SPOILAGE AND PATHOGENIC BACTERIA ISOLATED FROM TWO TYPES OF PORTUGUESE DRY SMOKED SAUSAGES AFTER SHELF-LIFE PERIOD IN MODIFIED ATMOSPHERE PACKAGE BACTERIAS DETERIORATIVASY PATÓGENAS AISLADAS DE DOS TIPOS DE CHORIZOS DESPUÉS DEL PERIODO DE VIDADEANAQUELENVASADAS ENATMÓSFERAS MODIFICADAS

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SPOILAGE AND PATHOGENIC BACTERIA ISOLATED FROM TWO TYPES OF PORTUGUESE DRY SMOKED SAUSAGES AFTER SHELF-LIFE PERIOD IN MODIFIED ATMOSPHERE PACKAGE BACTERIAS DETERIORATIVASY PATÓGENAS AISLADAS DE DOS TIPOS DE CHORIZOS DESPUÉS DEL PERIODO DE VIDADEANAQUELENVASADAS ENATMÓSFERAS MODIFICADAS J. S. T. Matos a; B. B. Jensen a; S. F. H. A. Barreto b; O. Hojberg a a Department of Animal Health, Welfare and Nutrition, Danish Institute of Agricultural Sciences, Tjele, Denmark b Faculdade de Medicina Veterinária, CIISA, UTL, R. Prof. Cid dos Santos - Polo Universitário, Alto da Ajuda, Lisboa, Portugal

To cite this Article Matos, J. S. T., Jensen, B. B., Barreto, S. F. H. A. and Hojberg, O.(2006) 'SPOILAGE AND

PATHOGENIC BACTERIA ISOLATED FROM TWO TYPES OF PORTUGUESE DRY SMOKED SAUSAGES AFTER SHELF-LIFE PERIOD IN MODIFIED ATMOSPHERE PACKAGE BACTERIAS DETERIORATIVASY PATÓGENAS AISLADAS DE DOS TIPOS DE CHORIZOS DESPUÉS DEL PERIODO DE VIDADEANAQUELENVASADAS ENATMÓSFERAS MODIFICADAS', CyTA - Journal of Food, 5: 3, 165 — 174 To link to this Article: DOI: 10.1080/11358120609487688 URL: http://dx.doi.org/10.1080/11358120609487688

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Cienc. Tecnol. Aliment. 5(3) 165-174 (2006) www.somenta.org/journal ISSN 1135-8122

CIENCIA Y TECNOLOGÍA ALIMENTARIA

SPOILAGE AND PATHOGENIC BACTERIA ISOLATED FROM TWO TYPES OF PORTUGUESE DRY SMOKED SAUSAGES AFTER SHELF-LIFE PERIOD IN MODIFIED ATMOSPHERE PACKAGE BACTERIAS DETERIORATIVAS Y PATÓGENAS AISLADAS DE DOS TIPOS DE CHORIZOS DESPUÉS DEL PERIODO DE VIDA DE ANAQUEL ENVASADAS EN ATMÓSFERAS MODIFICADAS

Matos, J. S. T.1#*; Jensen, B. B.1; Barreto, S. F. H. A.2; Hojberg, O.1 1 2

Danish Institute of Agricultural Sciences, Department of Animal Health, Welfare and Nutrition, 8830 Tjele, Denmark. Faculdade de Medicina Veterinária, CIISA, UTL, R. Prof. Cid dos Santos - Polo Universitário, Alto da Ajuda, 1300477 Lisboa, Portugal. # Present address: Instituto Superior de Agronomia; Departmento de Produção Agrícola e Animal - Secção de Produção Animal, Tapada da Ajuda, 1349-017 Lisboa, Portugal. Recibido/Received: 13-08-2006; aceptado/accepted: 07-11-2006 *Autor para la correspondencia. E-mail: [email protected]

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Abstract Members of the bacterial population of two types of Portuguese dry smoked sausages (chouriços) were identified and characterized. A total of 77 isolates were recovered from chouriços type Alentejano and Ribatejano, after 120 days at 20 ± 5 ºC in modified atmosphere package (55 % N2/45 % CO2) using non-selective and selective media. Based on phenotypic (cell morphology and fermentation profile) and genotypic (16SrDNA sequencing) characters, the isolates were grouped into the following genera: Bacillus, Enterococcus, Clostridium, Staphylococcus, Pediococcus, Lactobacillus and Paenibacillus. The species identified were: Enterococcus faecium (24.7 %), Bacillus subtilis (23.4 %), Staphylococcus epidermidis (14.3 %), B. cereus (7.8 %), Pediococcus acidilactici (6.5 %) Bacillus pumilus (6.5 %), Clostridium bifermentans (3.9 %), B. licheniformis, and Enterococcus faecalis (2.6 %). Major differences observed between the two varieties of chouriço were the absence of Pediococcus species and the presence of high numbers of isolates identified as Staphylococcus epidermidis in chouriço type Alentejano and, the high incidence of Bacillus species in sausage type Ribatejano. Differences found between products may be due to spices formulation in each type of product, the type of natural casings used and, the hygienic quality of the raw meat.

Resumen Se identificaron y caracterizaron los miembros de la población bacteriana de dos tipos de chorizos portugueses. Un total de 77 aislados fueron recuperados de los chouriços tipo Alentejano y Ribatejano, después de 120 días de almacenamiento con empaque en atmósfera modificada (55 % N2/ 45 % CO2) a 20 ± 5 °C usando medios de cultivo selectivos y no selectivos. Con base en caracteres del fenotipo (morfología celular y perfil de fermentación) y del genotipo (secuencia del 16SrDNA) los aislados fueron agrupado en los siguientes géneros: Bacilos, Enterococos, Clostridium, Staphylococos, Pediococos, Lactobacilos y Paenibacilos. Las especies identificadas fueron: Enterococcus faecium (24,7 %), Bacillus subtilis (23,4 %), Staphylococcus epidermidis (14,3 %), B. cereus (7,8 %), Pediococcus acidilactici (6,5 %) Bacillus pumilus (6,5 %), Clostridium bifermentans (3,9 %), B. licheniformis, and Enterococcus faecalis (2,6 %). Las principales diferencias observadas entre las dos variedades de chouriço fueron la ausencia de especies de Pediococcus y la presencia de un elevado número de aislados identificados como Staphylococcus epidermidis en chouriço tipo Alentejano y la alta incidencia de especies de Bacillus en el chouriços tipo Ribatejano. Las diferencias encontradas entre los productos pueden deberse a las especias incluidas en la formulación en cada tipo de producto, el tipo de recubrimiento natural y de la calidad higiénica de la carne cruda.

Keywords: Chouriço, modified atmosphere package, shelf life, bacteria, identification Palabras clave: Chorizo, envase en atmósferas modificadas, vida de anaquel, bacteria, identificación

INTRODUCTION

one very short maturation period, varying from one to three days. During this time all the meat, spices and curing ingredients are held at temperatures below 10 ºC. After this period the ingredients are stuffed into natural casings (salted pork intestine or beef dry casing), intensively smoked at high temperature for about 3 days. It is then considered a smoked sausage processed by heat (Sousa and Ribeiro, 1983).

Portuguese dry smoked sausage (chouriço) is very different from Spanish and other dry fermented sausages, not only in its taste but also in the processing technology. The Spanish sausage has a similar composition but is exposed to a long dry period at low temperature and is almost not smoked. Portuguese chouriço is exposed to

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The microbiology of dry smoked sausages is variable and complex and the rate of spoilage of these meat products can reduce the shelf life and cause substantial financial losses to manufacturing companies. The spoilage ecology is affected by a combination of intrinsic and extrinsic factors (Korkeala and Makela, 1989). Factors such as ripening technique (Cantoni and Comi, 1986), use of natural intestines of pork and beef to stuff meat batters (Byun et al., 2001), bacterial population of raw meat material (Nel et al., 2004; Sardinha et al., 2000), introduction of spices as formulation ingredients (Banerjee and Sarkar, 2003a), smoke (Wendorff, 1981), application of modified atmosphere and the CO2 concentrations (Stiles, 1991; Sivertsvik et al., 2002) and, storage temperature (Korkeala and Makela, 1989) all have significant influence on the microflora of these types of meat products and also on the growth and survival of spoilage microorganisms during shelf-life period. Several studies (Encinas et al., 1996; Garcia-Varona et al., 2000; te Giffel et al., 1996; Cocolin et al., 2001; Daporta Padin, 1988; Santos et al., 1998; Papamanoli et al., 2003; Hugas et al., 2003; Devriese et al., 1995) were conducted to identify bacterial populations on fermented dry sausages, however, despite increasing interest for modified atmosphere package (MAP), the effect of MAP on the spoilage bacteria of these meat products is not yet clear. Besides identifying the bacterial contamination during the manufacture processing, it is further important to identify the spoilage microorganisms that will remain or proliferate during the storage period in MAP, and might decrease shelf life compromising product safety. The type of spoilage microorganisms are primarily related to the extent and type of the initial contamination as affected by the resident «factory flora», the manufacturing site and the processing hygiene. The spoilage potential is not only dependent on the bacterial growth rates, but also on the specific metabolic activity of the different species growing on, or in, cooked meat products under certain storage conditions (Samelis et al., 2000). The purpose of this study was to determine late shelf life population in two types of Portuguese dry smoked sausages (chouriços) packaged in modified atmosphere, which do not yet show clear spoilage changes at the end of the producer-defined shelf life period (120 days at 20 ± 5 ºC). Tentative identification and characterization of these species were based on phenotypic (cell morphology and fermentation profile) and genotypic (16S rDNA sequencing) characters.

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smoked dry sausages consisted of a mixture of pork meat and fat minced (approx. 20 mm) with the formulation ingredients red pepper paste, water, salt, garlic paste, olive oil, spices, white wine, sugar, liquid smoke and additives (sodium nitrite and commercial sausage sodium polyphosphate) as outlined in Table 1. Major differences between the two products consisted in olive oil, spices, hot red pepper paste and white wine, besides the casings used. This paste is matured for one day at 5 ºC. The stuffing is filled into salted pork intestine in the Ribatejano sausage type and into beef dry casing for the Alentejano sausage type and, subject to the drying effect of smoke and temperature for about three days. The experiments were conducted at a commercial meat plant and the study was based on 12 samples (each sample was composed by a mixture of three sausages, a total of 36 sausages) randomly drawn from two batches. Representative sausages of each experiment were packed separately in modified atmosphere (55 % N2/45 % CO2) with a net weight of about 225g and stored at 20 ± 5 ºC for 120 days (shelf-life period determined by the manufacturing industry) for further analysis. Representative sausage samples presented pH values after production of 5.80 ± 0.17 and of 5.70 ± 0.10 and water activity varied between 0.93 ± 0.01 and 0.94 ± 0.01 [mean values ± standard deviation (n=6)] for sausage type A and R, respectively. After storage, pH values varied from 5.60 ± 0.00 and 5.43 ± 0.06 for sausage type A and R, respectively, and water activity remained with the same values (data not shown). Isolation of bacteria Microbial analyses were performed on triplicate samples. Each sample was prepared from three sausages. Twenty five grams were removed (approximately 8 g from each sausage) were cut in to pieces of 1 cm and homogenised into 225 ml of Peptone water (Merck; 7228). Ten fold dilutions were prepared in 0.85 % sodium chloride (Merck, 6404). For bacteria enumeration, 1 ml samples (0.1 ml for staphylococci) from the dilution tubes were inoculated onto non-selective and selective media. The ten fold dilutions used for isolation of spore forming bacteria, Clostridium perfringens and sulphite reduction clostridia were previously heated to 80 ºC for 10 minutes. Lactic acid bacteria were isolated from MRS-agar (Merck, 0660) plates incubated at 30 ºC in carbon dioxide atmosphere (Anaerocult, Merck, 1.13829) for 72 hours. Spore forming bacteria was performed using Tryptone Glucose Extract Agar (TGEA) (Oxoid; CM127). After cooling, the TGEA plates were sealed with 0.08 % of Agar (Himedia; RM026). The TGEA plates were incubated aerobically at 30 ºC for 120 h. The selective media used for isolation of staphylococci was Baird-Parker (BP) agar (Oxoid; CM275) supplemented with Egg Yolk – Tellurite emulsion (Oxoid; SR054C), incubated aerobically 37 ºC for 48 h. Staphylococci-like colonies were selected after application

MATERIALSAND METHODS Sausage manufacture and sampling Samples studied for this work were chouriços type Alentejano (A) and Ribatejano (R) produced by a large factory in Portugal. The recipe of these types of Portuguese

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Table 1. Composition of Alentejano and Ribatejano smoked dry sausages.

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Tabla 1. Composición de chorizos Alentejano y Ribatejano.

Formulation (g/kg) Meat raw material - Pork belly boneless, without rind - Pork picnic, boneless, without shank - Pork trimming 90/10 Sub-total Ingredients - Commercial sausage sodium polyphosphate, E452(i); (50% of P2O5) - Water - Sugar - Olive oil - Liquid smoke - Spice lourer - Sweet chilli - Powder clove - Garlic paste - Sweet red pepper paste - Special red pepper paste - Hot red pepper paste - White pepper (powder) - Sodium chloride with sodium nitrite, E250, (0.6% of NaNO2) - Salt - White wine Sub-total Total

Chouriço Alentejano

Chouriço Ribatejano

172.1 688.2 860.3

176.0 528.1 176.0 880.1

5.0

5.0

31.0 2.1 5.2 1.0 0.02 11.0 4.3 44.9 12.9 1.7 10.3

12.7 2.1 1.4 0.04 5.0 0.1 11.3 8.8 46.1 2.5 1.1 10.0

10.3 139.7 1000

10.0 3.9 119.9 1000

Viable counts were calculated as log CFU and colony forming unit presented as mean ± standard deviation, (n = 6). Results of search analysis (enterococci - EVA, sulphite reducing clostridia - OPSP and Clostridium perfringens - TSC) were reported as positive (presence of the organism) and as negative if not detected. From the highest dilution of each medium an average of 20 colonies were picked randomly, and transferred aseptically into tubes containing selective media according to the type of microorganism. Colonies isolated from TGEA and EVA-agar media were transferred aseptically into tubes containing PC medium with the following composition in distilled water (g l-1): 5.0 peptone of casein (Merck, 7213), 2.5 yeast extract (Oxoid, L21), 1.0 D(+)-glucose (Merck, 8337) incubated aerobically at 30 ºC (TGEA-colonies) and at 37 ºC (EVA-colonies). Isolates from MRS-agar were transferred into Hungate tubes containing MRS-broth (Merck, 0661) incubated at 30 ºC. Clostridia and staphylococci colonies were picked and transferred aseptically under anaerobic conditions into Hungate tubes containing Reinforced Clostridial-bouillon (Merck, 5411) added 0.005 g l-1 hemin and incubated at 37 ºC. After incubation of the cells overnight, the cell morphology was investigated by phase-contrast microscopy. Glycerol (20 %) was added to the remaining overnight cultures before storage at –80 ºC.

of Pastorex STAPH-PLUS test (Sanofi Pasteur; 56356), catalase test (20 vol. of H2O2); gram staining; haemolytic activity (Plates with Columbia with 5% of sheep blood; BioMerieux; 43041) and cell morphology observation by phase-contrast microscopy. Enterococci were picked from quantifying MRS plates and were determined using Azide Dextrose (Merck; 1590) after anaerobic incubation at 37 ºC for 24 h. From positive tubes (tubes with precipitate at the bottom of the tube), 1 ml was transferred aseptically into tubes with 10 ml of Ethyl Violet Azide broth (EVA-broth) (Difco; 060601-7). Positive results (violet precipitate at the bottom of the tube) were analyzed after anaerobic incubation at 37 ºC for 48 h. Colony isolation was performed by taking 1 ml from the highest dilution of EVA-broth positive tubes, poured it into Ethyl Violet Azide agar (EVA-broth with added 8 g l-1of agar) and incubate aerobically at 37 ºC for 48 h. The selective media used for isolation of Clostridium perfringens and sulphite reduction clostridia were Perfringens Agar Base (TSC) (Oxoid; CM 587), supplemented with cycloserine (Oxoid; SR088E) and Perfringens Agar Base (OPSP) (Oxoid; CM 543), supplemented with sodium sulphadiazine (Oxoid, SR076) and with oleandomyxine and polymyxine B (Oxoid, SR077), respectively. Both, TSC-plates and OPSP-plates were incubated at 37 ºC under anaerobic conditions for 120 h.

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Fermentation profiles The isolates were subcultured (1.0 ml inoculum of an overnight culture) in anaerobic (N2 atmosphere) roll tubes containing 9 ml of a pre-reduced sterilised peptone yeast glucose (PYG) medium described by Holdeman et al. (1977). After incubation at 30 ºC (lactic acid bacteria and spore forming bacteria) and at 37 ºC (staphylococci, enterococci and clostridia) for 48 h, the concentrations of fermentation products in terms of organic acids and gas were measured. Gas samples (500 µl) were removed from the headspace of the tubes with gas-tight syringes and analyzed for the concentration of CO2, N2 and H2 by gas chromatography using a thermal conductivity detector (TCD), according to the method described by Jensen and Jorgensen (1994). Subsequently, the tubes were thoroughly mixed, and samples (1.0 ml) were collected aseptically and the concentration of organic acids was quantified by gas chromatography using a flame ionization detector (FID) as outlined by Jensen et al. (1995). Studies were conducted in triplicate.

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DNA. The PCR program included 1 min of initial denaturation at 94 ºC, followed by 30 cycles of denaturation (30 s at 94 ºC), annealing (30 s at 60 ºC), and extension (45 s at 72 ºC), and a final extension at 72 ºC for 10 min. The PCR products were verified by electrophoresis in 1% agarose gel (w/v) stained with ethidium bromide and visualised by UV transillumination. Sequencing of 16S rDNA Prior to determination of the 16S rDNA sequences, the PCR products were purified on QIAquick PCR purification kit columns, according to the manufacturer‘s instructions (QIAGEN Gmbh, Hilden, Germany). The 16S rDNA sequences of the isolates were determined using the ABI BigDyeTM Terminator Cycle Sequencing Ready Reaction kit (PE Biosystems, Foster City) and monitored with an automatic sequence analyzer (ABI PRISM® 377 DNA sequencer PE Biosystems). Isolates were subjected to near-full-length 16S rDNA sequencing as described by Leser et al. (2002). To determine the closest relatives of the near-full-length 16S rDNA sequences retrieved, searches were conducted in GenBank using BLAST algorithm (Altschul et al., 1990).

DNA extraction Culture liquid (1.5 ml) was transferred from overnight cultures into sterile microfuge tubes and centrifuged at 13.000 x g for 5 min. The cell pellet was washed twice with 2.0 ml of TES-buffer (0.05 M Tris, 0.05 M NaCl, 0.005 M EDTA [pH 8.0]) and resuspended in 500 l TES-buffer. The nucleic acid extraction was initiated by incubating the suspension with 50 µg lysozyme (Sigma, St. Louis, USA) at 37 ºC for 30 min. After incubation, 50 µg each of Proteinase K and RNase (Sigma, St. Louis, USA) were added and the mixture was further incubated at 65 ºC for 60 min. Subsequently, 50 µg of 20 % sodium dodecyl sulphate was added, and incubation at 65 ºC continued for 10 min. After cooling to room temperature, the lysates were purified twice by extraction with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), and the residual phenol was removed by extraction with an equal volume of chloroform-isoamyl alcohol (24:1). After precipitation of the nucleic acids with ethanol, the DNA was dissolved in sterile water and stored at –20 ºC until further analysis.

Nucleotide sequence of the isolates The near-full-length sequences of the isolates have been deposited in GenBank under accession numbers AY587776 - AY587843. The phylogenetic relationship of the different species within the genus Enterococcus, Bacillus, Clostridium, Staphylococcus, Pediococcus, Lactobacillus and Paenibacillus was determined by comparative sequence analysis of their 16S rDNA genes. RESULTS AND DISCUSSION Characterization of isolates from two types of Portuguese smoked dry sausages A total of 77 isolates were obtained from Alentejano (35) and from Ribatejano (42) types of smoked dry sausage, and the distribution of recovery from the different media is given along with the viable counts and the search analysis results in Table 2. The 77 isolates were obtained from TGE-agar (35.0 %), MRS-agar (22.1 %), BP (22.1 %), TSC-agar (7.8 %), EVAagar (6.5 %), and finally, from OPSP-agar (6.5 %). The majority of the isolates recovered from TGE-medium were grouped as species of Bacillus (81.5 %), Enterococcus (14.8 %) and Paenibacillus (3.7 %). The isolates obtained from MRS-agar belonged to the genera Enterococcus (64.7 %), Pediococcus acidilactici (29.4 %) and Lactobacillus (5.9 %). The EVA-agar used showed, as expected, high selectivity for enterococci. From the OPSP- and TSC-agar isolates 60 % and 83.3 % respectively belonged to Clostridium. The remaining isolates recovered from OPSPagar belonged to Enterococcus (20 %) and Bacillus (20

PCR amplification Amplification of 16S rDNA was performed in a PCR Express thermal cycler (Hybaid, Teddington, U.K.), using the primers: S-D-Bact-0008-a-S-20 (5´-AGAG TTTGATCMTGGCTCAG-3´) and S-*-Univ-1492-a-A-19 (5´-GGTTACCTTGTTACGACTT-3´). The primers were purchased from DNA Technology (Aarhus, Denmark). The reaction mixture (50 µl) consisted of reaction buffer (final concentration, 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, [pH 8.8]), each deoxynucleoside triphosphate at concentration of 200 µM, 0.2 µM of each primer, 1.0 U of DyNAzyme TM II DNA polymerase (Finnzymes OY, Finland) and 2 µl (≈ 50 ng) of template

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C. aminovalericum 4

1 2

C. celerecrescens 1

B. subtilis type A

3

4 1 1

2

E. faecalis C. botulinum

C. bifermentans

+ B. subtilis

1 1 1

1

+

3

E. faecium 2 3

17

+ B. pumilus B. cereus

6

11 1 2

P. acidilactici 5

10 Total

E. faecium E. faecialis Total 3.4 ± 0.9 Ribatejano

L. sakei 1

4.3 ± 0.3

3

B. licheniformis B. pumilus B. subtilis type B

3.3 ± 0.3

4 1 10 3 2

1

S. epidermis 11 E. faecium 6 Alentejano

7 4 1

Clostridium sp. 1 E. faecium 2

B. cereus B. subtilis type B E. faecium Paenibacillus sp. 4

No

Log CFU g-1a 2.1 ± 0.3 16S identity No

Log CFU g-1a 4.9 ± 0.6 16S identity No

Log CFU g-1a 3.5 ± 0.8 Types of sausages

10

B. licheniformis C. subterminale

E. solitarius

16S identity OPSP No

Search analysisb + 16S identity TSC

No

Search analysisb + 16S identity No

EVA

Search analysisb + 16S identity

Media TGE BP MRS

%) and, from TSC-agar, the remaining isolates obtained belonged to Enterococcus (16.7 %) (Table 2). From the BP-medium, which was expected to be selective for staphylococci, only the isolates recovered from Alentejano (A) sausage type (64.7 %) were characterized as Staphylococcus. The isolates obtained from Ribatejano (R) sausage were grouped in the Bacillus genus (Table 2). Concerning to staphylococci, provisional grouping was based on phenotypic characteristics [morphology and fermentation profile (Table 3), catalase test, gram staining and haemolytic activity (data not shown)] and through application of Pastorex STAPH-PLUS test. From this group, two representative isolates, Te13a(A)BP and Te66a(A)BP, were selected and subjected to near-full-length 16S rDNA sequence analysis (Table 3). The relationship of representative isolates is depicted on a 16S rDNA-based phylogenetic tree (Figure 1). Based on these data, only one group of staphylococci was represented, namely Staphylococcus epidermidis. The lactic acid bacteria (Enterococcus species, Pediococcus acidilactici and Lactobacillus casei) presented similar fermentation profiles (Table 3) except for the isolate Te18(A)OPSP identified as Enterococcus solitarius, which produced higher amount of formic and acetic acid and less lactic acid. According to the genotypic characters, the enterococci belonged to three different groups or species: E. faecium, E. faecalis and E. solitarius. Among the E. faecium isolates one isolate, Te42(A)TGE, showed a divergence of more than 3% (Figure 1). Isolates identified as E. faecium and E. faecalis presented the same fermentation profiles and cell morphology as outlined by Knarreborg (2002). On the basis of comparative 16S rDNA sequence analysis, the genus Enterococcus belongs to the Gram-positive bacteria with low (< 50 mol %) G + C content in the DNA. Other typical members of this phylum are clostridia and bacilli (Franz et al., 2003). Within the Bacillus genus and based on the relationship of species reflected on the 16S rDNAphylogenetic tree (Figure 1), the following related species were distinguished: Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus and Bacillus cereus. According to the results presented in Table 3, B. pumilus KL-052 isolate-like [Te34(R)TGE] was the only that produced H2 as well as higher amount of total organic acids and formic acid when compared with the other related isolates of Bacillus pumilus species. The isolates Te31(R)TGE and Te76(R)TGE, identified as B. subtilis type A (Table 3), despite the close relationship between these and the other ones identified as B. subtilis (Figure 1, Table 3), higher amounts of total organic acids and lactic acid were produced by these two isolates. Among the B. subtilis isolates, Te36(A)TGE was the only one which produced H2. Among the clostridia genus all isolates produced H2 except the isolate Te11(R)TSC, tentatively identified as C. botulinum. Clostridium bifermentans isolates were the

Tabla 2. Identidad 16S de los 77 aislados obtenidos de chorizos portuguesas variedad Alentejano (A) y Ribatejano (R) y distribución de la recuperación de distintos medios. aPromedio ± desviación estándar (n=6) del log UFC g-1 de muestras obtenidas de medios MRS, BP y TGE. bLos resultados del análisis de búsqueda se muestran como + (presencia del organismo) en 1 g de muestra (TSC), en 0,1 g de muestra (OPSP) y en 0,001 g de muestra (EVA).

Matos et al.: Spoilage and pathogenic bacteria... Table 2. 16S Identity of the 77 isolates obtained from Alentejano (A) and Ribatejano (R) Portuguese smoked dry sausage and distribution of recovery from the different media. aMean values ± standard deviation (n=6) of log CFU g-1 of sample obtained from MRS, BP and TGE media. bResults of search analyses are presented as + (presence of the organism) in 1 g of sample (TSC), in 0.01 g of sample (OPSP) and in 0.001 g of sample (EVA).

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only ones producing isocapronic acid. Genotypic characterization of Clostridium bifermentans isolates was supported by phenotypic characters, which showed typical fermentation profiles as described by Chamkha et al. (2001). Among all clostridia isolates, isobutiric acid was not produced only by the isolates Te15(A)TSC, Te63(R)OPSP and Te16(R)OPSP. Higher amounts of isovaleric acid were detected in Te11(R)TSC and Te48(R)OPSP. Considering the fermentation profiles and the relationship of species based on the 16S rDNAphylogenetic tree, clostridia species were divided into 6 groups: Clostridium bifermentans, C. aminovalericum, C. celerecrescens, C. sp., C. subterminale, and C. botulinum.

©2006 SOMENTA

flora that will growth differentially (Sousa and Ribeiro, 1983). Dry-cured sausages such as the Spanish and Italian sausages are characterized by a long ripening period (30 to 45 days) at low temperatures (from 11 to 25 oC) and at relative humidity of 60 to 90 %. This long processing time, when compared with the short process applied in Portuguese smoked sausages (1 to 3 days) at temperatures below 10 ºC, could explain the lower values of pH promoted through lactic fermentation from meat sugars and sugar formulation ingredients (Sousa and Ribeiro, 1983). LAB species have also been identified as major spoilage populations of vacuum-packaged emulsion-type sausages and other processed meats stored at refrigeration temperatures (Samelis et al., 2000; Borch et al., 1996; Korkeala and Bjorkroth, 1997). Controlling the spoilage progress by LAB is difficult as these bacteria are psychotropic, microaerophilic, and resistant to nitrite, salt, and smoke (Franz and von Holy, 1996). Non lactic acid bacteria were characterized as Bacillus and Clostridium species in both types of sausages and also as Staphylococcus epidermidis in sausage type Alentejano. Identification of Bacillus strains isolated from Spanish fermented sausages during the manufacture and ripening performed by Encinas et al. (1996) revealed, in a total of 69 isolates, Bacillus subtilis (37), B. megaterium (22), B. pumilus (5), B. circulans (3) and 2 unidentified species. These authors, therefore, considered Bacillus the second most important group of spoilage bacteria after the lactic acid bacteria. Also te Giffel et al. (1996) showed the high prevalence of Bacillus species (Bacillus subtilis and Bacillus cereus) in meat products compared with raw meat (Konuma et al., 1988; Kramer and Gilbert, 1989) supporting the role played by meat additives as source of contamination of these species. Pafumi (1986) appointed spices as the principal source of spore-forming bacteria in large volumes of foods and, Trigo and Fraqueza (1998) discussed that mesophilic spores such as Bacillaceae species were dominant in beef dry casings. Also, previous studies on the microbiology of spices have demonstrated that Bacillus cereus and Clostridium perfringens are the sporeformers most commonly found (Antai, 1988; Banerjee and Sarkar, 2003b; Kneifel and Berger, 1994; Powers et al., 1976). The presence of aerobic Bacillus related species in these MAP chouriços, where the air in the package was replaced by a specific mixture of carbon dioxide and nitrogen (45 % CO2 and 55 % N2), can be explained considering that oxygen is not always completely removed and may also permeate through the packaging material (Vermieren et al., 1999). The level of residual oxygen in MAP packs may be due to factors such as oxygen permeability of the packaging material, the ability of the food to trap air, poor heat sealing ability of the packaging material causing air to leak in ineffective evacuation and/ or ineffective gas flushing (Smith et al., 1986), the storage

The late shelf life bacteria of chouriços type Alentejano and Ribatejano, after 120 days at 20 ± 5 ºC in modified atmosphere package (55 % N2 / 45 % CO2) The majority of the species isolated from type A sausage were assigned to the species Enterococcus faecium (34.3 %), Staphylococcus epidermidis (31.4 %), Bacillus cereus (11.4 %), Clostridium bifermentans (8.6 %) and sporadic isolates of Lactobacillus casei, Enterococcus solitarius, Paenibacillus sp., Clostridium sp. and Bacillus subtilis. In type R sausage the microflora included mainly Bacillus subtilis (30.9 %), Enterococcus faecium (16.7 %), Bacillus pumilus (11.9 %), Pediococcus acidilactici (11.9 %), Clostridium spp. (9.5 %), Bacillus cereus (4.7 %) and Enterococcus faecalis, Bacillus licheniformis and Bacillus subtilis type A (4.8 %). The lactic acid bacteria (LAB) species identified (Pediococcus acidilactici, Lactobacillus casei, Enterococcus faecium and Enterococcus faecalis) are recognized among the most frequently isolated LAB from dry sausages processed with different technologies (Santos et al., 1998; Papamanoli et al., 2003; Hugas et al., 2003; Devriese et al., 1995) and packaged in modified atmosphere (Samelis et al., 2000; Pexara et al., 2002). The persistence of enterococci during ripening of fermented sausages can be attributed to their wide range of growth temperatures and their high tolerance to salt (Hugas et al., 2003). It seems that in sausages, no hurdles are found for their growth, allowing them to coexist with lactobacilli as the dominant population. In fact, in sausages with less content of fermentable sugars and higher pH (> 5.0), enterococci become more competitive and also find better conditions for survival and growth. Presence of enterococci in fermented foods might also reflect higher level of contamination or poorer curing process (Hugas et al., 2003). In the present work, the pH of sausages (5.70 and 5.80 after production) indicates that no relevant lactic acid fermentation had taken place when compared with other types of dry fermented sausages. In fact, Fanco et al. (2002), González and Díez (2002), and Ferrer and Arboix (1986), in several types of dry-cured sausages, reported pH values in the range of 4.42 - 4.50. These differences may be related to the maturation period and the microbial

170

171 1458

1 1

Paenibacillus sp. [Te79(A)TGE]

1482

3 5 1

C. bifermentans [Te19(A)TSC; Te20(A)TSC; Te62(A)TSC]

Lactic acid bacteriab Pediococcus acidilactici [Te02(R)MRS; Te03(R)MRS; Te53(R)MRS; Te56(R)MRS; Te57(R)MRS Lactobacillus sakei [Te52(A)MRS]

Staphylococcib Staphylococcus epidermidis [Te13a(A)BP; Te13b(A)BP; Te13c(A)BP; Te13d(A)BP; Te13e(A)BP; Te66a(A)BP; Te66b(A)BP; Te66c(A)BP; Te66d(A)BP; Te66e(A)BP; Te66f(A)BP]

Enterococcus faecalis [Te14(R)MRS; Te65(R)TSC] Enterococcus faecium [Te01(A)MRS; Te06(A)MRS; Te32(A)TGE; Te43(A)TGE; Te49(A)MRS; Te50(A)MRS; Te51(A)MRS; Te82(A)MRS; Te91(A)TGE; Te92(A)EVA; Te95(A)EVA; Te37(R)MRS; Te45(R)EVA; Te47(R)EVA; Te58(R)MRS; Te59(R)MRS; Te61(R)MRS; Te93(R)EVA] Enterococcus solitarius [Te18(A)OPSP]

519-1503

1

C. aminovalericum [Te16(R)OPSP]

1344

1393

1

1494-1503

1372-1500

18

11

1462-1492

2

1434-1441

1439

1469

1 1

1450

1379

C. celerecrescens [Te63(R)OPSP]

1

C. subterminale [Te48(R)OPSP]

Clostridium sp. [Te15(A)TSC]

1

C. botulinum [Te11(R)TSC]

Clostridiab

938-1501

13

1493

1475-1504 1468-1495

1481

1 6

1477-1497

2

1445-1491

4

Bpc

2

Number of isolates

Bacillus licheniformis [Te21(R)BP; Te17(R)OPSP] Bacillus pumilus strain M1-9-1 [Te23(R)BP; Te68(R)BP; Te26(R)TGE; Te29(R)TGE] Bacillus pumilus strain KL-052 [Te34(R)TGE] Bacillus cereus [Te27(R)TGE; Te72(R)TGE; Te28(A)TGE; Te30(A)TGE; Te71(A)TGE; Te73(A)TGE] Bacillus subtilis type A [Te31(R)TGE; Te76(R)TGE] Bacillus subtilis type B [Te22(R)BP; Te25(R)BP; Te35(R)TGE; Te40(R)TGE; Te41(R)TGE; Te70(R)BP; Te81(R)TGE; Te83(R)TGE; Te84(R)TGE; Te85(R)TGE; Te87(R)TGE; Te89(R)TGE; Te90(R)TGE] Bacillus subtilis [Te36(A)TGE]

Bacillib

16S Identity

a

98-99

98

97-99

98-99

97

96-99

98

98

99

98

99

98

99

97

97-99

98-99

97-99

98

98-99

98-99

Similard

Coccus

Coccus

Coccus

Coccus

Rod

Coccus

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Morphology

47.6

35.9

48.9

51,4

68.8

63.3

58.1

14.8

26.1

33.8

41.6

57.2

23.0

18.0

5.9

39.6

35.6

21.4

6.8

36.5

Total (mM)

5.3

23.6

3.1

7.3

1.6

1.6

15.6

8.2

11.8

33.5

4.5

1.5

63.1

52.2

20.0

3.7

13.3

67.4

17.7

9.6

F

11.3

22.8

8.2

15.4

14.3

13.2

62.3

72.6

58.3

59.6

59.6

70.8

28.0

30.0

32.9

3.3

12.6

20.9

26.7

2.7

A

0.3

0.3

0.2

0.2

0.1

0.1

4.7

0.0

0.4

0.3

0.6

0.6

0.3

0.5

1.7

0.2

0.1

0.3

1.5

0.0

P

0.0

0.0

0.0

0.0

0.0

0.0

3.0

0.0

0.0

0.0

5.9

4.9

0.0

0.0

0.8

0.0

0.1

0.0

0.8

0.0

IB

0.4

0.4

0.3

0.3

0.2

0.2

0.5

1.1

0.5

0.5

13.3

10.2

0.6

0.8

2.7

0.4

0.3

0.6

2.4

0.3

B

0.1

0.0

0.0

0.0

0.0

0.0

0.2

0.0

0.0

0.0

11.6

8.8

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

IV

0.0

0.0

0.0

0.0

0.0

0.0

0.1

0.4

0.0

0.0

0.0

0.0

0.2

0.2

0.0

0.1

0.0

0.0

0.0

0.0

V

Organic acidse Molar ratio (%)

0.0

0.0

0.0

0.0

0.0

0.0

10.5

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

IC

0.3

0.2

0.2

0.2

0.1

0.1

0.1

0.6

0.3

0.2

0.2

0.1

0.4

0.5

1.4

0.2

0.2

0.3

1.2

0.2

C

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.5

0.7

0.0

0.2

0.0

0.2

0.0

0.0

H

81.0

50.7

86.7

75.4

82.6

83.5

1.9

12.4

25.8

3.9

2.6

2.1

4.7

11.8

29.6

90.4

71.2

7.8

40.0

85.6

L

1.6

1.8

1.3

1.4

1.0

1.1

1.1

4.6

2.9

1.9

1.6

1.1

2.5

3.3

10.8

1.6

2.2

2.6

9.6

1.8

S

0.0

0.0

0.0

0.0

0.0

0.0

5.9

12.0

14.1

14.2

2.3

0.0

9.2

1.3

0.0

0.0

0.0

3.9

0.0

0.0

H2

27.9

21.9

18.8

24.4

18.8

18.6

38.7

45.4

36.6

49.7

22.7

36.6

31.0

10.9

11.4

13.6

17.7

28.1

9.8

35.3

CO2

Gas (mM)

Tabla 3. Agrupamiento, basado en el análisis de la secuencia con longitud casi completa de 16S rDNA, morfología celular y perfil del producto de la fermentación de la glucosa, de aislados obtenidos de dos tipos de chorizos portuguesas, Alentejano (A) y Ribatejano (R). aResultados mas similares a la secuencia de longitud casi completa a 16S rDNA obtenidas en GenBank usando el algoritmo BLAST (Altschul et al., 1990). GenBank accession AY587776-AY587843. bAislados obtenidos de salchichas Alentejano (A), de salchichas de Ribatejano(R) y de los medios: BP-Baird Parker; TGE-Extracto de Triptona Glucosa agar; MRS-agar; EVA-Agar Etil Violeta Azida; OPSP-agar base Perfringens suplementedo con cycloserins; OPSP-agar base Perfringens adicionado con sulfadiazina de sodio y oleandomixina y polimixina B. cRango de la longitud de la longitud-cercana-completa de la secuencia 16S rDNA. dRango del porcentaje de similitud en la secuencia al BLAST mas cercano. ePromedio de la cantidad total (mM) y relación molar (%) de ácidos orgánicos producidos: (F: ácido fórmico, A: ácido acético, P: ácido propionico, IB: ácido isobutírico, B: ácido n-butírico, IV: ácido isovalérico, V: ácido n-valérico, IC: ácido isocaproico, C: ácido n-caproico, H: ácido heptanoico, L: ácido DL-láctico, S: ácido succínico).

Table 3. Grouping, based on near-full-length 16S rDNA sequence analysis, cell morphology and product profiles from glucose fermentation, of isolates obtained from two types of smoked dry Portuguese sausages, Alentejano (A) and Ribatejano (R). aClosest relatives of the near-full-length 16S rDNA sequences obtained in GenBank using BLAST algorithm (Altschul et al., 1990), GenBank accession AY587776-AY587843. bIsolates obtained from Alentejano sausage (A), from Ribatejano sausage (R) and from media: BP-Baird Parker; TGE-Tryptone Glucose Extract agar; MRS-agar; EVA-Ethyl Violet Azide agar; OPSP-Perfringens agar base supplemented with cycloserine; OPSP-Perfringens agar base supplemented with sodium sulphadiazine and oleandomyxine and polymyxine B. cRange of the length of the near-full-length 16S rDNA sequences. dRange of the percentage sequence similarity to the closest BLAST match. eMean values of total amount (mM) and molar ratio (%) of organic acids produced: (F: Formic acid, A: Acetic acid, P: Propionic acid, IB: Isobutyric acid, B: n-butyric acid, IV: Isovaleric acid, V: n-Valeric acid, IC: Isocapronic acid, C: n-capronic acid, H: Heptanoic acid, L: DL-lactic acid, S: Succinic acid).

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Te2 (R) MRS Te3 (R) MRS Te53 (R) MRS Pediococcus acidilactici DSM20284T (AJ305320) Te56 (R) MRS Te57 (R) MRS Te52 (A) MRS Lactobacillus sakei DSM20017T (M58829) Te11 (R) TSC Clostridium subterminale DSM758 (AF241843) Te48 (R) OPSP Clostridium subterminale DSM6970T (AF241844) Te19 (A) TSC Clostridium bifermentans DSM13560 (AF320283) Te20 (A) TSC Te62 (A) TSC Te15 (A) TSC Te63 (R) OPSP Clostridium sphenoides ATCC19403T (AB075772) Clostridium celerecrescens DSM5628T (X71848) Te16 (R) OPSP Clostridium aminovalericum DSM1283T (X73436) Te79 (A) TGE Paenibacillus peoriae DSM8320T (AJ320494) Te13(A) BP Te66 (A) BP Staphylococcus epidermis KL-004 (AY030340) Te17 (R) OPSP Te21 (R) BP Bacillus licheniformis DSM13T (X68416) Te22 (R) BP Te90 (R) TGE Te40 (R) TGE Te35 (R) TGE Te89 (R) TGE Te70 (R) BP Te31 (R) TGE Te41 (R) TGE Te76 (R) TGE Te83 (R) TGE Te87 (R) TGE Te25 (R) BP Te85 (R) TGE Te84 (R) TGE Te36 (A) TGE Te81 (R) TGE Bacillus subtilis IAM12118T (AB042061) Te23 (R) BP Te26 (R) TGE Te29 (R) TGE Te34 (R) TGE Te68 (R) BP Bacillus pumilus c10 (AY373359) Te27 (R) TGE Te30 (A) TGE Te71 (A) TGE Te73 (A) TGE Te72 (R) TGE Te28 (A) TGE Bacillus cereus G3317 (AY138278) Te18 (A) OPSP Enterococcus solitarius NCIMB 12902T (Y18338)

Te14 (R) MRS Te65 (R) TSC Enterococcus faecalis CECT481T (AJ420803) Enterococcus durans CECT411T (AJ420801) Te6 (A) MRS Te47 (R) EVA Te82 (A) MRS Te37 (R) MRS Te92 (A) EVA Te42 (A) TGE Te59 (R) MRS Te51 (A) MRS Te32 (A) TGE Te61 (R) MRS Te45 (R) EVA Te1 (A) MRS Te43 (A) TGE Te49 (A) MRS Te50 (A) MRS Te58 (R) MRS Te91 (A) TGE Te93 (R) EVA Te95 (A) EVA Enterococcus faecium CECT410 T (AJ420800)

0.10

Figure 1. 16S rDNA-based tree reflecting the relationship of species. The length bar indicates 10% estimated sequence divergence. Figura 1. Árbol basado en 16S rDNA reflejando la relación entre las especies. La longitud de la barra indica el 10 % estimado de la divergencia en la secuencia

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period and temperature (Smiddy et al., 2002). Smiddy et al. (2002), in twenty-five packs of 13 processed cooked meats packaged in modified atmospheres of 70 % N2 and 30 % CO2, observed that oxygen was present in 88 % of packs 24 h after packaging. Also, studies conducted in vacuum-packaged Vienna sausages, with in-package pasteurization stored at 8 ºC for 128 days (Franz and von Holy, 1996); and for 71 days (Dykes et al., 1996), showed that the bacterial populations during storage period were predominantly LAB (Pediococcus species) and further, the non-lactic acid bacteria were dominated by Bacillus isolates as in this study. The presence of staphylococci in Portuguese chouriço is in agreement with other authors whom also identified Staphylococcus strains in different varieties of spanish chorizo (García-Varona et al., 2000; Daporta Padín, 1988), and in naturally fermented Italian sausages (Cocolin et al., 2001). García-Varona et al. (2000) and Cocolin et al. (2001) characterised the predominant Micrococcaceae species involved in fermented sausage production as Staphylococcus xylosus. Daporta Padín (1988) and GarcíaVarona et al. (2000) also identified species of S. intermedius, S. saprophyticus, S. hominis, S. epidermidis and S. aureus in samples obtained from chorizo. Sausages with longer ripening times contain high numbers of Micrococcaceae in the early stages of fermentation (Cantoni and Comi, 1986) and, studies of the microbial composition of sausages have shown that staphylococci greatly exceed micrococci right from the beginning of fermentation (Bersani et al., 1991; Kloos et al., 1974; Seager et al., 1986). However, both microbial genera play a role in the fermentation of sausages as they contribute to color stability, reduce nitrates to nitrite, break down triglycerides and proteins and participate in aroma development (Montel et al., 1998). Major differences observed between the two varieties of chouriço were the absence of Pediococcus species and the presence of high numbers of isolates identified as Staphylococcus epidermidis in product A and, the high incidence of Bacillus species in sausage type R. Considering that MAP applied to both types of product was constituted by the same percentage of gases and, the storage conditions, maturation period and smoke treatment were the same, differences found between products may be due to spices formulation in each type of product, the type of natural casings used and, the hygienic quality of the raw meat. Also Hugas et al. (2003) showed that the number of enterococci during fermentation in natural fermented sausages of high pH (> 5.0), varied during ripening in the same manufacturing plant with the same formulation and technology. These results indicate that the hygienic quality of the raw meat may be essential as a source of microorganisms in these types of fermented meat products.

CONCLUSIONS Although contributions from other bacteria than those isolated can not be excluded, Enterococcus faecium (24.7 %), Bacillus subtilis (23.4 %), Staphylococcus epidermidis (14.3 %), B. cereus (7.8 %), Pediococcus acidilactici (6.5 %) Bacillus pumilus (6.5 %), Clostridium bifermentans (3.9 %), Bacillus licheniformis, and Enterococcus faecalis (2.6 %) were found in varieties Alentejano and Ribatejano of Portuguese chouriço after 120 days at 20 ± 5 ºC in MAP. No pathogens strains of Enterobacteriaceae were found. Further studies must be carried out to confirm the results obtained in this work in order to characterize the bacterial spoilage population which might decrease shelf-life and compromise product safety ACKNOWLEDGEMENT The authors wish to express their gratitude to the technicians at Danish Institute of Agricultural Sciences, Mona Dinesen, Karin Durup and Thomas Rebsdorf for excellent technical assistance. We are grateful to the Danish Institute of Agricultural Sciences (Denmark) and to the Foundation for Science and Technology (Portugal) for financial support. REFERENCES Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. 1990. Basic Local Alignment Search Tool. Journal of Molecular Biology 215, 403-410. Antai, S. P. 1988. Study of the Bacillus flora of Nigerian spices. International Journal of Food Microbiology 6, 259-261. Banerjee, M.; Sarkar, P. K. 2003. Microbiological quality of some retail spices in India. Food Research International 36, 469-474. Banerjee, M.; Sarkar, P. K. 2003. Growth and enterotoxin production by sporeforming bacterial pathogens from spices. Food Control 15(6), 491-496. Bersani, C.; Cantoni, C.; D’Aubert, S. 1991. Osservazioni sugli staphylococci coagulasi-negativi (CNS) presenti negli insaccati crudi stagionati. Industrie Alimentari 30, 12-14. Borch, E.; Kant-Muermans, M-L.; Blixt, Y. 1996. Bacterial spoilage of meat and cured meat products. International Journal of Food Microbiology 33, 103-120. Byun, M. W.; Lee, J. W.; Jo, C.; Yook, H. S. 2001. Quality properties of sausage made with gamma-irradiated natural pork and lamb casing. Meat Science 59, 223-228. Cantoni, C.; Comi, G. 1986. I Micrococchi degli insaccati crudi stagionati. Industrie Alimentari 25, 780-782. Chamkha, M.; Patel, B. K. C.; Garcia, J. L.; Labat, M. 2001. Isolation of Clostridium bifermentans from oil mill waste waters converting cinnamic acid to 3-phenylpropionic acid and emendation of the species. Anaerobe 7, 189-197. Cocolin, L.; Manzano, M.; Aggio, D.; Cantoni, C.; Comi, G. 2001. A novel polymerasechain reaction (PCR) – denaturing gradient gel electrophoresis (DGGE) for the identification of Micrococcaceae strains involved in meat fermentations. Its application to naturally fermented Italian sausages. Meat Science 57, 59-64.

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Cienc. Tecnol. Aliment. 5(3) 165-174 (2006)

ISSN 1135-8122

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