Psychrotrophic bacteria

Pseudomonad species usually constitute the largest percentage of lipolytic psychrotrophs in raw milk and cream and hence have attracted most attention. Lipases have been purified from P. fluorescens (Sugiura et al., 

1977 Severina and Bashkatova, 1979 Andersson, 1980 Dring and Fox, 1983 Stepaniak et al., 1987a), and P. fragi (Lawrence et al., 1967 Lu and Liska, 1969). The isolation and molecular characteristics of lipases from psychrotrophic bacteria have been reviewed by Fox et al. (1989). 

One of the most important properties of these lipases is their heat stability (Stepaniak et al., 1995). This varies with the species and strain (Fitz-Gerald et al., 1982) and also with the medium in which they are heated (Andersson et al., 1979). Many are sufficiently stable to retain at least some activity after pasteurization (Law et al., 1976 Fitz-Gerald et al., 1982 Kalogridou-Vassiliadou, 1984), and even after UHT treatment (Kishonti, 1975 Mottar, 1981 Christen et al., 1986). Milk proteins, except K-casein, 

A low level of NaCl (10 mM) may cause activation (Khan et al., 1967), although a high concentration inhibits the lipases. However, more than half of the activity remains in the presence of NaCl (2 M), a level similar to that in the aqueous phase of salted butter (Fitz-Gerald and Deeth, 1983). 

Unlike milk LPL, microbial lipases do not require a fatty acid acceptor such as BSA (Bengtsson and Olivecrona, 1980). Blood serum has been found to activate some of these enzymes (Fitz-Gerald and Deeth, 1983), including P. fluorescens lipases, and these have, consequently, been designated lipoprotein lipases (Aisaka and Tarada, 1979 Stepaniak and Sorhaug, 1989). 

---

McClements, J. M. J., Patterson, M. F., Linton, M. (2001). The effect of growth stage and growth temperature on high hydrostatic pressure inactivation of some psychrotrophic bacteria in milk. J. Food Pro., 64, 514-522. 

The increased use of tanks for the storage of raw milk on the farm between pickups has introduced the danger of potential off-flavor development caused by lipases that are produced by certain microorganisms (psychrotrophs) at low temperatures. The exocellular lipases of psychrotrophic bacteria are extremely heat resistant, and although the microorganisms are killed, the enzymes survive pasteurization and sterilization temperatures. Rancidity may become noticeable when cell counts exceed 106 or 107/ml. Downey (1975) has summarized the potential contribution of enzymes to the lipolysis of milk (Table 5.1). 

Hydrolytic rancidity flavor defects in Swiss, brick, and Cheddar cheeses have been linked to high concentrations of individual short chain free fatty acids (Woo et al 1984). Lipases from psychrotrophic bacteria have been implicated in causing rancidity in cheese (Cousin 1982 Kuzdzal-Savoie 1980), although most starter streptococci and lactobacilli isolated from cheese are also capable of hydrolyzing milk fat (Paulsen et al. 1980 Umemoto and Sato 1975). Growth of Clostridium tyrobutyricum in Swiss cheese causes the release of butyric acid and subsequent rancid-off flavors (Langsrud and Reinbold 1974). The endogenous lipoprotein lipase is also responsible for hydrolytic rancidity in nonpasteurized milk. 

Cousin, M. A. and Marth, E. H. 1977. Cheddar cheese made from milk that was precultured with psychrotrophic bacteria. J. Dairy Sci. 60, 1048-1056. 

Law, B. A. 1979. Reviews of the progress of dairy science Enzymes of psychrotrophic bacteria and their effects on milk and milk products. J. Dairy Res. 46, 573-588. Law, B. A. 1981. The formation of aroma and flavor compounds in fermented dairy products. Dairy Sci. Abstr. 43, 143-154. 

The proteolytic systems of psychrotrophic bacteria selectively attack /3- and as-caseins (Cousin and Marth 1977A), whereas whey proteins are relatively unaffected. Growth of psychrotrophic bacteria in milk results in decreased stability of casein, as measured by rennet coagulation time and heat stability (Cousin and Marth 1977B). Growth of psychrotrophs in milk also causes an increased rate of acid production by starter cultures as a result of increased quantities of readily available nitrogen compounds (Cousin and Marth 1977C.D). 

Many lipases produced by psychrotrophic bacteria retain activity after pasteurization and ultra-high-temperature (UHT) heat treatments (Cousin 1982 Adams and Brawly 1981). Butter made from cream which supported growth of lipase-producing psychrotrophs became rancid within two days (Kishonti and Sjostrom 1970). UHT milk processed from raw milk contaminated with lipase from a Pseudomo-... 

Adams, D. M., Barach, J. T. and Speck, M. L. 1975. Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin. J. Dairy Sci. 58, 828-834. 

Griffiths, M. W., Phillips, J. D. and Muir, D. D. 1981. Thermostability of proteases and lipases from a number of species of psychrotrophic bacteria of dairy origin. J. Appl. Bacteriol. 50, 289-303. 

Hosono, A. and Elliott, J. A. 1974. Properties of crude ethylester-forming enzyme preparations from some lactic acid and psychrotrophic bacteria. J. Dairy Sci. 57, 1432-1437. 

Kishonti, E. and Sjostrom, G. 1970. Influence of heat resistant lipases and proteases in psychrotrophic bacteria on product quality. 18th Int. Dairy Congr. IE, 501. 

Law, B. A. 1979A. Enzymes of psychrotrophic bacteria and their effects on milk and milk products. J. Dairy Res. 46, 573-588. 

Richardson, B. C. and Te Wheuti, I. E. 1978. Peirtied Cheiracterization of heat-stable ex-tracelluleir proteases of some psychrotrophic bacteria from raw milk. N.Z. J. Dairy Sci. Technol 13, 172-176. 

Quantification of the degradation products of raw-milk proteins by HPLC may furnish information on the shelf life of subsequently prepared UHT milk. Based on two earlier methods, Mottar et al. (127) applied HPLC to determine the specific proteolytic components that provide information concerning the presence and activity of gram-negative psychrotrophic bacteria. 

Bjorck, L. 1978. Antibacterial effect of the lactoperoxidase system on psychrotrophic bacteria in milk. J. Dairy Res. 45, 109-118. 

The enzymes responsible for the detrimental effects of lipolysis are of two main types those indigenous to milk, and those of microbial origin. The major indigenous milk enzyme is lipoprotein lipase. It is active on the fat in natural milk fat globules only after their disruption by physical treatments or if certain blood serum lipoproteins are present. The major microbial lipases are produced by psychrotrophic bacteria. Many of these enzymes are heat stable and are particularly significant in stored products. 

There were several new developments during the 1970s. Of particular importance was the purification and characterization of a lipoprotein lipase (LPL) and the acceptance of the postulate that this was the major, if not the only, lipase in cows milk (Olivecrona, 1980). Similarly, the elucidation of the lipase system in human milk as consisting of an LPL and a bile salt-stimulated lipase, and the possible role of the latter in infant nutrition, were noteworthy (Fredrikzon et al, 1978). Also, microbial lipolysis assumed substantial significance with the widespread use of low-temperature storage of raw milk and the recognition that heat-stable lipases produced by psychrotrophic bacteria were a major cause of flavor problems in stored dairy products (Law, 1979). 

Several psychrotrophic bacteria produce extracellular phospholipases, the most prevalent in milk being pseudomonads (particularly P. fluorescens), Alcaligenes, Acinetobacter, and Bacillus species (Fox et al., 1976 Owens, 1978a Phillips et al., 1981). Most of these produce phospholipase C, some produce phopholipase Ai and some produce both types (Deeth, 1983). Ser-ratia spp. have been shown to produce only phospholipase A (Deeth, 1983), while P. fragi has been reported not to produce phospholipases (Kwan and Skura, 1985). Phospholipase C from some pseudomonads has been purified and characterised (Doi and Nojima, 1971 Sonoki and Ikezawa, 1975 Stepa-niaketa/., 1987a Ivanov etal., 1996). Like the lipases, many of these enzymes have considerable heat stability and are not destroyed by pasteurization... 

Barach, J.T., Adams, D.M., Speck, M.L. 1976. Low temperature inactivation in milk of heat-resistant proteases from psychrotrophic bacteria. J. Dairy Sci. 59, 391-395. 

Deeth, H.C. 1983. Phospholipid degradation by phospholipases of some psychrotrophic bacteria. In Fats for the Future. Proc. Int. Conf. Oils Fats Waxes, 132-135, Duromark Publications, Auckland. 

Fitz-Gerald, C.H., Deeth, H.C., Coghill, D.M. 1982. Low temperature inactivation of lipases from psychrotrophic bacteria. Aust. J. Dairy Technol. 37, 51-54. 

Griffiths, M.W. 1989. Effect of temperature and milk fat on extracellular enzyme synthesis by psychrotrophic bacteria during growth in milk. Milchwissenschaft 44, 539-543. 

Kumura, H., Mikawa, K., Saito, Z. 1991. Influence of concomitant protease on the thermostability of lipase of psychrotrophic bacteria. Milchwissenschaft 46, 144-148. 

O Donnell, E.T. 1978. Heat resistance of lipase enzymes produced by psychrotrophic bacteria. 

Rowe, M.T., Johnston, D.E., Kilpatrick, D.J., Dunstall, G., Murphy, R.J. 1990. Growth and extracellular enzyme production by psychrotrophic bacteria in raw milk stored at a low temperature. Milchwissenschaft 45, 495 199. 

Shelley, A.W., Deeth, H.C., MacRae, I.C. 1987. A numerical taxonomic study of psychrotrophic bacteria associated with lipolytic spoilage of raw milk. J. Appl. Bacteriol. 62, 197-207. 

Stead, D. 1987. Production of extracellular lipases and proteinases during prolonged growth of strains of psychrotrophic bacteria in whole milk. J. Dairy Res. 54, 535 43. 

---