Detection of protease and lipase activity in Pseudomonas fluorescens isolated from milk and surfaces as milk deteriorating indicators

1. Pseudomonas fluorescens is a predominant bacterium causing milk spoilage.
2. Pseudomonas fluorescens is widely distributed in environments
3. Pseudomonas fluorescens excreted deteriorating exoenzymes affecting milk quality.

Introduction

1. fluorescens represents a saprophytic nonpathogenic with poor nutritional requirements and great diversity enabling them to survive in different environments including utensils used in the dairy production chain (1,2). These bacteria cause dairy product spoilage through their ability to produce some exoenzymes mainly proteases and lipases (3,4). Sometimes pseudomonas counts aren’t indicative of the assessment of milk spoilage (5,6). Although some pseudomonas species become inactive after the thermal processing of milk like pasteurization their exoenzymes revealed high resistance to heat and affected milk shelf-life by casein and fat degradation (7,8). One of these enzymes is Alkaline zinc metalloprotease which consists of one zinc atom with eight atoms of calcium and a high molecular weight ranging between 21-25 kilodaltons their activity depends on the presence of calcium ions (9,10). AprX hydrolysez k-casein, para k-casein and beta-casein releasing plasmin and plasminogen which create unacceptable changes in milk (11). P. fluorescens produce lipolytic enzymes giving the rancid odor and flavor of dairy products (12,13). Lipases have a molecular mass between 30-50 KDa with optimum alkaline PH (14). The production of lipase is affected by the concentration and the type of nitrogen, iron and carbon whereas the presence of magnesium, zinc and iron decreases the activity of lipase (15,16). Molecular methods including polymerase chain reaction assay are used to monitor the impact of protease and lipase by detecting related genes represented by aprX and lip genes as spoilage indicators to evaluate milk quality (17). Local research studies the molecular features and antimicrobial sensitivity of different bacterial isolates in some dairy products including some Pseudomonas spp. especially P. aeruginosa (18-20).

A few of them investigate the impact of P. fluorescens exoenzymes on milk quality therefore this study aims to screen the presence of aprX and lip genes in P. fluorescens isolated from cow milk teat surfaces and milk tanks in dairy farms in Nineveh province.

Material and methods

Ethical approval

The research was conducted considering the ethical approval of the institutional Animal Care and Use Committee at the College of Veterinary Medicine, University of Mosul and included an authorized ID of UM.VET. 2023.102. Mosul, Iraq,2024.

Samples

The study included collecting one hundred fifty samples of cows' raw milk, teat surfaces swabs and milk tank swabs from Nineveh province dairy farms to detect the presence of P. fluorescens Thirty-nine positive isolates of P. fluorescens were distributed as 19,11, and 9 isolates from raw milk, teat surfaces and milk tanks respectively were examined to investigate their abilities to produce exoenzymes as spoilage indicators.

Isolation and identification

samples were cultivated on Cetrimide agar (Neogen/USA) incubated at 25°C for 24-48 hours then purified on Cetrimide agar and followed by biochemical tests (21). The P. fluorescens isolates were confirmed using a polymerase chain reaction depending on the16srRNA gene.

DNA extraction

1. fluorescens colonies were subjected to DNA extraction depending on the bacterial DNA preparation kit (AddBio, Korea) following the manufacturer’s instruction protocol.

Polymerase chain reaction (PCR)

1. fluorescens protease and lipase activity were investigated using PCR assay depending on the detecting aprX and lip genes respectively. A specific primer was provided by (Macrogen/Korea). The primer consists of forward and reverse primers (22) with a molecular weight of 1434 bp and 1422 bp to aprX and lip gene respectively (Table1). The thermal profile included an initial denaturation of 10 min. at 95ºC followed by 35 cycles of 95ºC for 45s, then annealing 58ºC, 55ºC for 45 sec. for aprX and lip gene respectively, extension at 72ºC for 1 min. and final extension of 72ºC for 5 min. with cooling at 4ºC. PCR products were analyzed by electrophoresis (1.5% agarose gel) (AddBio, Korea) with 3 μl GelRed dye (AddBio, Korea). 5 μl of each PCR product was loaded into the well of agarose gel. The electrophoresis was carried out at 75 volts for 1 hour. The band was identified using the Gel doc EZ image (Bio-Rad, USA).

Table 1: Oligonucleotide primers sequence of P. fluorescens exoenzymes genes used in the current study

Results

Results showed the detection of protease and lipase exoenzymes genes of P. fluorescens strains isolated from cow raw milk, teat surfaces and milk tanks including aprX and lip genes. The aprX gene was detected in 58.9% (23/39) of isolates. The highest rate of aprX genes prevalence was found in the P. fluorescens isolates from teat surfaces (9/11) which was 81.8% compared to isolates from raw milk (7/19) which was 36.8%. The lip gene was detected in 23.1% (9/39) of isolates. P. fluorescens isolates from milk tanks revealed a higher percentage of the presence of lip gene (3/9) at 33.3% followed by isolates from raw milk (5/19) at 26.3% compared to P. fluorescens isolates from teat surfaces (1/11) 9.1% (Table 2 and Figure 1). The aprX and lip genes were detected at 1434 bp and 1422 bp respectively as shown in (Figures 2 and 3).

Table 2: Detections of protease and lipase activity in milk, teat surfaces and milk tanks

Figure 1: Percentage of protease and lipase genes in P. fluorescens isolates from milk, teat surfaces, and milk tanks.

Figure 2: Amplified products of aprX gene of P. fluorescens, Lanes M represent 100 bp DNA marker, lane 1-2; negative samples, lane 3-11; positive samples at 1434 bp product size, lane 12; negative control.

Figure 3: Amplified products of lip gene of P. fluorescens, Lanes M represent 100 bp DNA marker; lanes 1, 2, 4, 5, 6, 11, 12, 13, 14, 15, 18; negative samples, lanes 3, 7, 8, 9, 10, 16, 17; positive samples at 1422 bp product size lane 19; negative control.

Discussion

36. fluorescens is commonly isolated from soil, water and surfaces as a saprophytic bacterium (23,24) these species can secrete some enzymes including proteases and lipases exoenzymes and a number of these secreted enzymes depends on the P. fluorescens strain which highlighted the vast diversity of P. fluorescens isolates (25-27). aprX gene and lip gene were used as a marker to detect P. fluorescens protease and lipase activity in milk depending on PCR technique and distinguish proteolytic and lipolytic strains (28,29), to reduce the time for detecting P. fluorescens as deteriorating agents in raw milk and providing more flexible work to the dairy manager to assess milk quality (23,30). Therefore, PCR assay was used as a good and sensitive approach to detect the presence of protease and lipase enzymes as spoilage indicators to estimate the degradation of milk components including casein protein and lipids (31). The results of this study revealed the presence of the aprX gene in 36.8% of isolates from raw milk These results agree with the results obtained by De Longhi (32) who showed the presence of the aprX gene in 37% of P. fluorescens strains isolated from milk which indicates this bacterium’s presence in farm animals’ milking environments (33,34). aprX gene was observed in 71.2% of P. fluorescens isolates in bovine milk and the high presence of the aprX gene in cow raw milk is associated with sensory defects affecting the shelf life of milk (35). The presence of calcium is an essential factor for the potency and resistance of proteases (36,37). Also increased proteolytic activity in P. fluorescens may be related to the type of N-acyl homoserine lactone (AHL) either directly or indirectly (38). Although the lip gene was detected in raw milk and milk tank and teat surfaces their presence is at a lower limit than that of the aprX gene. These results disagree with Ribeiro (35) who revealed the lipolytic activity in 25% of P. fluorescens isolates from cow milk. In comparison the lipolytic activity of cow milk was evaluated in 9.3% of P. fluorescens isolates (39). It may be because protease was more resistant at a wide range of PH and temperatures (25,40,41) suggesting the adaptation of P. fluorescens strains to various environmental circumstances previous studies referred to the proteolytic activity of P. fluorescens in the late exponential and early stationary phase of growth (42-44). However, rapid and sensitive methods are essential to ensure product safety and perform risk assessment Preventive measures should be taken to reduce aprX biosynthesis in milk and dairy products.

Conclusion

Early detection of milk spoilage due to P. fluorescens is a significant predictive factor. This bacterium secretes exoenzymes represented by protease and lipase enzymes by identifying specific genes. Therefore, the activity of these exoenzymes is a limiting factor maintaining milk quality.

Acknowledgments

This research was supported using resources from the University of Mosul's College of Veterinary Medicine in Mosul, Iraq.

Conflict of interest

The authors confirm there was no conflict of interest.