PATRIMONIO CULTURAL

Since yeasts are able to grow well at low pH, they commonly cause fruity or yeasty odour and/or gas formation of fermented milk products such as cultured milks (e.g. yoghurt and butter milk) and fresh cheeses (e.g. cottage cheese), that provide a highly specialized ecological niche for yeasts that can use lactose or lactic acid and tolerate high salt concentrations (Fleet 1990). Yeasts that are able to produce proteolytic or lipolytic enzymes may also have a selective advantage in milk products, even those with low aw.

Growth of spoilage molds on cheese is a problem that dates back to prehistory. Control measures such as pasteurization, added liquid smoke, the use of antimycotic chemicals and specialized packaging don‟t seem to be completely effective.

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When it comes to restraining bacteriological spoilage of milk and dairy products, it is important to know exactly what spoilage agent we are dealing with so that a justifiable course of action can be set up to limit the initial contamination and control the outgrowth of the responsible bacteria. This implicates not only a thorough identification of the responsible bacteria, but also a clear insight in the issue.

It is generally accepted that aerobe spore-formers are the main spoilers of pasteurized milk and dairy products, as their spores survive this heat treatment whereas Pseudomonas„ thermoduric enzymes cause spoilage of milk and dairy products with a long shelf life (i.e., UHT treated or powdered products) as the activity of Pseudomonas enzymes is thought to be too low to affect pasteurized milk during its shelf life. But is the spoilage issue really this straightforward? Figure 7 shows an overview of the complexity of the milk spoilage issue.

For pseudomonads, spoilage of heat treated milk and milk products can be attributed to heat-stable enzymes produced by vegetative cells in the raw milk, or to enzymes (both heat-stable and heat labile) that are secreted by

Pseudomonas bacteria that entered the product due to post processing

contamination. These two spoilage processes can take place in both UHT treated and pasteurized samples. However, it needs to be remarked that aseptic filling of UHT products limits the possibility of post processing contamination considerably. Although aerobe spore-formers are thought to be more important in spoilage of pasteurized products because of the supposed higher activity of their spoilage enzymes, Pseudomonas species that entered the milk through PPC will easily overgrow these organisms because of their much larger growth rates and much shorter lag phases under refrigeration temperatures (i.e., the temperatures applied for storage of pasteurized products) (Sørhaug and Stepaniak 1997). Still, the importance of other genera might be underestimated as concluded by Nörnberg et al. (2010) who demonstrated marked proteolytic activity in strains of Burkholderia, Klebsiella and Aeromonas.

When it comes to aerobe spore-formers such as Bacillus s.l., the generally accepted idea that they are the most important spoilers of pasteurized milk products because their spores survive pasteurization, originates from actual spoiled pasteurized dairy products from which Bacillus species, mainly

Figure 7. Flowchart of the milk spoilage issue. The interference points of Bacillus s.l. and Pseudomonas bacteria and spoilage enzymes are represented on the left and on the right, respectively. The relative importance of each step is reflected in the magnitude of the arrows. White arrows represent the spoilage issue caused by post processing contamination, whereas the black arrows represent spoilage issues caused by enzymes from vegetative cells that are already present prior to processing. For Bacillus s.l., grey arrows represent spores already present prior to processing. PPC: post processing contamination.

However, studies on the heat resistance of their spoilage enzymes, indicated that they are equally resistant as Pseudomonas enzymes, able to withstand pasteurization and treatments applied during commercial milk powder manufacture (Chen et al. 2004) (spoilage route no2 in Figure 7) (however, data on their resistance to UHT treatment are lacking (spoilage route no3 in Figure 7)). This implicates that not only the spores that germinate upon these processing treatments can cause spoilage in the retail product, but that possibly also vegetative cells secrete thermoduric spoilage enzymes in the raw milk upon cold storage prior to heat treatment (Chen et al. 2004). The vegetative cells can be released from biofilms (in the milking equipment at the farm, in the pumping installation of the milk tanker and the pipelines in the dairy plant) or maybe a minor fraction from spores that were able to germinate upon cold storage of the raw milk - provided that the vegetative cells are psychrotolerant and therefore able to grow.

As aerobe spore-formers tend to be present as spores in biofilms and generation times and lag phases of psychrotolerant Bacillus s.l. members at 2- 7°C are considerably longer than those of Pseudomonas spp. (Chandler and McMeekin, 1985), this might nevertheless be a relatively less frequent phenomenon. However, under suboptimal storage temperatures, growth of vegetative Bacillus cells might be considerable. Indispensable knowledge to determine the importance of these spore-forming groups for spoilage of milk products may therefore be their possibility to grow out throughout the dairy chain (prior to processing). Although the predominant raw milk species B.

licheniformis, B. subtilis and B. pumilus are generally regarded as mesophilic

(Pacova et al. 2003), a fraction of their isolates show psychrotolerant traits that would enable them to grow out during cold storage of the raw milk. However, the presence of these strains in the form of vegetative cells has not yet been investigated in raw milk.

Furthermore, spores of certain Bacillus s.l. members can survive UHT treatment, thus ending up in a competition-free niche that is stored at room temperature, enabling them to grow out (and maybe produce spoilage enzymes) without restraints (spoilage route no4 in Figure 7). However, except for B. sporothermodurans, no UHT-resistant bacilli have been linked to spoiled UHT-products up to now except in the event of PPC (a minor phenomenon because UHT milk is aseptically filled), which complicates the

Bacillus s.l. spoilage route even more (spoilage route no5 in Figure 7). Still, as UHT-products are required to be microbiologically stable upon storage at room temperature, outgrowth of HRS in itself might be enough to render these products unacceptable for consumption.

This raises the question as to the identity of the true culprit(s) when it comes to milk spoilage. Since growth rates of Pseudomonas members are much higher and lag phases much shorter at low storage temperatures of raw