Avaliação, identificação e atividade enzimática de bactérias psicotróficas presentes no leite cru refrigerado

Extended storage of refrigerated milk can lead to reduced quality of raw and processed milk, which is a consequence of the growth and metabolic activities of psychrotrophic bacteria, able to grow under 7oC or lower temperatures. Although most of these microorganisms are destroyed by heat treatment, some have the potential to produce termoresistant proteolytic and lipolytic enzymes that can survive even UHT processing and reduce the processed products quality. Recently, the IN 51 determineds that milk should be refrigerated and stored at the farm what increased the importance of this group of microorganisms. In this work, psychrotrophic bacteria were isolated from 20 communitarian bulk tanks and 23 individual bulk tanks from dairy farms located at Zona da Mata region of Minas Gerais State and from southeastern Rio de Janeiro. Selected milk dilutions were plated on standard agar and after incubation for 10 days at 7oC, five colonies were isolated, firstly using nutrient agar and after using McConkey agar for 24 hours at 21oC. The isolates were identified by morphology, Gram stain method, catalase production, fermentative/oxidative metabolism and by API 20E, API 20NE, API Staph, API Coryne or API 50 CH (BioMerieux). In order to ensure reproductibility, API was repeated for 50% of the isolates. Species identification was considered when APILAB indexes reached 75% or higher. 309 strains were isolated, 250 Gram negative and 59 Gram positive. 250 Gram negative isolates were identified as: Acinetobacter spp. (39), Aeromonas spp. (07), A. Hydrophila (16), A. sobria (1), A. caviae (1), Alcaligenes feacalis (1), Burkholderia cepacia (12), Chryseomonas luteola (3), Enterobacter sp. (1), Ewingella americana(6), Hafnia alvei (7), Klebsiella sp. (1), Klebsiella oxytoca (10), Yersinia spp. (2), Methylobacterium mesophilicum (1), Moraxella spp. (4), Pantoea spp. (16), Pasteurella sp. (1), Pseudomonas spp. (10), P. fluorescens (94), P. putida (3), Serratia spp. (3), Sphigomonas paucomobilis (1). Five isolates kept unidentified. Pseudomonas was the predominant bacteria found (43%) and P. fluorescens the predominant species (37.6%), in accordance with previous reports. Qualitative analysis of proteolytic and lipolytic activity was based on halo formation using caseinate agar and tributirina agar during 72 hours at 21oC and during 10 days at 4°C, 10oC and 7°C. Among 250 Gram negative bacteria found, 104 were identified as Pseudomonas spp. and 60,57% of this group showed proteolytic and lipolytic acitivities over all four studied temperatures. 20% of Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Chryseomonas, Methylobacterium, Moraxella presented only lipolytic activity. Some isolates presented enzymatic activity in one or more studied temperatures. Among Gram positive bacteria, 30.51% were proteolytic and lipolytic at 10oC, 8.47% were proteolytic at 7oC, 10oC, and 21oC, 8.47% were proteolytic at all studied temperatures (4oC, 7oC, 10oC and 21oC) and 3.38% were proteolytic only at 21oC. At 4oC, only one isolate showed proteolytic activity and six isolates were lipolytic. In relation to Gram negative microorganisms, 4% were proteolytic and lipolytic at 7oC, 10oC and 21oC, 10% were proteolytic at 10oC and 4.4% were lipolytic at 4oC, 7oC, 10oC and 21oC, while 6.4% of all isolates were proteolytic and lipolytic at 10oC and 21oC as well as lipolytic at 4oC and 7oC. These findings are in accordance with previous researches that pointed out Pseudomonas as the predominant psycrotrophic flora in stored refrigerated raw milk

Diagnóstico regional do processo de queijo coalho comercializado em Natal/RN

Coalho cheese is a typical product of the Northeastern Brazil, which is consumed both raw and cooked. The present work aimed to study the characteristics of artisanal and industrial processes in the production of coalho cheese sold in Natal / RN in order to evaluate its quality and consumer s profile. Four artisanal cheeses plants were monitored and a questionnaire was sent to different cheese industries. Besides this, eight cheese samples (four artisanal and four industrial) were evaluated in regard to the microbiological quality, physical-chemical and sensory attributes. The sensory acceptance was evaluated by using 108 non-trained panelists by using the hedonic scale. The consumer s profile survey was applied to 400 consumers of coalho cheese. The lack of hygiene control was detected at the artisanal cheese production, which uses raw milk as its raw material. Research has shown that the industrialized cheeses are made from pasteurized milk provided by their own production or by a third party, as observed in cheese making dairies. In general, the results indicate variation in the manufacturing process of coalho cheese, which results in the lack of product standardization. Regarding the physical-chemical analysis, most artisanal and industrial samples presented moisture content between 36 and 40 %, classified as medium moisture cheese, which is the only parameter that showed no significant difference (p>0.05). However, the water activity (Aw), pH and acidity results differed significantly. All artisanal samples showed coliform contamination at 35 °C, which confirms the poor hygienic conditions. In regard to coliforms at 45 °C, 75 % of artisanal coalho cheese samples had value higher than 103 MPN / g, a value above the lawful limits determined by RDC nº 12. Fifty percent of industrial coalho cheese samples showed coagulase-positive Staphylococcus values above the limit allowed by the RDC nº 12, indicating poor handling. The sensory evaluation revealed that the taste was the only parameter that showed significant difference, and this difference was only between two industrial brands. The consumer s survey showed that the coalho cheese flavor is the most important reason for buying this kind of cheese. Although coalho cheese is mainly bought in supermarkets, open street markets and country shops are still important selling points. It is concluded that there is no coalho cheese standardization in the RN state, which leads to variations in physical-chemical and sensory attributes. Moreover, it is necessary greater hygiene control in the production and handling procedures of coalho cheese.

Avaliação de métodos de análises para determinação da contagem de células somáticas no leite cru, mantido em tanque de resfriamento

We analyzed the quality of raw milk from eight dairy farms in Rio Grande do Norte stored in a cooling tank , in order to evaluate methods for determining somatic cell counts (SCC). The Somaticell® kit and a portable Direct Cell Counter (DCC) were compared with each other and with the MilkoScanTM FT+ (FOSS Denmark), which uses Fourier Transform Infrared (FTIR) spectroscopy). Direct cell counter data were processed for somatic cell scores (log-transformed somatic cell count) and analyzed with the SAS®, statistical package , Statistical Analysis System, (SAS, INSTITUTE, 1998). Comparison of means and correlation of somatic cell scores were conducted using Pearson s correlation coefficient and the Tukey Test at 1 %. No significant difference was observed for comparison of means. The correlation between somatic cell scores was significant, that is, 0.907 and 0.876 between the MilkoScanTM FT+ and the Somaticell® kit and Direct Cell Count (DCC) respectively, and 0.943 between the Somaticell® kit and Direct Cell Count (DCC). The methods can be recommended for monitoring the quality of raw milk kept in a cooling tank in the production unit

Fatores de influência na produção de leite bovino sobre os níveis da contagem bacteriana total

The objective of this study was to evaluate the influence of milking procedures on the levels of total bacterial count (TBC) in bovine milk. In the first study the influences of procedures for hygienic milking, cleaning of milking equipment and milk cooling tanks on the TBC levels were evaluated. Four bulk samples of milk were collected from each tank in eight properties for TBC analysis, employing the flow cytometry method. A questionnaire was applied in each property to assess the current situation of milking procedures on each production system that took part on this research, followed by training of employees in good agricultural practices in the production of milk and monitoring of the TBC measurements. The methodology for analysis of longitudinal data was considered, focusing on random effects models. The results showed that the handling procedures for milking and the cleanliness of the cooling tank contributed to a further reduction in the levels of TBC raw milk cooling tanks. The second study aimed to describe the percentage of the properties that comply with the Normative Instruction Nº 51 (Brazil s IN 51) with regard to total bacterial count (TBC) in bovine milk. The study was conducted from January 2010 to July 2011. Milk samples were collected from the eight properties selected for TBC analysis by the flow cytometry method. Again, on each property a questionnaire was applied to assess the current situation of milking procedures on each production system that took part on this research, followed by training of employees in good agricultural practices in the production of milk and monitoring of the TBC measurements. The methodology of marginal models based on Generalized Estimate Equations (GEEs) was followed in the statistical analysis. The results showed that the handling procedures of the milking and the cleanliness of the cooling tanks contributed to a considerable percentage of the properties that reached the limits of TBC established by IN 51

Leite cru de quatro regiões leiteiras brasileiras: perspectivas de atendimento dos requisitos microbiológicos estabelecidos pela Instrução Normativa 51

Evidências que o leite produzido e consumido no Brasil nem sempre apresenta a qualidade desejada têm gerado a discussão e desenvolvimento de novas políticas de incentivo à produção leiteira, resultando no desenvolvimento do Programa Nacional de Melhoria da Qualidade do Leite. em complementação, em 2002 o Ministério da Agricultura publicou a Instrução Normativa 51 (IN51), com importantes inovações em relação à conservação e transporte do leite cru, além de estabelecimento de um padrão de qualidade para esse tipo de leite (10(6) UFC/mL), a ser implantado em diferentes prazos nas diferentes regiões do país, a partir de 2005. O presente trabalho teve como objetivo verificar se o leite cru produzido em quatro áreas de quatro estados produtores de leite no Brasil estaria, nesse momento, em condições de cumprir o estabelecido na IN 51, especialmente quanto ao atendimento dos padrões microbiológicos previstos. Amostras de leite cru, coletadas em 210 diferentes propriedades nas regiões de Viçosa, MG (47), Pelotas, RS (50), Londrina, PR (63) e Botucatu, SP (50), foram analisadas quanto aos níveis de contaminação por aeróbios mesófilos, utilizando o PetrifilmTM AC. Parcela significativa das amostras (48,6%) apresentaram contagens acima do determinado pela IN51, sendo 21,3% na região de Viçosa (MG), 56,0% na região de Pelotas (RS), 47,6% na região de Londrina (PR) e 68,0% na região de Botucatu (SP). Considerando as diferenças de cada região, foi possível observar a importância da refrigeração na conservação e transporte da produção, bem como da implantação de boas práticas e assistência técnica nas propriedades. Os resultados obtidos permitem concluir que a adequação às normas estabelecidas pela IN51 pode ser mais difícil em algumas regiões do que em outras, sendo fundamental a adoção da refrigeração na conservação e no transporte da produção, e de programas regionais de assistência a produtores leiteiros.

Evolução do índice proteolítico e do comportamento reológico durante a vida de prateleira de leite UAT/UHT

A proteólise do leite UAT/UHT durante a estocagem à temperatura ambiente é um dos fatores limitantes de sua vida de prateleira. Neste trabalho, dois lotes de leite cru contendo 10 amostras cada e, posteriormente ao processamento, dois lotes de leite UAT/UHT contendo 25 amostras cada foram colhidos em um laticínio para a contagem de microrganismos psicrotróficos (leite cru) e para o estudo do comportamento reológico e o índice proteolítico (leite UAT/UHT durante 120 dias de estocagem). Para a contagem de microrganismos psicrotróficos, foi utilizada a técnica da contagem padrão em placas. Para a determinação do índice proteolítico, foi determinada a presença de glicomacropeptídeo livre por espectrofotometria a 470 nm. A determinação dos parâmetros reológicos foi efetuada à temperatura ambiente, em quintuplicata em um reômetro de cone e placa. Houve aumento da proteólise no decorrer do armazenamento e aumento da viscosidade aparente após 60 dias de estocagem, provavelmente relacionados à presença de proteases de bactérias psicrotróficas do leite cru.

Efeito do processamento UAT (Ultra Alta Temperatura) sobre as características físico-químicas do leite

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Correlação entre métodos tradicionais e espectroscopia de ultra-som na determinação de características físico-químicas do leite

Compararam-se a espectroscopia de ultra-som (US) e os métodos tradicionais (MT) utilizados para a determinação de características físico-químicas do leite e estimaram-se as correlações entre esses métodos e suas acurácias. As características densidade, extrato seco desengordurado (ESD), teor de proteínas e teor de gordura foram determinadas em 65 amostras de leite cru por ambos os métodos. As densidades médias determinadas pelo US e pelos MT não diferiram entre si (P=0,14), e a correlação encontrada entre os dois métodos para a determinação da densidade não foi significativa (P= 0,08). Os teores médios de ESD, proteína e gordura encontrados pelo US e pelos MT foram diferentes (P=0,04, P<0,0001 e P<0,0001, respectivamente), as correlações entre os dois métodos utilizados para a análise dessas características foram positivas e significativas (r=0,0109, r=0,0007, r= <0,0001, respectivamente) e as acurácias dos métodos para essas determinações foram de 0,160, 0,062 e 0,145, respectivamente. Foi determinada a equação de regressão linear, que associa o teor de gordura obtido no método de espectroscopia de ultra-som ao do método butirométrico, que apresentou coeficiente de determinação de 0,5936.

Características microbiológicas de amostras de leite de tanque comunitário

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Seroepidemiology of Toxoplasma gondii, Neospora caninum, and Leishmania spp. infections and risk factors for cats from Brazil

The seroprevalence of infection by Toxoplasma gondii, Neospora caninum, and Leishmania spp. was detected through an indirect immunofluorescence in 70 cats from the Andradina Municipality, São Paulo State, Brazil. Anti-T. gondii antibodies (titer >64) were detected in 15.7% (11/70) of animals, whereas positivity for N. caninum (titer 16) was not observed in any animal. of the cats from urban and rural areas, 10.4% (5/48) and 27.2% (6/22) were positive for T. gondii, respectively. Breed, age, food, and contact with animals of other species were significant for considering the positivity for T. gondii (P <= 0.0001). Cats having access to streets (17.1%, 11/64), cats cohabiting with rats (19.6%, 10/51), and cats feeding on homemade food and raw milk (27.2%, 6/22) were positive for T. gondii. In addition, 4.2% (3/70) of the cats were positive for Leishmania spp. by ELISA technique and negative by IFAT without coinfection with T. gondii and Leishmania spp. There was no serological positivity against feline immunodeficiency virus or feline leukemia virus. In conclusion, T. gondii infection in part of the feline population from Andradina is not linked to immunosuppressions or coinfections but probably to postnatal infection in association with the type of diet and presence of rats.

CONDICOES BACTERIOLOGICAS DE LEITE CRU E DE LEITE PASTEURIZADO NA REGIAO DE ARARAQUARA, SP, BRASIL

Milk can be an important spreading vehicle of pathogenic agents mainly for young children who are an important group of milk consumers. 135 milk samples (77 of raw milk and 58 of pasteurized milk) were analysed in order to verify the number of heterotrophic bacteria, of Staphylococcus aureus and of total coliforms, as well as to determine presence of Salmonella, Shigella, enteropathogenic Escherichia coli (EPEC) and enteroinvasive Escherichia coli (EIEC). The results were negative for Salmonella, Shigella and EIEC. EPEC serotypes 0:28, 0:111 and 0:55 were isolated in 4 of raw milk samples. The heterotrophic bacteria was found in counts over 30.000 UFC/mL in 91%, 25% and 68,75% respectively of raw milk, milk grade B and grade C. Counts over 30.000 coliforms/mL were found in 70,13%, 6,25% and 6,24% of the raw milk, B and C, respectively. 32,40% of the raw milk had counts of S. aureus over 3.000 UFC/mL.

Determinação de GMP e CMP* no leite por métodos espectrofotométrico (ANSM) e cromatográfico (HPLC) - parâmetros metodológicos (*glicomacropeptídeo e caseinomacropeptídeo)

Glycomacropeptide is a glycosilated fraction of bovine kappa-casein that remains soluble when milk is clotted by rennin. Determinations of milk sialic acid content are useful because its concentration reflects the amount of free GMP of milk. In normal milk these amounts are very low, 12 to 16 times lower than in sweet whey. Therefore, its determination may be applied to verify possible frauds with whey addictions, since it works as a fingerprint. With the description of a new spectrophotometric method for determination of free GMP (ANSM) occurred a simplification of procedures, being faster than others (HPLC method), without loss of accuracy. However, due to variations of glycosilation in kappa-casein between animals, during the lactation period, due to mastitis and yet due to proteolysis on milk, it was necessary to know these variations to interpret correctly the analytical results. It was analyzed 1,703 samples of producer's raw milk and 1,189 samples of processed milk (HTST and UHT). The results showed that normal milk from herd (producer's milk) have only small amounts of free GMP, with A470nm = 0.232±0.088 or 3.89±1.25 mg of sialic acid/L. The upper limit of this distribution was A = 0.496; thus every bigger value may represent a problem, being outside of normal distribution.

Eficácia do tetra-test como ferramenta de gestão da qualidade do leite cru refrigerado

The effectiveness of a new management tool for screening refrigerated raw milk quality in a dairy industry is provided. Three hundred and seventy-four samples of raw milk from individual producers and 125 samples from bulk milk carriers from the same producers were analyzed. Producers' samples underwent a fast tube reduction test, or Tetra-Test, to estimate total bacteriological charge and their predominant microbiota. Bulk milk samples underwent standard plate count (SPC) as reference method. Tetra-test was effective in screening and assessing the microbiological quality of refrigerated raw milk, since 82.4% of samples with more than 106UFC/mL were readily detected (5 minutes to 2h). The test may be employed as a management tool either in screening or in interventions on the detected problem. In fact, its results provided complementary information on the characteristics of dominant microbiota. Most samples failed in compliance to IN 51 requirements. Further, 211 samples (56.4%) showed high microbial charge and, consequently, poor quality raw milk. Psychrotrophic groups were not yet the main degrading factors of cooled raw milk, albeit present in 76.8% of samples.

Liberação das enzimas LDH e PepX e evolução da maturação de queijo parmesão adicionado de culturas autolíticas de Lactobacilus helveticus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Características fenotípicas e genotípicas de Escherichia coli isoladas de queijos produzidos a partir de leite não pasteurizado

Pós-graduação em Medicina Veterinária - FCAV