Resistance. left at room temperature for 30 min to reach osmotic. yolk used as the heating menstruum was prepared in

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1983, p /83/ $02.00/0 Vol. 45, No. 1 Effects of Egg Yolk and Salt on Micrococcaceae Heat Resistance THEO VERRIPS* AND RENEE VAN RHEE Unilever Research Laboratorium, Vlaardingen, The Netherlands Received 12 April 1982/Accepted 6 October 1982 The heat resistance and growth possibilities of various members of the Micrococcaceae in egg yolk and egg yolk with added salt were determined. Egg yolk alone protected members of the Micrococcaceae considerably against heat. Whereas in water Staphylococcus aureus S6 had a decimal reduction time (D) value of 66 s at 55 C, its D value in egg yolk at the same temperature was 246 s. In salted egg yolk (water activity, 0.95), S. aureus S6 had a D value of 180 s at 66 C and was largely inactivated during the pasteurization processes currently applied. Micrococcus saprophyticus and S. epidermidis (D value of each under the same conditions, 390 s) could survive such treatments to a certain extent and can thus spoil commercial egg yolk. Pasteurization of whole eggs or egg yolk is legally required in many countries, mainly because of the high risk of contamination with members of the Enterobacteriaceae, including Salmonella spp., and members of the Micrococcaceae (proposed pasteurization conditions for whole eggs, egg yolk, and salted egg yolk: 61 to 63 C for 1.75 to 6.2 min). However, when pasteurization is carried out at too high a temperature (>70'C), some functional properties of egg yolk, such as emulsifying capacity, are at least partly lost. A common method for stabilizing pasteurized egg yolk against the outgrowth of various types of bacteria is the addition of a large amount of salt or a combination of a lower amount of salt and preservatives. It is well known that members of the Enterobacteriaceae cannot grow in medium with more than 8% salt (5). Staphylococci and micrococci, however, can grow at relatively high salt concentrations (2). Therefore, we studied the heat resistance of members of the Micrococcaceae, especially Staphylococcus aureus and S. epidermidis, in egg yolk with various amounts of salt added. We also studied the growth of members of the Micrococcaceae in egg yolk with various amounts of salt and with a combination of salt and potassium sorbate. MATERIALS AND METHODS Strains. Micrococcus lactis was isolated from egg yolk, M. saprophyticus was isolated from salted margarine, S. aureus S6 was obtained from the American Type Culture Collection (ATCC 13566), and S. epidermidis were isolated from sausage rolls. To determine growth possibilities in salted egg yolk, we used two additional S. aureus strains, Oxford and Bladel. All strains were classified according to the scheme of Baird-Parker (1). Preparation of inocula and heating experiments. All strains were cultivated in brain heart infusion medium (Difco Laboratories) for 48 h at 30 C under mild aeration (Aquatherm water bath shaker [New Brunswick Scientific Co.], 150 rpm). Stationary-phase cells were collected by centrifugation at 9000 x g for 15 min in a Superspeed RC-5 centrifuge (Ivan Sorvall, Inc.) and washed twice with distilled water. Finally, the pellet was suspended in the substrate to be tested (density, -109 cells per ml), and the suspension was left at room temperature for 30 min to reach osmotic equilibrium with the substrate. Egg yolk or salted egg yolk used as the heating menstruum was prepared in the laboratory from fresh eggs. The water activity (a,) of the heating medium was measured with a Sinascope (type SMT-B; Sina AG, Zurich, Switzerland) at 25 C. We then added 0.3 ml of the cell suspension to 30 ml of the same heated menstruum and thoroughly mixed the two. The temperature of the heating menstruum was controlled within 0.25 C. During heating, the menstruum was incubated under static conditions. At preset intervals, 1 ml of the heating menstruum was removed and suspended into 9 ml of diluent containing 1 g of peptone (Difco) and 8.5 g of sodium chloride per liter. The experiments were carried out at least twice, and the numbers of surviving microorganisms were determined in duplicate by dissemination of appropriate serial decimal dilutions into brain heart infusion agar. Colonies were counted after 3 days of incubation at 30 C. In the growth experiments, the number of cells was determined in the same way. The inactivation rates (k values) were determined from the logarithmic inactivation phase by the least-squares method to draw the best fitting line (J. P. P. M. Smelt, Ph.D. thesis, University of Utrecht, The Netherlands, 1980). If one assumes first-order inactivation kinetics, one can calculate the k value from N, = No (e-&') (7). RESULTS AND DISCUSSION The heat resistance data for the various micrococci in salted water (aw, 0.95) at various tem-

2 2 VERRIPS AND VAN RHEE TABLE 1. Micrococcaceae heat resistance in salted water (aw, 0.95) Organism Temp k (MC) D value (s) value (10-2 S-1) M. saprophyticus M. lactis S. aureus S S. epidermidis peratures are summarized in Table 1. M. saprophyticus was slightly more heat resistant than was S. epidermidis, whereas M. lactis and S. aureus S6 were much more heat sensitive. Unfortunately, in all of the heating menstrua tested, 0_o incubation time(nin) FIG. 1. Heat inactivation of staphylococci. Symbols: 0, S. epidermidis at 60 C in water and salt (aw, 0.95); *, S. aureus at 66 C in egg yolk and salt (aw, 0.95). N, Number of survivors. k(s-1) 100 E 1o-1 APPL. ENVIRON. MICROBIOL. egg yolk salt concentration (M) FIG. 2. Influence of salt concentration in egg yolk on S. epidermidis heat resistance. The temperature was 62 C. the Micrococcaceae inactivation curves had quite irregular shapes. Notable shoulder phases, tail phases, or both were nearly always found (Fig. 1), although we did not find the tail phases very pronounced, as was found in previous heating experiments with S. epidermidis in sucrose solutions (8). To compare k values, we always used the logarithmic parts of the inactivation curves to calculate decimal reduction time (D) and k values. The D values for S. aureus S6 in water were slightly lower than those found by Walker and Harmon (9) for the organism in phosphate buffer. That Walker and Harmon did not observe the shoulder phases we noticed can probably be explained by the fact that their sampling intervals were considerably longer than ours. Neglecting the shoulder phase will result in a D value of the logarithmic part of the inactivation curve that is slightly too high. When S. epidermidis was heated in egg yolk with the highest salt level tested (1.83 M NaCl),

3 VOL. 45, 1983 k(s-l) 10-1 lo-, \0 ifn3l lo, r7c) MICROCOCCACEAE HEAT RESISTANCE 3 TABLE 3. S. aureus S6 heat resistance Temp D value k value Heating menstruum (OC) (s) (10-2 s-,) Water Water + salt (aw, 0.95) Egg yolk Egg yolk + salt (aw, 0.95) K-1) obtained after heating in salted water (aw, 0.95) FIG. 3. Arrhenius curves of S. epide.rmidis heat and the Ea value obtained after heating in water inactivation in various heating menstrua. ' Symbols: A, with sucrose (aw, 0.95), and MJ/mol, water; A, egg yolk; 0, salt and water (aw, = 0.95);, ' respectively, suggests that the nature of the salt and egg yolk (aw, 0.95). T, Temperatiu 5re. ' agent that protects microbial cells against heat inactivation by lowering the a, of the heating menstruum is important and depends strongly the heat stability decreased slightly, whereas at on the temperature. 0 to 1.49 M NaCI, it increased accor-ding to the S. epidermidis in egg yolk with salt was exequation In k[naci] = In ko - a[nacl] ], with a = tremely heat resistant (a,, 0.95); even at 68 C, 2.37 (Fig. 2). The value a was intr*oduced by the D value was almost 5 min. Corry (3) to relate heat resistance inc:rease with Compared with S. epidermidis, S. aureus S6 solute concentration. Compared withlethe previ- was rather heat sensitive (Table 3), but the heat ously reported a value of about 0.9)9 for heat resistance increased significantly when egg yolk inactivation of S. epidermidis in succrose solu- or egg yolk with salt was used as the heating tions (8), the a value for the organismi in NaCl is menstruum. Because egg yolk has an a, of rather high, indicating a great depe-ndence of nearly 0.99, it should have protective activity. heat resistance on salt concentratic ;n Similar Walker and Harmon (9) also found that r S. aureonella us has a higher heat resistance in milk, skim dependence has been found for Salm (3), Citrobacter freundii (7), and Kiebsiella milk, or whey than in phosphate buffer. It can pneumoniae (6). not, therefore, be excluded that even extracellu- in various lar proteins protect microbial cells against heat Heat inactivation of S. epidermidisv media at a wide range of temperaturess is summa- inactivation. rized in Fig. 3; Table 2 gives the activation Considering the rather high heat resistance of energies (Ea values) of these reaction s. Whereas members of the Micrococcaceae in salted egg Ea value very often decreases when the solute yolk, we also studied the growth possibility of concentration is increased (4, 6-8), tlhe addition these microorganisms in egg yolk with salt or a of egg yolk protects the cells more efrectively at combination of salt and potassium sorbate (Fig. low temperatures than at higher tennperatures. 4 and 5). We found that even 12% salt was not In addition, the difference between tihe E15 value enough to inhibit the micrococcus growth, whereas the S. aureus mix did not grow in egg yolk with 211% salt. A combination of 7% salt TABLE 2. Ea values for S. epidernyuis heat and 1% potassium sorbate inhibited the growth inactivation of both staphylococci and micrococci. This Heating menstruum Temp ( C) E (MJ/mol) means that potassium sorbate in rather high Ea(MJ/mol) concentrations is also an active inhibitor of Water microbial growth at a high ph (ph of egg yolk, Water + salt (aw, 0.95) ca. 6.5). Studies of the influence of temperature Water + sucrose (aw, 0.95) indicate that neither micrococci nor staphylo- Egg yolk cocci can grow in salted egg yolk at tempera- Egg yolk + salt (a,, 0.95) tures lower than 8 C (unpublished data).

4 4 VERRIPS AND VAN RHEE APPL. ENVIRON. MICROBIOL L incubation time(week) FIG. 4. Growth of S. aureus mix in egg yolk at 25 C. Symbols: 0, 10% NaCl; *, 11% NaCl; A, 12% NaCl; A, 7% NaCl-1% potassium sorbate. N, Number of survivors. Although our results indicate that there is no real danger that S. aureus will survive normal pasteurization (61 to 63 C, 1.75 to 6.2 min), survival and subsequent growth of S. epidermidis and M. saprophyticus are possible. Growth of these bacteria in egg yolk can cause the loss of emulgation properties and can give the product an unpleasant rancid taste. The rather high Ea values for members of the Micrococcaceae heated in salted egg yolk indicate that a short, high-temperature pasteurization seems to be more adequate for preventing Micrococcaceae spoilage of salted egg yolk than are the currently used processes. This has also been suggested in regard to heat inactivation of S. epidermidis in sucrose solutions (9). Egg yolk ' l incubation time week) FIG. 5. Growth of micrococcus mix in egg yolk at 25 C. Symbols: 0, 10% NaCl; 0, 11% NaCl; A, 12% NaCl; A, 7% NaCl-potassium sorbate. N, Number of survivors. stabilization can be achieved by addition of 7% salt-1% potassium sorbate or by storage below 80C. LITERATURE CITED 1. Baird-Parker, A. C Family I. Micrococcaceae Pribram 1929, 385, p In R. F. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The William & Wilkins Co., Baltimore. 2. Christian, J. H. B., and J. A. Waltho The water relation of staphylococci and micrococci. J. Appl. Bacteriol. 25: Corry, J. E. L The effect of sugars and polyols on the heat resistance of Salmonellae. J. Appl. Bacteriol. 37: Dega, C. A., J. M. Goepfert, and C. H. Amundson

5 VOL. 45, 1983 Heat resistance of Salmonellae in concentrated milk. Appl. Microbiol. 23: Matches, J. R., and J. Liston Effects of incubation temperature on the salt tolerance of Salmonellae. J. Milk Food Technol. 35: Verrips, C. T., R. Glas, and R. H. Kwast Heat resistance of Klebsiella pneumoniae in media with various sucrose concentrations. Eur. J. AppI. Microbiol. Biotechnol. 8: MICROCOCCACEAE HEAT RESISTANCE 5 7. Verrips, C. T., and R. H. Kwast Heat resistance of Citrobacterfreundii in media with various water activities. Eur. J. Appl. Microbiol. Biotechnol. 4: Verrips, C. T., and R. van Rhee Heat inactivation of Staphylococcus epidermidis at various water activities. Appl. Environ. Microbiol. 41: Walker, G. C., and L. G. Harmon Thermal resistance of Staphylococcus aureus in milk, whey and phosphate buffer. Appl. Microbiol. 14: