The aim of this study was to identify Pseudomonas spp. in raw milk the production of extracellular enzymes (e.g., peptidases and lipases). Keywords: Pseudomonas, hydrolases, protease, lipase, glycosidase The strain 1A4R was isolated from refrigerated raw milk in Plate Count Agar (PCA; Mast . Extracellular enzyme activities produced by Pseudomonas sp. during growth on. The LipM lipase had a maximum activity at 25 °C and a broad pH optimum ranging from to In Brazil, the practice of refrigerating raw milk at the dairy farm Many of these enzymes are produced by Pseudomonas fluorescens Genetic diversity and spoilage potentials among Pseudomonas spp.

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The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise byppseudomonas effective strategy to avoid dairy spoilage.

Species of YersiniaPseudomonasSerratiaand Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens d.

Lipolytic activity was high in isolates of Acinetobacterand the highest in Acinetobacter guillouiae. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. For these reasons, thermo-stable spoilage mjlk produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry.

The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce. Raw milk serves as an ideal medium for the growth of bacteria due to its high nutritional value Champagne et al.

A cold chain system is commonly used for controlling the growth of bacteria in raw milk during storage and transportation. Raw milk can be contaminated with psychrotrophic bacteria from a variety of sources including air, water, soil, and milking equipment Vacheyrou et al. Among psychrotrophic bacteria, genera of PseudomonasAcinetobacterFlavobacteriumChryseobacteriumand Serratia are the most frequently isolated from raw milk Vithanage et al.

Increasing global demand for dairy products requires dairy manufacturers to produce products with high quality and prolonged shelf-life. The dairy industry is facing constraints related to maintaining high quality and avoiding losses as a result of microbial spoilage. Mesophilic and thermophilic spore forming bacteria have the potential to contaminate processed ny products, which may then fail to comply with specifications for spore content Sadiq et al.

However, psychrotrophic bacteria present more serious challenges to the dairy industry.

Raw milk is contaminated with heat-stable enzymes produced by a broad spectrum of psychrotrophic bacteria at low temperatures, which can survive all successive processing conditions and bypseudomonass active in processed dairy products Vithanage et al. For example, the decimal reduction time D- value of proteases produced by Pseudomonas spp. Consequently, these heat-stable enzymes may lead to unacceptable biochemical changes, a decrease in nutritional value, and reduced prodkction of dairy products Stoeckel et al.

Lipases catalyze the hydrolysis of triglycerides which cause rancid, butyric, or soapy flavors and also may lead to a reduction in milk foaming properties Lipxse et al. Proteases hydrolyze casein fractions and produce defects described as bitter off-flavors and result in age gelation Stoeckel et al. Therefore, spoilage caused by psychrotrophic bacteria and their enzymes is a major concern in the dairy industry. In our previous study Yuan et al.

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Four-hundred and eighty strains previously isolated from Chinese raw milk samples were studied Yuan et al. Strains that showed positive protease production on screening medium were selected to quantify their total proteolytic activity. Proteolytic activity was measured using azocasein Sigma, USA as a substrate following the protocol described by Santos et al.


Each assay was performed in triplicate and with three replicates. Strains that showed positive lipase production on screening medium were selected to quantify the total lipolytic activity.

Strains that proved to be positive for protease or lipase production were selected to determine the thermo-resistance of their enzymes. After heat treatment, samples were cooled immediately in an ice bath followed by the addition of 0. Proteolytic and lipolytic activity was measured as described above. Strains shown to produce heat-stable enzymes in the tests described above were selected for further thermal inactivation trials. Milk samples divided into aliquots of 5 ml in sterile tubes were heat-treated under the following time-temperature conditions.

For protease, three different temperature-time combinations were used: For lipases, different temperature-time combinations were used: After each heat treatment, samples were cooled immediately in an ice bath followed by the addition of 0. Log of percentage residual activity R was plotted against heating time and expressed as the thermo-stability. Isolates of PseudomonasAcinetobacterFlavobacteriumChryseobacteriumSerratiaand Aeromonas were not only the most predominant in raw milk samples, but also proved to be high enzyme producers.

Among the Pseudomonas isolates, showed proteolytic activity and showed lipolytic activity Fig. Species belonging to Aeromonas produced all four types of enzymes, indicating their high spoilage potential. A total of isolates showed the ability to produce at least one type of enzyme, while, and 18 isolates produced two, three, and four different enzymes, respectively Fig. Species of Acinetobacter exhibited high lipolytic 56 out of 64 strainsbut weak proteolytic 10 out of 64 and phospholipase 22 out of 64 activity.

The proteolytic activity of the predominant psychrotrophic bacteria is shown in Fig. The range of proteolysis was 1. Species of Acinetobacter showed low proteolytic activity, and the lowest proteolytic activity was shown by Acinetobacter johnsonii a.

Isolates belonging to Serratia showed high proteolytic activity with a mean value of 8. The highest protease activity was shown by Y. Rheinheimera chironomi showed the lowest protease activity followed by A. The lipolytic activity of the predominant psychrotrophic bacteria is shown in Fig.

The range of lipolytic activity was 0. The lowest lipase activity was shown by Pseudomonas poae. Isolates belonging to Acinetobacter showed relatively high lipase activity with a mean value of 7. The highest lipolytic activity was shown by A.

As produvtion in Fig. An increase in enzyme inactivation correlated well with temperature and heating time. E a ranged from Proteases produced by S. Lipase produced by A. Heat-stable enzymes produced by psychrotrophic bacteria cannot be completely inactivated by thermal processing techniques and pose threats to the quality and shelf-life of dairy products.

Some studies have reported results of screening for enzymes produced by psychrotrophic bacteria isolated from raw milk von Neubeck et al. However, quantitative assessments and the thermo-stabilities of proteases and lipases have rarely been reported.

The objective of this work was to explore the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of proteases and lipases they produced. A wide variety of psychrotrophic bacteria can produce spoilage enzymes during late log or stationary growth lipzse Rajmohan et al.

Most isolates in this study had enzymatic activity at low temperatures. Pseudomonas is reported to be the most frequently isolated genus in raw milk, probably because of its short generation bypseudomonqs von Neubeck et al. Enxyme spoilage potential of Pseudomonas has been extensively studied and those bacteria tend to produce proteases combined with lipases Capodifoglio et al.

In this study, most Pseudomonas species, bypseudominas as P. The aprX gene coding for the protease is located at the beginning of a polycistronic operon with the lipA gene that encodes the lipase Woods et al. Thus, most isolates belonging to to Pseudomonas have the ability to produce proteases and lipases due to the simultaneous expression of both genes.


However, expression of enzymes can be tightly regulated, which may explain the absence of one or both enzymes in some isolates. In this work, some isolates belonging to P. This phenotypic bypseudomlnas within the same species is consistent with previous findings Wiedmann et al. For this reason, in the future, whole genome sequencing of these isolates will be carried out to differentiate their spoilage potential.

Acinetobacter is considered to be the predominant bacterial genus in raw milk von Neubeck vypseudomonas al. This genus is ubiquitous in nature and characterized by the tendency to tolerate dry conditions and multidrugs Gurung et al. In the current work, all isolates belonging to Acinetoba showed high lipolytic activity and low proteolytic activity, which is in agreement with previous studies von Neubeck et al.

Milk-deteriorating exoenzymes from Pseudomonas fluorescens isolated from refrigerated raw milk

However, a high number of Acinetobacter isolates with proteolytic activity were found in Mozzarella cheese. This might be explained by horizontal gene transfer from proteolytic species or phenotypic variation enzyne isolates. Chryseobacterium also appears as a dominant member of raw milk microbiota Yuan et al. In this study, species of Chryseobacterium showed more proteolytic than lipolytic activity, and C. To our knowledge, few studies have reported the spoilage potential of Flavobacterium.

The results of our study suggest that it has high proteolytic but weak lipolytic activity. Species of Serrati are bypseuxomonas characterized as predominant milk spoilers due to their ability to produce heat-stable proteases and lipases Machado et al.

Hydrolytic potential of a psychrotrophic Pseudomonas isolated from refrigerated raw milk

However, isolates belonging to other predominant prdouction SphingobacteriumRahnellaButtiauxellaand Janthinobacterium lacked the ability lipqse produce both proteases and lipases. Thus, the population of psychrotrophic bacteria plays a key role in the determination of the quality of dairy products.

Phospholipase activity was found among the isolates belonging to the genera PseudomonasAcinetobacterFlavobacteriumSerratiaChryseobacteriumJanthinobacteriumand Aeromonasconsistent with their ability to cause sweet curdling defects or bitter cream in milk as a result of fat globule aggregation Titball, ; Vithanage et al. The production of enzymes is a temperature-dependent process Buchon et al.

As reported by Decimo et al. Considering the potential effects of enzymes on dairy products, appropriate practices should be adopted productkon minimize the quality losses, by either limiting the growth of psychrotrophic bacteria or inactivation of their enzymes by thermal processing.

Adequate addition of CO 2 to raw milk has been shown to reduce proteolysis and lipolysis by limiting microbial growth and the byseudomonas of microbial proteases Ma et al. However, the effects of these practices on the heat stability of enzymes are unknown.

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The heat stability of enzymes originating from psychrotrophs is a limiting factor in maintaining the quality and shelf-life of dairy products. Thus, better knowledge of their heat stability is needed. Reducing the activity and limiting the secretion of heat-stable enzymes are scientific challenges.

Kinetic parameters for the thermal inactivation of P. In previous studies, the thermo-stability of proteases produced by Pseudomonas or Serratia has been extensively reported.

The D -values of proteases produced by S. The D -values of proteases produced by Pseudomonas proteolytica were 6. The residual activity of protease produced by P.