Effects of lipolytic enzymes Pseudomonas fluorescens on liberation of fatty acids from milk fat

Effects of thermostable lipolytic enzymes Pseudomonas fluorescens 66 ZB in pasteurized milk on concentration of free fatty acids (VMK) in milk were studied in selected milk samples. Identical bulk milk samples were analysed by the method specified in previous papers (Vyletělová et al. 1999a, b, 2000). Reference milk samples (without bacterial strains) and the experimental ones (containing Ps. fl. 150 th. CFU/ml and 2800 th. CFU/ml, resp.) were stored at 6.5°C and 14°C and analysed at regular time intervals (24 h) - Table 1. An extractive-titric method (Kadlec et al. 1996; Table 2 and Fig. 2) was used for monitoring of fatty acid (MK) liberation. Precise analyses of MK and VMK were made by the chromatographic method (Figs. 1, 3 and 4). Medium-chain fatty acids (C12-C16) are liberated first of all; short-chain acids (C6-C10) were found sporadically or in very small quantities (Table 2). Dissociation constant of the specific fatty acid liberated from milk fat affects principally relationships between pH and free fatty acid concentration. The predominating proportion of long-chain acids in liberated fatty acid formation is associated with lower reduction of pH as compared to the predomination of fatty acids with shorter chains associated with more substantial reduction of pH. In our study, a rapid decrease of pH was noted before 168 h (Table 24); this corresponds to low concentrations of short-chain free fatty acids. Vyletělová et al. (2000) found significant relations between pH and contents of VMK (measured by the extractive-titric method); in some samples, correlation coefficients amounted to r = -0.93*** (P £ 0.001). The extractive-titric method analysing VMK concentrations (mmol/kg milk fat) provides results characterized by a systematic rise (e.g., 32.0 mmol/kg instead of 13.0 mmol/kg in raw milk). According to Kratochvíl (1992) 20 mmol VMK/kg milk fat signalized the starting point characterizing flavour degradation of milk caused by activities of fatty acids C12-C14 above all; the transformed value (respecting specifics of the extractive-titric method) amounts to 49 mmol/kg. In case of higher storage temperature a significant break is found after 144 h; in case of lower temperature this break is after 192 h (Table 2). Limits determining potential lipolytic modifications of milk flavour (RLZCHV) as related to specific samples and temperatures at VMK levels amounting to 49 mmol/kg or 20 mmol/kg are outlined in Fig. 2. Milk samples No. 5 and No. 6 stored at higher temperature surpassed this risk limit at 56 h and 64 h, respectively (Table 2, Fig. 2). On the contrary, milk samples stored temperatures corresponding to the standard storage temperature (storage of raw milk, transport, storage of pasteurized milk) surpass the mentioned risk level after 90 h and 140.5 h. Obtained results document the predominant role of storage temperature in the whole complex (production and processing of milk as a raw material or an intermediate product); evident differences in contamination rates (105 an 106) can be characterized as secondary effects in this case (Table 2). As related to practical conditions, the mentioned facts imply immediate processing of raw milk and pasteurized milk. This postulate must be respected namely by dairy plants producing delicate milk products. Vyletělová et al. (2000) found a notable VMK increase/24 h (7-11 mmol/kg milk fat) under specific temperatures and microbial contamination.