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Addition of 2.5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed, sensory-acceptable servelat sausage and cooked ham stored at 4°C
International Journal of Food Microbiology 38 (1997) 71–76
Short communication
Addition of 2.5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed, sensory-acceptable servelat sausage and cooked ham stored at 48C Hans Blom
a,b,
*, Eva Nerbrink c , Richard Dainty a , Therese Hagtvedt a , Elisabeth Borch c , Hilde Nissen a , Truls Nesbakken d , e
˚ , Norway Norwegian Food Research Institute, Oslovn. 1, N-1430 As b Tine, Norwegian Dairies, Breigata 10, Oslo, Norway c ¨ Swedish Meat Research Institute, Box 504, S-244 24 Kavlinge , Sweden d Norwegian Meat Co-operative, Department of Research and Development, P.O. Box 360 Økern, 0513 Oslo, Norway e ¨ The Royal Veterinary and Agricultural University, Department of Veterinary Microbiology, Bulowsvej 13, 1870 Fredriksberg C, Denmark a
Received 10 February 1997; received in revised form 9 July 1997; accepted 21 July 1997
Abstract A study of the inhibitory effects of propylparaben and of a combination of lactate and acetate against growth of Listeria monocytogenes in inoculated liquid medium, sliced servelat sausage and cooked ham, were performed using rifampicin resistant Listeria strains in inoculation experiments. A consumer acceptance test of products produced with and without the compounds was also performed. Propylparaben was found to be effective in a model liquid non-fat medium, but was without effect in the actual products. This illustrates the potential pitfalls in translating results from studies in liquid media to fat-containing food products. The combined inhibitory and sensory results showed that a mixture of 2.5% lactate and 0.25% acetate (w / w, calculated on the water phase), could be used to increase the margins of safety for sliced and spreadable vacuum-packed ready-to-eat cooked meat products stored for 4–6 weeks. In addition, strict control of temperature during production and storage is very important. 1997 Elsevier Science B.V. Keywords: Listeria monocytogenes; Propylparaben; Lactate; Acetate; Cooked meat products
1. Introduction Vacuum-packed sliced meat products in Norway typically have pH values of around 6.2, salt contents of around 3% (calculated on water phase) and water *Corresponding author.
activities of 0.97 or higher. These products, therefore, have little if any inherent stability against the growth of Listeria monocytogenes, even under good refrigeration (ICMSF, 1996). Practical means of reducing the susceptibility of such products to growth of L. monocytogenes are therefore needed. There is ample documentation from tests in lab-
0168-1605 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0168-1605( 97 )00088-3
72
H. Blom et al. / International Journal of Food Microbiology 38 (1997) 71 – 76
oratory media of the anti-listeria effects of a range of permitted food additives including acetate, lactate (Ahamad and Marth, 1989; Ita and Hutkins, 1991; El-Shenawy and Marth, 1992; Houtsma et al., 1993; Golden et al., 1995) and parabens (Payne et al., 1989; Moir and Eyles, 1992; Yousef et al., 1991). In complimentary studies in foods, effective inhibition of L. monocytogenes was obtained for up to 4 weeks by incorporating 2% (w / w) sodium lactate in vacuum-packed meat products stored at temperatures from 1–78C (Unda et al., 1991; Qvist et al., 1994). In another study, 3% but not 2% (w / w) sodium lactate prevented growth of the pathogen in pork liver sausage at 58C (Weaver and Shelaf, 1993). On the other hand, 2.5% (w / w) sodium lactate failed to prevent growth of L. monocytogenes in turkey slurries stored at 48C, but did so for at least 6 weeks when used in combination with 0.1% (w / v) diacetate (Schlyter et al., 1993); diacetate (0.1% w / v) was ineffective when used on its own. Qvist et al. (1994), showed that 2% (w / w) sodium lactate in combination with 0.25% (w / w) glucono-d-lactone (GDL) suppressed growth of L. monocytogenes at 58C better than when either compound was used alone. Furthermore the mixture, in contrast to lactate alone, also inhibited L. monocytogenes in bologna stored at 108C (Qvist et al., 1994). At this temperature Miller and Acuff (1994) found 4% (w / v) sodium lactate to be necessary to inhibit L. monocytogenes on cooked beef. The results of a study of the inhibitory effects of propylparaben and of a combination of lactate and acetate against growth of L. monocytogenes in sliced servelat sausage and cooked ham are presented below. A consumer acceptance test of products produced with and without the acid mixture was also done.
2. Materials and methods
2.1. Bacteria Three different strains of L. monocytogenes were used. Strain 2230 / 92, serotype 1 was isolated from a refrigerated sample of cooked sausage implicated in a listeriosis outbreak in Norway (Nesbakken, 1995); strain 167, serotype 4b from a knife in a meat production plant; and strain 187, serotype 4b from a
routine quality control sample of a second type of cooked sausage. Primary stock cultures were maintained at 2 808C in Brain Heart Infusion broth (BHI, Difco) containing 20% (v / v) glycerol. Working stock cultures were prepared by incubating overnight in BHI at 378C without shaking. Rifampicin-resistant mutants (rif-mutants) of each strain were obtained according to Foegeding et al. (1992). Rif-mutants were maintained and grown as described for the parent strains. For experimental use, all strains were grown individually overnight in BHI at 378C. Cocktails were prepared by mixing equal culture volumes of appropriate strains.
2.2. Study of inhibitory factors in liquid media A basal medium (0.2 M Na 2 HPO 4 ? 2H 2 O / NaH 2 PO 4 ? H 2 O buffer of required pH; 2.0% tryptone; 0.2% D-glucose; 0.05% sodium ascorbate and 70 ppm sodium nitrite) (details of varied factors given in Table 1) was inoculated with | 10 7 cells / ml of a cocktail comprising equal numbers of the three strains. Aliquots (400 m l) were dispensed into microtitre plate wells, covered with 50 m l of liquid paraffin and incubated at 98C for 30 days. Absorbance at 420–580 nm was measured every 2 h and growth estimated by calculation of the area under the growth curve using a Bioscreen C instrument fitted with Biolink software (Labsystems Co., Helsinki, Finland).
2.3. Product inoculation experiments Servelat and cooked ham were produced commercially according to current recipes. Sodium salts of lactate (PurasalS / SP 60, Purac Biochem, Gorinchem, Holland), acetate and propylparaben (Fluka, Germany) were mixed with the ingredients to give the required nominal concentrations (calculated in % weight of the water content of the product). The products were sliced and vacuum-packed within 24 h of production, inoculated by injection of 0.1 ml of a cocktail of the three rif-mutants through gas probe self-sealing tape (TORAY ENGINEERING Co. Ltd., England) and stored at 4 or 98C. For microbial analysis, aliquots of appropriate dilutions were plated on MRS agar (CM359, Oxoid) pH 5.7 for lactic acid bacteria and on blood agar containing 50 m g / ml
H. Blom et al. / International Journal of Food Microbiology 38 (1997) 71 – 76
rifampicin. Hemolytic colonies on blood agar were registered as L. monocytogenes.
2.4. Consumer acceptance trial Consumer acceptance of servelat and cooked ham produced for the third inoculation experiment, i.e. controls containing 3% (w / w) salt and test products containing all three compounds, was tested at a local supermarket. In the course of a single day, 171 persons were presented with slices of the samples and asked the questions: — which sample of each product do you like best, why do you prefer it, would you buy it at today’s price, how often do you buy the product and what is your age and gender?
3. Results and discussion Growth of the cocktail of L. monocytogenes at 98C was effectively prevented by either 125 or 250 ppm propylparaben irrespective of levels of other media components or pH value (Table 1). Tested individually at pH 5.5, 3.5% sodium lactate or 0.5% sodium acetate inhibited growth of the pathogen strongly (Table 1). When tested at pH 6.3, the value expected in the products of interest, the individual acids each gave only small degrees of inhibition
73
(Table 1). However, a combination of 1.75% sodium lactate and 0.25% sodium acetate in the presence of 2.75% salt gave complete inhibition at pH 5.5, a high degree of inhibition at pH 5.9 and moderate inhibition at pH 6.3. To minimize the perceived increase in salt / sour taste associated with the acid mixture, the salt content of the standard product formulations was reduced from 3 to 2.5% (w / w), the lowest feasible on technological grounds. The original level of 3% was maintained in all controls. In a storage trial with servelat, propylparaben had no measurable effect on the growth of L. monocytogenes, its numbers increasing in test and control samples from initial numbers of | 300 cfu / g to | 10 8 cfu / g within 10 and 21 days at 98C and 48C, respectively (results not shown). A possible explanation is the hydrophobicity of parabens, favouring its partition into the fat phase. Indeed chemical analysis revealed its presence only in the product’s lipid phase. Complete inhibition of L. monocytogenes was observed for the whole of the 5-week storage period at 48C in both servelat and cooked, sliced ham formulated with the acid mixture (Fig. 1a and c) while there was good growth of the pathogen in the respective control samples (Fig. 1a and c). The acid mixture inhibited growth of L. monocytogenes
Table 1 Inhibition of growth of Listeria monocytogenes in liquid media by combinations of salt, pH, sodium lactate, sodium acetate and propylparaben pH
NaCl
Sodium lactate
Sodium acetate
Growth a
Propylparaben
Growth a
5.5 6.3 5.9 5.5 6.3 5.9 5.5 5.9 5.9 5.9 6.3 5.9 5.5 6.3 5.9 5.5 6.3
4.5 1.0 2.75 1.0 4.5 2.75 2.75 1.0 2.75 4.5 2.75 2.75 1.0 4.5 2.75 4.5 1.0
0 0 0 0 0 1.75 1.75 1.75 1.75 1.75 1.75 1.75 3.5 3.5 3.5 3.5 3.5
0 0 0.25 0.5 0.5 0 0.25 0.25 0.25 0.25 0.25 0.5 0 0 0.25 0.5 0.5
18.9 20.1 16.1 7.1 15.5 14.5 5.3 12.0 8.2 7.9 10.4 6.0 6.4 15.1 5.0 5.5 13.3
0 0 125 250 250 0 125 125 125 125 125 250 0 0 125 250 250
18.1 18.7 5.0 4.9 5.0 13.1 5.0 5.0 5.0 5.0 5.2 5.0 4.8 12.9 4.9 5.0 5.2
a
Area under growth curve determined turbidometrically. Values over 6 represent growth.
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H. Blom et al. / International Journal of Food Microbiology 38 (1997) 71 – 76
Fig. 1. Growth of Listeria monocytogenes (closed symbols) and lactic acid bacteria (open symbols) in vacuum-packed, sliced servelat with 2.5% w / v NaCl stored at (A) 48C and (B) 98C or in vacuum-packed, sliced, cooked ham stored at (C) 48C and (D) 98C. (s,d) controls and (h,j) products made with 2.5% sodium lactate and 0.25% sodium acetate.
throughout 5 weeks storage at 98C (Fig. 1b and d), while numbers reached 10 8 cfu / g in controls. However, in sliced, cooked ham, inhibition of the pathogen by the acid mixture was only maintained for the first 2–3 weeks storage (Fig. 1d). There was good growth of lactic acid bacteria in control and acidcontaining samples at both temperatures (Fig. 1a–d). By the end of storage, pH values had fallen from around 6.4 to 5.5 in control servelat samples, while the pH in the acid-containing samples was unchanged. The results from the control samples again illustrate the potential for growth should L. monocytogenes survive the processing of, or contaminate vacuum-packed, sliced, cooked meat products after processing. However, even at pH values above 6.0, a mixture of 2.5% lactate and 0.25% acetate was a substantial hurdle to the growth of this pathogen, the effects being more reproducible and long lasting at 4 than at 98C. The findings of inhibition by the acid mixture are consistent, at least in part, with those of Schlyter et al. (1993) who showed that 2.5% lactate
in combination with 0.1% diacetate, a compound consisting in the solid state of equimolar amounts of acetic acid and sodium acetate, not only prevented growth of L. monocytogenes in turkey slurries for up to 6 weeks at 48C, but resulted in a small decrease in numbers (0.3 log cycles). In a parallel study, 0.1% acetate in combination with 2.5% lactate only slowed, but did not prevent growth of L. monocytogenes (results not shown). One factor contributing to the observed differences could have been that the mixture containing 0.1% diacetate lowered the pH of the turkey slurries from 6.2 to 6.0 (Schlyter et al., 1993) while there was no measurable difference in initial pH between control and acid-containing samples in the present study. Qvist et al. (1994) made use of the pH lowering effect of GDL to enhance the inhibitory effects of lactate against L. monocytogenes in vacuum-packed, sliced bologna sausage. In that study 2% lactate in combination with 0.25% GDL, which resulted in a fall in pH from 6.6 to 6.3, inhibited its growth for at least 7 weeks both at 5 and 108C (Qvist et al., 1994). At this level of GDL,
H. Blom et al. / International Journal of Food Microbiology 38 (1997) 71 – 76
sensory scores remained above the acceptability level throughout storage and there were no significant differences from control samples (Qvist et al., 1994). In the present study, consumer testing of fresh, uninoculated samples of the servelat and cooked, sliced ham was done at a local supermarket. Of 171 participants, 85 preferred the control ham and 86 the ham with the added acid mixture, while 111 preferred the control servelat and 60 the product with the added acid mixture, the choice mainly being based on taste. The majority were frequent or occasional buyers of both products and would have purchased the preferred product and in some cases both. Age and gender had no measurable effect on the responses. The equal preferences for ham samples produced with and without the acids indicates that their inclusion in this product would not produce any undue degree of customer dissatisfaction. In the case of the relatively mild tasting servelat, this may not be so. Assuming the reason for the greater consumer preference for the standard servelat lies with the increased salt / sour taste associated with the use of the acids, the possibility of minimizing the effect needs to be investigated. This could involve a more critical examination of the minimal salt concentration necessary for the product’s technological properties, a possible small decrease in pH of the product and / or use of alternative salts of lactate and acetate. The findings presented confirm and reinforce the previously reported inhibitory effects of mixtures of lactate and acetate against growth of L. monocytogenes in turkey slurries with pH values greater than 6.0 (Schlyter et al., 1993). Explanations for these effects at pH values well above the respective pK values of the acids, and therefore at relatively low concentrations of the undissociated acid with which inhibition is normally associated, await further studies. At the same time, the results provide an alternative solution to the combination of lactate and glucono-d-lactone shown to be effective against L. monocytogenes in bologna sausage (Qvist et al., 1994). Although the combination did not adversely affect the sensory properties of the bologna, the acidifying effects of the lactone could lead to sensory problems in some products. The combined inhibitory and sensory results suggest that a mixture of 2.5% (w / v) lactate and 0.25% (w / v) acetate, could be used to increase the margins
75
of safety for sliced and spreadable vacuum-packed ready-to-eat cooked meat products with similar characteristics to servelat and cooked ham. Strict temperature control of production and storage however, are still a very important hurdle against the growth of this organism. Finally, the results with propylparaben illustrate the potential pitfalls in translating results from studies in liquid media to fatcontaining food products.
References Ahamad, N., Marth, E.H., 1989. Behaviour of Listeria monocytogenes at 7, 13, 21 and 358C in tryptose broth acidified with acetic, citric or lactic acid. J. Food Prot. 52, 688–695. El-Shenawy, M.A., Marth, E.H., 1992. Behavior of Listeria monocytogenes in the presence of sodium propionate together with food acids. J. Food Prot. 55, 241–245. Foegeding, P.M., Thomas, A.B., Pilkington, D.H., Klaenhammer, T.R., 1992. Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry-fermented sausage production. Appl. Environ. Microbiol. 58,(3) 884–890. Golden, M.H., Buchanan, R.L., Whiting, R.C., 1995. Effect of sodium acetate or sodium propionate with EDTA and ascorbic acid on the inactivation of Listeria monocytogenes. J. Food Safety 15, 53–65. Houtsma, P.C., de Wit, J.C., Rombouts, F.M., 1993. Minimum inhibitory concentration (MIC) of sodium lactate for pathogens and spoilage organisms occurring in meat products. Int. J. Food Microbiol. 20, 247–257. ICMSF., 1996. International Commission on Microbiological Specifications for Foods. Micro-organisms in Foods. 5. Microbiological Specifications of Food Pathogens. Blackie, London, pp. 141–182. Ita, P.S., Hutkins, R.W., 1991. Intracellular pH and survival of Listeria monocytogenes Scott A in tryptic soy broth containing acetic, lactic, citric and hydrochloric acids. J. Food Prot. 54, 15–19. Miller, R.K., Acuff, G.R., 1994. Sodium lactate affects pathogens in cooked beef. J. Food Sci. 59, 15–19. Moir, C.J., Eyles, M.J., 1992. Inhibition, injury, and inactivation of four psycrotrophic food-borne bacteria by the preservatives methyl p-hydroxybenzoate and potassium sorbate. J. Food Prot. 55,(5) 360–366. Nesbakken, T., 1995. Listeria monocytogenes in the food chain — a foodborne infection problem common to the Nordic countries. Tema Nord. The Nordic Council of Ministers 635, 48 p. Payne, K.D., Rico-Munoz, E., Davidson, P.M., 1989. The antimicrobial activity of phenolic compounds against Listeria monocytogenes and their effectiveness in a model milk system. J. Food Prot. 52, 151–153. Qvist, S., Sehested, K., Zeuthen, P., 1994. Growth suppression of Listeria monocytogenes in a meat product. Int. J. Food Microbiol. 24, 283–293.
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Schlyter, J.H., Glass, K.A., Loeffelholz, J., Degnan, A.J., Luchansky, J.B., 1993. The effects of diacetate with nitrite, lactate, or pediocin on the viability of Listeria monocytogenes in turkey slurries. Int. J. Food Microbiol. 19, 271–281. Unda, J.R., Molins, R.A., Walker, H.W., 1991. Clostridium sporogenes and Listeria monocytogenes: survival and inhibition in microwave-ready beef roasts containing selected antimicrobials. J. Food Sci. 56, 198–205.
Weaver, R.A., Shelaf, L.A., 1993. Antilisterial activity of sodium, potassium or calcium lactate in pork liver sausage. J. Food Safety 13, 133–146. Yousef, A.E., Gajewski II, R.J., Marth, E.H., 1991. Kinetics of growth and inhibition of Listeria monocytogenes in the presence of antioxidant food additives. J. Food Sci. 56, 10–17.
Short communication
Addition of 2.5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed, sensory-acceptable servelat sausage and cooked ham stored at 48C Hans Blom
a,b,
*, Eva Nerbrink c , Richard Dainty a , Therese Hagtvedt a , Elisabeth Borch c , Hilde Nissen a , Truls Nesbakken d , e
˚ , Norway Norwegian Food Research Institute, Oslovn. 1, N-1430 As b Tine, Norwegian Dairies, Breigata 10, Oslo, Norway c ¨ Swedish Meat Research Institute, Box 504, S-244 24 Kavlinge , Sweden d Norwegian Meat Co-operative, Department of Research and Development, P.O. Box 360 Økern, 0513 Oslo, Norway e ¨ The Royal Veterinary and Agricultural University, Department of Veterinary Microbiology, Bulowsvej 13, 1870 Fredriksberg C, Denmark a
Received 10 February 1997; received in revised form 9 July 1997; accepted 21 July 1997
Abstract A study of the inhibitory effects of propylparaben and of a combination of lactate and acetate against growth of Listeria monocytogenes in inoculated liquid medium, sliced servelat sausage and cooked ham, were performed using rifampicin resistant Listeria strains in inoculation experiments. A consumer acceptance test of products produced with and without the compounds was also performed. Propylparaben was found to be effective in a model liquid non-fat medium, but was without effect in the actual products. This illustrates the potential pitfalls in translating results from studies in liquid media to fat-containing food products. The combined inhibitory and sensory results showed that a mixture of 2.5% lactate and 0.25% acetate (w / w, calculated on the water phase), could be used to increase the margins of safety for sliced and spreadable vacuum-packed ready-to-eat cooked meat products stored for 4–6 weeks. In addition, strict control of temperature during production and storage is very important. 1997 Elsevier Science B.V. Keywords: Listeria monocytogenes; Propylparaben; Lactate; Acetate; Cooked meat products
1. Introduction Vacuum-packed sliced meat products in Norway typically have pH values of around 6.2, salt contents of around 3% (calculated on water phase) and water *Corresponding author.
activities of 0.97 or higher. These products, therefore, have little if any inherent stability against the growth of Listeria monocytogenes, even under good refrigeration (ICMSF, 1996). Practical means of reducing the susceptibility of such products to growth of L. monocytogenes are therefore needed. There is ample documentation from tests in lab-
0168-1605 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0168-1605( 97 )00088-3
72
H. Blom et al. / International Journal of Food Microbiology 38 (1997) 71 – 76
oratory media of the anti-listeria effects of a range of permitted food additives including acetate, lactate (Ahamad and Marth, 1989; Ita and Hutkins, 1991; El-Shenawy and Marth, 1992; Houtsma et al., 1993; Golden et al., 1995) and parabens (Payne et al., 1989; Moir and Eyles, 1992; Yousef et al., 1991). In complimentary studies in foods, effective inhibition of L. monocytogenes was obtained for up to 4 weeks by incorporating 2% (w / w) sodium lactate in vacuum-packed meat products stored at temperatures from 1–78C (Unda et al., 1991; Qvist et al., 1994). In another study, 3% but not 2% (w / w) sodium lactate prevented growth of the pathogen in pork liver sausage at 58C (Weaver and Shelaf, 1993). On the other hand, 2.5% (w / w) sodium lactate failed to prevent growth of L. monocytogenes in turkey slurries stored at 48C, but did so for at least 6 weeks when used in combination with 0.1% (w / v) diacetate (Schlyter et al., 1993); diacetate (0.1% w / v) was ineffective when used on its own. Qvist et al. (1994), showed that 2% (w / w) sodium lactate in combination with 0.25% (w / w) glucono-d-lactone (GDL) suppressed growth of L. monocytogenes at 58C better than when either compound was used alone. Furthermore the mixture, in contrast to lactate alone, also inhibited L. monocytogenes in bologna stored at 108C (Qvist et al., 1994). At this temperature Miller and Acuff (1994) found 4% (w / v) sodium lactate to be necessary to inhibit L. monocytogenes on cooked beef. The results of a study of the inhibitory effects of propylparaben and of a combination of lactate and acetate against growth of L. monocytogenes in sliced servelat sausage and cooked ham are presented below. A consumer acceptance test of products produced with and without the acid mixture was also done.
2. Materials and methods
2.1. Bacteria Three different strains of L. monocytogenes were used. Strain 2230 / 92, serotype 1 was isolated from a refrigerated sample of cooked sausage implicated in a listeriosis outbreak in Norway (Nesbakken, 1995); strain 167, serotype 4b from a knife in a meat production plant; and strain 187, serotype 4b from a
routine quality control sample of a second type of cooked sausage. Primary stock cultures were maintained at 2 808C in Brain Heart Infusion broth (BHI, Difco) containing 20% (v / v) glycerol. Working stock cultures were prepared by incubating overnight in BHI at 378C without shaking. Rifampicin-resistant mutants (rif-mutants) of each strain were obtained according to Foegeding et al. (1992). Rif-mutants were maintained and grown as described for the parent strains. For experimental use, all strains were grown individually overnight in BHI at 378C. Cocktails were prepared by mixing equal culture volumes of appropriate strains.
2.2. Study of inhibitory factors in liquid media A basal medium (0.2 M Na 2 HPO 4 ? 2H 2 O / NaH 2 PO 4 ? H 2 O buffer of required pH; 2.0% tryptone; 0.2% D-glucose; 0.05% sodium ascorbate and 70 ppm sodium nitrite) (details of varied factors given in Table 1) was inoculated with | 10 7 cells / ml of a cocktail comprising equal numbers of the three strains. Aliquots (400 m l) were dispensed into microtitre plate wells, covered with 50 m l of liquid paraffin and incubated at 98C for 30 days. Absorbance at 420–580 nm was measured every 2 h and growth estimated by calculation of the area under the growth curve using a Bioscreen C instrument fitted with Biolink software (Labsystems Co., Helsinki, Finland).
2.3. Product inoculation experiments Servelat and cooked ham were produced commercially according to current recipes. Sodium salts of lactate (PurasalS / SP 60, Purac Biochem, Gorinchem, Holland), acetate and propylparaben (Fluka, Germany) were mixed with the ingredients to give the required nominal concentrations (calculated in % weight of the water content of the product). The products were sliced and vacuum-packed within 24 h of production, inoculated by injection of 0.1 ml of a cocktail of the three rif-mutants through gas probe self-sealing tape (TORAY ENGINEERING Co. Ltd., England) and stored at 4 or 98C. For microbial analysis, aliquots of appropriate dilutions were plated on MRS agar (CM359, Oxoid) pH 5.7 for lactic acid bacteria and on blood agar containing 50 m g / ml
H. Blom et al. / International Journal of Food Microbiology 38 (1997) 71 – 76
rifampicin. Hemolytic colonies on blood agar were registered as L. monocytogenes.
2.4. Consumer acceptance trial Consumer acceptance of servelat and cooked ham produced for the third inoculation experiment, i.e. controls containing 3% (w / w) salt and test products containing all three compounds, was tested at a local supermarket. In the course of a single day, 171 persons were presented with slices of the samples and asked the questions: — which sample of each product do you like best, why do you prefer it, would you buy it at today’s price, how often do you buy the product and what is your age and gender?
3. Results and discussion Growth of the cocktail of L. monocytogenes at 98C was effectively prevented by either 125 or 250 ppm propylparaben irrespective of levels of other media components or pH value (Table 1). Tested individually at pH 5.5, 3.5% sodium lactate or 0.5% sodium acetate inhibited growth of the pathogen strongly (Table 1). When tested at pH 6.3, the value expected in the products of interest, the individual acids each gave only small degrees of inhibition
73
(Table 1). However, a combination of 1.75% sodium lactate and 0.25% sodium acetate in the presence of 2.75% salt gave complete inhibition at pH 5.5, a high degree of inhibition at pH 5.9 and moderate inhibition at pH 6.3. To minimize the perceived increase in salt / sour taste associated with the acid mixture, the salt content of the standard product formulations was reduced from 3 to 2.5% (w / w), the lowest feasible on technological grounds. The original level of 3% was maintained in all controls. In a storage trial with servelat, propylparaben had no measurable effect on the growth of L. monocytogenes, its numbers increasing in test and control samples from initial numbers of | 300 cfu / g to | 10 8 cfu / g within 10 and 21 days at 98C and 48C, respectively (results not shown). A possible explanation is the hydrophobicity of parabens, favouring its partition into the fat phase. Indeed chemical analysis revealed its presence only in the product’s lipid phase. Complete inhibition of L. monocytogenes was observed for the whole of the 5-week storage period at 48C in both servelat and cooked, sliced ham formulated with the acid mixture (Fig. 1a and c) while there was good growth of the pathogen in the respective control samples (Fig. 1a and c). The acid mixture inhibited growth of L. monocytogenes
Table 1 Inhibition of growth of Listeria monocytogenes in liquid media by combinations of salt, pH, sodium lactate, sodium acetate and propylparaben pH
NaCl
Sodium lactate
Sodium acetate
Growth a
Propylparaben
Growth a
5.5 6.3 5.9 5.5 6.3 5.9 5.5 5.9 5.9 5.9 6.3 5.9 5.5 6.3 5.9 5.5 6.3
4.5 1.0 2.75 1.0 4.5 2.75 2.75 1.0 2.75 4.5 2.75 2.75 1.0 4.5 2.75 4.5 1.0
0 0 0 0 0 1.75 1.75 1.75 1.75 1.75 1.75 1.75 3.5 3.5 3.5 3.5 3.5
0 0 0.25 0.5 0.5 0 0.25 0.25 0.25 0.25 0.25 0.5 0 0 0.25 0.5 0.5
18.9 20.1 16.1 7.1 15.5 14.5 5.3 12.0 8.2 7.9 10.4 6.0 6.4 15.1 5.0 5.5 13.3
0 0 125 250 250 0 125 125 125 125 125 250 0 0 125 250 250
18.1 18.7 5.0 4.9 5.0 13.1 5.0 5.0 5.0 5.0 5.2 5.0 4.8 12.9 4.9 5.0 5.2
a
Area under growth curve determined turbidometrically. Values over 6 represent growth.
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H. Blom et al. / International Journal of Food Microbiology 38 (1997) 71 – 76
Fig. 1. Growth of Listeria monocytogenes (closed symbols) and lactic acid bacteria (open symbols) in vacuum-packed, sliced servelat with 2.5% w / v NaCl stored at (A) 48C and (B) 98C or in vacuum-packed, sliced, cooked ham stored at (C) 48C and (D) 98C. (s,d) controls and (h,j) products made with 2.5% sodium lactate and 0.25% sodium acetate.
throughout 5 weeks storage at 98C (Fig. 1b and d), while numbers reached 10 8 cfu / g in controls. However, in sliced, cooked ham, inhibition of the pathogen by the acid mixture was only maintained for the first 2–3 weeks storage (Fig. 1d). There was good growth of lactic acid bacteria in control and acidcontaining samples at both temperatures (Fig. 1a–d). By the end of storage, pH values had fallen from around 6.4 to 5.5 in control servelat samples, while the pH in the acid-containing samples was unchanged. The results from the control samples again illustrate the potential for growth should L. monocytogenes survive the processing of, or contaminate vacuum-packed, sliced, cooked meat products after processing. However, even at pH values above 6.0, a mixture of 2.5% lactate and 0.25% acetate was a substantial hurdle to the growth of this pathogen, the effects being more reproducible and long lasting at 4 than at 98C. The findings of inhibition by the acid mixture are consistent, at least in part, with those of Schlyter et al. (1993) who showed that 2.5% lactate
in combination with 0.1% diacetate, a compound consisting in the solid state of equimolar amounts of acetic acid and sodium acetate, not only prevented growth of L. monocytogenes in turkey slurries for up to 6 weeks at 48C, but resulted in a small decrease in numbers (0.3 log cycles). In a parallel study, 0.1% acetate in combination with 2.5% lactate only slowed, but did not prevent growth of L. monocytogenes (results not shown). One factor contributing to the observed differences could have been that the mixture containing 0.1% diacetate lowered the pH of the turkey slurries from 6.2 to 6.0 (Schlyter et al., 1993) while there was no measurable difference in initial pH between control and acid-containing samples in the present study. Qvist et al. (1994) made use of the pH lowering effect of GDL to enhance the inhibitory effects of lactate against L. monocytogenes in vacuum-packed, sliced bologna sausage. In that study 2% lactate in combination with 0.25% GDL, which resulted in a fall in pH from 6.6 to 6.3, inhibited its growth for at least 7 weeks both at 5 and 108C (Qvist et al., 1994). At this level of GDL,
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sensory scores remained above the acceptability level throughout storage and there were no significant differences from control samples (Qvist et al., 1994). In the present study, consumer testing of fresh, uninoculated samples of the servelat and cooked, sliced ham was done at a local supermarket. Of 171 participants, 85 preferred the control ham and 86 the ham with the added acid mixture, while 111 preferred the control servelat and 60 the product with the added acid mixture, the choice mainly being based on taste. The majority were frequent or occasional buyers of both products and would have purchased the preferred product and in some cases both. Age and gender had no measurable effect on the responses. The equal preferences for ham samples produced with and without the acids indicates that their inclusion in this product would not produce any undue degree of customer dissatisfaction. In the case of the relatively mild tasting servelat, this may not be so. Assuming the reason for the greater consumer preference for the standard servelat lies with the increased salt / sour taste associated with the use of the acids, the possibility of minimizing the effect needs to be investigated. This could involve a more critical examination of the minimal salt concentration necessary for the product’s technological properties, a possible small decrease in pH of the product and / or use of alternative salts of lactate and acetate. The findings presented confirm and reinforce the previously reported inhibitory effects of mixtures of lactate and acetate against growth of L. monocytogenes in turkey slurries with pH values greater than 6.0 (Schlyter et al., 1993). Explanations for these effects at pH values well above the respective pK values of the acids, and therefore at relatively low concentrations of the undissociated acid with which inhibition is normally associated, await further studies. At the same time, the results provide an alternative solution to the combination of lactate and glucono-d-lactone shown to be effective against L. monocytogenes in bologna sausage (Qvist et al., 1994). Although the combination did not adversely affect the sensory properties of the bologna, the acidifying effects of the lactone could lead to sensory problems in some products. The combined inhibitory and sensory results suggest that a mixture of 2.5% (w / v) lactate and 0.25% (w / v) acetate, could be used to increase the margins
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of safety for sliced and spreadable vacuum-packed ready-to-eat cooked meat products with similar characteristics to servelat and cooked ham. Strict temperature control of production and storage however, are still a very important hurdle against the growth of this organism. Finally, the results with propylparaben illustrate the potential pitfalls in translating results from studies in liquid media to fatcontaining food products.
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