Study of Proteolytic and Lipolytic Activities of Pseudomonas spp . Isolated From Pasteurized Milk in Tunisia

Psychrotrophic bacteria have been recognized as a recurring problem in refrigerated storage and distribution of perishable food products. This study was performed to isolate, characterize and evaluate the prevalence of Pseudomonas spp. in pasteurized milk. Thirty (30) pasteurized milk samples were collected aseptically from different dairy shops, supermarkets and groceries in different areas in Sfax Governorate, Tunisia and analyzed for total viable count and Pseudomonas spp. occurrence and explain their proteolytic and lipolytic activities. The results showed that Pseudomonas aeruginosa was found to be the predominant from the Pseudomonas spp. followed by Pseudomonas fluorescens. The total viable count was (3.25 ± 0.02) × 10 CFU/ml. Out of 8 Pseudomonas spp. isolates that were examined for proteolytic and lipolytic activities; two isolates had proteolytic activity, while two had lipolytic activity and four isolates had both.


Introduction
Milk has many nutritious qualities that make it an important part of children's diet.To produce the best quality milk and to achieve all the nutritious benefits of it, the highest quality raw milk must be obtained.Pasteurization and shelf stable milk products through ultra high temperature (UHT) continuous flow sterilization are available (Goff & Griffiths, 2006).The conditions of heat treatment used for pasteurization depend on the final product; lower temperatures are used for refrigerated products and higher heat treatments are used for products stored at room temperature (United States Code of Federal Regulation [USCFR], 2006).Psychrotrophic bacteria have been recognized as a recurring problem in the refrigerated storage and distribution of fluid milk, and perishable dairy products for several decades (Singh, Wani, Karim, & Langowski, 2012).So, the psychrotrophic have received increased attention by investigators during recent years, because modern developments in the handling and transportation of milk have resulted in milk being held for longer period at refrigeration temperature before processing, manufacturing or consumption.Some psychrotrophic bacteria may grow at a temperature of 7°C although their optimum temperature is higher.Rapid cooling and cold storage of raw milk favor the growth of psychrotrophic bacteria in milk (Barbano, Ma, & Santos, 2006).They become dominant microflora during cold storage of milk and their extracellular enzymes, particularly proteases and lipases, contribute to the spoilage of milk products (Hantis-Zacharov & Halpern, 2007).
Microbial growth and metabolism shorten the shelf life of milk by producing undesirable changes in aroma and taste attributes that influence consumer acceptability of the products (Fromm & Boor, 2004).Factors limiting milk stability are well established: bacterial contamination (Kabir & Niar, 2013), inadequate packaging system (Singh et al., 2012) and improper temperature control (Ben Moussa, Mankai, Ben Fekih, & Hassouna, 2013).Vulnerability of milk's fat and protein to physical-chemical alterations can also lead to deterioration, thus, reducing its quality.Cromie (1991) reported that the factors which influence the shelf life of pasteurized milk include the quality of the raw material, the binomial temperature/time pasteurization, resistant microorganisms to pasteurization (particularly Psychrotrophic), the presence and activity of post pasteurization contaminants, the packaging system and storage temperature post pasteurization which had the greatest impact on the stability of the product as shelf life of pasteurized milk.Extra cellular lipolytic enzymes produced from psychrotrophics can produce deteriorate effects as rancid flavours and odours in milk and dairy products that make a subsequent reduction of shelf-life and the products become unacceptable to consumers (Downey, 1980).
The proteolytic enzymes produced by psycrotrophics in milk are more powerful in its action on milk proteins than that naturally present in milk and that produced by leucocytes even if present by great amount (Grieve & Kitchen, 1985).Pseudomonas spp.produces a large number of extracellular toxins, which include phytotoxic factor, pigments, hydrocyanic acid, proteolytic enzymes (Baglinière et al., 2013), phospholipase and enterotoxins.Exotoxins are responsible for Pseudomonas spp.pathogenicity because it can produce leucopoenia, circulatory collapse, necrosis of liver, pulmonary edema, hemorrhage and kidney tubular necrosis.The enterotoxin produced is responsible for diarrhea disease.
High temperature for short times was less effective in destruction of the Pseudomonas spp.than that of temperature for long times because the condition created in the milk at high temperatures may response of injured cells (Cousin, 1982).Generally, they are considered incapable of surviving pasteurization and they may be post-processing contamination.Although, they can cause gastroenteritis if ingested in large number (>10 6 cell), but also the food would be clearly spoiled before such numbers would reached (Johnson, 1990).Moreover, its presence in milk and its products was considered as a possible indicator of fecal contamination.Also Pseudomonas aeruginosa has been implicated in epidemics of moderate to severe diarrhea in children in the form of enteric fever (Todar, 2004).
This study aimed to isolate, characterize and evaluate the prevalence of Pseudomonas spp. in pasteurized milk sold in Sfax Governorate, Tunisia.Also, observation of the proteolytic and lipolytic behavior of it in the pasteurized milk was investigated.

Materials and Methods
Thirty (30) samples of pasteurized milk were collected randomly from various local milk supermarkets in different areas in Sfax Governorate, Tunisia.Collected samples were examined for total viable cells and occurrence and behavior of Pseudomonas organisms.Psychrotrophic isolates were obtained from pasteurized milk by pouring plating appropriately diluted sample using nutrient agar.Incubation was done at 7°C for 10 days (Cousin, 2001).The psychrotrophic isolates were subjected to biochemical characterization after activating the culture in nutrient broth at 37°C for 24 h (Barrow & Feltham, 1993).

Identification of Pseudomonas Isolates
The representative suspected colonies were purified and then identified according to Bergey's Manual of Systemic Bacteriology (1984) and API 20 NE kit (England).Pseudomonas isolates were subcultured onto nutrient agar (NA) plates and incubated at 30°C for 24 h.Pure cultures were inoculated into nutrient broth and incubated overnight at 30°C prior to testing.

Oxidation Fermentation Test
Lactose negative, blue-black and greenish bright dark colonies were lactose positive as a result of incubation during 48 h at 30°C.Among these, continuous studies were carried out on the lactose negative ones and these colonies were applied on the oxidation fermentation test.For Pseudomonas oxidative reaction, among the same samples of catalase (+), motility (+) and oxidase (+) activities, colonies were studied on growth at 4 and 41°C which were first taken into consideration.So, Pseudomonas, which incubated into yeast extract medium were left at 4°C for 7 to 10 days and at 41°C for 24 h, respectively (Mickova, Lukasova, & Konencny, 1989).

Determination of the Proteolytic and Lipolytic Activities of Pseudomonas spp.
Eight (8) strains belonging to Pseudomonas spp.Isolated previously from pasteurized milk samples were investigated for their proteolytic and lipolytic activities as described by Harrigan and McCance (Harrigan & MacCance, 1976).

Proteolytic Activity Using Skim Milk Agar
Overnight cultures were spot inoculated onto milk agar, standard plate count agar supplemented with 10% sterile skim milk.The inoculated plates were incubated at 20 and 4°C for 10 days.The presence of transparent zones around the spots was recorded as positive strains referring to protease production, and subsequently flooded with 10% v/v acetic acid solution.Clear zone around the colonies after 1 min exposure were regarded as positive (Harrigan & MacCance, 1976).

Lipolytic Activity
Lipolytic counts (LP) were determined using NA containing tributyrin.The medium was prepared using 10 g of tributyrin and 28 g of NA.Plates were incubated at 20 and 4°C for 72 h to determine viable LP.Lipolytic activity was determined by measuring clear zone around each colony (Harrigan & MacCance, 1976).

Incidence of Pseudomonas spp. among the Examined Samples
The samples showed an average of total viable count of (3.25 ± 0.02) × 10 4 CFU/ml.According to Prevention of Food Adulteration Act (PFA, 2007), total plate count of pasteurized milk should not exceed 30.000CFU/ml.However, it was observed that all the standard plate count were characterized by the predominance of typical small circular entire smooth and shining uncolored colonies.Furthermore, only this type of colony was evident in the plates used for taking psychrotrophic count (Table 1).The Pseudomonas spp.count was (1.2 ± 0.01) × 10 2 CFU/ml among different samples and the isolates were subjected to bacteriological examination as shown in Table 2.
The results showed that 8 samples were positive for occurrence of Pseudomonas spp., with a percentage of 26.66%.Identity of the isolates was confirmed by using API 20 NE for detection and isolation of Pseudomonas spp.; 6 isolates of P. aeruginosa and 2 isolates of Pseudomonas fluorescens were detected.Many investigators indicated that some Pseudomonas spp.could survive heat treatment used in pasteurization of milk (Abad, Villafafila, Frias, & Rodriguez-Fernandez, 1993).Incidence of P. aeruginosa in pasteurized milk has been reported by Kumaresan and Annalvilli (2008).According to them, post processing contamination led to the predominance of heat sensitive Pseudomonas spp. in pasteurized milk.
Quality defects in pasteurized milk are most often the result of microbial contamination, growth and spoilage.Microbial defects usually become evident in the finished product through shelf-life evaluations or consumer complaints.Post-pasteurization contamination with psychrotrophic spoilage bacteria is most detrimental.In most cases, product contamination is the result of insufficient cleaning and sanitation of the processing equipment and plant environment.Practices that are being followed in the dairy processing unit would have singled out psychrotrophics as the major category of spoilage organism (Alatossava & Alatossava, 2007).

The Proteolytic and Lipolytic Activities of Pseudomonas spp.
The high spoilage potential of Pseudomonas spp. is not only because of its ability to multiply at refrigeration temperatures but also because of their ability to produce thermostable proteases and lipases (Sorhaug & Stepaniak 1997).The importance of proteases and lipases, which causes bacterial virulence, was proven in several studies.However, there was less study in the protease and lipase activities of Pseudomonas spp.This study showed that the Pseudomonas spp.isolates did not show proteolytic and lipolytic activities at 4°C, while at 20°C the P. aeruginosa and P. fluorescens isolates showed these activities as shown in Table 3: 2 isolates were positive for proteolytic; 2 isolates for lipolytic and 4 isolates for both proteolytic and lipolytic.This finding agrees with the findings of Dogan and Boor (2003).Psychrotrophics were implicated in many defects in milk and dairy products which are a problem resulting from prolonged refrigeration storage (Garg, 1990) and distribution of perishable food products.Pseudomonas spp.have been implicated in the spoilage of processed milk kept under chilled condition because of their capacity to multiply under refrigeration with the production of thermostable proteases and lipases (Rajmohan, 2002) which plays an important role in milk spoilage.

Conclusion
Raw milk deteriorates in only a few days even when stored under refrigeration temperatures.In this study, the results cleared that after pasteurization, 8 samples from 30 samples were positive for occurrence of Pseudomonas spp., with a percentage of 26.66% that may be due to insufficient pasteurization or the post contamination by this genus and the temperature which milk and dairy products are exposed influences the type of microorganisms that will grow in them.As dairy equipment and utensils constitute the major source of many types of psychrotrophics in milk, so special attention should be considered in their cleaning and sanitation to produce milk of low bacterial count or even completely free of psychrotropics bacteria.The previous information indicated that psychrotrophics are still inevitable because they are widely distributed in nature, withstand sanitizers and can liberate heat stable enzyme causing spoilage of food and some of them considered as food borne pathogens.This knowledge increase attention toward the way by which the restriction of these microorganisms must be done.

Table 1 .
Prevalence and count (CFU/ml) of Pseudomonas spp. in examined pasteurized milk samples

Table 2 .
The bacteriological examination of Pseudomonas spp. in examined pasteurized milk samples

Table 3 .
Proteolytic and lipolytic activities of Pseudomonas spp.isolated from pasteurized milk samples.