Gelation of ultra-high-temperature-sterilized milk by proteases from a strain of<i>Pseudomonas fluorescens</i>isolated from raw milk

Law, B. A.; Andrews, Anthony T.; Sharpe, M. Elisabeth

doi:10.1017/s0022029900020057

Cited by 101 publications

(58 citation statements)

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“…Porém, pode ocasionar problemas tecnológicos associados à atividade de enzimas proteolíticas e lipolíticas de bactérias psicrotróficas. Muitas destas enzimas são termorresistentes [23,33] e estão relacionadas às perdas de qualidade e à redução da vida de prateleira do leite UHT e de outros produtos lácteos [8,9,11,17,18,22,24,28,35,39,40].…”

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Qualidade microbiológica de leite cru refrigerado e isolamento de bactérias psicrotróficas proteolíticas

Pinto¹,

Martins²,

Vanetti³

2006

Ciênc. Tecnol. Aliment.

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RESUMOA estocagem do leite cru refrigerado na fonte de produção reduz perdas econômicas por atividade acidificante de bactérias mesofílicas, mas permite a seleção de bactérias psicrotróficas relacionadas a problemas tecnológicos e econômicos na indústria de laticínios. Com o objetivo de avaliar a qualidade microbiológica de leite cru, foram analisadas amostras provenientes de tanques de refrigeração individual, coletivos e do silo de uma indústria processadora de leite. Além disso, bactérias psicrotróficas proteolíticas foram isoladas do leite cru refrigerado e caracterizadas quanto à reação ao Gram e fermentação de glicose. O leite cru refrigerado mantido no silo industrial não atendeu ao padrão microbiológico legal e apresentou contagens microbianas significativamente superiores às do leite mantido em tanques individuais ou coletivos. Diferença significativa na contaminação por mesófilos e psicrotrófilos proteolíticos e não proteolíticos e por Pseudomonas foi observada entre as amostras coletadas nos tanques de refrigeração e no silo industrial. A microbiota Gram-negativa foi isolada com maior freqüência, especialmente bactérias Gram-negativas não fermentadoras de glicose. Palavras-chave: leite refrigerado, microbiota psicrotrófica, qualidade microbiológica. SUMMARYMICROBIAL QUALITY OF RAW REFRIGERATED MILK AND ISOLATION OF PSYCHROTROPHIC PROTEOLYTIC BACTERIA. Storing refrigerated raw milk in the source of production reduces economic losses which occur due to the acidifying activity of mesophilic bacteria. Nevertheless, this process selects psychrotrophic bacteria that cause technological and economic problems to the dairy industry. Samples of individual and collective refrigerated tanks and samples from an industrial silo in a dairy industry were collected and analyzed to evaluate the microbiological quality of raw milk. Furthermore, proteolytic psychrotrophic bacteria were isolated from refrigerated raw milk and characterized according to the Gram reaction and glucose fermentation. The refrigerated raw milk from the industrial silo was not compatible with legal microbiological standards, and its microbial count was significantly higher than milk from individual and collective tanks. A significant difference of contamination by mesophiles, proteolytic and non-proteolytic psychrotrophs, and Pseudomonas was observed in samples collected from refrigeration tanks compared to samples collected from the industrial silo. The Gram-negative microbiota was isolated more often, especially the glucose non-fermentative Gram-negative bacteria.

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Qualidade microbiológica de leite cru refrigerado e isolamento de bactérias psicrotróficas proteolíticas

Pinto¹,

Martins²,

Vanetti³

2006

Ciênc. Tecnol. Aliment.

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“…Lipases degrade the milk fat, causing rancid, soapy, and occasional bitter off-flavors through the formation of medium-chain fatty acids. Proteases that degrade casein cause a gray color, bitter off-flavors, and gelation of ultra high-temperature (UHT) products (7,19,21).…”

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Influence of Storage Conditions on the Growth of Pseudomonas Species in Refrigerated Raw Milk

Jonghe¹,

Coorevits

Hoorde

et al. 2011

Appl Environ Microbiol

160

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The refrigerated storage of raw milk throughout the dairy chain prior to heat treatment creates selective conditions for growth of psychrotolerant bacteria. These bacteria, mainly belonging to the genus Pseudomonas, are capable of producing thermoresistant extracellular proteases and lipases, which can cause spoilage and structural defects in pasteurized and ultra-high-temperature-treated milk (products). To map the influence of refrigerated storage on the growth of these pseudomonads, milk samples were taken after the first milking turn and incubated laboratory scale at temperatures simulating optimal and suboptimal preprocessing storage conditions. The outgrowth of Pseudomonas members was monitored over time by means of cultivation-independent denaturing gradient gel electrophoresis (DGGE). Isolates were identified by a polyphasic approach. These incubations revealed that outgrowth of Pseudomonas members occurred from the beginning of the dairy chain (farm tank) under both optimal and suboptimal storage conditions. An even greater risk for outgrowth, as indicated by a vast increase of about 2 log CFU per ml raw milk, existed downstream in the chain, especially when raw milk was stored under suboptimal conditions. This difference in Pseudomonas outgrowth between optimal and suboptimal storage was already statistically significant within the farm tank. The predominant taxa were identified as Pseudomonas gessardii, Pseudomonas gessardii-like, Pseudomonas fluorescens-like, Pseudomonas lundensis, Pseudomonas fragi, and Pseudomonas fragi-like. Those taxa show an important spoilage potential as determined on elective media for proteolysis and lipolysis.

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“…Pseudomonas fluorescens is well known as a major psychrotrophic contaminant of raw milk stored in refrigerated tanks (16). Many studies of this bacterium have been concerned with the production of deleterious extracellular enzymes, such as thermostable proteases (8,15,27).…”

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Effect of growth temperature on several exported enzyme activities in the psychrotrophic bacterium Pseudomonas fluorescens

Gügi¹,

Orange²,

Hellio³

et al. 1991

J Bacteriol

108

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In accordance with previous results, the activity of extracellular proteases from Pseudomonas fluorescens MFO is maximal at a growth temperature of 17.5°C, well below the optimal growth temperature. In addition, the activities of three periplasmic phosphatases display the same growth temperature optimum. Chemostat experiments have shown that it is the growth temperature itself and not the value of the growth rate that regulates these activities. In contrast, a foreign periplasmic phosphatase, expressed under the control of its own promoter, displays a different sensitivity toward temperature. We conclude that in the psychrotrophic strain P. fluorescens MFO, growth temperature exerts a specific control upon the activity of certain enzymes. The critical temperature (17.5C) is within the range of normal growth, suggesting that this control is probably different from a cold shock or heat shock response.Pseudomonas fluorescens is well known as a major psychrotrophic contaminant of raw milk stored in refrigerated tanks (16). Many studies of this bacterium have been concerned with the production of deleterious extracellular enzymes, such as thermostable proteases (8,15,27). Among the numerous observations concerning these enzymes, it has been repeatedly shown that most strains maximally produce proteases at a temperature (15 to 20°C) well below the optimal growth temperature (25 to 30°C) (13,19,25). However, no studies have yet dealt with the mechanism of regulation of protease production with regard to temperature.At this stage two main questions can be raised regarding the elucidation of this mechanism. The first one relates to the specificity of this temperature effect, i.e., whether it is restricted to the production of proteases or extended to the production of other enzymes. To this end, the activities of extracellular proteases as a function of growth temperature were compared with those of several periplasmic phosphatases. The exported enzymes all showed the same regulation by temperature even though they are clearly differentially regulated by other growth conditions. Thus, it was important to determine whether this temperature effect might involve protein export through the cytoplasmic membrane. If so, any foreign exported protein should be submitted to the same effect. The expression of a gene from the mesophilic species Escherichia coli, under the control of its own promoter, was studied in P. fluorescens at different growth temperatures. In this case, a temperature effect similar to that observed with the native enzymes was not demonstrated.The second question is whether the temperature itself is the direct cause of the regulation or an indirect effector acting through the growth rate variation; such an indirect effect has indeed been demonstrated for several activities or proteins in mesophilic bacteria (5). To answer this question, the activities of the two acidic phosphatases were assayed in cells grown in a chemostat at two different temperatures and * Corresponding author. several dilution rates. The ...

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Gelation of ultra-high-temperature-sterilized milk by proteases from a strain ofPseudomonas fluorescensisolated from raw milk

Cited by 101 publications

References 8 publications

Qualidade microbiológica de leite cru refrigerado e isolamento de bactérias psicrotróficas proteolíticas

Qualidade microbiológica de leite cru refrigerado e isolamento de bactérias psicrotróficas proteolíticas

Influence of Storage Conditions on the Growth of Pseudomonas Species in Refrigerated Raw Milk

Effect of growth temperature on several exported enzyme activities in the psychrotrophic bacterium Pseudomonas fluorescens

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