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Keywords:

  • LAB;
  • PCR detection;
  • rDNA identification;
  • vacuum-packed meat products spoilage

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aim:  To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products.

Methods and Results:  A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, ‘morcilla’ and ‘fiambre de magro adobado’ obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing.

Conclusions:  Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products.

Significance and Impact of the Study:  Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cooked meat products are frequently commercialized by packaging under vacuum or in a modified atmosphere and are expected to maintain their sensory qualities for long storage times under refrigerated conditions. It is well known that the composition of the gaseous-phase changes during storage and that the bacterial microbiota undergoes selection towards CO2-tolerant but slowly growing species, mainly represented by psychrotrophic leuconostocs and ‘atypical’ lactobacilli (Lactobacillus curvatus/Lactobacillus sakei) (Reuter 1981; Nissen et al. 2001; Hammes and Hertel 2003). These have been reported to be the major spoilage bacteria in meat product ecosystems (Dainty and Mackey 1992; Borch et al. 1996; Korkeala and Björkroth 1997; Björkroth et al. 1998) causing defects such as souring, off-flavours, discolouration, gas production, slime production and a decrease in pH (Egan et al. 1980; Egan 1983; Schillinger and Lücke 1987; Borch and Agerhem 1992). Although lactic acid bacteria (LAB) populations in the original product are usually below the detection limit (<10 CFU g−1), they increase during storage to 108 CFU g−1 and cause spoilage (Nerbrink and Borch 1993; Björkroth and Korkeala 1996a; Hamasaki et al. 2003). However, the composition and evolution of the LAB populations may vary depending on the matrix and should be investigated in order to improve the commercial shelf life of these products.

The characterization and study of the development of LAB populations during storage of vacuum-packed meat products has been approached mainly by culture methods (Barakat et al. 2000; Susiluoto et al. 2002; Jones 2004). In these studies, de Man, Rogosa and Sharpe (MRS) is usually utilized as the isolation medium which might result in the selection of species, as it has been observed that some LAB are not able to grow on it, i.e. most of the Carnobacterium species (Hammes and Hertel 2003), some Lactococcus (Barakat et al. 2000) and some Lactobacillus (Falsen et al. 1999). Subsequently, recovered isolates are further identified by biochemical, morphological and physiological tests that are time consuming and may yield uncertain results or misidentifications. Although LAB species of different genera frequently share many phenotypical traits, some species show high intraspecific physiological variation (Parente et al. 2001; Björkroth and Holzapfel 2003; Hammes and Hertel 2003). These methods also have limitations because of their tendency to relatively poor reproducibility (Gonzalez et al. 2000; Corsetti et al. 2001; Muyanja et al. 2003; Temmerman et al. 2004).

During the last few years, several molecular methods have been applied to the identification and typing of LAB such as rapid amplified ribosomal DNA restriction analysis (ARDRA) (Ventura et al. 2000), specific PCR (Berthier and Ehrlich 1998; Yost and Nattress 2000; Scarpellini et al. 2002; Macián et al. 2004), internal spacer region (ISR) and ISR restriction profiles (Chenoll et al. 2003), ribotyping (Björkroth and Korkeala 1996b), repetitive extragenic palindromic (rep)-PCR (Gevers et al. 2001) or random amplification of polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE) (Björkroth et al. 1996). However, data derived from their application in the analysis of LAB populations associated with vacuum-packed meat spoilage are still limited. Ribotyping has been very useful in differentiating between Lact. sakei starter and spoiler strains (Björkroth and Korkeala 1996b), in the evaluation of the LAB contamination in vacuum-packed sliced cooked meat products (Björkroth and Korkeala 1997) or in the identification of the organisms associated to spoilage of herring filets in acetic acid marinade (Lyhs et al. 2004). Recently, ribotyping used in combination with phenotypic methods allowed the identification of most of the LAB isolated from ‘morcilla de Burgos’ (Santos et al. 2005). Multiplex PCR and RAPD analysis demonstrated that a mixed community of Lact. sakei/Lact. curvatus and Leuconostoc spp. in vacuum-packed beef changed to one in which a single Leuconostoc strain dominated (Yost and Nattress 2002); however, only these LAB were investigated. Among the molecular techniques available to study bacterial communities, denaturing gel electrophoresis has been applied for the rapid detection of previously identified bacteria responsible for spoilage in meat processing plants and meat products (Takahashi et al. 2004). Another approximation, based on PFGE and API 50CH profile identification, was applied by Jones (2004) who analysed the succession dynamics of LAB populations in chill-stored vacuum-packaged beef.

In the present work, we have analysed the presence of LAB associated with a variety of vacuum-packed fresh and spoiled meat products. LAB levels in food have been evaluated using three different culture media in order to achieve a broader range of recovery of LAB species. Identification of isolates used rDNA-based molecular approaches that we previously developed (Chenoll et al. 2003; Macián et al. 2004). The results have provided useful information about the species composition and development of LAB populations related to bloating spoilage of vacuum-packed cooked meat products.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Products analysed

In all, 18 samples corresponding to 13 vacuum-packed meat products were analysed, including cooked ham (one) and chopped turkey (two) both bought at supermarkets, and ‘fiambre de magro adobado’ (FMA) (nine) and ‘morcilla’ (six) obtained from production lines. FMA is a typical Spanish marinated pork product. The marinade contains salt, vegetable proteins, powdered milk, lactose and dextrose sugars and some preservatives as additives. The marinated meat pieces are stuffed into an artificial casing and cooked. After chilling, the product is cold-stored before slicing and vacuum-packaging. ‘Morcilla’ is a typical Spanish sausage that consists of a mixture of onion, pork fat, blood, salt and different spices, stuffed into a natural casing. All meat products were stored in our laboratory at 4°C before analysis. Of the 13 samples, ten showed an acidic and strong but not disgusting odour (sour odour) as a symptom of spoilage, and nine of them that were analysed after the commercial shelf life period had expired showed bloated packs derived from gas accumulation. Discolouration was only observed in ‘morcilla’. Liquid appeared in all packages. Cooked ham and chopped turkey B (i.e. sample B) showed package bloating during the commercial shelf life period. Chopped turkey B that was sampled during the shelf life period was incubated at 30°C for 24 h in buffered peptone water (BPW; Merck, Darmstadt, Germany) prior to analysis, as a LAB enrichment step. Sensory evaluation of the products is shown in Table 1.

Table 1.   Microbiological analysis of fresh and spoiled vacuum-packed meat products: plate counts in three media (MRS, TSYE, YGLP) and identification of LAB isolates by rDNA-based methods. Time of sampling after manufacturing and spoilage symptoms are indicated
 Cooked ham*Chopped turkey‘Morcilla’‘Fiambre de magro adobado’
A†B*ABCA*BCDEFG
  1. nt, not tested; MRS, medium of de Man, Rogosa and Sharpe; YGLP, yeast glucose lactose peptone; TSYE, trypticase soy yeast extract.

  2. *Product analysed within the commercial shelf life.

  3. †Product sampled before expiry date, incubated for 24 h at 30°C prior to analysis.

  4. ‡Time passed between packaging and sampling.

  5. §Liquid, gas and sour odour appeared in all spoiled products. Discolouration was only observed in ‘morcilla’ after shelf life period.

  6. ¶Differences in numbers between the recovered and identified isolates are due to lose of viability.

Time (months)‡1112341233388
Spoilage§+++++++++++
Counts (CFU g−1)
 MRS7·6 × 1062·4 × 1067·6 × 1076 × 1073·3 × 1073·3 × 1073·1 × 1031·7 × 1081·4 × 1081·5 × 1087·5 × 1081·5 × 1081·1 × 107
 TSYE9·6 × 1074·7 × 1078·5 × 1075·4 × 1074·5 × 1073·5 × 1078·5 × 1039·3 × 1071·5 × 1082·5 × 1087·7 × 1081·6 × 1081·9 × 107
 YGLPnt6·5 × 1078·3 × 1075·1 × 1075·1 × 1075 × 107nt1·3 × 108Nt1·8 × 1086·6 × 1061·5 × 1089·7 × 106
Total of recovered isolates17482327383716301737373030
Carnobacterium3/1737/481/23   1/16      
C. maltaromaticum 2    1      
C. divergens3            
C. gallinarum             
C. mobile 351          
Leuconostoc14/17¶1/485/23¶5/2725/38¶31/3711/1625/3017/1718/37¶12/371/30 
Leuc. Mesenteroides814523312251617121 
Leuc. Carnosum      5      
Nonregistered profiles3   1 4 1    
Lactobacillus 9/48¶9/2311/2713/38¶5/37¶2/163/30 17/37¶14/37¶29/3030/30
Lact. Plantarum    3    742830
Lact. curvatus  1    1 45  
Lact. sakei  8118211 11  
Other species 5   2 1 111 
Nonregistered profiles         31  
Enterococcus  7/237/27   1/30 1/37   
Lactococcus lactis     1/37   1/377/37  
Nonregistered profiles 1/481/234/27  2/161/30  4/37  

Sample preparation and lactic acid bacteria isolation

Food (40 g) was aseptically cut and homogenized in 360 ml of BPW for 1–2 min in a sterile plastic bag with lateral filter (BagPage S 400, BagSystem, Interscience, St-Nom-la-Breteche, France) using a stomacher (Stomacher Lab-Blender 400, Seward, London, UK). One millilitre of the resulting mixture was taken from the filter side and tenfold serial dilutions were prepared in sterile saline solution (0·8% w/v NaCl) up to 10−7. A 100 μl aliquot was spread in duplicate on agar plates for LAB enumeration. Plates were incubated in micro-aerobic conditions at 30°C for 7 days and LAB were enumerated by counting colonies on three media. MRS (Oxoid, Madrid, Spain) was used as the most common medium utilized for isolating and propagating lactobacilli (de Man et al. 1960; Hammes and Hertel 2003). Yeast glucose lactose peptone (YGLP; http://www.cect.org) and trypticase soy yeast extract (TSYE; Collins et al. 1987) were used for nonselective recovery of carnobacteria. For each sample, up to ten colonies per medium were randomly picked from enumeration plates and cultured on the corresponding medium. Gram determination, oxidase and catalase tests were performed using standard procedures. Those isolates that were Gram-positive, catalase- and oxidase-negative were considered as LAB. They were stored for a long term at −20°C in a 10% (w/v) dilution of the corresponding broth medium supplemented with 20% (w/v) glycerol, for further analysis.

DNA isolation

DNA from pure cultures was extracted following the guanidium thiocyanate method (Pitcher et al. 1989), spectrophotometrically quantified (Ultrospec 2000 spectrophotometer, Amersham Biosciences UK Limited, Buckinghamshire, UK) and adjusted to a final concentration of 40 ng μl−1 in ultra-pure water (Sigma-Aldrich, Madrid, Spain). Total DNA was extracted from 1 ml aliquots of the homogenized food using the DNeasy® Tissue Kit (Qiagen GmbH, Hilden, Germany) and purified DNA was recovered in 100 μl of ultra-pure water. For colony PCR, isolated colonies were picked from plates and resuspended in 50 μl of ultra-pure water, heated for 10 min at 100°C and centrifuged at 13 000 rev min−1 for 15 min. The supernatant was used as template for PCR amplification.

Internal spacer region and rapid amplified ribosomal DNA restriction analysis profiles

PCR amplification of both the 16S rDNA gene and the ISR between the 16S and 23S rDNA genes, as well as restriction analysis of amplicons using DdeI and HaeIII (Roche Diagnostics, Barcelona, Spain), was carried out as previously described by Chenoll et al. (2003).

PCR detection and identification of lactic acid bacteria

Homogenized food samples were analysed by PCR using specific primers for the genera Leuconostoc and Carnobacterium, and species Carnobacterium divergens, Carnobacterium gallinarum, Carnobacterium maltaromaticum, Lact. curvatus, Lactobacillus plantarum and Lact. sakei.

Previously described 16S rDNA genus- and species-specific primers for Leuconostoc and Carnobacterium (Macián et al. 2004), as well as ISR species-specific primers for Lact. curvatus, Lact. sakei and Lact. plantarum (Berthier and Ehrlich 1998), were used in this study. PCR amplification was carried out according to Berthier and Ehrlich (1998) and Macián et al. (2004), unless otherwise stated. Briefly, PCR cocktails contained 1× PCR buffer (10 mmol l−1 Tris–HCl, pH 8·8; 1·5 mmol l−1 MgCl2, 50 mmol l−1 KCl, 0·1% Triton X-100), 100 μmol l−1 of each dNTP, appropriate primer concentration and 0·5–1 U of thermostable DNA polymerase (Bioron Taq DNA polymerase, Bonsai Technologies, Madrid, Spain) in a final volume of 50 μl. DNA template for amplification was supplied as (i) purified DNA (200 ng), (ii) 5 μl of DNA from homogenized food or (iii) 5 μl of a supernatant from single colony extract. For specificity reasons, the annealing temperature used for Lact. curvatus was 42°C, and for Lact. sakei it was 58°C. Reactions were carried out in a GeneAmp PCR System 9700 (PE Applied Biosystems, Norwalk, CT, USA) Thermal Cycler.

Electrophoresis

Fifteen microlitres of ISR amplification products were electrophoresed on a 2·5% (low electroendosmosis) agarose gel in TBE buffer (45 mmol l−1 Tris-borate, pH 7·6 and 1 mmol l−1 Na2EDTA) at 80 V for 105 min. Twenty microlitres of samples of restricted 16S rDNA were electrophoretically separated through a 2% agarose gel in TBE buffer at 50 V for 150 min. Five microlitres of specific amplifications were electrophoretically separated through a 2% agarose gel in TAE buffer (40 mmol l−1 Tris-acetate, pH 7·6 and 1 mmol l−1 Na2EDTA) at 100 V for 25 min. Gels were stained with ethidium bromide and photographed under UV light. Gel images were recorded using a video camera (GELPRINTER PLUS, TDI, Madrid, Spain) and stored as TIFF files.

Banding pattern analysis

Digitized images were converted, normalized, analysed and combined using the Software package BioNumerics 2.5 (Applied Maths, Kortrijk, Belgium). In order to normalize the banding patterns, molecular weight markers were included every seventh track. The levels of similarity between pairs of traces were computed using the Jaccard coefficient. Data were clustered using the unweighted pair group method with arithmetic mean algorithm. Identification of profiles was carried out by comparison with a database previously generated in our laboratory with the aid of the BioNumerics software, containing ISR and ARDRA profiles corresponding to 91 reference strains (Chenoll et al. 2003). In this study, the database has been increased to 124 strains including species belonging to other LAB and related genera: Carnobacterium (11 strains representing 11 species), Enterococcus (ten strains corresponding to ten species), Lactobacillus (61 strains, including 45 species), Lactococcus (seven strains representing four species), Leuconostoc (13 strains representing 11 species), Pediococcus (nine strains including six species), Vagococcus (two strains of Vagococcus salmoniarum), Weissella (five strains corresponding to five species) and Staphylococcus (six strains representing six species).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Microbial analysis of vacuum-packed meat products

Table 1 summarizes the results of microbial counts that were found to fall in the range 106–108 CFU g−1 in samples analysed after the expiry of their commercial shelf life or those showing spoilage symptoms. The lowest LAB counts were obtained in FMA sample A (103 CFU g−1) that was analysed 1 month after packaging and did not show spoilage symptoms. Two FMA samples of the same batch, one analysed before slicing (As) and the other after packaging (Ap), showed no growth in any of the media used (data not shown). Three samples of ‘morcilla’ analysed 4–7 days after packaging and not showing spoilage symptoms, had microbial counts around 104–105 CFU g−1, but LAB were not recovered (data not shown). Chopped turkey A was analysed after 24 h incubation at 30°C and its microbial counts were found to be of the same logarithmic order as spoiled products. In general, microbial counts were of the same logarithmic order in the three independent media used. Exceptions were cooked ham and chopped turkey A, which showed counts 1 log unit lower in MRS than in the other two media, and FMA sample E in which counts were 2 log units lower in YGLP than in MRS or TSYE.

Identification of isolates following an rDNA-based molecular approach

A total of 387 colonies recovered from the enumeration plates were analysed applying the molecular identification schedule previously proposed (Chenoll et al. 2003). Table 1 summarizes the results of isolate identification and the ratios genus/isolates and species/genus for each product. First, ISR profiles were obtained applying the PCR reaction directly to colony extracts, which allowed the assignment of 142 (37%) isolates to the genera Lactobacillus/Pediococcus, 165 (43%) to the genus Leuconostoc, 42 (11%) to the genus Carnobacterium, 16 (4%) to the genus Enterococcus and nine (2%) to the genus Lactococcus. Thirteen isolates (3%) showed ISR profiles not registered in our database and remained unidentified. Seventeen of the 387 isolates (11 identified as Lactobacillus/Pediococcus and six identified as Leuconostoc) were unrecoverable after a second streaking, and thus could not be further identified at species level. Of the remaining 357 isolates, 329 (92%) were identified to the species level. The ISR profile analysis allowed direct identification of the 42 Carnobacterium isolates as C. maltaromaticum (three), C. divergens (three) and Carnobacterium mobile (36), all of which were recovered from product samples analysed prior to expiry of their commercial shelf life and/or previously incubated at 30°C for 24 h (Table 1). Carnobacterium isolates were confirmed by specific PCR. The ARDRA profile analysis revealed that all Lactobacillus/Pediococcus isolates actually belonged to Lactobacillus species. They were identified as Lact. plantarum (72), Lact. curvatus/sakei (44), Lactobacillus collinoides (three), Lactobacillus murinus (two), Lactobacillus suebicus (one), Lactobacillus jensenii (two), Lactobacillus coryniformis (two) and Lactobacillus salivarius (one), and four isolates could not be ascribed to any of the profiles recorded in our database. Differentiation between these Lact. curvatus/sakei was carried out by specific PCR using the primers described by Berthier and Ehrlich (1998). Of the 44 Lact. curvatus/sakei isolates, 11 showed a 305-bp amplification product corresponding to Lact. curvatus and 33 yielded a 290-bp amplification product corresponding to Lact. sakei. Isolates identified as Lact. plantarum were confirmed by specific PCR (primers described by Berthier and Ehrlich 1998). Of 159 Leuconostoc isolates, 145 (91%) were identified as Leuconostoc mesenteroides and five (3%) as Leuconostoc carnosum by combining ARDRA-DdeI and ARDRA-HaeIII profiles, whereas nine isolates (6%) showed nonregistered profiles. Nine isolates belonged to the species Lactococcus lactis as revealed by 16S rDNA sequence analysis performed according to Chenoll et al. (2003).

Global results concerning the distribution of isolates into genera according to the isolation medium are shown in Fig. 1. Lactobacillus spp. were mainly recovered from MRS (47%); leuconostocs were obtained at similar numbers in the three media but at slightly higher frequency in TSYE (38%); carnobacteria were recovered in very low numbers from MRS (12%) when compared with TSYE and YGLP (45% and 43%, respectively); enterococci were mainly recovered from MRS and TSYE; lactococci only from TSYE and YGLP, and the LAB isolates showing nonregistered patterns arose mainly from YGLP. Regarding Carnobacterium species recovery, C. divergens was isolated from MRS and TSYE, C. maltaromaticum from TSYE and YGLP only and C. mobile mainly from TSYE and YGLP.

image

Figure 1.  Distribution of isolates into genera according to the enumeration media. (bsl00001) MRS, (bsl00008) TSYE, (bsl00036) YGLP.

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Lactic acid bacteria populations in the vacuum-packed meat products analysed: species composition and evolution

Table 1 shows the population species composition for the different meat products analysed. Cooked ham that was gas spoiled was dominated by Leuc. mesenteroides but C. divergens was also present. In chopped turkey sample A, analysed after incubation at 30°C for 24 h, the LAB composition was mainly dominated by carnobacteria; sample B, which was gas spoiled, was dominated by Lact. sakei, Enterococcus spp. and Leuc. mesenteroides. The compositions of LAB populations in ‘morcilla’ and FMA were analysed at different times after packaging and are shown in Fig. 2(a,b), respectively. Leuconostoc mesenteroides, Lact. plantarum and Lact. sakei were the predominant species in both products. In ‘morcilla’ (Fig. 2a), the 2-month storage sample (A) contained Leuc. mesenteroides, members of Lact. sakei and Enterococcus spp. After 3 months storage (sample B) Leuc. mesenteroides was the dominant species, followed by Lact. sakei; the former subsequently became the unique Leuconostoc species recovered after 4 months (sample C). No Lact. curvatus isolates appeared. In FMA (Fig. 2b), Leuc. mesenteroides was the predominant species 2 and 3 months after packaging, reaching a maximum at 2 months at the time when Lact. plantarum, Lact. curvatus and Lact. sakei scored the lowest numbers. Only in samples F and G, corresponding to an unusually longer storage period (8 months), did Lact. plantarum reach the highest numbers and become almost the unique species present.

image

Figure 2.  Evolution of the predominant species in (a) ‘morcilla’ and (b) ‘fiambre de magro adobado’ during storage. Percentages of isolates recovered in successive samplings are represented at different times after packaging.

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Table 2 shows the results of LAB detection by PCR using total DNA extracted from the homogenized samples as template. Carnobacterium was detected by PCR in cooked ham, chopped turkey and ‘morcilla’ samples B and C, but isolates were only recovered from products within the shelf life period. In FMA, PCR positive results were only obtained in Ap and A samples, but only after incubation in MRS broth at 30°C for 24 h. The remaining samples were negative for Carnobacterium by PCR, and one isolate was recovered from sample A (within the shelf life period). The genus Leuconostoc was detected by PCR in all samples analysed directly from the homogenized food except in FMA sample As. FMA samples A and Ap were positive after incubation at 30°C in MRS. Isolates were recovered from all samples except in FMA sample G, analysed 8 months after packaging. The genus Lactobacillus was detected in all samples except cooked ham. Lactobacillus curvatus was detected by PCR in ‘morcilla’ and FMA, and isolates were only recovered in chopped turkey B and FMA samples B, D and E. Lactobacillus sakei was detected by PCR in all samples except cooked ham, and isolates were recovered in chopped turkey B, ‘morcilla’ and FMA samples A, B, D and E. Lactobacillus plantarum was detected by PCR in ‘morcilla’ samples B and C, and FMA samples A, B, D, E, F and G. Isolates were recovered from ‘morcilla’ B and FMA samples D, E, F and G.

Table 2.   PCR detection of Carnobacterium (genus and nonmotile species), Leuconostoc (genus), Lactobacillus curvatus, Lactobacillus sakei and Lactobacillus plantarum in vacuum-packed meat products using genus/group/species specific primers, and comparison with the results obtained after identification of isolates (PCR detection/presence of isolates)
 Cooked hamChopped turkey‘Morcilla’‘Fiambre de magro adobado’
ABABCAs*Ap*A*BCDEFG
  1. As, sample A before slicing; Ap, sample A just after packaging; nt, not tested; w, weak amplification band.

  2. *Samples As, Ap and A were analysed by PCR after homogenization and also after 24 and 48 h incubation at 30°C in MRS broth.

  3. †Positive after 24 h incubation at 30°C in MRS broth.

  4. ‡Nonmotile group of Carnobacterium species.

Carnobacterium+/++/++/+−/−+/−+/−−/−+†/−+†/+−/−−/−−/−−/−−/−−/−
Cnmg‡+/++/++/−−/−−/−+/−−/−+†/−+†/−−/−−/−−/−−/−−/−−/−
C. maltaromaticum+/−+/++/−nt/−−/−+/−−/−+†/−+†/+nt/−−/−−/−−/−nt/−nt/−
C. divergens+/+−/−+/−nt/−−/−+/−−/−+†/−+/−nt/−+/−−/−−/−nt/−nt/−
C. callinarum−/−−/−−/−nt/−−/−−/−−/−−/−−/−nt/−−/−−/−−/−nt/−nt/−
Leuconostoc+/++/++/++/++/++/+−/−+†/−+†/++/++/++/++/++/++/−
Lact. curvatus−/−−/−−/++/−+/−+/−w/−w/−+/−+/+w/−+/++/+w/−w/−
Lact. sakei−/−w/−+/++/++/++/+w/−w/−+/++/+w/−+/++/+w/−w/−
Lact. plantarum−/−−/−−/−−/−+/+w/−−/−−/−+/−+/−−/−+/++/++/++/+

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Studies on the characterization and development of LAB populations associated with vacuum-packed meat products are usually carried out by culture methods. However, the difficulties of LAB identification using morphological, physiological and biochemical traits hamper a detailed description of the species composition. As an alternative, molecular techniques have been recognized as very useful tools for obtaining accurate identification of LAB species as well as for the detection of species present in low numbers. However, data from studies based on molecular techniques are still very scarce.

In the present study, we have analysed four types of meat products that differ in their manufacturing characteristics but have in common that they are vacuum-packed and commercialized refrigerated. We used three isolation media including the generally recommended MRS, together with YGLP and TSYE, to improve the recovery of species other than Lactobacillus and Leuconostoc, i.e. Carnobacterium and Lactococcus spp. (Collins et al. 1987; Barakat et al. 2000).

On the basis of microbial counts, LAB were not detectable in samples processed close to the date of packaging. However, after storage under vacuum and refrigeration, LAB counts reached 108 CFU g−1, which is in agreement with data reported in the literature (Björkroth and Korkeala 1997; Hamasaki et al. 2003; Jones 2004). Following the rDNA-based molecular approach previously developed by our group (Chenoll et al. 2003; Macián et al. 2004), 97% of isolates were identified at the genus level and 92% at the species level. To our knowledge, studies on the identification of LAB exclusively based on molecular techniques, have been carried out by ribotyping (Lyhs et al. 2004) and rep-PCR (Gevers et al. 2001), and our results are in accordance with the LAB identification levels reported in them. Previous studies based on morphological and physiological characterization failed in the identification of LAB-spoilage-related strains (Mäkeläet al. 1992; Samelis et al. 1998) or only a low percentage of strains were ascribed to species (Gonzalez et al. 2000; Corsetti et al. 2001). By combining physiological tests with molecular techniques, i.e. DNA–DNA hybridization, DNA sequencing and rep-PCR, LAB isolates have been successfully identified in species by other authors (Cai et al. 1998; Corsetti et al. 2001; Tamang et al. 2005). Compared with previous studies based on the isolation and phenotypic characterization of LAB, the approach applied here, exclusively based on molecular techniques, has greatly improved the proportion of isolates accurately identified at species level. In addition, as the isolates are obtained from three different media, our results allow a better approximation to the real LAB species composition.

Comparison of the species recovered from the three isolation media confirmed that MRS yields a higher recovery of Lactobacillus but fails in the isolation of some Carnobacterium species such as C. maltaromaticum and C. mobile, which are not able to grow in the presence of acetate (Collins et al. 1987). Similarly, Lactococcus isolates were recovered on TSYE and YGPL plates but not on MRS plates. The lack of growth of Lactococcus on MRS has been previously described (Barakat et al. 2000). The greatest diversity of LAB species was observed among isolates recovered from TSYE.

Despite LAB counts being of the same log order in samples stored for more than 2 months, they did not reflect species diversity and evolution during storage. Similar observations have also been made in other studies of vacuum-packed meat products (Egan et al. 1980; Jones 2004). The LAB isolates belonged mainly to the genera Carnobacterium, Lactobacillus and Leuconostoc in increasing order of abundance. Carnobacteria were only recovered in samples analysed after 1 month of storage, despite using TSYE and YGLP media. The presence of Carnobacterium in fresh and short-term storage meat products and their absence in spoiled products have already been described (von Holy et al. 1991; Barakat et al. 2000; Jones 2004). Indeed, most isolates of the genus Carnobacterium corresponded to samples that were analysed before expiry of the shelf life date and processed after an incubation of 24 h at 30°C. In addition, Carnobacterium spp. were detected by PCR in some samples of ‘morcilla’ and FMA. Lactobacillus and Leuconostoc species dominated the LAB population in the four meat products analysed during storage and showing spoilage symptoms, which is in accordance with the results reported by other authors regarding the occurrence of these organisms in spoiled meat products (Borch et al. 1996; Björkroth and Korkeala 1997; Björkroth et al. 2000; Santos et al. 2005). Our results suggest that while Carnobacterium occurs in fresh meat products and can be enriched using appropriate culture conditions, it becomes overgrown and substituted by Leuconostoc and Lactobacillus during storage of vacuum-packed meat products. This succession dynamics of LAB populations has already been observed by Jones (2004) for chill-stored vacuum-packed beef.

In the cases of FMA and ‘morcilla’, samples were taken after different storage times allowing us to analyse the changes in LAB species composition. In FMA, 1–2 months after packaging, Leuconostoc (various species) were present in greater numbers than Lactobacillus spp. After 3 months of storage, almost equal numbers of the genera Lactobacillus and Leuconostoc were present but Leuc. mesenteroides was the only species of this genus recovered. The dominance of Leuc. mesenteroides in spoiled vacuum-packed meat products has already been reported by Samelis et al. (1998, 2000) and Hamasaki et al. (2003). Only in samples stored for an unusually long time (8 months) did Lactobacillus plantarum became the dominant species. In ‘morcilla’, Lact. sakei reached a maximum after 2 months of storage while Leuc. mesenteroides increased during storage and replaced the other species after 3–4 months. The dominance of Leuc. mesenteroides in both products can be explained by the rapid growth of this species, as observed by Hamasaki et al. (2003), as well as by the ability of leuconostocs to produce antagonist substances towards other LAB, as noted by Yost and Nattress (2002).

Although Leuconostoc became the dominant population in spoiled long-storage samples, members of this genus were not recovered at the time of packaging. However, they were detected by PCR after enrichment, thus proving their presence below the detection threshold of culture methods. The FMA sample taken before slicing was the only one that remained negative both by PCR and by recovery of isolates, which indicates that the slicing blade is the source of Leuconostoc contamination. This hypothesis has been confirmed in a parallel study using RAPD characterization that revealed the presence of Leuc. mesenteroides among the isolates recovered from the slicing blade (data not shown).

In general, Leuc. mesenteroides, Lact. plantarum, Lact. sakei and Lact. curvatus were the species recovered in the greatest numbers. Of them Leuc. mesenteroides has heterofermentative metabolism and produces gas from glucose (a substrate present in meat), whereas the three lactobacilli are facultatively heterofermentative (Hammes and Hertel 2003), thus being able to produce CO2 via the phosphogluconate pathway from gluconate and pentoses (not present in the meat products analysed). It supports the main role of Leuc. mesenteroides in the bloating spoilage by the production of CO2.

As a conclusion, the combination of three isolation media and the use of rDNA-based molecular techniques have provided a reliable description of LAB species composition in spoiled vacuum-packed cooked meat products. Our results point to Leuc. mesenteroides as being the main species responsible for bloating spoilage in these products. Leuconostoc mesenteroides was below the detection limit of culture methods but became the dominant species when bloating appeared. PCR detection has proved to be a sensitive technique capable of detecting this spoilage bacterium at very low levels. Thus, PCR-based methods targeted to Leuc. mesenteroides are promising tools for the prediction of shelf life in these products.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by ‘Comisión Interministerial de Ciencia y Tecnología’ (CICYT) grant AGL2000-1462. EC is the recipient of a PhD fellowship I3P-BPD2001-1 from the ‘Consejo Superior de Investigaciones Científicas’ (CSIC). PE is the recipient of a postgraduate fellowship TS/03/UVEG/16 from the ‘Generalitat Valenciana’. We thank Alicia Quiñonero for technical assistance, the CECT for reference cultures and CS for supplying the samples for this research. We thank A.P. MacCabe for critical reading of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Barakat, R.K., Griffiths, M.W. and Harris, L.J. (2000) Isolation and characterization of Carnobacterium, Lactococcus, and Enterococcus spp. from cooked, modified atmosphere packed, refrigerated, poultry meat. Int J Food Microbiol 62, 8394.
  • Berthier, F. and Ehrlich, S.D. (1998) Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region. FEMS Microbiol Lett 161, 97106.
  • Björkroth, K.J. and Korkeala, H.J. (1996a) Evaluation of Lactobacillus sake contamination in vacuum-packaged sliced cooked meat products by ribotyping. J Food Prot 59, 398401.
  • Björkroth, K.J. and Korkeala, H.J. (1996b) rRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime. Int J Food Microbiol 30, 293302.
  • Björkroth, K.J. and Korkeala, H.J. (1997) Use of rRNA gene restriction patterns to evaluate lactic acid bacterium contamination of vacuum-packaged sliced cooked whole-meat product in a meat processing plant. Appl Environ Microbiol 63, 448453.
  • Björkroth, K.J. and Holzapfel, W.H. (2003) Genera Leuconostoc, Oenococcus and Weissella. In The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, 3rd edn, release 3.12 ed. Dworkin, M. New York: Springer-Verlag. http://link.springer-ny.com/link/service/books/10125/ .
  • Björkroth, K.J., Ridell, J. and Korkeala, H.J. (1996) Characterization of Lactobacillus sake strains associated with production of ropy slime by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) patterns. Int J Food Microbiol 31, 5968.
  • Björkroth, K.J., Vandamme, P. and Korkeala, H.J. (1998) Identification and characterization of Leuconostoc carnosum, associated with production and spoilage of vacuum-packaged, sliced, cooked ham. Appl Environ Microbiol 64, 33133319.
  • Björkroth, K.J., Geisen, R., Schillinger, U., Weiss, N., De Vos, P., Holzapfel, W.H., Korkeala, H.J. and Vandamme, P. (2000) Characterization of Leuconostoc gasicomitatum sp. nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions. Appl Environ Microbiol 66, 37643772.
  • Borch, E. and Agerhem, H. (1992) Chemical, microbial and sensory changes during the anaerobic cold storage of beef inoculated with a homofermentative Lactobacillus sp. or a Leuconostoc sp. Int J Food Microbiol 15, 99108.
  • Borch, E., Kant-Muermans, M.L. and Blixt, Y. (1996) Bacterial spoilage of meat and cured meat products. Int J Food Microbiol 33, 103120.
  • Cai, Y., Benno, Y., Ogawa, M., Ohmomo, S., Kumai, S. and Nakase, T. (1998) Influence of Lactobacillus spp. from an inoculant and of Weissella and Leuconostoc spp. from forage crops on silage fermentation. Appl Environ Microbiol 64, 29822986.
  • Chenoll, E., Macián, M.C. and Aznar, R. (2003) Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rRNA-based techniques. Syst Appl Microbiol 26, 546556.
  • Collins, M.D., Farrow, A.E., Phillips, B.A. and Jones, D. (1987) Classification of Lactobacillus divergens, Lactobacillus piscicola, and some catalase-negative, asporogenous rod-shaped bacteria from poultry in a new genus, Carnobacterium. Int J Syst Bacteriol 37, 310316.
  • Corsetti, A., Lavermicocca, P., Morea, M., Baruzzi, F., Tosti, N. and Gobbetti, M. (2001) Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat (species Triticum durum and Triticum aestivum) sourdoughs of Southern Italy. Int J Food Microbiol 64, 95104.
  • Dainty, R.H. and Mackey, B.M. (1992) The relationship between the phenotypic properties of bacteria from chill-stored meat and spoilage processes. J Appl Bacteriol 73, 103S114S.
  • De Man, J.C., Rogosa, M. and Sharpe, M.E. (1960) A medium for the cultivation of lactobacilli. J Appl Bacteriol 23, 130135.
  • Egan, A.F. (1983) Lactic acid bacteria of meats and meat products. Antonie Van Leeuwenhoek 49, 327336.
  • Egan, A.F., Ford, A.L. and Shay, B.J. (1980) A comparison of Microbacterium thermosphactum and lactobacilli as spoilage organisms of vacuum-packaged sliced luncheon meats. J Food Sci 45, 17451748.
  • Falsen, E., Pascual, C., Sjöden, B., Ohlén, M. and Collins, M.D. (1999) Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human sources: description of Lactobacillus iners sp. nov. Int J Syst Bacteriol 49, 217221.
  • Gevers, D., Huys, G. and Swings, J. (2001) Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 205, 3136.
  • Gonzalez, C.J., Encinas, J.P., Garcia-Lopez, M.L. and Otero, A. (2000) Characterization and identification of lactic acid bacteria from freshwater fishes. Food Microbiol 17, 383391.
  • Hamasaki, Y., Ayaki, M., Fuchu, H., Sugiyama, M. and Morita, H. (2003) Behavior of psychrotrophic lactic acid bacteria isolated from spoiling cooked meat products. Appl Environ Microbiol 69, 36683671.
  • Hammes, W.P. and Hertel, C. (2003) The genera Lactobacillus and Carnobacterium. In The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, 3rd edn, release 3.15. ed. Dworkin, M. New York: Springer-Verlag. http://link.springer-ny.com/link/service/books/10125/ .
  • Von Holy, A., Cloete, T.E. and Holzapfel, W.H. (1991) Quantification and characterization of microbial populations associated with spoiled, vacuum-packed Vienna sausages. Food Microbiol 8, 95104.
  • Jones, R.J. (2004) Observations on the succession dynamics of lactic acid bacteria populations in chill-stored vacuum-packaged beef. Int J Food Microbiol 90, 273282.
  • Korkeala, H.J. and Björkroth, K.J. (1997) Microbiological spoilage and contamination of vacuum-packed cooked sausages. J Food Prot 60, 724731.
  • Lyhs, U., Koort, J.M.K., Lundström, H.S. and Björkroth, K.J. (2004) Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated the lactic acid bacterium population associated with strong slime formation in an acetic-acid herring preserve. Int J Food Microbiol 90, 207218.
  • Macián, M.C., Chenoll, E. and Aznar, R. (2004) Simultaneous detection of Carnobacterium and Leuconostoc in meat products by multiplex PCR. J Appl Microbiol 97, 384394.
  • Mäkelä, P.M., Korkeala, H.J. and Laine, J.J. (1992) Ropy slime-producing lactic acid bacteria contamination at meat processing plants. Int J Food Microbiol 17, 2735.
  • Muyanja, C., Narvhus, J.A., Treimo, J. and Langsrud, T. (2003) Isolation, characterisation and identification of lactic acid bacteria from bushera: a Ugandan traditional fermented beverage. Int J Food Microbiol 80, 201210.
  • Nerbrink, E. and Borch, E. (1993) Evaluation of bacterial contamination at separate processing stages in emulsion sausage production. Int J Food Microbiol 20, 3744.
  • Nissen, H., Holck, A. and Dainty, R.H. (2001) Identification of Carnobacterium spp. and Leuconostoc spp. in meat by genus-specific 16S rRNA probes. Lett Appl Microbiol 19, 165168.
  • Parente, E., Grieco, S. and Crudele, M.A. (2001) Phenotypic diversity of lactic acid bacteria isolated from fermented sausages produced in Basilicata (Southern Italy). J Appl Microbiol 90, 943952.
  • Pitcher, D.G., Saunders, N.A. and Owen, R.J. (1989) Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151156.
  • Reuter, G. (1981) Psychrotrophic lactobacilli in meat products. In Psychrotrophic Microorganisms in Spoilage and Pathogenicity ed. Robots, T.A., Hobbs, G., Christian, J.H.B. and Skovgaard, N. pp. 253258. London: Academic Press.
  • Samelis, J., Kakouri, A., Georgiadou, K.G. and Metaxopoulos, J. (1998) Evaluation of the extent and type of bacterial contamination at different stages of processing of cooked ham. J Appl Microbiol 84, 649660.
  • Samelis, J., Kakouri, A. and Rementzis, J. (2000) Selective effect of the product type and the packaging conditions on the species of lactic acid bacteria dominating the spoilage microbial association of cooked meats at 4°C. Food Microbiol 17, 329340.
  • Santos, E.M., Jaime, I., Rovira, J., Lyhs, U., Korkeala, H. and Björkroth, J. (2005) Characterization and identification of lactic acid bacteria in ‘‘morcilla de Burgos’’. Int J Food Microbiol 97, 285296.
  • Scarpellini, M., Mora, D., Colombo, S. and Franzetti, L. (2002) Development of genus/species-specific PCR analysis for identification of Carnobacterium strains. Curr Microbiol 45, 2429.
  • Schillinger, U. and Lücke, F.K. (1987) Lactic acid bacteria on vacuum-packaged meat and their influence on shelf life. Fleischwirtschaft 67, 12441248.
  • Susiluoto, T., Korkeala, H. and Björkroth, J. (2002) Leuconostoc gasicomitatum is the dominating lactic acid bacterium in retailed modified-atmosphere-packaged marinated broiler meat strips on sell-by-day. Int J Food Microbiol 80, 8997.
  • Takahashi, H., Kimura, B., Yoshikawa, M., Gotou, S., Watanabe, I. and Fujii, T. (2004) Direct detection and identification of lactic acid bacteria in a food processing plant and in meat products using denaturing gradient gel electrophoresis. J Food Prot 67, 25152520.
  • Tamang, J.P., Tamang, B., Schillinger, U., Franz, C.M.A.P., Gores, M. and Holzapfel, W.H. (2005) Identification of predominant lactic acid bacteria isolated from traditionally fermented vegetable products of the Eastern Himalayas. Int J Food Microbiol 105, 347356.
  • Temmerman, R., Huys, G. and Swings, J. (2004) Identification of lactic acid bacteria: culture-dependent and culture-independent methods. Trends Food Sci Technol 15, 348359.
  • Ventura, M., Casas, I.A., Morelli, L. and Callegari, M.L. (2000) Rapid amplified ribosomal DNA restriction analysis (ARDRA) identification of Lactobacillus spp. isolated from fecal and vaginal samples. Syst Appl Microbiol 23, 504509.
  • Yost, C.K. and Nattress, F.M. (2000) The use multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage. Lett Appl Microbiol 31, 129133.
  • Yost, C.K. and Nattress, F.M. (2002) Molecular typing techniques to characterize the development of a lactic acid bacteria community on vacuum-packaged beef. Int J Food Microbiol 72, 97105.