Since the adoption of refrigerated bulk tanks for the collection and storage of milk, the predominant organisms in milk are now psychrotrophic bacteria. These are organisms that are able to grow at temperatures below 7 °C, although their optimum growth temperature may lie between 20 and 30 °C. The majority of the psychrotrophic bacteria (excluding Bacillus spp.) are destroyed by pasteurization, but they produce extracellular enzymes that are extremely thermostable. The most important of these enzymes from the commercial viewpoint are the proteases and lipases, both of which are able to withstand high temperature short time and UHT treatments. Proteolysis in UHT milk can cause the development of bitter flavor and lead to an increase in viscosity, with eventual formation of a gel during storage, which is a major factor limiting its shelf life and market potential. The enzymes responsible for the proteolysis are the native milk alkaline proteinase, plasmin, and heat-stable extracellular bacterial proteinases produced by psychrotrophic bacterial contaminants in raw milk. Proteolysis of casein caused by plasmin is responsible for gelation and bitterness of UHT milk during storage.
Psychrotrophic bacteria in milk
Psychrotrophic microorganisms that are capable of growing in milk at temperatures close to 0 °C are represented by both Gram-negative bacteria (such as Pseudomonas, Achromobacter, Aeromonas, Serratia, Alcaligenes, Chromobacterium, and Flavobacterium spp.) and Gram-positive bacteria (such as Bacillus, Clostridium, Corynebacterium, Streptococcus, Lactobacillus, and Microbacterium spp.). Furthermore, refrigeration conditions under which raw milk is stored selects for growth of psychrotrophs, many of which produce heat-stable enzymes while milk awaits processing. Following heat treatment, these enzymes can continue to degrade milk in the absence of viable bacterial cells. A variety of psychrotrophic organisms, including P. fluorescens, P. putida, P. fragi, P. putrefaciens, Acinetobacter spp., Achromobacter spp., Flavobacterium spp., Aeromonas spp., and Serratia marcescens, produce heat-stable extracellular proteases and some of them also produce heat-stable extracellular lipases. Pseudomonas spp. usually represents no more than 10% of the microflora of freshly drawn milk; however, they are the most important of the psychrotrophs that dominate the microflora of raw or pasteurized milk at the time of spoilage. This genus is also known to be strongly lipolytic (Sørhaug and Stepaniak 1997).
Thermoresistant psychrotrophs Among the microorganisms that survive pasteurization (the thermoduric microorganisms), sporeforming Bacillus spp. dominate those that are also psychrotrophs. Other thermoduric psychrotrophs are represented in the genera Arthrobacter, Microbacterium, Streptococcus, Corynebacterium, and Clostridium. Clostridium is also sporeformer and anaerobic. Psychrotrophic Bacillus spp. secrete heat-resistant extracellular proteinases, lipases, and phospholipase (lecithinase) that are of comparable heat resistance to those of Pseudomonads. Bacillus sporothermodurans is a mesophilic spore-former that produces highly heat-resistant spores and was first detected in UHT milk in 1985 in southern Europe and in UHT milk in Germany in 1990 (Pettersson and others 1996). The spores survive the heat process and then multiply to a maximum of about 105/mL of milk during incubation at 30 °C for 5 d, but cause no noticeable spoilage. The spores of B. sporothermodurans are more resistant than the spores of many thermophiles (Brown 2000).
Plasmin, plasminogen, plasminogen activators (PAs), PA inhibitors (PAIs), and plasmin inhibitors (PIs)
Plasmin (EC 126.96.36.199), the main native protease in milk, is part of a complex system consisting of plasminogen, PA, PAI, and PI (Crudden and Kelly 2003). An important part of the potential plasmin activity is present as plasminogen. In fresh milk, the concentration of plasminogen is much higher than that of plasmin (Nielsen 2003). Plasmin is an alkaline serine proteinase with pH optimum of 7.5, which readily hydrolyzes β-casein, αs2-casein, and (more slowly) αs1-casein (Fox and McSweeney 1996). Its enzymatic reactions result in desired and undesired effects on dairy products. Plasmin plays a positive role in cheese ripening for many varieties of cheeses (such as Emmental, Romano, Swiss, and Gouda); however, its enzymatic action during milk clotting and storage of UHT milk can affect the products adversely.
Plasmin activity is higher in mastitis and late-lactation milk due to the increased level of PAs. The levels of plasmin and plasminogen can vary considerably with the stage of lactation, breed, age, and presence of mastitis (Grufferty and Fox 1986). The final plasmin activity of milk and, subsequently, milk casein hydrolysis, would depend not only on the amount of plasminogen and PA but also on the quantity of PAI. The occurrence in milk of blood serum trypsin inhibitors with both high and low molecular weight has been documented. They presumably would interfere with the function of serine proteinases and therefore, with plasmin and PA activity. However, other milk constituents, for example, β-lactoglobulin also could inhibit these enzymes (Korycka-dahl and others 1983). Many interactions between plasminogen, plasmin, PA, PAI, and PI characterize the plasmin system (Figure 5A). The role of PA in the system is to mediate plasminogen conversion into plasmin, whereas PAI and PI inhibit PA and plasmin activities, respectively. PAs are likely native to milk or produced by microorganisms (Deharveng and Nielsen 1991). Two major types of PA are known to be present in fresh milk: a tissue type associated with casein and a urokinase type associated with the somatic cells. PIs and PAI are located mainly in milk serum; however, the inhibitors might appear in several different forms, possibly due to formation of complexes with other milk proteins (Precetti and others 1997). α1-Antitrypsin, α2-antiplasmin, and PAI have been isolated from milk serum and partially characterized (Weber and Nielsen 1991).
Little is known about the heat stability of PAI and PI, and it has been generally proposed that the inhibitors are thermally unstable. Richardson (1983) suggested that PAI is inactivated by mild thermal treatments. An increase in activity of PL and a subsequent decrease in concentration of PG were observed in pasteurized milk compared to raw milk after incubation at 37 °C for up to 80 h. Effect of heat treatment on inhibitors of both PA and plasmin was studied by (Prado and others 2006) and fractions of milk with inhibitory activities against PA and plasmin were isolated. Thermal inactivation of PA inhibitor (81.1%) was considerably different from that of PI (35.8%) in milk after heat treatment at 75 °C for 15 s. The PI in the isolated fractions was suggested to be α2-antiplasmin, since it reacted immunochemically with polyclonal goat antihuman α2-antiplasmin and competitively inhibited plasmin. Results showed that PA inhibitor is less heat stable than PI, indicating that plasminogen activation could overcome any inhibition of plasmin resulting after milk pasteurization.
Hard gel formation was caused by Pseudomonas proteinase and partial digestion of the casein by plasmin in the samples incubated at 40 °C for 3 h. The clarified samples indicated extensive breakdown of casein as evident by turbidity, while the gelled samples indicated limited proteolysis. The characteristics of hard gel are similar to that caused by rennet, which, such as Pseudomonas proteinase, preferentially hydrolyzes the hydrophilic glycomacropeptide from κ-casein on the outside of the micelle. This leaves the casein micelle largely intact and also reduces steric repulsion between micelles, allowing the formation of a more compact gel. By contrast, plasmin attacks κ-casein located inside the micelle, thereby disrupting the micelle and inhibiting the formation of a strong gel. Extensive proteolysis of skim milk by plasmin completely clarifies skim milk (Datta and Deeth 2003).
Plasmin itself is a heat-stable enzyme that survives pasteurization and many UHT processes (D140 °C is 32 s) and the initial (1 d) plasmin activity of UHT milk containing KIO3 was significantly higher than that in raw milk (Kennedy and Kelly 1997). The inhibitors present in fresh milk are heat labile, whereas the activators are known to be heat stable (Richardson 1983; Lu and Nielsen 1993). Consequently, heat treatment of milk alters the natural balance between the activators and inhibitors in favor of the activators. This can lead to enhanced proteolysis in heated milk (Deharveng and Neilsen 1991). Driessen and van der Waals (1978) reported the D142 °C for milk proteinase (plasmin) to be 18 s. Rollema and Poll (1986) reported 28%, 6%, 4%, and 1.3% plasmin/plasminogen remaining after indirect heating for 5 s at 110, 120, 140, and 147 °C, respectively, but none after heating at 147 °C for 10 s.
If proteases survive UHT sterilization, milk containing them and stored without refrigeration would be a good environment for their activity. The optimum pH for one protease ranges from pH 7 to 8 with 85% to 90% of maximum activity at pH 6.5, the pH of milk (Speck and Adams 1976). The production of different phospholipases has been reported for Gram-negative and Gram-positive psychrotrophs. The “bitty cream” defect (floating clumps of fat) occurs in products that contain high numbers of Bacillus cells, suggesting a unique ability of phospholipases from this microorganism to damage the fat globule membrane (Sørhaug and Stepaniak 1997).