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Prevention of ropiness in cooked pork by bacteriocinogenic cultures
International Dairy Journal 12 (2002) 239–246
Prevention of ropiness in cooked pork by bacteriocinogenic cultures M.T. Aymerich, M. Garriga*, S. Costa, J.M. Monfort, M. Hugas IRTA, Meat Technology Center, Granja Camps i Armet, s/n, 17121 Monells, Spain Received 17 April 2001; accepted 3 September 2001
Abstract Several bacteriocinogenic lactic acid bacteria and some bacteriocins were assessed as biopreservatives on the prevention of the slime production in sliced vacuum-packed cooked pork. Enterococcus faecium CTC492 and Lactobacillus sakei CTC494, bacteriocin producers, prevented ropiness due to Lb. sakei CTC746 to a different extent. Ent. faecium CTC492 plus enterocins and Lb. sakei CTC494 plus sakacin prevented slime production by Lb. sakei CTC746 until 21 days of storage at 81C as well as enterocins did. Nisin inhibited the defect when Lc. carnosum CTC747 was the slime producer strain. The capability of the bacteriocins to resist the pasteurization protocol and their interaction with the meat matrix were studied. The interaction with the soluble meat matrix did not alter the activity of sakacin and nisin, but affected the activity of enterocins by a 2 log2 AU mL1 decrease. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ropy slime; Bacteriocin; Lactic acid bacteria; Meat products
1. Introduction Sliced cooked ham is a highly perishable meat product. The low salt content (2% on average), a pH of around 6.0 and a water activity higher than 0.945 are only small hurdles to inhibit the usual types of microorganisms associated with post-processing contamination. Several investigations on the improvement of the shelf-life of cooked meat products, have shown that low oxygen atmospheres increase their shelf life (Mol, Hietbring, Mollen, & van Tinteren, 1971; Silla & Simonsen, 1985). Vacuum and modified atmosphere (MA) packaging with several CO2 and N2 rates are the most widely used packaging techniques for cooked meat products. Some authors have reported that there is no difference in the shelf-life of the products in the different atmospheres assayed (Simard, Lee, Laleye, & Holley, 1983; Boerema, Penny, Cummings, & Bell, 1993) while others reported that a MA with 100% of N2 or a MA with concentrations of CO2 lower than 50% (Blickstad & Molin, 1983; *Corresponding author. Tel.: +34-972-630052; fax: +34-972630373. E-mail address: [email protected] (M. Garriga).
Ahvenainen, Skytta, & Kivikataja, 1989; Borch & Nerbrink, 1989) were the best. Kotzekidou and Bloukas (1996) showed that some bacterial preparations (Flora Carn L2 from Chr. Hansen) used as protective cultures could extend the shelf life of certain meat products, although slime production could not be avoided . (Bjorkroth & Korkeala, 1997). Lactic acid bacteria (LAB) are generally regarded as safe (GRAS) microorganisms with many applications in the food industry. Bacteriocinogenic strains of Lactobacillus sakei, Lb. curvatus and Pediococcus acidilactici have been used as starter cultures in several meat products for their ability to inhibit the growth of some pathogenic and spoilage bacteria (Schillinger, 1990; Hammes, Bantleon, & Min, 1990; Hugas, Garriga, Aymerich, & Monfort, 1995). Strains of LAB are also the major group of spoilage bacteria developing on various types of vacuum-packed meats (Kitchell & Shaw, 1975; Gill & Newton, 1978; Dainty, Shaw, Harding, & Michanie, 1979; Beverley, Hitchener, Egan, & Rogers, 1982), the main strains belonging to the genus Leuconostoc (Lc. mesenteroides, Lc. carnosum) and Lactobacillus (Lb. viridescens, Lb. brevis, Lb. sakei and Lb. plantarum). In these products, LAB produce their typical sensory changes, such as
0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 1 4 3 - 1
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souring, gas, SH2 and slime (Kempton & Bobier, 1970; . Dainty & Mackey, 1992; Bjorkroth & Korkeala, 1997) therefore spoilage can occur during the shelf-life period. In many countries, the formation of ropy slime on vacuum-packed cooked meat products has been a common spoilage problem, with high financial losses. LAB mainly Lactobacillus and Leuconostoc are responsible for this defect (Korkeala, Suortti, & M.akel.a, 1988). The slime is often formed before the sell-by date and consumers find the appearance of slimy products very offensive, although no off-odours are detected. Several studies have shown that cooked meat products become contaminated with LAB during handling after cooking (i.e, slicing and packing) (Borch, Nerbrink, & Svensson, 1988; M.akel.a & Korkeala, 1992; M.akel.a, Korkeala, & Laine, 1992). Formation of ropy slime on the surface of salted dry products (speck and seasoned ham) by Lb. sakei has also been reported (Cantoni, Bersani, & d’Aubert, 1992). Slime formation is due to the secretion by LAB of long-chain, high-molecular-mass, viscosifying or gelling exocellular polysaccharides (EPS) into the environment. They are thought to play a role in the protection of the microbial cell against desiccation, phagocytosis, phage attack, antibiotics or toxic compounds, predation by prototozoans, osmotic control, adhesion to surfaces, and also in cellular recognition (Dudman, 1977). The objectives of this study were to develop a meat system where slime could be reproduced, as a model to evaluate the effectiveness of several bacteriocinogenic cultures and some bacteriocins on the prevention of the slime production in sliced vacuum-packed cooked meat products.The ability of the slime producer strains to produce slime in in situ assays from different carbohydrates, packaging atmospheres and storage temperatures were assayed. The behaviour of the bacteriocins and the slime producer strains to the pasteurization process were also tested.
2. Material and methods 2.1. Bacterial strains and bacteriocins The LAB slime producers: Lb. sakei CTC746, Lc. carnosum CTC747 were isolated from ropy slime sliced cooked ham (Garriga, Aymerich, Costa, Gou, Monfort, & Hugas, 1998). The bioprotective cultures used were: Lb. sakei CTC494, sakacin K producer (Hugas et al., 1995) and Ent. faecium CTC492, enterocin A and B producer (Aymerich, Holo, Havarstein, Hugas, Garriga, & Nes, 1996; Aymerich, Artigas, Garriga, Monfort, & Hugas, 2000). All strains were from meat origin. The strains were grown overnight in MRS at 301C, harvested by centrifugation (5000 rpm 10 min), resuspended in glycerol and MRS broth (1:1) and stored at 401C prior
to use. Before inoculation, viable counts were determined. Bacteriocins (enterocins A, B and sakacin K) were precipitated from the supernatant culture of the producer strains with ammonium sulphate 300 g L1 and the pellet was dissolved in 1:100 of the initial culture volume in phosphate buffer 50 mm pH 7.2. For Nisaplin (Danisco-Cultor), a commercially produced bacteriocin (nisin), 1 g of liophylized product was dissolved in 5 mL of phosphate buffer 50 mm pH 7.2 and centrifugated for 1 min at 3000 rpm in a microfuge (HeraeusTM) to remove the solid residues. The bacteriocins were tested against the slime producer strains by the method of the agar spot test (Tagg, Dajani, & Wannamaker, 1976). The minimum inhibitory concentration (MIC) and the minimum killing concentration (MKC) were determined by the method of Nielsen, Dickson, and Crouse (1990). 2.2. Manufacture of modelized cooked pork The meat was coarsely ground through a 12 mm plate and brined (150 g Kg1) for 48 h at 3–51C. The brine solution contained (in g Kg1): NaCl, 20; NaNO2, 0.15; pentasodium tripolyphosphate, 3; sodium ascorbate, 0.50; carbohydrate, 10; sodium glutamate, 1 and water, 115.35. The mixture was stuffed into 7 cm diameter cellulose casings and cooked until the internal temperature reached 671C (pasteurization factor P ¼ 25). After cooling it was sliced (10 g per slice). Two different assays were carried out. In the first assay five different lots were prepared, each one with a different carbohydrate (lactose, glucose, sucrose, maltodextrins and starch). For each lot two different types of packaging were applied (vacuum and MA (80% N2:20% CO2) and two storage temperatures (41C and 81C). The samples were stored up to 21 days. In the second assay, the best conditions for the development of the slime production, according to the results obtained in the first assay, were selected; that is, sucrose, vacuum-packaging and 81C for storage. The samples were then inoculated with LAB bioprotective cultures and bacteriocins. The ropy slime-producing strains (6 102 cells) and the bacteriocinogenic strains (2 105 cells) were inoculated between two slices of cooked pork of 10 g each. Each slime-producing strain was inoculated alone and in combination with bioprotective cultures and/or bacteriocins. The bacteriocins, titer determined against the slime producer strain Lb. sakei CTC746, were inoculated at 128 AU g1 (Arbitrary Units) for enterocins A and B and Nisaplin, and 9.5 AU g1 for sakacin K. AU was defined as the reciprocal of the highest dilution showing growth inhibition of the indicator lawn. A maximum volume of 200 mL (considering ropy slime-producing strains, bacteriocinogenic strains and bacteriocins) were inoculated between two slices in order not to significantly
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increase the water activity of the product. Triplicates from each treatment were sampled at selected times (0, 7, 15 and 21 days) to determine lactobacilli populations (MRS agar incubated at 301C in anaerobiosis for 72 h), pH (Ingold electrode 406-M4-S7/35) and slime (visually). The identity of the recovered strains was confirmed by checking their plasmid profile (Anderson & McKay, 1983) according to a progressive colony sampling plan to evaluate the competitiveness of the inoculated strains during the process (Garriga et al., 1996). 2.3. Behaviour of the slime-producing strains and the bacteriocins to a pasteurization heating process In order to assess the effectivenes of several postpasteurization processes on the inhibition of the slimeproducing strains, several heat treatments were assayed in modelized-cooked pork, 651C, 681C and 711C for 30, 60 and 90 min with a P range of 6.41–165.74. To assess the stability of the bacteriocins to the pasteurization protocol, the temperature profile was reproduced in a GeneAMp, PCR System 2400 (Perkin Elmer, Norwalk, USA). The bacteriocins were dissolved in potassium phosphate buffer 50 mm, in a soluble meat matrix and in the whole meat matrix. The meat matrix was obtained by homogenizing 10 g of cooked pork in 90 mL of dilution medium (0.1% Bactopeptone and 0.85% NaCl, pH 7.0) with an Ultra-Turrax T25 (Janke & Kunkel, IKA Labortechnik, Sweden); to obtain the soluble meat matrix, the former solution was filtrated. Duplicate samples were taken at the beginning of the process (RT), at 551C, at 671C and at the end of the process (551C) and the bacteriocin titer was determined by the agar spot test against Listeria innocua CTC1014. 2.4. Sensory evaluation After 15 days of storage at 81C a panel consisting of 4 trained members evaluated two different samples of cooked pork, from representative treatments, for odour characteristics, using a non-structured 10-cm long scale with anchor points 1 cm from each end (Stone, Sidel, Oliver, Woolsey, & Singleton, 1974). The descriptors were cured, tart, floral and flour/yeast odours. The mean score of the panel for each sample was recorded.
241
generated by the two fold dilution bacteriocin assay method, the data from bacteriocin activity were analysed as log2 AU/50.
3. Results 3.1. Slime production in modelized cooked pork with different carbohydrates Lb. sakei CTC746 produced slime in all the samples (Fig. 1), packed either with vacuum or MA (80% N2:20% CO2) and in both temperatures assayed (41C and 81C). When Lc. carnosum CTC747 was the inoculated strain in cooked pork, only the samples which contained sucrose as a carbohydrate became slimy. The presence of the strain was confirmed by identifying its plasmid profile. In the control lot, some of the samples with sucrose became slimy after 21 days of storage in both of the packaging atmospheres assayed. In the control lot LAB grew from 1.5 to 6.25 log CFU g1 during the 15 days of storage at 41C, in MA or vacuum packed for any of the carbohydrates used. When glucose was the carbon source, the counts reached 8 log CFU g1. In the lots where the slime producers, Lb. sakei CTC746 or Lc. carnosum CTC747, were inoculated, the LAB counts reached 8 log CFU g1 in each of the atmospheres or carbohydrates used. At 41C and 81C, the pH significatively decreased (Po0.05) from the beginning of the experiment to the end (21 days of storage), from 6.35 to an average of 5.95 in all the treatments. The packaging atmosphere was not determinant. At 81C, the pH in the treatments inoculated with the slime producer strains were significantly lower in the lots with glucose (pH 5.41) and maltodextrins (pH 5.40) as a carbon source than in the lots with starch (pH 6.13) and lactose (pH 6.04). In the control lot, with glucose, the pH (5.53) was significantly lower
2.5. Statistics Analysis of variance (ANOVA) was performed using the General Linear Model procedure of the SAS software. Least-significance difference (LSD) test (SAS, 1988) was used for pH, odour descriptors in the sensorial analyses, and bacteriocin activity when considering their resistance to the cooking protocol. To unify the experimental error into the exponential scale
Fig. 1. Slime formation in cooked pork by Lactobacillus sakei CTC746.
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when compared with the treatment with starch (6.10). At 41C there were no differences on the slime formation between the different carbohydrates assayed. 3.2. Resistance of the slime-producing strains to the heat treatment After 21 days of storage at 81C all the inoculated samples with the slime producer strains were slimy. There was no relationship between the degree of ropiness and the inocula (from 2.5 101 to 2.5 105 CFU g1), or between the vacuum and MA. Different post-pasteurization treatments were assayed in order to test the heat resistance of the slime-producer strains Lb. sakei CTC746 and Lc. carnosum CTC747. A heat treament of 651C 30 min (P=6.10) was enough to prevent the slime production by Lc. carnosum CTC747 after 21 days of storage. Lb. sakei CTC746 needed a longer heat treatment, 651C 60 min (P=18) to avoid ropiness after 21 days of storage. 3.3. Determination of MIC and MKC The MIC of sakacin K against Lb. sakei CTC746 was estimated as a mean value of 19 AU mL1 and the MKC in 37.5 AU mL1. For enterocins A and B, the MIC was estimated in 37.5 AU mL1 and the MKC in 50 AU mL1. For nisin, the MIC was estimated in 287 AU mL1 and the MKC in 574 AU mL1. The estimated MIC and MKC for nisin against Lc. carnosum
CTC747 was 12.5 AU mL1. Lc. carnosum CTC747 was not sensitive to sakacin K and enterocins A and B.
3.4. Effect of the bioprotective cultures and bacteriocins on the inhibition of the slime formation The assays with bioprotective cultures and/or their bacteriocins were carried out, as determined previously, in vacuum-packed samples, stored at 81C and manufactured with sucrose as a carbon source in order to facilitate the production of slime by any of the slime producers. The bioprotective cultures (Ent. faecium CTC492 and Lb. sakei CTC494) did not produce slime when inoculated in cooked pork, at 81C for 21 days. Ent. faecium CTC492 prevented the ropiness due to Lb. sakei CTC746 during the first week of storage at 81C and Lb. sakei CTC494 during the first 15 days of storage at 81C. However, none of the bacteriocinogenic strains assayed could prevent the defect when Lc. carnosum CTC747 was the slime-producer strain (Table 1). Ent. faecium CTC492 plus enterocins A and B at 128 AU g1 and Lb. sakei CTC494 plus sakacin K at 9.5 AU g1 were able to prevent slime production by Lb. sakei CTC746 until 21 days of storage, however the slime production from Lc. carnosum CTC747 was not delayed. The bacteriocins were able to inhibit the growth of the slime-producing strains (Table 1). Enterocins A and B were able to inhibit the production of slime by Lb. sakei CTC746 for 21 days. Sakacin and nisin were not able to inhibit the production of the slime by Lb. sakei CTC746.
Table 1 Effect of some bacteriocinogenic lactic acid bacteria and their bacteriocins against Lactobacillus sakei CTC746 and Leuconostoc carnosum CTC747, ropy slime-producing bacteria, inoculated in modelized cooked pork and stored at 81C Slime productiona during storage period (days) LOT
7
15
21
Control Ent. faecium CTC492 (Bac+) Lb. sakei CTC494 (Bac+) Lb. sakei CTC746 Lb. sakei CTC746+ Ent. faecium CTC492 Lb. sakei CTC746+Ent. faecium CTC492+enterocins A+B Lb. sakei CTC746+enterocins A+B Lb. sakei CTC746+Lb. sakei CTC494 Lb. sakei CTC746+Lb. sakei CTC494+sakacin K Lb. sakei CTC746+sakacin K Lb. sakei CTC746+nisin Lc. carnosum CTC747 Lc. carnosum CTC747+Ent. faecium CTC492 Lc. carnosum CTC747+Ent. faecium CTC492+enterocins A+B Lc. carnosum CTC747+enterocins A+B Lc. carnosum CTC747+Lb. sakei CTC494 Lc. carnosum CTC747+Lb. sakei CTC494+sakacin K Lc. carnosum CTC747+sakacin K Lc. carnosum CTC747+nisin
+++ + +++ +++ +++ +++ +++ +++ +++ +++ +++
+++ +++ + +++ +++ +++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +
a
Three different samples were visually evaluated for each treatment and sampling time. +, slime produced; , not slime produced.
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The inhibition of ropiness by Lc. carnosum CTC747 during the 21-day period of storage was only achieved by nisin. The pH of the samples significatively ðPo0:05Þ decreased during the 21-day period of storage of the product at 81C. The samples containing the slime producer Lb. sakei CTC746 and the bioprotective culture Ent. faecium CTC492 plus enterocins had the highest drop in pH (5.3). The pH of the rest of the samples decreased from 6.15 to an average of 5.5.
3.5. Sensory evaluation No significant differences ðP > 0:05Þ were obtained between the control and most of the treatments with bacteriocins. In the treatments where Lb. sakei CTC494 and/or sakacin K were inoculated as bioprotectors, a cured odour was detected; this odour was not observed by the trained panel in the control lot and in the treatments containing the bioprotective culture Ent. faecium CTC492 and/or the enterocins A and B and nisin (Table 2). The tart odour was predominantly detected in the lots containing Ent. faecium CTC492 and enterocins. With this descriptor, no significant differences were observed between the control treatment and the rest of the treatments, the sakacin treatment giving lower scores than the control lot. No significant flour or yeast odour was detected in any of the samples. A floral odour was only detected in the samples inoculated with Ent. faecium CTC492.
3.6. Influence of the thermal cooking gradient to bacteriocin activity The activity of enterocins A and B significantly ðPo0:05Þ decreased 2 log2 AU mL1 in the initial interaction with the soluble meat matrix and 4 log2 AU mL1 in the initial interaction with the whole meat matrix when compared to a phosphate buffer;
although it was kept stable during the cooking protocol in each of the matrixes. The activity of sakacin K did not decrease in the initial interaction with the soluble meat matrix, but it decreased 2.5 log2 AU mL1 when resuspended in the whole meat matrix and compared to the activity in phosphate buffer. The initial activity in each of the matrixes kept stable during the cooking protocol. Nisin activity was not reduced by the initial interaction with the soluble or the whole meat matrix when compared to phosphate buffer, but its activity decreased by 1.5 log2 AU mL1 along the cooking protocol in the whole meat matrix. In the whole meat matrix the activity around the solid meat residues was higher than in the rest of the halus for all the bacteriocins tested, indicating adsorption to meat particles.
4. Discussion The ropy slime production in vacuum-packed, cooked meat products has been attributed to homofermentative Lactobacillus spp. and Leuconostoc spp. by several authors (Korkeala et al., 1988; von Holy, Cloete, & Holzapfel, 1991; Dykes, Cloete, & von Holy, 1994; Garriga et al., 1998). In this study Ent. faecium CTC492 could inhibit the production of slime due to Lb. sakei CTC746 up to 7 days of storage at 81C, and Lb. sakei CTC494 up to 15 days of storage at 81C. Thus, Lb. sakei CTC494 was more effective as a bioprotective culture than Ent. faecium CTC492. Sakacin production has been described at low temperatures (41–151C) and at acidic pH’s with a maximum production at 41C after 10 days and at 101C after 3 days (Hugas, Page! s, Garriga, & Monfort, 1998). On the contrary, enterocin production by Ent. faecium CTC492 was enhanced at neutral pH (6–7.5) and mild temperatures (25–351C). The maximum enterocins production at 41C was after 18 days of incubation and eight times lower than the overnight
Table 2 Sensory evaluation of cooked pork inoculated with slime-producing strains, bioprotective cultures and/or bacteriocins after 15 days of storage at 81C Odoura LOT
Cured
Tart
Yeast/flour
Floral
Control Lb. sakei CTC746+enterocins CTC746+Ent. faecium CTC492+enterocins A+B CTC746+sakacin K CTC746+Lb. sakei CTC494+sakacin K CTC746+nisin
0.4c70.5 0.4c70.5 0.6c71.1 2.3b71.5 4.1a71.2 1.0c70.9
2.5bc72.1 4.3a71.8 3.3ab72.8 0.6d70.7 1.3cd71.2 1.8bcd72.3
0.6ab70.7 0.1b70.4 0.5ab70.8 1.1a71.3 0.8ab71.2 0.8ab71.2
0.0b70.0 0.0b70.0 2.7a71.5 0.0b70.0 0.0b70.0 0.0b70.0
a
Values are expressed as means7standard deviations. Means within the same column with different superscripts differ ðPo0:05Þ:
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production at 301C. At 151C, the maximum production was observed after 5 days and it was 2.5 times lower than at 301C, explaining the failure to control the slimeproducer strain at chilling temperatures. Nitrite and NaCl at the concentrations used in cooked meat pork did not affect enterocin production (Aymerich et al., 2000). Few reports on the use of bioprotective cultures in vacuum-packed meat products have been published. Kotzekidou and Bloukas (1996) reported 1 week extension of the shelf-life of vacuum-packed sliced cooked ham stored at 41C when inoculated with the bioprotective culture Lb. alimentarius FloraCarn L2. . However, according to Bjorkroth and Korkeala (1997), the same strain could not avoid slime production in frankfurters inoculated with several slime-producing Lb. sakei strains and stored at 61C during 28 days, leading to spoilage in almost all packages. Lb. sakei CTC494 and Ent. faecium CTC492 have been successfully reported as bioprotective cultures according to their antilisterial effect in fresh meat (pork and poultry), sliced cooked meat products and fermented sausages (Hugas et al., 1995, 1998; Aymerich et al., 2000). The addition of the bioprotective cultures Lb. sakei CTC494 or Ent. faecium CTC492 plus their bacteriocins, resulted in an extension of the shelf-life through the prevention of the slime production by Lb. sakei CTC746 after 21 days of storage. This may be explained by the increase of the antimicrobial activity and also by the induction of the bacteriocin synthesis, the bacteriocins acting as inductors as has been previously reported (Eijsink, Brurberg, Middelhoven, & Nes, 1996; Nilsen, Nes, & Holo, 1998). Enterocins A and B could prevent the slime formation by Lb. sakei CTC746 until 21 days of vacuum-storage at 81C while sakacin K and nisin could not. The ratio between the added units and the MKC of the different bacteriocins applied was not the same due to bacteriocin concentration and the maximum volume allowed to be inoculated in order to avoid a significative increase in the water activity of the product. The ratio between the added units and the MKC was more or less 0.2 for sakacin and nisin and 2.5 for enterocins. This could explain why the enterocins were more effective than sakacin K and nisin. However, from the results obtained and despite the low ratio between sakacin K and its MKC, it could be considered that the bioprotective culture Lb. sakei CTC494 alone or with their bacteriocin sakacin was able to produce enough antimicrobial activity to inhibit slime production. The bacteriocin nisin was able to inhibit slime production from Lc. carnosum CTC747. Nisin had a four-fold higher titer against Lc. carnosum CTC747 than against Lb. sakei CTC746 in the in vitro agar spot test assays. The ratio between the nisin dosage applied and its MKC was 41 for Lc. carnosum CTC747, while it was
only 0.2 for Lb. sakei CTC746. This increased ratio could explain the slime inhibition produced by Lc. carnosum CTC747 observed in the in situ experiments. In previous reports some authors have reported a lack of solubility, uneven distribution and lack of stability for the effective use of nisin in meat products (DelvesBroughton, 1990). In this study, the inhibition of the slime production by nisin in a cooked meat product could be related to a good ratio between the dosage applied and the MKC of nisin for the particular strain tested. This may also partially explain some of the effective uses of nisin reported in frankfurters, raw meat and pork slurries (Caserio, Stecchini, Pastore, & Gennari, 1979; Chung, Dickson, & Crouse, 1989; Cutter & Siragusa, 1994). The possibility of adding bacteriocins in the meat before the cooking process due to their thermotolerance is of great interest. The microbial growth after processing may be prevented at the most critical step, slicing, where the total microbiota is very low and may facilitate the growth of a particular strain. In this sense, although the thermoresistance of enterocins, sakacin and nisin to 1001C for 10 min is generally known (Aymerich, Hugas, & Monfort, 1998), their ability to resist the pasteurization protocol and their interaction with the soluble meat matrix and the whole meat matrix have not been reported yet. The interaction with the soluble meat matrix did not alter the activity of sakacin K and nisin, but affected the activity of enterocins A and B by a 2 log2 AU mL1 decrease; the reported decrease might be due to the interaction with the soluble meat matrix. To keep an effective ratio between bacteriocin dosage and MKC futher pre-cooking applications will have to be considered. In the whole meat matrix a 4 log2 AU mL1 reduction for enterocins A and B activity and a 2.5 log2 AU mL1 reduction for sakacin K activity were observed compared to the phosphate buffer. This decrease in activity may be the result of adsorption to the meat particulates. Nonetheless, this may also represent an uneven homogeneity in the food system and explain ropiness observed in the enterocins lot after 15 days of storage. No unusual odours were detected in the nisin samples compared to the control treatment. Concerning the samples where sakacin and enterocins have been added, the cured odour of the former and the tart and floral odours of the latter drove the panelists to give a low score to the samples. The differences observed could be due to the processing steps of the bacteriocins sakacin K and enterocins, the odours observed could be avoided by improving the purification methods of the bacteriocins applied. In conclusion, it is not advisable to use sucrose in the formulation of cooked pork products since this is the most suitable carbon source for EPS production by slime-producing LAB strains. To avoid the development
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of slime, it is important to assure a good microbial quality of fresh meat and ingredients, good hygiene in slicing and packaging techniques together with good manufacturing practices. The use of bioprotective cultures and their metabolites could help in delaying the growth of spoiling slime-producing LAB beyond the shelf-life.
Acknowledgements The authors gratefully acknowledge Dolors Gua" rdia for Sensorial Analyses. This work was supported by CICYT, project no ALI97-0411.
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M.akel.a, P. M., Korkeala, H. J., & Laine, J. J. (1992). Ropy slime producing lactic acid bacteria contamination at meat processing plants. International Journal of Food Microbiology, 17, 27–35. Nielsen, J. W., Dickson, J. S., & Crouse, J. D. (1990). Use of a bacteriocin produced by Pediococcus acidilactici to inhibit Listeria monocytogenes associated with fresh meat. Applied and Environmental Microbiology, 56, 2142–2145. Nilsen, T., Nes, I. F., & Holo, H. (1998). An exported inducer peptide regulates bacteriocin production in Enterococcus faecium CTC492. Journal of Bacteriology, 180, 1848–1854. SAS. (1988). SAS/STAT User’s guide. Release 6.03. Statistical Analysis System. SAS Institute INC., Cary, NC. Schillinger, U. (1990). In D. D. Bils, & S. S. Kung (Eds.), Biotechnology and Food Safety, Bacteriocins of lactic acid bacteria (pp. 55–74). Boston: Butterworth-Heinemann.
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Prevention of ropiness in cooked pork by bacteriocinogenic cultures M.T. Aymerich, M. Garriga*, S. Costa, J.M. Monfort, M. Hugas IRTA, Meat Technology Center, Granja Camps i Armet, s/n, 17121 Monells, Spain Received 17 April 2001; accepted 3 September 2001
Abstract Several bacteriocinogenic lactic acid bacteria and some bacteriocins were assessed as biopreservatives on the prevention of the slime production in sliced vacuum-packed cooked pork. Enterococcus faecium CTC492 and Lactobacillus sakei CTC494, bacteriocin producers, prevented ropiness due to Lb. sakei CTC746 to a different extent. Ent. faecium CTC492 plus enterocins and Lb. sakei CTC494 plus sakacin prevented slime production by Lb. sakei CTC746 until 21 days of storage at 81C as well as enterocins did. Nisin inhibited the defect when Lc. carnosum CTC747 was the slime producer strain. The capability of the bacteriocins to resist the pasteurization protocol and their interaction with the meat matrix were studied. The interaction with the soluble meat matrix did not alter the activity of sakacin and nisin, but affected the activity of enterocins by a 2 log2 AU mL1 decrease. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ropy slime; Bacteriocin; Lactic acid bacteria; Meat products
1. Introduction Sliced cooked ham is a highly perishable meat product. The low salt content (2% on average), a pH of around 6.0 and a water activity higher than 0.945 are only small hurdles to inhibit the usual types of microorganisms associated with post-processing contamination. Several investigations on the improvement of the shelf-life of cooked meat products, have shown that low oxygen atmospheres increase their shelf life (Mol, Hietbring, Mollen, & van Tinteren, 1971; Silla & Simonsen, 1985). Vacuum and modified atmosphere (MA) packaging with several CO2 and N2 rates are the most widely used packaging techniques for cooked meat products. Some authors have reported that there is no difference in the shelf-life of the products in the different atmospheres assayed (Simard, Lee, Laleye, & Holley, 1983; Boerema, Penny, Cummings, & Bell, 1993) while others reported that a MA with 100% of N2 or a MA with concentrations of CO2 lower than 50% (Blickstad & Molin, 1983; *Corresponding author. Tel.: +34-972-630052; fax: +34-972630373. E-mail address: [email protected] (M. Garriga).
Ahvenainen, Skytta, & Kivikataja, 1989; Borch & Nerbrink, 1989) were the best. Kotzekidou and Bloukas (1996) showed that some bacterial preparations (Flora Carn L2 from Chr. Hansen) used as protective cultures could extend the shelf life of certain meat products, although slime production could not be avoided . (Bjorkroth & Korkeala, 1997). Lactic acid bacteria (LAB) are generally regarded as safe (GRAS) microorganisms with many applications in the food industry. Bacteriocinogenic strains of Lactobacillus sakei, Lb. curvatus and Pediococcus acidilactici have been used as starter cultures in several meat products for their ability to inhibit the growth of some pathogenic and spoilage bacteria (Schillinger, 1990; Hammes, Bantleon, & Min, 1990; Hugas, Garriga, Aymerich, & Monfort, 1995). Strains of LAB are also the major group of spoilage bacteria developing on various types of vacuum-packed meats (Kitchell & Shaw, 1975; Gill & Newton, 1978; Dainty, Shaw, Harding, & Michanie, 1979; Beverley, Hitchener, Egan, & Rogers, 1982), the main strains belonging to the genus Leuconostoc (Lc. mesenteroides, Lc. carnosum) and Lactobacillus (Lb. viridescens, Lb. brevis, Lb. sakei and Lb. plantarum). In these products, LAB produce their typical sensory changes, such as
0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 1 4 3 - 1
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souring, gas, SH2 and slime (Kempton & Bobier, 1970; . Dainty & Mackey, 1992; Bjorkroth & Korkeala, 1997) therefore spoilage can occur during the shelf-life period. In many countries, the formation of ropy slime on vacuum-packed cooked meat products has been a common spoilage problem, with high financial losses. LAB mainly Lactobacillus and Leuconostoc are responsible for this defect (Korkeala, Suortti, & M.akel.a, 1988). The slime is often formed before the sell-by date and consumers find the appearance of slimy products very offensive, although no off-odours are detected. Several studies have shown that cooked meat products become contaminated with LAB during handling after cooking (i.e, slicing and packing) (Borch, Nerbrink, & Svensson, 1988; M.akel.a & Korkeala, 1992; M.akel.a, Korkeala, & Laine, 1992). Formation of ropy slime on the surface of salted dry products (speck and seasoned ham) by Lb. sakei has also been reported (Cantoni, Bersani, & d’Aubert, 1992). Slime formation is due to the secretion by LAB of long-chain, high-molecular-mass, viscosifying or gelling exocellular polysaccharides (EPS) into the environment. They are thought to play a role in the protection of the microbial cell against desiccation, phagocytosis, phage attack, antibiotics or toxic compounds, predation by prototozoans, osmotic control, adhesion to surfaces, and also in cellular recognition (Dudman, 1977). The objectives of this study were to develop a meat system where slime could be reproduced, as a model to evaluate the effectiveness of several bacteriocinogenic cultures and some bacteriocins on the prevention of the slime production in sliced vacuum-packed cooked meat products.The ability of the slime producer strains to produce slime in in situ assays from different carbohydrates, packaging atmospheres and storage temperatures were assayed. The behaviour of the bacteriocins and the slime producer strains to the pasteurization process were also tested.
2. Material and methods 2.1. Bacterial strains and bacteriocins The LAB slime producers: Lb. sakei CTC746, Lc. carnosum CTC747 were isolated from ropy slime sliced cooked ham (Garriga, Aymerich, Costa, Gou, Monfort, & Hugas, 1998). The bioprotective cultures used were: Lb. sakei CTC494, sakacin K producer (Hugas et al., 1995) and Ent. faecium CTC492, enterocin A and B producer (Aymerich, Holo, Havarstein, Hugas, Garriga, & Nes, 1996; Aymerich, Artigas, Garriga, Monfort, & Hugas, 2000). All strains were from meat origin. The strains were grown overnight in MRS at 301C, harvested by centrifugation (5000 rpm 10 min), resuspended in glycerol and MRS broth (1:1) and stored at 401C prior
to use. Before inoculation, viable counts were determined. Bacteriocins (enterocins A, B and sakacin K) were precipitated from the supernatant culture of the producer strains with ammonium sulphate 300 g L1 and the pellet was dissolved in 1:100 of the initial culture volume in phosphate buffer 50 mm pH 7.2. For Nisaplin (Danisco-Cultor), a commercially produced bacteriocin (nisin), 1 g of liophylized product was dissolved in 5 mL of phosphate buffer 50 mm pH 7.2 and centrifugated for 1 min at 3000 rpm in a microfuge (HeraeusTM) to remove the solid residues. The bacteriocins were tested against the slime producer strains by the method of the agar spot test (Tagg, Dajani, & Wannamaker, 1976). The minimum inhibitory concentration (MIC) and the minimum killing concentration (MKC) were determined by the method of Nielsen, Dickson, and Crouse (1990). 2.2. Manufacture of modelized cooked pork The meat was coarsely ground through a 12 mm plate and brined (150 g Kg1) for 48 h at 3–51C. The brine solution contained (in g Kg1): NaCl, 20; NaNO2, 0.15; pentasodium tripolyphosphate, 3; sodium ascorbate, 0.50; carbohydrate, 10; sodium glutamate, 1 and water, 115.35. The mixture was stuffed into 7 cm diameter cellulose casings and cooked until the internal temperature reached 671C (pasteurization factor P ¼ 25). After cooling it was sliced (10 g per slice). Two different assays were carried out. In the first assay five different lots were prepared, each one with a different carbohydrate (lactose, glucose, sucrose, maltodextrins and starch). For each lot two different types of packaging were applied (vacuum and MA (80% N2:20% CO2) and two storage temperatures (41C and 81C). The samples were stored up to 21 days. In the second assay, the best conditions for the development of the slime production, according to the results obtained in the first assay, were selected; that is, sucrose, vacuum-packaging and 81C for storage. The samples were then inoculated with LAB bioprotective cultures and bacteriocins. The ropy slime-producing strains (6 102 cells) and the bacteriocinogenic strains (2 105 cells) were inoculated between two slices of cooked pork of 10 g each. Each slime-producing strain was inoculated alone and in combination with bioprotective cultures and/or bacteriocins. The bacteriocins, titer determined against the slime producer strain Lb. sakei CTC746, were inoculated at 128 AU g1 (Arbitrary Units) for enterocins A and B and Nisaplin, and 9.5 AU g1 for sakacin K. AU was defined as the reciprocal of the highest dilution showing growth inhibition of the indicator lawn. A maximum volume of 200 mL (considering ropy slime-producing strains, bacteriocinogenic strains and bacteriocins) were inoculated between two slices in order not to significantly
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increase the water activity of the product. Triplicates from each treatment were sampled at selected times (0, 7, 15 and 21 days) to determine lactobacilli populations (MRS agar incubated at 301C in anaerobiosis for 72 h), pH (Ingold electrode 406-M4-S7/35) and slime (visually). The identity of the recovered strains was confirmed by checking their plasmid profile (Anderson & McKay, 1983) according to a progressive colony sampling plan to evaluate the competitiveness of the inoculated strains during the process (Garriga et al., 1996). 2.3. Behaviour of the slime-producing strains and the bacteriocins to a pasteurization heating process In order to assess the effectivenes of several postpasteurization processes on the inhibition of the slimeproducing strains, several heat treatments were assayed in modelized-cooked pork, 651C, 681C and 711C for 30, 60 and 90 min with a P range of 6.41–165.74. To assess the stability of the bacteriocins to the pasteurization protocol, the temperature profile was reproduced in a GeneAMp, PCR System 2400 (Perkin Elmer, Norwalk, USA). The bacteriocins were dissolved in potassium phosphate buffer 50 mm, in a soluble meat matrix and in the whole meat matrix. The meat matrix was obtained by homogenizing 10 g of cooked pork in 90 mL of dilution medium (0.1% Bactopeptone and 0.85% NaCl, pH 7.0) with an Ultra-Turrax T25 (Janke & Kunkel, IKA Labortechnik, Sweden); to obtain the soluble meat matrix, the former solution was filtrated. Duplicate samples were taken at the beginning of the process (RT), at 551C, at 671C and at the end of the process (551C) and the bacteriocin titer was determined by the agar spot test against Listeria innocua CTC1014. 2.4. Sensory evaluation After 15 days of storage at 81C a panel consisting of 4 trained members evaluated two different samples of cooked pork, from representative treatments, for odour characteristics, using a non-structured 10-cm long scale with anchor points 1 cm from each end (Stone, Sidel, Oliver, Woolsey, & Singleton, 1974). The descriptors were cured, tart, floral and flour/yeast odours. The mean score of the panel for each sample was recorded.
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generated by the two fold dilution bacteriocin assay method, the data from bacteriocin activity were analysed as log2 AU/50.
3. Results 3.1. Slime production in modelized cooked pork with different carbohydrates Lb. sakei CTC746 produced slime in all the samples (Fig. 1), packed either with vacuum or MA (80% N2:20% CO2) and in both temperatures assayed (41C and 81C). When Lc. carnosum CTC747 was the inoculated strain in cooked pork, only the samples which contained sucrose as a carbohydrate became slimy. The presence of the strain was confirmed by identifying its plasmid profile. In the control lot, some of the samples with sucrose became slimy after 21 days of storage in both of the packaging atmospheres assayed. In the control lot LAB grew from 1.5 to 6.25 log CFU g1 during the 15 days of storage at 41C, in MA or vacuum packed for any of the carbohydrates used. When glucose was the carbon source, the counts reached 8 log CFU g1. In the lots where the slime producers, Lb. sakei CTC746 or Lc. carnosum CTC747, were inoculated, the LAB counts reached 8 log CFU g1 in each of the atmospheres or carbohydrates used. At 41C and 81C, the pH significatively decreased (Po0.05) from the beginning of the experiment to the end (21 days of storage), from 6.35 to an average of 5.95 in all the treatments. The packaging atmosphere was not determinant. At 81C, the pH in the treatments inoculated with the slime producer strains were significantly lower in the lots with glucose (pH 5.41) and maltodextrins (pH 5.40) as a carbon source than in the lots with starch (pH 6.13) and lactose (pH 6.04). In the control lot, with glucose, the pH (5.53) was significantly lower
2.5. Statistics Analysis of variance (ANOVA) was performed using the General Linear Model procedure of the SAS software. Least-significance difference (LSD) test (SAS, 1988) was used for pH, odour descriptors in the sensorial analyses, and bacteriocin activity when considering their resistance to the cooking protocol. To unify the experimental error into the exponential scale
Fig. 1. Slime formation in cooked pork by Lactobacillus sakei CTC746.
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when compared with the treatment with starch (6.10). At 41C there were no differences on the slime formation between the different carbohydrates assayed. 3.2. Resistance of the slime-producing strains to the heat treatment After 21 days of storage at 81C all the inoculated samples with the slime producer strains were slimy. There was no relationship between the degree of ropiness and the inocula (from 2.5 101 to 2.5 105 CFU g1), or between the vacuum and MA. Different post-pasteurization treatments were assayed in order to test the heat resistance of the slime-producer strains Lb. sakei CTC746 and Lc. carnosum CTC747. A heat treament of 651C 30 min (P=6.10) was enough to prevent the slime production by Lc. carnosum CTC747 after 21 days of storage. Lb. sakei CTC746 needed a longer heat treatment, 651C 60 min (P=18) to avoid ropiness after 21 days of storage. 3.3. Determination of MIC and MKC The MIC of sakacin K against Lb. sakei CTC746 was estimated as a mean value of 19 AU mL1 and the MKC in 37.5 AU mL1. For enterocins A and B, the MIC was estimated in 37.5 AU mL1 and the MKC in 50 AU mL1. For nisin, the MIC was estimated in 287 AU mL1 and the MKC in 574 AU mL1. The estimated MIC and MKC for nisin against Lc. carnosum
CTC747 was 12.5 AU mL1. Lc. carnosum CTC747 was not sensitive to sakacin K and enterocins A and B.
3.4. Effect of the bioprotective cultures and bacteriocins on the inhibition of the slime formation The assays with bioprotective cultures and/or their bacteriocins were carried out, as determined previously, in vacuum-packed samples, stored at 81C and manufactured with sucrose as a carbon source in order to facilitate the production of slime by any of the slime producers. The bioprotective cultures (Ent. faecium CTC492 and Lb. sakei CTC494) did not produce slime when inoculated in cooked pork, at 81C for 21 days. Ent. faecium CTC492 prevented the ropiness due to Lb. sakei CTC746 during the first week of storage at 81C and Lb. sakei CTC494 during the first 15 days of storage at 81C. However, none of the bacteriocinogenic strains assayed could prevent the defect when Lc. carnosum CTC747 was the slime-producer strain (Table 1). Ent. faecium CTC492 plus enterocins A and B at 128 AU g1 and Lb. sakei CTC494 plus sakacin K at 9.5 AU g1 were able to prevent slime production by Lb. sakei CTC746 until 21 days of storage, however the slime production from Lc. carnosum CTC747 was not delayed. The bacteriocins were able to inhibit the growth of the slime-producing strains (Table 1). Enterocins A and B were able to inhibit the production of slime by Lb. sakei CTC746 for 21 days. Sakacin and nisin were not able to inhibit the production of the slime by Lb. sakei CTC746.
Table 1 Effect of some bacteriocinogenic lactic acid bacteria and their bacteriocins against Lactobacillus sakei CTC746 and Leuconostoc carnosum CTC747, ropy slime-producing bacteria, inoculated in modelized cooked pork and stored at 81C Slime productiona during storage period (days) LOT
7
15
21
Control Ent. faecium CTC492 (Bac+) Lb. sakei CTC494 (Bac+) Lb. sakei CTC746 Lb. sakei CTC746+ Ent. faecium CTC492 Lb. sakei CTC746+Ent. faecium CTC492+enterocins A+B Lb. sakei CTC746+enterocins A+B Lb. sakei CTC746+Lb. sakei CTC494 Lb. sakei CTC746+Lb. sakei CTC494+sakacin K Lb. sakei CTC746+sakacin K Lb. sakei CTC746+nisin Lc. carnosum CTC747 Lc. carnosum CTC747+Ent. faecium CTC492 Lc. carnosum CTC747+Ent. faecium CTC492+enterocins A+B Lc. carnosum CTC747+enterocins A+B Lc. carnosum CTC747+Lb. sakei CTC494 Lc. carnosum CTC747+Lb. sakei CTC494+sakacin K Lc. carnosum CTC747+sakacin K Lc. carnosum CTC747+nisin
+++ + +++ +++ +++ +++ +++ +++ +++ +++ +++
+++ +++ + +++ +++ +++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +
a
Three different samples were visually evaluated for each treatment and sampling time. +, slime produced; , not slime produced.
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The inhibition of ropiness by Lc. carnosum CTC747 during the 21-day period of storage was only achieved by nisin. The pH of the samples significatively ðPo0:05Þ decreased during the 21-day period of storage of the product at 81C. The samples containing the slime producer Lb. sakei CTC746 and the bioprotective culture Ent. faecium CTC492 plus enterocins had the highest drop in pH (5.3). The pH of the rest of the samples decreased from 6.15 to an average of 5.5.
3.5. Sensory evaluation No significant differences ðP > 0:05Þ were obtained between the control and most of the treatments with bacteriocins. In the treatments where Lb. sakei CTC494 and/or sakacin K were inoculated as bioprotectors, a cured odour was detected; this odour was not observed by the trained panel in the control lot and in the treatments containing the bioprotective culture Ent. faecium CTC492 and/or the enterocins A and B and nisin (Table 2). The tart odour was predominantly detected in the lots containing Ent. faecium CTC492 and enterocins. With this descriptor, no significant differences were observed between the control treatment and the rest of the treatments, the sakacin treatment giving lower scores than the control lot. No significant flour or yeast odour was detected in any of the samples. A floral odour was only detected in the samples inoculated with Ent. faecium CTC492.
3.6. Influence of the thermal cooking gradient to bacteriocin activity The activity of enterocins A and B significantly ðPo0:05Þ decreased 2 log2 AU mL1 in the initial interaction with the soluble meat matrix and 4 log2 AU mL1 in the initial interaction with the whole meat matrix when compared to a phosphate buffer;
although it was kept stable during the cooking protocol in each of the matrixes. The activity of sakacin K did not decrease in the initial interaction with the soluble meat matrix, but it decreased 2.5 log2 AU mL1 when resuspended in the whole meat matrix and compared to the activity in phosphate buffer. The initial activity in each of the matrixes kept stable during the cooking protocol. Nisin activity was not reduced by the initial interaction with the soluble or the whole meat matrix when compared to phosphate buffer, but its activity decreased by 1.5 log2 AU mL1 along the cooking protocol in the whole meat matrix. In the whole meat matrix the activity around the solid meat residues was higher than in the rest of the halus for all the bacteriocins tested, indicating adsorption to meat particles.
4. Discussion The ropy slime production in vacuum-packed, cooked meat products has been attributed to homofermentative Lactobacillus spp. and Leuconostoc spp. by several authors (Korkeala et al., 1988; von Holy, Cloete, & Holzapfel, 1991; Dykes, Cloete, & von Holy, 1994; Garriga et al., 1998). In this study Ent. faecium CTC492 could inhibit the production of slime due to Lb. sakei CTC746 up to 7 days of storage at 81C, and Lb. sakei CTC494 up to 15 days of storage at 81C. Thus, Lb. sakei CTC494 was more effective as a bioprotective culture than Ent. faecium CTC492. Sakacin production has been described at low temperatures (41–151C) and at acidic pH’s with a maximum production at 41C after 10 days and at 101C after 3 days (Hugas, Page! s, Garriga, & Monfort, 1998). On the contrary, enterocin production by Ent. faecium CTC492 was enhanced at neutral pH (6–7.5) and mild temperatures (25–351C). The maximum enterocins production at 41C was after 18 days of incubation and eight times lower than the overnight
Table 2 Sensory evaluation of cooked pork inoculated with slime-producing strains, bioprotective cultures and/or bacteriocins after 15 days of storage at 81C Odoura LOT
Cured
Tart
Yeast/flour
Floral
Control Lb. sakei CTC746+enterocins CTC746+Ent. faecium CTC492+enterocins A+B CTC746+sakacin K CTC746+Lb. sakei CTC494+sakacin K CTC746+nisin
0.4c70.5 0.4c70.5 0.6c71.1 2.3b71.5 4.1a71.2 1.0c70.9
2.5bc72.1 4.3a71.8 3.3ab72.8 0.6d70.7 1.3cd71.2 1.8bcd72.3
0.6ab70.7 0.1b70.4 0.5ab70.8 1.1a71.3 0.8ab71.2 0.8ab71.2
0.0b70.0 0.0b70.0 2.7a71.5 0.0b70.0 0.0b70.0 0.0b70.0
a
Values are expressed as means7standard deviations. Means within the same column with different superscripts differ ðPo0:05Þ:
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production at 301C. At 151C, the maximum production was observed after 5 days and it was 2.5 times lower than at 301C, explaining the failure to control the slimeproducer strain at chilling temperatures. Nitrite and NaCl at the concentrations used in cooked meat pork did not affect enterocin production (Aymerich et al., 2000). Few reports on the use of bioprotective cultures in vacuum-packed meat products have been published. Kotzekidou and Bloukas (1996) reported 1 week extension of the shelf-life of vacuum-packed sliced cooked ham stored at 41C when inoculated with the bioprotective culture Lb. alimentarius FloraCarn L2. . However, according to Bjorkroth and Korkeala (1997), the same strain could not avoid slime production in frankfurters inoculated with several slime-producing Lb. sakei strains and stored at 61C during 28 days, leading to spoilage in almost all packages. Lb. sakei CTC494 and Ent. faecium CTC492 have been successfully reported as bioprotective cultures according to their antilisterial effect in fresh meat (pork and poultry), sliced cooked meat products and fermented sausages (Hugas et al., 1995, 1998; Aymerich et al., 2000). The addition of the bioprotective cultures Lb. sakei CTC494 or Ent. faecium CTC492 plus their bacteriocins, resulted in an extension of the shelf-life through the prevention of the slime production by Lb. sakei CTC746 after 21 days of storage. This may be explained by the increase of the antimicrobial activity and also by the induction of the bacteriocin synthesis, the bacteriocins acting as inductors as has been previously reported (Eijsink, Brurberg, Middelhoven, & Nes, 1996; Nilsen, Nes, & Holo, 1998). Enterocins A and B could prevent the slime formation by Lb. sakei CTC746 until 21 days of vacuum-storage at 81C while sakacin K and nisin could not. The ratio between the added units and the MKC of the different bacteriocins applied was not the same due to bacteriocin concentration and the maximum volume allowed to be inoculated in order to avoid a significative increase in the water activity of the product. The ratio between the added units and the MKC was more or less 0.2 for sakacin and nisin and 2.5 for enterocins. This could explain why the enterocins were more effective than sakacin K and nisin. However, from the results obtained and despite the low ratio between sakacin K and its MKC, it could be considered that the bioprotective culture Lb. sakei CTC494 alone or with their bacteriocin sakacin was able to produce enough antimicrobial activity to inhibit slime production. The bacteriocin nisin was able to inhibit slime production from Lc. carnosum CTC747. Nisin had a four-fold higher titer against Lc. carnosum CTC747 than against Lb. sakei CTC746 in the in vitro agar spot test assays. The ratio between the nisin dosage applied and its MKC was 41 for Lc. carnosum CTC747, while it was
only 0.2 for Lb. sakei CTC746. This increased ratio could explain the slime inhibition produced by Lc. carnosum CTC747 observed in the in situ experiments. In previous reports some authors have reported a lack of solubility, uneven distribution and lack of stability for the effective use of nisin in meat products (DelvesBroughton, 1990). In this study, the inhibition of the slime production by nisin in a cooked meat product could be related to a good ratio between the dosage applied and the MKC of nisin for the particular strain tested. This may also partially explain some of the effective uses of nisin reported in frankfurters, raw meat and pork slurries (Caserio, Stecchini, Pastore, & Gennari, 1979; Chung, Dickson, & Crouse, 1989; Cutter & Siragusa, 1994). The possibility of adding bacteriocins in the meat before the cooking process due to their thermotolerance is of great interest. The microbial growth after processing may be prevented at the most critical step, slicing, where the total microbiota is very low and may facilitate the growth of a particular strain. In this sense, although the thermoresistance of enterocins, sakacin and nisin to 1001C for 10 min is generally known (Aymerich, Hugas, & Monfort, 1998), their ability to resist the pasteurization protocol and their interaction with the soluble meat matrix and the whole meat matrix have not been reported yet. The interaction with the soluble meat matrix did not alter the activity of sakacin K and nisin, but affected the activity of enterocins A and B by a 2 log2 AU mL1 decrease; the reported decrease might be due to the interaction with the soluble meat matrix. To keep an effective ratio between bacteriocin dosage and MKC futher pre-cooking applications will have to be considered. In the whole meat matrix a 4 log2 AU mL1 reduction for enterocins A and B activity and a 2.5 log2 AU mL1 reduction for sakacin K activity were observed compared to the phosphate buffer. This decrease in activity may be the result of adsorption to the meat particulates. Nonetheless, this may also represent an uneven homogeneity in the food system and explain ropiness observed in the enterocins lot after 15 days of storage. No unusual odours were detected in the nisin samples compared to the control treatment. Concerning the samples where sakacin and enterocins have been added, the cured odour of the former and the tart and floral odours of the latter drove the panelists to give a low score to the samples. The differences observed could be due to the processing steps of the bacteriocins sakacin K and enterocins, the odours observed could be avoided by improving the purification methods of the bacteriocins applied. In conclusion, it is not advisable to use sucrose in the formulation of cooked pork products since this is the most suitable carbon source for EPS production by slime-producing LAB strains. To avoid the development
M.T. Aymerich et al. / International Dairy Journal 12 (2002) 239–246
of slime, it is important to assure a good microbial quality of fresh meat and ingredients, good hygiene in slicing and packaging techniques together with good manufacturing practices. The use of bioprotective cultures and their metabolites could help in delaying the growth of spoiling slime-producing LAB beyond the shelf-life.
Acknowledgements The authors gratefully acknowledge Dolors Gua" rdia for Sensorial Analyses. This work was supported by CICYT, project no ALI97-0411.
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