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Combined effect of nisin and high hydrostatic pressure on destruction of Listeria innocua and Escherichia coli in liquid whole egg
International Journal of Food Microbiology 43 (1998) 15–19
Combined effect of nisin and high hydrostatic pressure on destruction of Listeria innocua and Escherichia coli in liquid whole egg E. Ponce, R. Pla*, E. Sendra, B. Guamis, M. Mor-Mur ` , Universitat Autonoma ` Unitat de Tecnologia dels Aliments, Ce.R.T. A., Facultat de Veterinaria de Barcelona, 08193 Bellaterra, Barcelona, Spain Received 27 May 1997; received in revised form 11 May 1998; accepted 4 June 1998
Abstract High hydrostatic pressure inactivation of Escherichia coli and Listeria innocua inoculated in liquid whole egg was improved significantly (P , 0.05) with nisin addition at concentrations of 1.25 and 5 mg / l. A reduction of almost 5 log 10 units in E. coli counts and more than 6 log 10 units for L. innocua was obtained at 450 MPa and 5 mg / l of nisin. For this treatment, the two microorganisms were not detectable after 1 month of storage at 48C. The amount of nisin added did not affect E. coli inactivation at 300 MPa. For L. innocua, 5 mg / l of nisin was more effective than 1.25 mg / l. Nisin showed no effect when samples were stored at 208C after pressurization, except for samples with L. innocua containing 5 mg / l of nisin and treated with 450 MPa. 1998 Elsevier Science B.V. All rights reserved. Keywords: High hydrostatic pressure; Nisin; Listeria innocua; Escherichia coli; Whole egg
1. Introduction The shelf-life of liquid egg ranges from a few days to several weeks depending on initial bacterial load, the pasteurization process employed, and temperature of storage (Schafi et al., 1970; Cunningham, 1977; Payne et al., 1979; Ball et al., 1987). Nisin is an antibacterial peptide produced by Lactococcus lactis subsp. lactis, which could be added to the liquid egg prior to the pasteurization *Corresponding author. Tel.: 1 34 3 5811446; fax: 1 34 3 5812006; e-mail: [email protected]
process due to its heat resistance (Lueck, 1980). Nisin is active principally against Gram-positive bacteria, but Gram-negative bacteria, damaged cells in particular, may be sensitive to nisin (Kordel and Sahl, 1986; Kalchayanand et al., 1992). High hydrostatic pressure has been demonstrated to inactivate microorganisms (Farr, 1990; Cheftel, 1992; Ponce et al., 1998a,b), and emerges as an alternative to the usual heat treatments, with the possibility of improving the microbiological quality of egg products without impairing their functional properties, flavour and colour. The purpose of this study was to examine the
0168-1605 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 98 )00088-9
16
E. Ponce et al. / International Journal of Food Microbiology 43 (1998) 15 – 19
potential for inactivation of Listeria innocua and Escherichia coli in liquid whole egg by high hydrostatic pressure combined with the addition of nisin, and the effect on shelf-life under storage at 4 and 208C.
2. Materials and methods Preparation of the liquid egg was performed according to Ponce et al. (1998a). Blended whole eggs were pasteurized at 608C for 3.5 min and pH was adjusted to 8.0 by the addition of 1 N NaOH or 1 N HCl. Liquid egg was separated in two batches, one was inoculated with E. coli and the other with L. innocua both at a level of 10 6 CFU / ml. Samples from each batch were taken as untreated controls. Each inoculated batch was divided in three portions. One portion was kept as a control for pressurization process and the other two were added nisin (1.25 and 5 mg / l, respectively). Samples were distributed in sterile polyester bottles of 40 ml (Bibby Sterilin Ltd, Stone, UK), which were completely filled and the caps sealed with Teflon film (Kartell, Italy). Bottles were then pressurized at 350 and 450 MPa, respectively, for 10 min at 208C. Counts were determined immediately after pressurization and also after 18 h storage at 48C to evaluate the presence of injured cells. Then, half of the bottles for each treatment were maintained at 48C and the other identical half at 208C for different periods of time. Escherichia coli CECT 405 and Listeria innocua CECT 910 were obtained from Spanish Type Culture Collection (Universidad de Valencia, Spain) and revived according to Ponce et al. (1998a), (1998b). Nisin was obtained from Aplin & Barret Ltd. (Trowbridge, UK). Appropriate decimal dilutions were made in Ringer solution (9 ml) for both microorganisms. Viable counts were determined in Violet Red Bile Agar (VRBA; Oxoid Ltd., Hampshire, UK) and Palcam Agar (Palcam, Biokar, Beauvais, France) for E. coli and L. innocua, respectively. VRBA plates were incubated at 378C for 24 h and Palcam plates at 378C for 48 h. High-pressure treatments of 300 and 450 MPa (at 208C for 10 min) were applied in a discontinuous isostatic press from Gec Alsthom ACB (Nantes, France). The pressure chamber filled with 50% water–alcohol solution was maintained at 208C by a
constant flow of 80% water–alcohol solution through the jacket of the chamber (Ponce et al., 1998a). All experiments were run twice. Analysis of variance was performed using the General Linear Models procedure of Statistical Analysis System (SAS Institute Inc., 1982, Cary, USA). Duncan’s new multiple range test was used to obtain pairwise comparisons among sample means. Evaluations were based on a 5% significance level (P , 0.05).
3. Results and discussion Pressurization induced cell stress, as can be seen comparing counts at 0 and 18 h post-treatment (Table 1). The results indicate the presence of injured cells which are able to grow 18 h after pressure treatment, but not in selective media right after the treatment. Pressurization significantly (P , 0.05) affected E. coli and L. innocua recovery. It is seen that the Gram-negative microorganism (E. coli) is more sensitive to pressurization than the Grampositive (L. innocua). Nisin addition to liquid egg Table 1 Evaluation of injured cells of E. coli and L. innocua caused by pressurization Storage time at 48C (h) 0
E. coli Control 300 MPa 300 MPa 1 A( 2 ) 300 MPa 1 B ( 2 ) 450 MPa 450 MPa 1 A 450 MPa 1 B L. innocua Control 300 MPa 300 MPa 1 A 300 MPa 1 B 450 MPa 450 MPa 1 A 450 MPa 1 B
18
Log 10
S.D.
Log 10
S.D.
6.31 a( 1 ) 5.92 b 5.28 d 5.54 c 1.80 fg 1.51 h 1.51 h
0.19 0.08 0.13 0.06 0.25 0.32 0.13
5.97 b 5.87 b 5.38 cd 5.41 cd 2.35 e 1.93 f 1.56 gh
0.35 0.22 0.18 0.22 0.25 0.24 0.14
6.63 a( 1 ) 5.79 b 5.93 b 5.92 b n.d.f n.d.f n.d.f
0.25 0.40 0.01 0.07 — — —
6.43 a 6.41 a 4.86 c 2.89 d 4.93 c 1.71 e n.d.f
0.15 0.11 0.05 0.32 0.08 0.40 —
Counts were made in selective media at 0 and 18 h post-treatment. (1) Means with the same letter are not significantly different (P , 0.05). S.D., standard deviation; n.d., not detected. Means from four replicates, in Log 10 CFU / ml. (2) Nisin concentration: A 5 1.25 mg / l; B 5 5 mg / l.
E. Ponce et al. / International Journal of Food Microbiology 43 (1998) 15 – 19
increased significantly (P , 0.05) the effect of pressure on both microorganisms. L. innocua suffered larger reductions at the higher concentration of nisin, and the effect was most pronounced (P , 0.05) at 450 MPa. Eventhough E. coli is a Gram-negative microorganism its inactivation was enhanced (P , 0.05) by nisin, but the amount added seemed not to have a significant effect. As seen from Table 2 the evolution of E. coli and L. innocua in liquid egg stored at 48C followed different patterns, as a function of pressure treatment and concentration of nisin. E. coli counts in pressurized samples decreased significantly (P , 0.05) with storage time, while L. innocua counts remained unaffected or even increased after 30 days. The reason could be that it is a psycrotroph able to recover after storage at low temperatures, as reported by Benkerroum and Sandine (1988) for L. monocytogenes. E. coli was not affected by the amount of nisin added because the most important factor involved in its inactivation is the injury suffered under high pressure. Results obtained after storage at 208C are shown in Table 3. After 3 days of storage at 208C, E. coli counts were above 10 8 CFU / ml regardless of the treatment. On the contrary, L. innocua evolution was
17
significantly (P , 0.05) affected by the treatment applied. However, in all cases growth exceeding 10 6 CFU / ml was observed after 5 days of storage, except in the treatment with 450 MPa and 5 mg / l of nisin that took 9 days to reach such counts (data not shown). The primary site of action of nisin against vegetative cells is considered to be the cytoplasmatic membrane, acting as a depolarizing agent in a voltage-dependent fashion (Ray, 1992; Montville et al., 1995; Muriana, 1996). Nisin has a bactericide effect principally on Gram-positive microorganisms but also has a mild effect in Gram-negative microorganisms, even when intact cells are generally resistant. E. coli becomes sensitive to nisin when the outer cell membrane is disrupted as reported by Kordel and Sahl (1986). Kalchayanand et al. (1992) also observed that bacteriocins are more effective on sublethally injured Gram-negative microorganisms. Microbial cellular membrane is affected by high pressure, resulting in osmotic changes, lysis, alterations of nuclear material, and other modifications which can result in cell death (Mackey et al., 1994). High pressure produces, on Gram-negative cells such as E. coli, sublethal injury in the outer membrane (Hauben et al., 1996), and so sensitizes E. coli to
Table 2 Evolution of L. innocua and E. coli inoculated in liquid egg under storage at 48C Storage time 18 h
E. coli Control 300 MPa 300 MPa 1 A( 2 ) 300 MPa 1 B ( 2 ) 450 MPa 450 MPa 1 A 450 MPa 1 B L. innocua Control 300 MPa 300 Mpa 1 A 300 MPa 1 B 450 MPa 450 MPa 1 A 450 MPa 1 B
8 days
15 days
30 days
Log 10
S.D.
Log 10
S.D.
Log 10
S.D.
Log 10
S.D.
5.97 b( 1 ) 5.87 b 5.38 c 5.41 c 2.35 f 1.93 ghi 1.56 ij
0.35 0.22 0.18 0.22 0.25 0.24 0.14
6.51 a 5.27 c 4.32 d 4.29 d 1.82 hi 1.37 jk 1.09 kl
0.03 0.24 0.24 0.06 0.38 0.25 0.09
6.27 a 4.19 d 3.12 e 3.25 e 1.10 l 0.22 m n.d.m
0.09 0.06 0.13 0.22 0.31 0.25 —
6.27 ab 3.02 e 2.23 fg 2.12 fgh n.d.m n.d.m n.d.m
0.37 0.17 0.30 0.25 — — —
6.43 d( 1 ) 6.41 d 4.86 g 2.89 i 4.93 g 1.71 l n.d.n
0.15 0.11 0.05 0.32 0.08 0.40 —
7.14 c 6.05 e 4.25 h 2.11 k 5.02 g 1.45 l n.d.n
0.24 0.15 0.08 0.39 0.08 0.07 —
7.53 a 6.24 de 4.34 h 1.93 k 4.82 g 1.20 m n.d.n
0.12 0.06 0.06 0.10 0.11 0.37 —
7.36 ab 7.23 bc 5.31 f 3.03 i 6.08 e 2.24 j n.d.n
0.18 0.07 0.21 0.18 0.09 0.16 —
Combined effect of nisin and pressure (MPa). (1) Means with the same letter are not significantly different (P , 0.05). S.D., standard deviation; n.d., not detected. Means from four replicates, in Log 10 CFU / ml. (2) Nisin concentration: A 5 1.25 mg / l; B 5 5 mg / l.
18
E. Ponce et al. / International Journal of Food Microbiology 43 (1998) 15 – 19
Table 3 Evolution of L. innocua and E. coli inoculated in liquid egg under storage at 208C following 18 h at 48C Storage time 18 h (48C)
E. coli Control 300 MPa 300 MPa 1 A( 2 ) 300 MPa 1 B ( 2 ) 450 MPa 450 MPa 1 A 450 MPa 1 B L. innocua Control 300 MPa 300 MPa 1 A 300 MPa 1 B 450 MPa 450 MPa 1 A 450 MPa 1 B
3 days
5 days
Log 10
S.D.
Log 10
S.D.
Log 10
5.97 c( 1 ) 5.87 c 5.38 d 5.41 d 2.35 e 1.93 f 1.56 f
0.35 0.22 0.18 0.22 0.25 0.24 0.14
8.73 a 8.84 a 8.56 b 8.63 b 8.91 a 8.85 a 8.80 a
0.08 0.13 0.21 0.10 0.24 0.18 0.09
not not not not not not not
6.43 e( 1 ) 6.41 e 4.86 g 2.89 i 4.93 g 1.71 k n.d.l
0.15 0.11 0.05 0.32 0.08 0.40 —
7.34 ab 7.03 cd 6.36 e 5.70 f 6.94 d 4.45 h 1.37 k
0.21 0.33 0.29 0.30 0.06 0.07 0.06
7.24 bcd 7.51 ab 6.91 d 6.29 e 7.70 a 6.21 e 2.25 j
S.D.
determined determined determined determined determined determined determined 0.14 0.18 0.30 0.44 0.22 0.14 0.11
Combined effect of nisin and pressure (MPa). (1) Means with the same letter are not significantly different (P , 0.05). S.D., standard deviation; n.d., not detected. Means from four replicates, in Log 10 CFU / ml. (2) Nisin concentration: A 5 1.25 mg / l; B 5 5 mg / l.
nisin and also inhibits murein synthesis (Reisinger et al., 1980; Henning et al., 1986). This phenomenon could help nisin penetration and its consequent action on the cytoplasmatic membrane, and in combinations with adverse temperature conditions (48C) could partially explain our results for E. coli inactivation. Nisin effect could have been enhanced by its possible synergism with lysozyme (a natural component of egg white) which increases the permeability of the outer membrane, thus, allowing nisin access to the cytoplasmatic membrane. Actual commercial pasteurization conditions do not inactivate lysozyme, since the thermal stability of lysozyme is remarkably high. Durance (1994) reported that lysozyme remained stable after 30 min at 718C. The effect of nisin could have been better in not so basic conditions (pH 8.0) since the nisin molecule is acidic in nature and exhibits the greatest stability and solubility under acidic conditions (Ray, 1992). Moreover, it would be interesting to know nisin activity after pressurization. The loss of nisin activity is function of the initial concentration, pH and storage temperature. Delves-Broughton (1990) has already indicated that losses are more pronounced at high pH and high temperatures, supporting the lower
effectiveness of nisin addition when pressurized samples were held at 208C. Nisin addition to liquid egg combined with high hydrostatic pressure presented a synergistic effect that allows their less intensive usage to enhance shelf-life-maintaining biophysical properties. Future work should be directed to optimize combinations of pressure and food additives with the goal of eradicating pathogens and spoilage microorganisms in liquid whole egg.
Acknowledgements We would like to thank the Spanish Type Culture Collection (CECT) for providing the strains and Aplin & Barret Ltd. (Trowbridge, UK) for providing the nisin.
References Ball, Jr. H.R., Hamid-Samimi, M., Foegeding, P.M., Swartzel, K.R., 1987. Functionality and microbial stability of ultrapasteurized, aseptically packaged refrigerated whole egg. J. Food Sci. 52, 1212–1218.
E. Ponce et al. / International Journal of Food Microbiology 43 (1998) 15 – 19 Benkerroum, N., Sandine, W.E., 1988. Inhibitory action of nisin against Listeria monocytogenes. J. Dairy Sci. 71, 32–37. Cheftel, J.C., 1992. Effects of high hydrostatic pressure on food constituents: an overview. In: Balny, C., Heremans, K., Masson, P. (Eds.), High Pressure and Biotechnology. Colloque INSERM. John Libbey Eurotext Ltd., London, pp. 195–209. Cunningham, F.E., 1977. Egg product pasteurisation. In: Stadelman, W.S., Cotterill, O.J. (Eds.), Egg Science and Technology, 2nd ed. AVI Pub. Co., CT, pp. 161–186. Delves-Broughton, J., 1990. Nisin and its uses as a food preservative. Food Technol. 44, 100–112, 117. Durance, T.D., 1994. Separation, purification, and thermal stability of lysozyme and avidin from chicken egg white. In: Sim, J.S., Nakai, S. (Eds.), Egg Uses and Processing Technologies. New Developments. Cab Int., Wallingford, UK, pp. 77–85. Farr, D., 1990. High pressure technology in the food industry (Review). Trends Food Sci. Technol. 1, 14–16. Hauben, K.J.A., Woytack, E.Y., Soontjens, C.C.F., Michiels, C.W., 1996. High pressure transient sensibilization of Escherichia coli to nisin and lysozyme by disruption of outer-membrane permeability. J. Food Prot. 59, 350–355. Henning, S., Metz, R., Hammes, W.P., 1986. Studies on the mode of action of nisin. Int. J. Food Microbiol. 3, 121–134. Kalchayanand, N., Hanlin, M.B., Ray, B., 1992. Sublethal injury makes Gram-negative and resistant Gram- positive bacteria sensitive to the bacteriocins, pediocin AcH and Nisin. Lett. Appl. Microbiol. 15, 239–243. Kordel, M., Sahl, H.G., 1986. Susceptibility of bacterial, eukaryotic and artificial membranes to the disruptive action of cationic peptide pep5 and Nisin. FEMS Microbiol. Lett. 34, 139.
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Lueck, E., 1980. Nisin. In: Antimicrobial Food Additives. Springer-Verlag, Berlin, pp. 252–256. ` K., Isaacs, N.S., Stenning, R., Brooker, Mackey, B.M., Forestiere, B., 1994. The effect of high hydrostatic pressure on Salmonella Thompson and Listeria monocytogenes examined by electron microscopy. Lett. Appl. Microbiol. 19, 429–432. Montville, T.J., Winkowski, K., Ludescher, R.D., 1995. Models and mechanisms for bacteriocin action and application. Int. Dairy J. 5, 797–814. Muriana, P.M., 1996. Bacteriocins for control of Listeria spp. in food. J. Food Prot. Suppl., 54–63. Payne, J., Gooch, J.E.T., Barnes, E.M., 1979. Heat resistant bacteria in pasteurised whole egg. J. Appl. Bacteriol. 46, 601–613. Ponce, E., Pla, R., Capellas, M., Guamis, B., Mor-Mur, M., 1998a. Inactivation of Escherichia coli inoculated in liquid whole egg by high hydrostatic pressure. Food Microbiol. (in press). Ponce, E., Pla, R., Mor-Mur, M., Gervilla, R., Guamis, B., 1998. Inactivation of Listeria innocua inoculated in liquid whole egg by high hydrostatic pressure. J. Food Prot. 61, 119–122. Ray, B., 1992. Nisin of Lactococcus lactis spp lactis as a food preservative. In: Food Biopreservatives of Microbial Origin. CRS Press, Boca Raton, FL, pp. 207–264. Reisinger, P., Seidel, H., Tschesche, H., Hammes, W.P., 1980. The effect of nisin on murein synthesis. Arch. Microbiol. 127, 187–193. SAS Institute Inc., 1982. SAS User’s Guide: Statistics, 1982 ed. SAS Institute Inc. Cary, NC. Schafi, R., Cotterill, O.J., Nicholas, M.C., 1970. Microbial flora of commercially pasteurized egg products. Poultry Sci. 49, 578– 585.
Combined effect of nisin and high hydrostatic pressure on destruction of Listeria innocua and Escherichia coli in liquid whole egg E. Ponce, R. Pla*, E. Sendra, B. Guamis, M. Mor-Mur ` , Universitat Autonoma ` Unitat de Tecnologia dels Aliments, Ce.R.T. A., Facultat de Veterinaria de Barcelona, 08193 Bellaterra, Barcelona, Spain Received 27 May 1997; received in revised form 11 May 1998; accepted 4 June 1998
Abstract High hydrostatic pressure inactivation of Escherichia coli and Listeria innocua inoculated in liquid whole egg was improved significantly (P , 0.05) with nisin addition at concentrations of 1.25 and 5 mg / l. A reduction of almost 5 log 10 units in E. coli counts and more than 6 log 10 units for L. innocua was obtained at 450 MPa and 5 mg / l of nisin. For this treatment, the two microorganisms were not detectable after 1 month of storage at 48C. The amount of nisin added did not affect E. coli inactivation at 300 MPa. For L. innocua, 5 mg / l of nisin was more effective than 1.25 mg / l. Nisin showed no effect when samples were stored at 208C after pressurization, except for samples with L. innocua containing 5 mg / l of nisin and treated with 450 MPa. 1998 Elsevier Science B.V. All rights reserved. Keywords: High hydrostatic pressure; Nisin; Listeria innocua; Escherichia coli; Whole egg
1. Introduction The shelf-life of liquid egg ranges from a few days to several weeks depending on initial bacterial load, the pasteurization process employed, and temperature of storage (Schafi et al., 1970; Cunningham, 1977; Payne et al., 1979; Ball et al., 1987). Nisin is an antibacterial peptide produced by Lactococcus lactis subsp. lactis, which could be added to the liquid egg prior to the pasteurization *Corresponding author. Tel.: 1 34 3 5811446; fax: 1 34 3 5812006; e-mail: [email protected]
process due to its heat resistance (Lueck, 1980). Nisin is active principally against Gram-positive bacteria, but Gram-negative bacteria, damaged cells in particular, may be sensitive to nisin (Kordel and Sahl, 1986; Kalchayanand et al., 1992). High hydrostatic pressure has been demonstrated to inactivate microorganisms (Farr, 1990; Cheftel, 1992; Ponce et al., 1998a,b), and emerges as an alternative to the usual heat treatments, with the possibility of improving the microbiological quality of egg products without impairing their functional properties, flavour and colour. The purpose of this study was to examine the
0168-1605 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 98 )00088-9
16
E. Ponce et al. / International Journal of Food Microbiology 43 (1998) 15 – 19
potential for inactivation of Listeria innocua and Escherichia coli in liquid whole egg by high hydrostatic pressure combined with the addition of nisin, and the effect on shelf-life under storage at 4 and 208C.
2. Materials and methods Preparation of the liquid egg was performed according to Ponce et al. (1998a). Blended whole eggs were pasteurized at 608C for 3.5 min and pH was adjusted to 8.0 by the addition of 1 N NaOH or 1 N HCl. Liquid egg was separated in two batches, one was inoculated with E. coli and the other with L. innocua both at a level of 10 6 CFU / ml. Samples from each batch were taken as untreated controls. Each inoculated batch was divided in three portions. One portion was kept as a control for pressurization process and the other two were added nisin (1.25 and 5 mg / l, respectively). Samples were distributed in sterile polyester bottles of 40 ml (Bibby Sterilin Ltd, Stone, UK), which were completely filled and the caps sealed with Teflon film (Kartell, Italy). Bottles were then pressurized at 350 and 450 MPa, respectively, for 10 min at 208C. Counts were determined immediately after pressurization and also after 18 h storage at 48C to evaluate the presence of injured cells. Then, half of the bottles for each treatment were maintained at 48C and the other identical half at 208C for different periods of time. Escherichia coli CECT 405 and Listeria innocua CECT 910 were obtained from Spanish Type Culture Collection (Universidad de Valencia, Spain) and revived according to Ponce et al. (1998a), (1998b). Nisin was obtained from Aplin & Barret Ltd. (Trowbridge, UK). Appropriate decimal dilutions were made in Ringer solution (9 ml) for both microorganisms. Viable counts were determined in Violet Red Bile Agar (VRBA; Oxoid Ltd., Hampshire, UK) and Palcam Agar (Palcam, Biokar, Beauvais, France) for E. coli and L. innocua, respectively. VRBA plates were incubated at 378C for 24 h and Palcam plates at 378C for 48 h. High-pressure treatments of 300 and 450 MPa (at 208C for 10 min) were applied in a discontinuous isostatic press from Gec Alsthom ACB (Nantes, France). The pressure chamber filled with 50% water–alcohol solution was maintained at 208C by a
constant flow of 80% water–alcohol solution through the jacket of the chamber (Ponce et al., 1998a). All experiments were run twice. Analysis of variance was performed using the General Linear Models procedure of Statistical Analysis System (SAS Institute Inc., 1982, Cary, USA). Duncan’s new multiple range test was used to obtain pairwise comparisons among sample means. Evaluations were based on a 5% significance level (P , 0.05).
3. Results and discussion Pressurization induced cell stress, as can be seen comparing counts at 0 and 18 h post-treatment (Table 1). The results indicate the presence of injured cells which are able to grow 18 h after pressure treatment, but not in selective media right after the treatment. Pressurization significantly (P , 0.05) affected E. coli and L. innocua recovery. It is seen that the Gram-negative microorganism (E. coli) is more sensitive to pressurization than the Grampositive (L. innocua). Nisin addition to liquid egg Table 1 Evaluation of injured cells of E. coli and L. innocua caused by pressurization Storage time at 48C (h) 0
E. coli Control 300 MPa 300 MPa 1 A( 2 ) 300 MPa 1 B ( 2 ) 450 MPa 450 MPa 1 A 450 MPa 1 B L. innocua Control 300 MPa 300 MPa 1 A 300 MPa 1 B 450 MPa 450 MPa 1 A 450 MPa 1 B
18
Log 10
S.D.
Log 10
S.D.
6.31 a( 1 ) 5.92 b 5.28 d 5.54 c 1.80 fg 1.51 h 1.51 h
0.19 0.08 0.13 0.06 0.25 0.32 0.13
5.97 b 5.87 b 5.38 cd 5.41 cd 2.35 e 1.93 f 1.56 gh
0.35 0.22 0.18 0.22 0.25 0.24 0.14
6.63 a( 1 ) 5.79 b 5.93 b 5.92 b n.d.f n.d.f n.d.f
0.25 0.40 0.01 0.07 — — —
6.43 a 6.41 a 4.86 c 2.89 d 4.93 c 1.71 e n.d.f
0.15 0.11 0.05 0.32 0.08 0.40 —
Counts were made in selective media at 0 and 18 h post-treatment. (1) Means with the same letter are not significantly different (P , 0.05). S.D., standard deviation; n.d., not detected. Means from four replicates, in Log 10 CFU / ml. (2) Nisin concentration: A 5 1.25 mg / l; B 5 5 mg / l.
E. Ponce et al. / International Journal of Food Microbiology 43 (1998) 15 – 19
increased significantly (P , 0.05) the effect of pressure on both microorganisms. L. innocua suffered larger reductions at the higher concentration of nisin, and the effect was most pronounced (P , 0.05) at 450 MPa. Eventhough E. coli is a Gram-negative microorganism its inactivation was enhanced (P , 0.05) by nisin, but the amount added seemed not to have a significant effect. As seen from Table 2 the evolution of E. coli and L. innocua in liquid egg stored at 48C followed different patterns, as a function of pressure treatment and concentration of nisin. E. coli counts in pressurized samples decreased significantly (P , 0.05) with storage time, while L. innocua counts remained unaffected or even increased after 30 days. The reason could be that it is a psycrotroph able to recover after storage at low temperatures, as reported by Benkerroum and Sandine (1988) for L. monocytogenes. E. coli was not affected by the amount of nisin added because the most important factor involved in its inactivation is the injury suffered under high pressure. Results obtained after storage at 208C are shown in Table 3. After 3 days of storage at 208C, E. coli counts were above 10 8 CFU / ml regardless of the treatment. On the contrary, L. innocua evolution was
17
significantly (P , 0.05) affected by the treatment applied. However, in all cases growth exceeding 10 6 CFU / ml was observed after 5 days of storage, except in the treatment with 450 MPa and 5 mg / l of nisin that took 9 days to reach such counts (data not shown). The primary site of action of nisin against vegetative cells is considered to be the cytoplasmatic membrane, acting as a depolarizing agent in a voltage-dependent fashion (Ray, 1992; Montville et al., 1995; Muriana, 1996). Nisin has a bactericide effect principally on Gram-positive microorganisms but also has a mild effect in Gram-negative microorganisms, even when intact cells are generally resistant. E. coli becomes sensitive to nisin when the outer cell membrane is disrupted as reported by Kordel and Sahl (1986). Kalchayanand et al. (1992) also observed that bacteriocins are more effective on sublethally injured Gram-negative microorganisms. Microbial cellular membrane is affected by high pressure, resulting in osmotic changes, lysis, alterations of nuclear material, and other modifications which can result in cell death (Mackey et al., 1994). High pressure produces, on Gram-negative cells such as E. coli, sublethal injury in the outer membrane (Hauben et al., 1996), and so sensitizes E. coli to
Table 2 Evolution of L. innocua and E. coli inoculated in liquid egg under storage at 48C Storage time 18 h
E. coli Control 300 MPa 300 MPa 1 A( 2 ) 300 MPa 1 B ( 2 ) 450 MPa 450 MPa 1 A 450 MPa 1 B L. innocua Control 300 MPa 300 Mpa 1 A 300 MPa 1 B 450 MPa 450 MPa 1 A 450 MPa 1 B
8 days
15 days
30 days
Log 10
S.D.
Log 10
S.D.
Log 10
S.D.
Log 10
S.D.
5.97 b( 1 ) 5.87 b 5.38 c 5.41 c 2.35 f 1.93 ghi 1.56 ij
0.35 0.22 0.18 0.22 0.25 0.24 0.14
6.51 a 5.27 c 4.32 d 4.29 d 1.82 hi 1.37 jk 1.09 kl
0.03 0.24 0.24 0.06 0.38 0.25 0.09
6.27 a 4.19 d 3.12 e 3.25 e 1.10 l 0.22 m n.d.m
0.09 0.06 0.13 0.22 0.31 0.25 —
6.27 ab 3.02 e 2.23 fg 2.12 fgh n.d.m n.d.m n.d.m
0.37 0.17 0.30 0.25 — — —
6.43 d( 1 ) 6.41 d 4.86 g 2.89 i 4.93 g 1.71 l n.d.n
0.15 0.11 0.05 0.32 0.08 0.40 —
7.14 c 6.05 e 4.25 h 2.11 k 5.02 g 1.45 l n.d.n
0.24 0.15 0.08 0.39 0.08 0.07 —
7.53 a 6.24 de 4.34 h 1.93 k 4.82 g 1.20 m n.d.n
0.12 0.06 0.06 0.10 0.11 0.37 —
7.36 ab 7.23 bc 5.31 f 3.03 i 6.08 e 2.24 j n.d.n
0.18 0.07 0.21 0.18 0.09 0.16 —
Combined effect of nisin and pressure (MPa). (1) Means with the same letter are not significantly different (P , 0.05). S.D., standard deviation; n.d., not detected. Means from four replicates, in Log 10 CFU / ml. (2) Nisin concentration: A 5 1.25 mg / l; B 5 5 mg / l.
18
E. Ponce et al. / International Journal of Food Microbiology 43 (1998) 15 – 19
Table 3 Evolution of L. innocua and E. coli inoculated in liquid egg under storage at 208C following 18 h at 48C Storage time 18 h (48C)
E. coli Control 300 MPa 300 MPa 1 A( 2 ) 300 MPa 1 B ( 2 ) 450 MPa 450 MPa 1 A 450 MPa 1 B L. innocua Control 300 MPa 300 MPa 1 A 300 MPa 1 B 450 MPa 450 MPa 1 A 450 MPa 1 B
3 days
5 days
Log 10
S.D.
Log 10
S.D.
Log 10
5.97 c( 1 ) 5.87 c 5.38 d 5.41 d 2.35 e 1.93 f 1.56 f
0.35 0.22 0.18 0.22 0.25 0.24 0.14
8.73 a 8.84 a 8.56 b 8.63 b 8.91 a 8.85 a 8.80 a
0.08 0.13 0.21 0.10 0.24 0.18 0.09
not not not not not not not
6.43 e( 1 ) 6.41 e 4.86 g 2.89 i 4.93 g 1.71 k n.d.l
0.15 0.11 0.05 0.32 0.08 0.40 —
7.34 ab 7.03 cd 6.36 e 5.70 f 6.94 d 4.45 h 1.37 k
0.21 0.33 0.29 0.30 0.06 0.07 0.06
7.24 bcd 7.51 ab 6.91 d 6.29 e 7.70 a 6.21 e 2.25 j
S.D.
determined determined determined determined determined determined determined 0.14 0.18 0.30 0.44 0.22 0.14 0.11
Combined effect of nisin and pressure (MPa). (1) Means with the same letter are not significantly different (P , 0.05). S.D., standard deviation; n.d., not detected. Means from four replicates, in Log 10 CFU / ml. (2) Nisin concentration: A 5 1.25 mg / l; B 5 5 mg / l.
nisin and also inhibits murein synthesis (Reisinger et al., 1980; Henning et al., 1986). This phenomenon could help nisin penetration and its consequent action on the cytoplasmatic membrane, and in combinations with adverse temperature conditions (48C) could partially explain our results for E. coli inactivation. Nisin effect could have been enhanced by its possible synergism with lysozyme (a natural component of egg white) which increases the permeability of the outer membrane, thus, allowing nisin access to the cytoplasmatic membrane. Actual commercial pasteurization conditions do not inactivate lysozyme, since the thermal stability of lysozyme is remarkably high. Durance (1994) reported that lysozyme remained stable after 30 min at 718C. The effect of nisin could have been better in not so basic conditions (pH 8.0) since the nisin molecule is acidic in nature and exhibits the greatest stability and solubility under acidic conditions (Ray, 1992). Moreover, it would be interesting to know nisin activity after pressurization. The loss of nisin activity is function of the initial concentration, pH and storage temperature. Delves-Broughton (1990) has already indicated that losses are more pronounced at high pH and high temperatures, supporting the lower
effectiveness of nisin addition when pressurized samples were held at 208C. Nisin addition to liquid egg combined with high hydrostatic pressure presented a synergistic effect that allows their less intensive usage to enhance shelf-life-maintaining biophysical properties. Future work should be directed to optimize combinations of pressure and food additives with the goal of eradicating pathogens and spoilage microorganisms in liquid whole egg.
Acknowledgements We would like to thank the Spanish Type Culture Collection (CECT) for providing the strains and Aplin & Barret Ltd. (Trowbridge, UK) for providing the nisin.
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