Elsevier

Food Control

Volume 111, May 2020, 107063
Food Control

Milk substrates influence proteolytic activity of Pseudomonas fluorescens strains

https://doi.org/10.1016/j.foodcont.2019.107063Get rights and content

Highlights

  • Bacterial proteolysis in milk agar plates may differ from that in fluid milk.

  • Presence of fat influences proteolytic activity of Pseudomonas fluorescens in milk.

  • Pasteurized milk was the only substrate where proteolysis led to gelation onset.

  • Skim milk powder is not a suitable substrate for evaluating microbial proteolysis.

Abstract

Pseudomonas fluorescens spoiling raw milk produces a heat-stable protease, namely AprX, that may degrade k-casein with a chymosin-like activity thus causing gelation of commercial milk during storage. Four strains of P. fluorescens were selected for both the presence of aprX gene and proteolytic activity in milk agar plate (a negative control was included) and were incubated in various milk substrates, i.e. pasteurized milk, UHT milk and reconstituted milk powder, differing for heat-treatment and presence of fat, in order to evaluate whether the type of milk substrates could affect their growth and proteolytic activity. While bacterial growth was mainly influenced by temperature (4 or 25 °C) for all strains, HPLC and CZE patterns of incubated milk samples showed that the extent and trend of proteolysis were highly heterogeneous and not exclusively strain-dependent. Indeed, pasteurized milk was the only substrate where aprX-positive strains led to gelation onset whereas other milk types underwent different destabilization. Ultrastructural features observed by transmission electron microscopy for casein micelles, whey proteins and fat globules, where present, explained how the processing conditions, sometimes including repeated heat-treatments, may have influenced the extent of proteolysis operated by P. fluorescens strains in the tested milk substrates. This study has highlighted that different milk substrates may bring to different conclusions when used in experiments aiming to elucidate the mechanisms of bacterial proteolysis since both ultrastructural and compositional properties may impact on accessibility of cleavage sites to proteases.

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)

  • D. Ercolini et al.

    Molecular identification of mesophilic and psychrotrophic bacteria from raw cow's milk

    Food Microbiology

    (2009)
  • J. Janzen et al.

    Relationship of protease activity to shelf-life of skim and whole milk

    Journal of Dairy Science

    (1982)
  • J. Ji et al.

    Rehydration behaviours of high protein dairy powders: The influence of agglomeration on wettability, dispersibility and solubility

    Food Hydrocolloids

    (2016)
  • H. Lin et al.

    Prediction of growth of Pseudomonas fluorescens in milk during storage under fluctuating temperature

    Journal of Dairy Science

    (2016)
  • M. Lu et al.

    Spoilage of milk and dairy products

  • B. Malmgren et al.

    Changes in proteins, physical stability and structure in directly heated UHT milk during storage at different temperatures

    International Dairy Journal

    (2017)
  • S. Marchand et al.

    Heterogeneity of heat-resistant proteases from milk Pseudomonas species

    International Journal of Food Microbiology

    (2009)
  • N.H. Martin et al.

    Symposium review: Effect of post-pasteurization contamination on fluid milk quality

    Journal of Dairy Science

    (2018)
  • S. Masiello et al.

    Identification and characterization of psychrotolerant coliform bacteria isolated from pasteurized fluid milk

    Journal of Dairy Science

    (2016)
  • A. Matéos et al.

    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

    (2015)
  • R. McKellar

    Development of off-flavors in ultra-high temperature and pasteurized milk as a function of proteolysis

    Journal of Dairy Science

    (1981)
  • S. Nasser et al.

    Microstructure evolution of micellar casein powder upon ageing: Consequences on rehydration dynamics

    Journal of Food Engineering

    (2017)
  • Cited by (20)

    • An Illumina MiSeq sequencing-based method using the mreB gene for high-throughput discrimination of Pseudomonas species in raw milk

      2022, LWT
      Citation Excerpt :

      Which could become more low-temperature threatening species. Furthermore, the species possessed the highest protease expression (Colantuono et al., 2020) and biofilm-producing ability (Cruz & da Motta, 2019). This suggests that pollution control of this species should be considered first.

    • Whey protein influences the production and activity of extracellular protease from Pseudomonas fluorescens W3

      2022, LWT
      Citation Excerpt :

      Datta and Deeth (2003) also found that the protease AprX has the strongest ability to hydrolyze κ-casein, however, D'Incecco et al. (2019) found that the protease AprX has the strongest ability to hydrolyze β-casein. AprX has chymosin-like activity and can specifically hydrolyze the 105–106 peptide bonds of κ-casein to produce Casein Macro Peptide, after κ-casein is hydrolyzed, the casein micelles are exposed, and the damaged casein micelles aggregate with each other to form gelation (Colantuono et al., 2019; D'Incecco et al., 2019). The hydrolysis of κ-casein by protease AprX can promote the curdling of cheese and reduce the curdling time, however, excessive protease will cause harm to cheese quality, so it is very important to know the minimum protease concentration that harms the quality of dairy products (Paludetti, O'Callaghan, Sheehan, Gleeson, & Kelly, 2020).

    • Insight into antibacterial mechanism of polysaccharides: A review

      2021, LWT
      Citation Excerpt :

      For instance, Staphylococcus aureus produced staphylococcus enterotoxin in the contaminated food and caused serious gastroenteritis (Wu, Li, et al., 2019). Pseudomonas fluorescens can spoil the milk products by producing thermostable alkaline metallo-protease and then influence human digestion functions (Colantuono et al., 2020). Although the preservation methods of desiccation and refrigeration can effectively delay food spoilage, the flavor of foods would be altered after such processes (Lin, Gu, Li, Vittayapadung, & Cui, 2018).

    • Biomarkers associated with cheese quality uncovered by integrative multi-omic analysis

      2021, Food Control
      Citation Excerpt :

      However, the possibility that some of these S. thermophilus may have originated from post-pasteurisation contamination cannot be discounted either (Gobbetti et al., 2015). In addition to excessive growth of L. lactis, other genera such as Pseudomonas known to produce off-flavours in foods (Colantuono et al., 2020) were present in greater amounts in the low-quality cheese samples. Metabolome fingerprints also varied between the cheeses of different qualities.

    • Identification, subtyping, and tracking of dairy spoilage-associated Pseudomonas by sequencing the ileS gene

      2021, Journal of Dairy Science
      Citation Excerpt :

      In contrast, Pseudomonas spp. isolated from raw bovine milk exhibited much more variation than we observed in low-temperature proteolysis for these 3 species groups (Meng et al., 2017). Identification of spoilage-associated enzymatic activities in pure culture systems provides initial data on spoilage capabilities of isolates; however, it should be noted that spoilage expression in fluid milk may depend on additional variables, such as prior heat treatment, storage conditions, and presence of additional types of bacteria (Worm et al., 2000; Nicodème et al., 2005; Colantuono et al., 2020). Future studies may benefit from correlating spoilage phenotypes with presence and genotype of the aprX-lipA operon, which encodes extracellular protease and lipase enzymes that may be associated with product degradation (Woods et al., 2001; Zhang et al., 2019).

    View all citing articles on Scopus
    View full text