Milk substrates influence proteolytic activity of Pseudomonas fluorescens strains
Introduction
Psychrotrophic bacteria are ubiquitous and are able to grow at low temperatures (<7 °C), thus can easily prevail during the refrigerated storage of raw milk (Lu & Wang, 2017; Martin, Boor, & Wiedmann, 2018). Just after milking, psychrotrophic bacteria only account for a small fraction of raw milk microbial population, whose composition is closely related to the health status of the cows and hygienic conditions of milking equipment. Subsequently, their prevalence is influenced by transportation and storage conditions of milk (Lu & Wang, 2017; Vithanage et al., 2016). Raw milk is usually stored at refrigeration temperatures (<6–7 °C) until pasteurization or ultra-high temperature (UHT) treatment, in order to delay bacterial growth and preserve original quality attributes. However, it was estimated that psychrotrophic bacteria can increase up to 50% of the whole microbial population after one-day cold storage and more than 90% after two days cold storage (Lafarge et al., 2004). During their exponential growth phase, as well as in the early stationary phase, psychrotrophic bacteria produce heat-resistant proteolytic and lipolytic enzymes that, during storage, negatively affect quality attributes of milk and milk derived products, including milk powders (Alves et al., 2018; Chen, Daniel, & Coolbear, 2003; Stevenson, Rowe, Wisdom, & Kilpatrick, 2003). Specifically, proteolytic deterioration of milk has been related to development of astringent or bitter flavors and visually detectable alterations such as sediment formation, gelation or coagulation (D'Incecco et al., 2019; Marchand, Duquenne, Heyndrickx, Coudijzer, & De Block, 2017; Stoeckel et al., 2016). Similarly, bacterial lipases activity may cause off-flavors and rancid taste development (Bekker et al., 2016). The contamination by psychrotrophic bacteria is among the primary causes of premature physicochemical deterioration of drinking milk, and represents a relevant concern for the industry since it leads to reduced acceptance of the product, with significant economic losses and reputational damages for the companies.
The population of psychrotrophic bacteria contaminating raw milk is composed by different microbial genera, mainly including Acinetobacter spp., Achromobacter spp., Aeromonas spp., Alcaligenes spp., Bacillus spp. Enterobacter spp., Flavobacterium spp., Pseudomonas spp. and Serratia spp., with Pseudomonas spp. being the predominant one (Ercolini, Russo, Ferrocino, & Villani, 2009; Zhang, Palmer, Teh, Biggs, & Flint, 2019). The genus Pseudomonas is a heterogeneous group of aerobic, mesophilic and psychrotolerant, non spore-forming Gram-negative bacteria having a shorter generation time compared to other species of psychrotrophic bacteria (Sørhaug & Stepaniak, 1997). P. fluorescens can produce a thermostable alkaline metallo-protease named AprX, that plays a predominant role in spoilage of milk products (Dufour et al., 2008; Machado et al., 2017; Marchand et al., 2009; Matéos et al., 2015).
However, despite the advancements in elucidating the role of P. fluorescens enzymes in milk deterioration, literature data are sometimes contradictory and difficult to compare (Zhang, Bijl, Svensson, & Hettinga, 2019). A major reason for this situation could be the fact that studies are conducted using different milk substrates, where a multitude of aspects may interfere. Reconstituted skim milk powder (SMP) with further sterilization by autoclaving is often used as standard substrate, since commercial SMP is long shelf-stable and easy to use (Anema, 2017). Some authors studied the activity of AprX by growing P. fluorescens in UHT milk (Baglinière et al., 2012), or by adding the enzyme extract to single casein fractions (Recio, García-Risco, Ramos, & López-Fandiño, 2000; Stuknytė et al., 2016). Other authors inoculated the strains either in microfiltered pasteurized milk (Brasca et al., 2016; D'Incecco et al., 2019) or in microfiltered raw skim milk (Gaucher et al., 2011) before incubation and sterilization. These milk products undergo very different processing conditions during both preparation and storage. Principally, thermal treatments used may vary from very mild conditions, such as those of pasteurization (72–75 °C for few seconds), up to sterilization and drying, where temperatures exceeding 100 °C are reached. It is well known that heating induces changes to milk components such as protein glycosylation via the Maillard reaction, enzyme inactivation, whey protein denaturation and interaction with casein micelles (Cattaneo, Masotti, & Pellegrino, 2012). In turn, presence of fat makes also a difference since both casein and whey proteins may bind to fat globules to form large aggregates that can be further stabilized (or even disrupted) during high-pressure streaming through the homogenizer valve or the spray-dryer nozzle (D'Incecco, Rosi, Cabassi, Hogenboom, & Pellegrino, 2018a). Several studies evidenced that selected milk products have very different ultrastructure (Auty, 2011) and, recently, the presence of stable aggregates is reaching increasing attention (Raak, Abbate, Lederer, Rohm, & Jaros, 2018). However, to the authors knowledge, these aspects have been largely underestimated in studies intended to evaluate microbial metabolic diversities in milk.
In this context, this paper aimed to elucidate the possible effects of ultrastructure and composition of selected milk substrates on growth and proteolytic activity of P. fluorescens strains coming from different sources. Pasteurized milk and UHT milk, also having different fat contents, as well as SMP were considered in this study. Beside overall proteolysis, the main focus was on the specific activity of AprX on k-casein due to its role in inducing milk gelation. Because of its great power in spatial resolution, transmission electron microscopy (TEM) was used to support interpretation of the analytical results. Overall, the experimental plan was arranged to gain fundamental information for selecting the suitable substrate for studies dealing with microbial behavior in milk.
Section snippets
Chemicals
Water was obtained through a Milli-Q purification system (Millipore, USA). Trifluoroacetic acid (TFA), 10X reaction buffer, Taq polymerase, Gene-Ruler™ DNA ladder mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Osmium tetroxide and Spurr resin were obtained from EMS (Hatfield, PA, USA). Glutaraldehyde, paraformaldehyde and sodium cacodylate were purchased from Agar Scientific (Stansted, UK). Agarose was from VWR (Milan, Italy). Primers were obtained from Eurofins Scientific
Characterization and selection of Pseudomonas fluorescens strains
A total of 15 strains were identified as belonging to P. fluorescens spp. or genetically related species, based on the presence of fragments of 600 base pairs (bp) revealed by RSA analysis and the positive record of species-specific PCR. The results concerning the detection of the aprX gene and the evaluation of the proteolytic activity of strains in skim milk agar plates, are summarized in Table 1. The aprX gene was detected in all strains, with the exception of the WA10NA strain that,
Discussion
Due to its documented role in milk spoilage, P. fluorescens is an excellent test microorganism to investigate growth rate, proteolytic activity and promotion of instability signs in different milk substrates (Oliveira, Favarin, Luchese, & McIntosh, 2015). The proteolytic activity of this species is mainly associated to the production of an extracellular thermostable alkaline metallo-protease referred as AprX (Dufour et al., 2008; Machado et al., 2017; Marchand et al., 2009; Matéos et al., 2015
Author contributions
Conceived and designed the experiments: A.C., P.D., M.G.F., L.P. Performed the experiments: A.C., P.D., V.R., G.R. Analysed the data: A.C., P.D. Wrote the paper: A.C., P.D., M.G.F., L.P.
Declaration of competing interest
Authors declare no conflict of interest.
Acknowledgements
Microscopy observations were carried out at The Advanced Microscopy Facility Platform – UNItech NOLIMITS – University of Milan.
References (45)
- et al.
Temperature modulates the production and activity of a metalloprotease from Pseudomonas fluorescens 07A in milk
Journal of Dairy Science
(2018) Storage stability and age gelation of reconstituted ultra-high temperature skim milk
International Dairy Journal
(2017)Analytical methods | microscopy (microstructure of milk constituents and products)
- et al.
Quantitative and qualitative variability of the caseinolytic potential of different strains of Pseudomonas fluorescens: Implications for the stability of casein micelles of UHT milks during their storage
Food Chemistry
(2012) - et al.
Lipid breakdown and sensory analysis of milk inoculated with Chryseobacterium joostei or Pseudomonas fluorescens
International Dairy Journal
(2016) - et al.
Detection and impact of protease and lipase activities in milk and milk powders
International Dairy Journal
(2003) - et al.
Bacterial proteolysis of casein leading to UHT milk gelation: An applicative study
Food Chemistry
(2019) - et al.
Effect of temperature on the microstructure of fat globules and the immunoglobulin-mediated interactions between fat and bacteria in natural raw milk creaming
Journal of Dairy Science
(2018) - et al.
Microfiltration and ultra-high-pressure homogenization for extending the shelf-storage stability of UHT milk
Food Research International
(2018) - et al.
Molecular typing of industrial strains of Pseudomonas spp. isolated from milk and genetical and biochemical characterization of an extracellular protease produced by one of them
International Journal of Food Microbiology
(2008)
Molecular identification of mesophilic and psychrotrophic bacteria from raw cow's milk
Food Microbiology
Relationship of protease activity to shelf-life of skim and whole milk
Journal of Dairy Science
Rehydration behaviours of high protein dairy powders: The influence of agglomeration on wettability, dispersibility and solubility
Food Hydrocolloids
Prediction of growth of Pseudomonas fluorescens in milk during storage under fluctuating temperature
Journal of Dairy Science
Spoilage of milk and dairy products
Changes in proteins, physical stability and structure in directly heated UHT milk during storage at different temperatures
International Dairy Journal
Heterogeneity of heat-resistant proteases from milk Pseudomonas species
International Journal of Food Microbiology
Symposium review: Effect of post-pasteurization contamination on fluid milk quality
Journal of Dairy Science
Identification and characterization of psychrotolerant coliform bacteria isolated from pasteurized fluid milk
Journal of Dairy Science
Proteolysis of milk proteins by AprX, an extracellular protease identified in Pseudomonas LBSA1 isolated from bulk raw milk, and implications for the stability of UHT milk
International Dairy Journal
Development of off-flavors in ultra-high temperature and pasteurized milk as a function of proteolysis
Journal of Dairy Science
Microstructure evolution of micellar casein powder upon ageing: Consequences on rehydration dynamics
Journal of Food Engineering
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