Milk fat influences proteolytic enzyme activity of dairy Pseudomonas species

https://doi.org/10.1016/j.ijfoodmicro.2020.108543Get rights and content

Highlights

  • The proteolytic activity of all six dairy isolates of Pseudomonas strains varies with the strongest being P. lundensis DZ845.

  • Pseudomonas showed more proteolytic activity in milk medium than TSB.

  • Milk fat induces the activity or production of protease from Pseudomonas.

Abstract

This study investigated the effect of growth conditions on proteolytic activity of six Pseudomonas strains, (Pseudomonas fragi DZ1, Pseudomonas koreensis DZ138, Pseudomonas rhodesiae DZ351, Pseudomonas fluorescens DZ390, Pseudomonas synxantha DZ832 and Pseudomonas lundensis DZ845), isolated from raw milk. The proteolytic activity of all Pseudomonas strains in dairy media (skim milk and whole milk) was significantly higher (p < 0.05) than in non-dairy media (TSB), with most activity from Pseudomonas grown in whole milk. The proteolytic activity from P. lundensis DZ845 grown in TSB with the addition of 5% (w/v) butter was higher than other dairy ingredients added to TSB and the amount of proteolytic activity increased with increasing concentrations of butter (from 5 to 15%). P. rhodesiae DZ351 showed little proteolytic activity in all TSB supplemented with dairy ingredients. Only four of the six strains produced one protease of 47 kDa when grown in TSB. However, all six strains were able to produce at least one type of proteases in milk medium. For P. lundensis DZ845, a 12% casein zymography gel revealed that the presence of butter could induce proteolytic activity. This is the first study showing the effect of milk fat (butter) on the proteolytic activity of Pseudomonas. This highlights the greater vulnerability of whole milk compared to skim milk to proteolytic activity.

Introduction

Refrigerated storage of raw milk is a common practice to minimise the spoilage of milk caused by the growth of mesophilic microorganisms. However, this could enable the growth of psychrotrophic bacteria (psychrotrophs), which are able to produce proteolytic enzymes, many of which are resistant to heat treatment such as ultra-high temperature (UHT) processing (Hahne et al., 2019). Proteolytic enzymes hydrolyse peptide bonds in milk proteins, resulting in the alteration of physico-chemical, functional and sensory properties of milk and milk products. More specifically, proteolysis results in bitter flavours, gelation and spoilage in milk (Chen et al., 2003; Hantsis-Zacharov and Halpern, 2007). Pseudomonas species are the most commonly present psychrotrophs found in chilled raw milk at the time of spoilage (Ercolini et al., 2009; Raats et al., 2011). The Pseudomonas species like Pseudomonas fluorescens, Pseudomonas fragi and Pseudomonas lundensis constitute the predominant microorganisms limiting the shelf life of UHT milk (Marchand et al., 2009a). Most of their proteases are alkaline metalloproteases belonging to the serralysin family (AprX), with a molecular mass of about 50 kDa (Matéos et al., 2015). These enzymes are typically produced in the late log or early stationary growth phase. Ercolini et al. (2009) reported that milk products develop off-flavours when the number of Pseudomonas spp. reaches 2.2 × 106–3.6 × 107 cfu/mL, most likely due to these proteases. This suggests that the quality of the final product is negatively affected as time increases since the spoilage causing proteases produced by Pseudomonas correlates with the growth of the bacteria that increase with time. In order to maintain the quality and prolong the shelf life of the dairy products, controlling the growth of Pseudomonas and their proteolytic activity in raw milk prior to any heat treatment is of paramount importance.

There have been some studies investigating the effect of growth conditions including pH, temperature, and mineral concentrations on the proteolytic activity of Pseudomonas (Gügi et al., 1991; Hellio et al., 1993; Rahman et al., 2005; Zhang et al., 2015). For example, the growth rate and proteolytic activity of P. fluorescens grown in nutrient broth are less than when grown in skim milk (Zhang et al., 2015). The production of protease is known to require the presence of an inducer in the culture medium (Nicodème et al., 2005). Skim milk is an efficient inducer for some Pseudomonas strains, but the influence of whole milk is unknown. Furthermore, the effect of individual milk components on the bacterial growth and their proteolytic activity has not been well studied.

The most common species found in raw milk are P. fluorescens, following by P. fragi and P. lundensis, so they are the most studied species (von Neubeck et al., 2016; Zhang et al., 2015). Pseudomonas species P. rhodesiae and P. synxantha have also been frequently isolated from raw milk (von Neubeck et al., 2017; C. Zhang et al., 2019), but there is very little information on their growth and proteolytic activity which may potentially be involved in spoilage of milk products. This research investigated the effect of dairy ingredients on the bacterial growth and proteolytic activity of six Pseudomonas species in both dairy and non-dairy media. This knowledge will enable the dairy industry to understand the role of dairy ingredients in triggering bacterial proteolytic activity and provide information to help in controlling protease activity in dairy product manufacture.

Section snippets

Bacterial strains

In our previous study of New Zealand raw milk samples, a large set of Pseudomonas were isolated and identified by MALDI-TOF MS (D. Zhang et al., 2019). Six dominant Pseudomonas strains (P. fragi DZ1, P. koreensis DZ138, P. rhodesiae DZ351, P. fluorescens DZ390, P. synxantha DZ832 and P. lundensis DZ845) showing high proteolytic activity by agar diffusion at 7 °C for 7 days were selected for this particular study. All strains were stored in microbeads at −80 °C and were pre-incubated at 20 °C

Bacterial growth and proteolytic activity in TSB, skim milk and whole milk

The growth of six Pseudomonas strains at 7 °C for 7 days in TSB, skim milk and whole milk and their proteolytic activity is shown in Figs. S1–6. The viable counts increased as expected over the seven days incubation period. The strains used in this study had similar growth curves in all three media. The initial bacterial counts ranged from 2 to 4 log cfu/mL and the final counts for all the samples were around 9–10 log cfu/mL. All the strains reached late log phase or early stationary phase

Discussion

Pseudomonas species are the predominant species among the psychrotrophs in refrigerated milk due to their tendency to outgrow other bacteria present in refrigerated raw. They are known for their production of heat-resistant proteases in the refrigerated raw milk (Raats et al., 2011; Von Neubeck et al., 2015; Xin et al., 2017). These proteases are very heat-resistant and even survive after UHT processing, leading to spoilage of dairy products (Samaržija et al., 2012). In this study, we examined

Conclusions

The findings have satisfied the aim of this paper to determine the effects of dairy ingredients on the proteolytic activity of six Pseudomonas species at 7 °C for 7 days. The triggering of the proteolytic activity of Pseudomonas is a complex process influenced by medium composition, particularly milk fat.

Acknowledgements

This work was financially supported by Tetra Pak, New Zealand.

References (35)

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