la producción de proteasas.

Efeito da remoção de células somáticas pela microfiltração sobre a lipólise do leite / Effect of somatic cell removal by microfiltration on the lipolysis of milk

O objetivo do presente estudo foi avaliar o efeito da retirada mecânicadas células somáticas do leite cru sobre a taxa de lipólise durante o armazenamento refrigerado do leite pasteurizado. O delineamento experimental utilizado foi totalmente casualizado, com três repetições e arranjo fatorial de tratamentos 2 X 2 X 2, constituídos por dois nível de gordura do leite (desnatado e integral), dois níveis de contagem de células somáticas (CCS baixa - CCSB e CCS alta - CCSA), e pela aplicação ou não da microfiltração ao leite. Três lotes de leite cru CCSB (75.000 cél./mL) e CCSA (1.150.000 cél./mL) foram obtidos de vacas selecionadas e submetidos ao desnate centrífugo, à microfiltração em sistema a vácuo, sendo em seguida pasteurizados e armazenados por 21 dias sob refrigeração a 6ºC. Foram realizadas medidas repetidas no tempo, as quais corresponderam aos dias de coleta do leite pasteurizado durante o período de armazenamento (1, 7, 14 e 21). A microfiltração associada ao desnate foram eficazes na remoção das células somáticas do leite, com reduções de 92,5 e 99,5% para o leite CCSB e CCSA, respectivamente. A lipólise do leite aumentou em função do tempo de armazenamento e foi maior para o leite microfiltrado, contudo não sofreu influência da CCS. Independentemente da CCS, ocorreu aumento da lipólise do leite pasteurizado durante o período de armazenamento.

The objective of this study was to evaluate the effects of raw milk somatic cell removal by microfiltration on lipolysis of pasteurized milk during refrigerated storage. A completely randomized design was used, with 3 repetitions and a 2 X 2 X 2 factorial arrangement of treatments as follow: milk fat level (skimmed and whole milk), two different levels of somatic cell counts - low somatic cell count (LSCC) and high somatic cell count (HSCC) - and, use of microfiltration ornot. LSCC raw milk - 75,000 cells/ml - and HSCC raw milk - 1,150,000 cells/ml - were obtained from selected cows, skimmed and submitted to vacuum microfiltration. Milk from all treatments was pasteurized and kept refrigerated at 6ºC for 21 days. Repeated measures during storage time were taken from pasteurized milk at days 1, 7, 14 and 21. The application of milk microfiltration was efficient on somatic cell removal, with reduction of 92,5 and 99,5% for LSCC and HSCC,repectively. Lipolysis of milk was increased during storage period and was higher for milk submitted to icrofiltration, however no effect of SCC was observed. Lipolysis in pasteurized milk increased independently of SCC, during its refrigerated storage period.

Monitoramento da eficiência de pasteurização de leite de cabra, baseado no sistema de análise de perigos e pontos críticos de controle, em mini-usinas do Cariri Paraibano / Monitoring of the efficiency of pasteurization in goat´s milk based on the System of Analysis and Critical Control Point, in Cariri Paraibano

O leite de cabra pasteurizado no Brasil é hoje uma realidade. O controle da pasteurização é feito através da pesquisa de enzimas, fosfatase alcalina e peroxidase, usadas como parâmetro para distinguir o leite cru do pasteurizado corretamente, superaquecido ou recontaminado com leite cru. Diante destes aspectos este trabalho teve como objetivo avaliar se o leite processado nas mini usinas de leite de cabra da região do cariri paraibano estava sendo realmente bem pasteurizado, avaliando assim a eficiência da pasteurização através da pesquisa de enzimas fosfatase alcalina, indicada no Sistema de Analise de Perigos e Pontos Críticos de Controle – APPCC, dentro do fluxograma de leite pasteurizado e também a enzima peroxidase. Durante o período de janeiro a dezembro de 2004, foram acompanhadas 6 (seis) mini usinas no cariri paraibano, com o monitoramento da pasteurização, coletando-se amostras de leite embaladas, sendo realizadas 10 (dez) visitas, totalizando no final 10 (dez) amostras por mini-usina. As amostras foram coletadas em sua própria embalagem (sacos plásticos – tipo popular “barriga mole”), mantidas acondicionadas em caixas de isopor com gelo até o momento da análise, obedecendo todas as normas prescritas para coleta e envio de amostras para coleta e envio de amostras para a análises físico-químicas pelo Laboratório Nacional de Referência Animal – LANARA (BRASIL, 1981). Utilizou para pesquisa da enzima fosfatase o kit fosfatase alcalina da Diasys, de acordo com as recomendações do laboratório. Para a análise de peroxidase utilizou-se guaiacol e água oxigenada 10 volumes (BRASIL, 1981). Avaliando o efeito da pasteurização sobre as duas enzimas pesquisadas tem-se um resultado de apenas 66,66 por cento de amostras de leite devidamente pasteurizadas, estando assim o consumidor, além de fraudado por perdas nutricionais com o superaquecimento do leite, correndo riscos de adquirir diversas enfermidades veiculadas pelo consumo de leite cru.

Dissociation of milk xanthine oxidase and effect of some potent inhibitors on its catalytic function

Milk xanthine oxidase [XO] was prepared and purified by gel chromatographic analysis. Chromatographic runs were made on sephadex G200 column saturated with urea. The elution profile recorded two peaks: Dimeric and monomeric xanthine oxidase, respectively. The inhibitory effect of bisulfite, folate and ascorbate on the catalytic activity of xanthine oxidase was monitored by incubating the enzyme with different concentrations of these inhibitors. On assaying aliquot sample of the incubation mixture in a standardized method, a pronounced lag in rate production was recorded spectrophotometrically at 290 nm. XO activity increased from zero until substrate became limiting, but in case of inhibition the rate of catalysis never reached maximal velocity compared to control. Besides the inhibitory mechanism of morin and myricetin as flavonoids on XO activity were also studied by kinetic analysis. It was concluded that bisulfite, folate, ascorbate and flavonoids could be utilized in chemotherapy of hyperuricemia or gout

Antibacterial property of goat milk lactoperoxidase.

The goat milk lactoperoxidase was purified using CM sephadex C-50 and sephadex G 100. The purity of protein was confirmed by SDS-PAGE. The purified protein was found to have antibacterial action against most of the disease causing bacteria.

Recurrent mutation-selection to improve rennet production in Candida tsukubaensis

The yeast Candida tsukubaensis is of industrial importance for the production of microbial milk-clotting enzyme. Milk-clotting enzyme is an enzymatic complex capable of coagulating milk for cheese manufacturing. High clotting activity (CA) and low proteolytic activity (PA) are desirable qualities. To study the genetic nature of the CA and PA traits, we analyzed 179 colonies obtained after mutagenic treatment. Analysis of the data obtained for this populations showed that CA and PA are traits controlled by polygenes and that they are correlated (r = 0.3). The existence of a positive correlation indicates that selection for one trait without considering the other may alter one of the traits in an undesirable direction, since the objective of selection would be an increase in clotting activity and a decrease in proteolytic activity. Three cycles of recurrent mutation-selection were carried out to obtain improved strains. The ultraviolet light dose permitting a 5 per cent rate of cell survival was sufficient to generate genetic variability in the three selection cycles. At the end of the third cycle there was an increase of about 98 per cent in clotting activity and a decrease of about 20 per cent in proteolytic activity. Analysis of variance of the selective cycles showed that the linear effects were significant (P < 0.01) for both traits. Estimates of genetic variances and heritabilities of the three selection cycles are presented.

Avaliaçäo sensorial e metabólica de leite com baixo teor de lactose / Sensory and metabolic evaluation of low-lactose milk

Leite cuja concentraçäo de lactose foi reduzida por betagalactosidade foi submetido a análise sensorial por 57 sujeitos. Avaliaçäo metabólica desse leite foi realizada pelo teste de excreçäo de hidrogênio pulmonar com quatro indivíduos após ingestäo de leite com baixo teor de lactose e de leite integral. O hidrogênio foi determinado antes da ingestäo e 30, 60, 90, 120, 150 e 180 minutos após a ingestäo de 300ml de leite. O estudo mostra, ainda, que o leite com baixo teor de lactose apresenta boa avaliaçäo sensorial e metabólica quando comparado com o leite integral. Este trabalho sugere o uso de leite com baixo teor de lactose para indivíduos intolerantes e para pacientes com deficiência secundária de lactase.

Production of milk Clotting-enzyme by penicillium funiculosum during growth in whey permeate

A new milk clotting enzyme was produced from Penicillium funiculosum NRCE-629 in submerged cultivars using salted whey permeate supplemented with corn steep liquor [CSL] as nitrogen source. The optimum level of CSL supplementation was 500 mg N/L. The highest milk-clotting activity [MCA] was obtained with initial pH 3.0 after 6 days aerobic incubation by the tested fungus. Furthermore, the MCA produced by this fungus has low proteolytic activity [PA] which is a desirable property required for cheese curd formation to prevent soft texture or bitter taste. These desired properties of enzyme may increase the feasibility of production as a suitable fungal substitute for calf rennet

Extracción de la pepsina aviar (EC 3.423.1) y su utilización como substituto de la quimosina en la elaboración de queso blanco pasteurizado / Extraction of avian pepsin (EC 3.423.1) its utilization as chymosin substitute when the making white pasteurized cheese

Se extrajo pepsina aviar a partir de estómagos glandulares de aves, para ser utilizada como substituto de la quimosina en a elaboración de queso blanco pasteurizado. La enzima extraida demostró poseer un rendimiento de 4,3 g de pepsina aviar liofilizada por cada kilogramo de estómagos glandulares, y una actividad coagulante, sobre el substrato de Berridge, ocho veces superior a la quimosina utilizada como patrón de referencia. No se encontraron diferencias significativas en la composición química ni en la calidad organoléptica de los quesos fabricados, cuando se utilizó pepsina aviar, quimosina aviar, quimosina y la mezcla de ambos. Demostrándose que la substitución, tanto parcial como total, de la quimosina por la pepsina en queso blanco pasteurizado es factible, ya que ofrece una mayor actividad coagulante y un queso con características químicas y sensoriales similares a las obtenidas con el cuajo tradicional

Amino acids: modifiers of xanthine oxidase activity.

L-Glutamic acid has been found to be a positive and L-lysine a negative modifier of the xanthine oxidase activity at the optimum pH (7.4) of the enzyme. Increase in pH was observed to be associated with a progressive decrease in the inhibition produced by L-lysine.