MG/ML FRASCO GOTERO DE 10 ML

In milk produced under sanitary conditions, the typical bacteria of the udder surface, mainly Micrococcaceae, predominate and less than 10% of the total microbiota are psychrotolerant microorganisms, but this percentage can mount up to 75%-90% under unsanitary conditions (Adams et al. 1975;Kurzweil and Busse 1973;Thomas and Thomas 1973). The main psychrotolerant aerobic bacteria which contaminate raw and pasteurized milk are primarily aerobic Gram-negative rods belonging to the Pseudomonaceae with approximately 65-70% of psychrotolerant isolates from raw milk assigned to the genus Pseudomonas (Garcia et al. 1989). Other genera present include Aeromonas, Acinetobacter, Alcaligenes, Chromobacterium, Flavobacterium and Serratia (Champagne et al. 1994;Cousin 1982;Lafarge et

al. 2004). Under the low temperature conditions throughout the dairy chain, members of the genus Pseudomonas are able to grow out and dominate the microbiota found in raw milk (Sørhaug and Stepaniak 1997). This may be explained because Pseudomonas members show the shortest generation times at 0-7°C (Chandler and McMeekin 1985). Furthermore, Pseudomonas spp. are able to colonize the processing line by adhering strongly to the surface of the milk processing equipment. This may enable them to persist unless removed by proper cleaning and sanitizing procedures (Bishop and White 1986;Cousin 1982). In the summer season, there is an increase in total psychrotolerant count, but no typical seasonal pattern was observed in the incidence of

Pseudomonas (Garcia et al. 1989). However, a seasonal pattern in the

proteolytic capacity of Pseudomonas isolates from raw milk was demonstrated by Marchand et al. (2009a).

P. fluorescens has traditionally been accepted as the most important

spoilage organism (Dogan and Boor 2003;Jayarao and Wang 1999). Nowadays, the importance of P. fluorescens in milk spoilage is under debate as it seems to be overestimated in the past due to an incorrect identification (Marchand et al. 2009a).

Marchand et al. (2009a) identified Pseudomonas lundensis and

Pseudomonas fragi members as the most important proteolytic spoilers in raw

milk based on a thorough identification of the strains using a polyphasic approach. A recent study by De Jonghe et al. (2011) acknowledged the predominant presence and spoilage capacity of P. fluorescens-like and P.

gessardii-like organisms, being closely related but clearly distinct from the P. fluorescens type strain.

As pseudomonads are well-known spoilage organisms, a lot is documented about their spoilage enzymes. Though optimal enzyme synthesis occurs in the majority of psychrotrotolerant bacteria at 20-30°C, considerable synthesis occurs even at lower temperature, for example, production of extracellular protease by Pseudomonas fluorescens at 5°C was 55% of that produced at 20°C (McKellar 1982). Furthermore, the enzymes remain active at temperatures well under their optimum temperature, for instance even at 2°C for P. fluorescens (Braun et al. 1999).

In contrast to lipolytic enzymes, the majority of Pseudomonas species produce only one heat resistant type of protease that is thought to be responsible for the spoilage of milk (Dufour et al. 2008;Fairbairn and Law 1986;Marchand et al. 2009b): the alkaline metalloprotease AprX protease that is widespread throughout the genus Pseudomonas (Chabeaud et al. 2001;Kumeta et al. 1999;Liao and McCallus 1998;Marchand et al. 2009b). It has a molecular mass of approximately 45 kDa (Dufour et al. 2008;Koka and Weimer 2001;Marchand et al. 2009b) and it belongs to the highly conserved serralysin family that is characterized by a zinc binding motif, a calcium binding domain containing four glycine rich repeats (G-G-X-G-X-D), a high content of hydrophobic amino acids and no cysteine residues (Kumeta et al. 1999;Rawlings and Barrett 1995). It is encoded by the aprX gene which lies on the aprX-lipA operon as demonstrated for P. fluorescens strain B52 (McCarthy et al. 2004;Woods et al. 2001).

Even though Pseudomonas species are easily inactivated by various heat treatments, an important fraction of the spoilage enzymes that they produce during growth, remains active because of their resistance to high temperatures.

Pseudomonas species are known to produce heat-stable spoilage enzymes that

retain significant activity even after UHT processing and production of milk powders (Chen et al. 2003). These enzymes can then cause spoilage and structural defects in pasteurized and UHT-treated milk and milk-powder derived products (chocolate, deserts etc.).

Thermostability of P. fluorescens proteases is the most intensively studied but strains belonging to other Pseudomonas species have also been proven to retain approximately 10% of their original activity after exposure to 140°C for 5 s (Kroll 1989). A recent study by Marchand et al. (2009a) showed P. fragi and P. lundensis as the most important producers of heat-stabile proteases.

Even though the occurrence of heat-stable lipases is much less extensively studied than the occurrence of heat-stable proteases, heat-resistance is believed to be a common characteristic of lipases from psychrotolerant microorganisms (Andersson et al. 1979;Cogan 1977;Cousin 1982;Shelley et al. 1986;Shelley et

al. 1987). Griffiths et al. (1981) found residual lipase activity of over 10% (mean value 30%) after exposure to 140°C for 5 s in strains belonging to a wide variety of Pseudomonas species (P. fluorescens, P. stutzeri, P. putida and

P. fragi) (Griffiths et al. 1981). However, recent data do not support this

overall heat-stability of Pseudomonas lipases (unpublished data, De Jonghe et al.).

A unique feature of both proteases and lipases of psychrotolerant

Pseudomonas species, is their sensitivity toward low temperature inactivation

(LTI) (Kroll 1989), meaning that they are rapidly irreversibly inactivated just above the optimum temperature for activity.

For proteases, the formation of enzyme-casein aggregates is proposed as an explanation for this phenomenon rather than an autolytic mechanism due to unfolding of the protein chain into a more sensitive conformation (Chen et al. 2003). For lipases however, the mechanism for LTI still remains unclear (Kroll 1989;Sørhaug and Stepaniak 1997): hydrolysis by proteinases or inactivation by aggregation with caseins has been suggested (Gasincova et al. 1994). It seems that lipases are more sensitive to this type of inactivation than proteases (Griffiths et al. 1981).