Redalyc.Spoilage and Pathogenic Bacteria Isolated from Two Types Of

Ciencia y Tecnología Alimentaria ISSN: 1135-8122 [email protected] Sociedad Mexicana de Nutrición y Tecnología de Alimentos México

Matos, J. S. T.; Jensen, B. B.; Barreto, S. F. H. A.; Hojberg, O. Spoilage and pathogenic bacteria isolated from two types of portuguese dry smoked sausages after shelf-life period in modified atmosphere package Ciencia y Tecnología Alimentaria, vol. 5, núm. 3, 2006, pp. 165-174 Sociedad Mexicana de Nutrición y Tecnología de Alimentos Reynosa, México

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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 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 Danish Institute of Agricultural Sciences, Department of Animal Health, Welfare and Nutrition, 8830 Tjele, Denmark. 2 Faculdade de Medicina Veterinária, CIISA, UTL, R. Prof. Cid dos Santos - Polo Universitário, Alto da Ajuda, 1300- 477 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] 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 Portuguese dry smoked sausage (chouriço) is very ingredients are held at temperatures below 10 ºC. After this different from Spanish and other dry fermented sausages, period the ingredients are stuffed into natural casings not only in its taste but also in the processing technology. (salted pork intestine or beef dry casing), intensively The Spanish sausage has a similar composition but is smoked at high temperature for about 3 days. It is then exposed to a long dry period at low temperature and is considered a smoked sausage processed by heat (Sousa almost not smoked. Portuguese chouriço is exposed to and Ribeiro, 1983).

The microbiology of dry smoked sausages is smoked dry sausages consisted of a mixture of pork meat variable and complex and the rate of spoilage of these meat and fat minced (approx. 20 mm) with the formulation products can reduce the shelf life and cause substantial ingredients red pepper paste, water, salt, garlic paste, olive financial losses to manufacturing companies. The spoilage oil, spices, white wine, sugar, liquid smoke and additives ecology is affected by a combination of intrinsic and (sodium nitrite and commercial sausage sodium extrinsic factors (Korkeala and Makela, 1989). Factors such polyphosphate) as outlined in Table 1. as ripening technique (Cantoni and Comi, 1986), use of Major differences between the two products natural intestines of pork and beef to stuff meat batters consisted in olive oil, spices, hot red pepper paste and (Byun et al., 2001), bacterial population of raw meat material white wine, besides the casings used. This paste is matured (Nel et al., 2004; Sardinha et al., 2000), introduction of for one day at 5 ºC. The stuffing is filled into salted pork spices as formulation ingredients (Banerjee and Sarkar, intestine in the Ribatejano sausage type and into beef dry 2003a), smoke (Wendorff, 1981), application of modified casing for the Alentejano sausage type and, subject to atmosphere and the CO2 concentrations (Stiles, 1991; the drying effect of smoke and temperature for about three Sivertsvik et al., 2002) and, storage temperature (Korkeala days. The experiments were conducted at a commercial and Makela, 1989) all have significant influence on the meat plant and the study was based on 12 samples (each microflora of these types of meat products and also on sample was composed by a mixture of three sausages, a the growth and survival of spoilage microorganisms during total of 36 sausages) randomly drawn from two batches. shelf-life period. Representative sausages of each experiment were packed

Several studies (Encinas et al., 1996; Garcia-Varona separately in modified atmosphere (55 % N2/45 % CO2) with et al., 2000; te Giffel et al., 1996; Cocolin et al., 2001; a net weight of about 225g and stored at 20 ± 5 ºC for 120 Daporta Padin, 1988; Santos et al., 1998; Papamanoli et days (shelf-life period determined by the manufacturing al., 2003; Hugas et al., 2003; Devriese et al., 1995) were industry) for further analysis. Representative sausage conducted to identify bacterial populations on fermented samples presented pH values after production of 5.80 ± dry sausages, however, despite increasing interest for 0.17 and of 5.70 ± 0.10 and water activity varied between modified atmosphere package (MAP), the effect of MAP 0.93 ± 0.01 and 0.94 ± 0.01 [mean values ± standard on the spoilage bacteria of these meat products is not yet deviation (n=6)] for sausage type A and R, respectively. clear. Besides identifying the bacterial contamination After storage, pH values varied from 5.60 ± 0.00 and 5.43 ± during the manufacture processing, it is further important 0.06 for sausage type A and R, respectively, and water to identify the spoilage microorganisms that will remain or activity remained with the same values (data not shown). proliferate during the storage period in MAP, and might decrease shelf life compromising product safety. The type Isolation of bacteria of spoilage microorganisms are primarily related to the Microbial analyses were performed on triplicate extent and type of the initial contamination as affected by samples. Each sample was prepared from three sausages. the resident «factory flora», the manufacturing site and Twenty five grams were removed (approximately 8 g from the processing hygiene. The spoilage potential is not only each sausage) were cut in to pieces of 1 cm and dependent on the bacterial growth rates, but also on the homogenised into 225 ml of Peptone water (Merck; 7228). specific metabolic activity of the different species growing Ten fold dilutions were prepared in 0.85 % sodium chloride on, or in, cooked meat products under certain storage (Merck, 6404). conditions (Samelis et al., 2000). For bacteria enumeration, 1 ml samples (0.1 ml for The purpose of this study was to determine late staphylococci) from the dilution tubes were inoculated shelf life population in two types of Portuguese dry onto non-selective and selective media. The ten fold smoked sausages (chouriços) packaged in modified dilutions used for isolation of spore forming bacteria, atmosphere, which do not yet show clear spoilage changes Clostridium perfringens and sulphite reduction clostridia at the end of the producer-defined shelf life period (120 were previously heated to 80 ºC for 10 minutes. Lactic acid days at 20 ± 5 ºC). Tentative identification and bacteria were isolated from MRS-agar (Merck, 0660) plates characterization of these species were based on incubated at 30 ºC in carbon dioxide atmosphere phenotypic (cell morphology and fermentation profile) and (Anaerocult, Merck, 1.13829) for 72 hours. Spore forming genotypic (16S rDNA sequencing) characters. 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). MATERIALS AND METHODS The TGEA plates were incubated aerobically at 30 ºC for 120 h. The selective media used for isolation of Sausage manufacture and sampling staphylococci was Baird-Parker (BP) agar (Oxoid; CM275) Samples studied for this work were chouriços type supplemented with Egg Yolk – Tellurite emulsion (Oxoid; Alentejano (A) and Ribatejano (R) produced by a large SR054C), incubated aerobically 37 ºC for 48 h. factory in Portugal. The recipe of these types of Portuguese Staphylococci-like colonies were selected after application

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

Tabla 1. Composición de chorizos Alentejano y Ribatejano.

Formulation Chouriço Chouriço (g/kg) Alentejano Ribatejano Meat raw material - Pork belly boneless, without rind 172.1 176.0 - Pork picnic, boneless, without shank 688.2 528.1 - Pork trimming 90/10 - 176.0 Sub-total 860.3 880.1 Ingredients - Commercial sausage sodium polyphosphate, 5.0 5.0 E452(i); (50% of P2O5) - Water 31.0 12.7 - Sugar 2.1 2.1 - Olive oil 5.2 - - Liquid smoke 1.0 1.4 - Spice lourer 0.02 0.04 - Sweet chilli - 5.0 - Powder clove - 0.1 - Garlic paste 11.0 11.3 - Sweet red pepper paste 4.3 8.8 - Special red pepper paste 44.9 46.1 - Hot red pepper paste 12.9 2.5 - White pepper (powder) 1.7 1.1 - Sodium chloride with sodium nitrite, E250, (0.6% 10.3 10.0 of NaNO2) - Salt 10.3 10.0 - White wine - 3.9 Sub-total 139.7 119.9 Total 1000 1000

of Pastorex STAPH-PLUS test (Sanofi Pasteur; 56356), Viable counts were calculated as log CFU and catalase test (20 vol. of H2O2); gram staining; haemolytic colony forming unit presented as mean ± standard activity (Plates with Columbia with 5% of sheep blood; deviation, (n = 6). Results of search analysis (enterococci BioMerieux; 43041) and cell morphology observation by - EVA, sulphite reducing clostridia - OPSP and Clostridium phase-contrast microscopy. perfringens - TSC) were reported as positive (presence of Enterococci were picked from quantifying MRS the organism) and as negative if not detected. plates and were determined using Azide Dextrose (Merck; From the highest dilution of each medium an 1590) after anaerobic incubation at 37 ºC for 24 h. From average of 20 colonies were picked randomly, and positive tubes (tubes with precipitate at the bottom of the transferred aseptically into tubes containing selective tube), 1 ml was transferred aseptically into tubes with 10 media according to the type of microorganism. Colonies ml of Ethyl Violet Azide broth (EVA-broth) (Difco; 0606- isolated from TGEA and EVA-agar media were transferred 01-7). Positive results (violet precipitate at the bottom of aseptically into tubes containing PC medium with the the tube) were analyzed after anaerobic incubation at 37 following composition in distilled water (g l-1): 5.0 peptone ºC for 48 h. Colony isolation was performed by taking 1 ml of casein (Merck, 7213), 2.5 yeast extract (Oxoid, L21), 1.0 from the highest dilution of EVA-broth positive tubes, D(+)-glucose (Merck, 8337) incubated aerobically at 30 ºC poured it into Ethyl Violet Azide agar (EVA-broth with (TGEA-colonies) and at 37 ºC (EVA-colonies). Isolates from added 8 g l-1of agar) and incubate aerobically at 37 ºC for MRS-agar were transferred into Hungate tubes containing 48 h. The selective media used for isolation of Clostridium MRS-broth (Merck, 0661) incubated at 30 ºC. Clostridia and perfringens and sulphite reduction clostridia were staphylococci colonies were picked and transferred Perfringens Agar Base (TSC) (Oxoid; CM 587), aseptically under anaerobic conditions into Hungate tubes supplemented with cycloserine (Oxoid; SR088E) and containing Reinforced Clostridial-bouillon (Merck, 5411) Perfringens Agar Base (OPSP) (Oxoid; CM 543), added 0.005 g l-1 hemin and incubated at 37 ºC. After supplemented with sodium sulphadiazine (Oxoid, SR076) incubation of the cells overnight, the cell morphology was and with oleandomyxine and polymyxine B (Oxoid, SR077), investigated by phase-contrast microscopy. Glycerol (20 respectively. Both, TSC-plates and OPSP-plates were %) was added to the remaining overnight cultures before incubated at 37 ºC under anaerobic conditions for 120 h. storage at –80 ºC.

Fermentation profiles DNA. The PCR program included 1 min of initial The isolates were subcultured (1.0 ml inoculum of denaturation at 94 ºC, followed by 30 cycles of denaturation an overnight culture) in anaerobic (N2 atmosphere) roll (30 s at 94 ºC), annealing (30 s at 60 ºC), and extension (45 tubes containing 9 ml of a pre-reduced sterilised peptone s at 72 ºC), and a final extension at 72 ºC for 10 min. The yeast glucose (PYG) medium described by Holdeman et PCR products were verified by electrophoresis in 1% al. (1977). After incubation at 30 ºC (lactic acid bacteria agarose gel (w/v) stained with ethidium bromide and and spore forming bacteria) and at 37 ºC (staphylococci, visualised by UV transillumination. enterococci and clostridia) for 48 h, the concentrations of fermentation products in terms of organic acids and gas Sequencing of 16S rDNA were measured. Gas samples (500 µl) were removed from Prior to determination of the 16S rDNA sequences, the headspace of the tubes with gas-tight syringes and the PCR products were purified on QIAquick PCR analyzed for the concentration of CO2, N2 and H2 by gas purification kit columns, according to the manufacturer‘s chromatography using a thermal conductivity detector instructions (QIAGEN Gmbh, Hilden, Germany). The 16S (TCD), according to the method described by Jensen and rDNA sequences of the isolates were determined using Jorgensen (1994). Subsequently, the tubes were the ABI BigDyeTM Terminator Cycle Sequencing Ready thoroughly mixed, and samples (1.0 ml) were collected Reaction kit (PE Biosystems, Foster City) and monitored aseptically and the concentration of organic acids was with an automatic sequence analyzer (ABI PRISM® 377 quantified by gas chromatography using a flame ionization DNA sequencer PE Biosystems). Isolates were subjected detector (FID) as outlined by Jensen et al. (1995). Studies to near-full-length 16S rDNA sequencing as described by were conducted in triplicate. Leser et al. (2002). To determine the closest relatives of the near-full-length 16S rDNA sequences retrieved, DNA extraction searches were conducted in GenBank using BLAST Culture liquid (1.5 ml) was transferred from algorithm (Altschul et al., 1990). overnight cultures into sterile microfuge tubes and centrifuged at 13.000 x g for 5 min. The cell pellet was Nucleotide sequence of the isolates washed twice with 2.0 ml of TES-buffer (0.05 M Tris, 0.05 The near-full-length sequences of the isolates have M NaCl, 0.005 M EDTA [pH 8.0]) and resuspended in 500 been deposited in GenBank under accession numbers l TES-buffer. The nucleic acid extraction was initiated by AY587776 - AY587843. The phylogenetic relationship of incubating the suspension with 50 µg lysozyme (Sigma, the different species within the genus Enterococcus, St. Louis, USA) at 37 ºC for 30 min. After incubation, 50 µg Bacillus, Clostridium, Staphylococcus, Pediococcus, each of Proteinase K and RNase (Sigma, St. Louis, USA) Lactobacillus and Paenibacillus was determined by were added and the mixture was further incubated at 65 ºC comparative sequence analysis of their 16S rDNA genes. 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 RESULTS AND DISCUSSION were purified twice by extraction with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), and the Characterization of isolates from two types of Portuguese residual phenol was removed by extraction with an equal smoked dry sausages volume of chloroform-isoamyl alcohol (24:1). After A total of 77 isolates were obtained from Alentejano precipitation of the nucleic acids with ethanol, the DNA (35) and from Ribatejano (42) types of smoked dry sausage, was dissolved in sterile water and stored at –20 ºC until and the distribution of recovery from the different media further analysis. is given along with the viable counts and the search analysis results in Table 2. PCR amplification The 77 isolates were obtained from TGE-agar (35.0 Amplification of 16S rDNA was performed in a PCR %), MRS-agar (22.1 %), BP (22.1 %), TSC-agar (7.8 %), EVA- Express thermal cycler (Hybaid, Teddington, U.K.), using agar (6.5 %), and finally, from OPSP-agar (6.5 %). The the primers: S-D-Bact-0008-a-S-20 (5´-AGAG majority of the isolates recovered from TGE-medium were TTTGATCMTGGCTCAG-3´) and S-*-Univ-1492-a-A-19 grouped as species of Bacillus (81.5 %), Enterococcus (5´-GGTTACCTTGTTACGACTT-3´). The primers were (14.8 %) and Paenibacillus (3.7 %). The isolates obtained purchased from DNA Technology (Aarhus, Denmark). The from MRS-agar belonged to the genera Enterococcus (64.7 reaction mixture (50 µl) consisted of reaction buffer (final %), Pediococcus acidilactici (29.4 %) and Lactobacillus concentration, 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, (5.9 %). The EVA-agar used showed, as expected, high 0.1% Triton X-100, [pH 8.8]), each deoxynucleoside selectivity for enterococci. From the OPSP- and TSC-agar triphosphate at concentration of 200 µM, 0.2 µM of each isolates 60 % and 83.3 % respectively belonged to primer, 1.0 U of DyNAzymeTM II DNA polymerase Clostridium. The remaining isolates recovered from OPSP- (Finnzymes OY, Finland) and 2 µl (≈ 50 ng) of template agar belonged to Enterococcus (20 %) and Bacillus (20

%) 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 E. solitarius C. subterminale B. licheniformis C. celerecrescens celerecrescens C. from Alentejano (A) sausage type (64.7 %) were C. aminovalericum characterized as Staphylococcus. The isolates obtained No 16S identity No 16S Promedio ± desviación Mean values ± standard a

from Ribatejano (R) sausage were grouped in the Bacillus a genus (Table 2). b Concerning to staphylococci, provisional grouping Search was based on phenotypic characteristics [morphology and analysis 1 + 1 1 +

fermentation profile (Table 3), catalase test, gram staining sp. + 1 and haemolytic activity (data not shown)] and through application of Pastorex STAPH-PLUS test. From this group, E. faecalis C. botulinum Clostridium two representative isolates, Te13a(A)BP and Te66a(A)BP, C. bifermentans were selected and subjected to near-full-length 16S rDNA sequence analysis (Table 3). The relationship of identity 16S No b representative isolates is depicted on a 16S rDNA-based la recuperación de distintos medios. of recovery from the different media. of recovery from the different Search phylogenetic tree (Figure 1). Based on these data, only analysis + 1 + one group of staphylococci was represented, namely 1 + Staphylococcus epidermidis. The lactic acid bacteria (Enterococcus species, E. faecium Pediococcus acidilactici and Lactobacillus casei) E. faecium presented similar fermentation profiles (Table 3) except for No 16S identity No 16S the isolate Te18(A)OPSP identified as Enterococcus b 1 solitarius, which produced higher amount of formic and 3 Search

acetic acid and less lactic acid. According to the genotypic analysis characters, the enterococci belonged to three different Media sp. sp. + 3 +

groups or species: E. faecium, E. faecalis and E. solitarius. 1 1 + 2 + Among the E. faecium isolates one isolate, Te42(A)TGE, type A A type showed a divergence of more than 3% (Figure 1). Isolates type B B. cereus B. cereus B. subtilis B. subtilis B. subtilis B. pumilus B. E. faecium

identified as E. faecium and E. faecalis presented the same Paenibacillus fermentation profiles and cell morphology as outlined by No 16S identity identity 16S No

Knarreborg (2002). On the basis of comparative 16S rDNA en 1 g de muestra (TSC), Los resultados del análisis de búsqueda se muestran como + (presencia organismo) b -1a sequence analysis, the genus Enterococcus belongs to in 1 g of sample (TSC), 0.01 (OPSP) Results of search analyses are presented as + (presence the organism) b 10 the Gram-positive bacteria with low (< 50 mol %) G + C Log 3.3 ± 0.3 0.3 ± 3.3 3 content in the DNA. Other typical members of this phylum g CFU 0.3 ± 2.1 4 are clostridia and bacilli (Franz et al., 2003). 2 Within the Bacillus genus and based on the

relationship of species reflected on the 16S rDNA- type B B. subtilis B. subtilis B. pumilus B. S. epidermis phylogenetic tree (Figure 1), the following related species B. licheniformis were distinguished: Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus and Bacillus cereus. According identity 16S No to the results presented in Table 3, B. pumilus KL-052 -1a 3 isolate-like [Te34(R)TGE] was the only that produced H Log 2 CFU g as well as higher amount of total organic acids and formic 2 0.3 4.3 ± 1 0.6 4.9 ± 11 acid when compared with the other related isolates of 1 Bacillus pumilus species. The isolates Te31(R)TGE and L. sakei E. faecium E. faecium de muestras obtenidas de medios MRS, BP y TGE. y de muestras obtenidas medios MRS, BP

Te76(R)TGE, identified as B. subtilis type A (Table 3), E. faecialis -1 P. acidilactici P. acidilactici of sample obtained from MRS, BP and TGE media. and of sample obtained from MRS, BP despite the close relationship between these and the other -1 MRS MRS BP TGE EVA TSC OPSP

ones identified as B. subtilis (Figure 1, Table 3), higher identity 16S No amounts of total organic acids and lactic acid were -1a -1a 2 5 1 1 1 produced by these two isolates. Among the B. subtilis 4 Log Log 3.4 ± 0.9 0.9 ± 3.4 4 3.5 ± 0.8 0.8 ± 3.5 6 CFU g CFU isolates, Te36(A)TGE was the only one which produced H2.

Among the clostridia genus all isolates produced

16S Identity of the 77 isolates obtained from Alentejano (A) and Ribatejano (R) Portuguese smoked dry sausage distribution 16S Identity of the 77 isolates obtained from Identidad 16S de los 77 aislados obtenidos de chorizos portuguesas variedad Alentejano (A) y Ribatejano (R) distribución de Identidad 16S de los 77 aislados obtenidos chorizos portuguesas variedad

H2 except the isolate Te11(R)TSC, tentatively identified as C. botulinum. Clostridium bifermentans isolates were the Total 10 6 17 3 2 4 4 2 3 17 6 10 Total

Total 7 11 10 2 4 1 1 4 2 10 11 7 Total Ribatejano of Types sausages Alentejano

Table 2. Table and in 0.001 g of sample (EVA). 2. Tabla 0,1 g de muestra (OPSP) y en 0,001 (EVA). deviation (n=6) of log CFU g estándar (n=6) del log UFC g

169 Cienc. Tecnol. Aliment. 5(3) 165-174 (2006) ISSN 1135-8122 ©2006 SOMENTA only ones producing isocapronic acid. Genotypic flora that will growth differentially (Sousa and Ribeiro, characterization of Clostridium bifermentans isolates was 1983). Dry-cured sausages such as the Spanish and Italian supported by phenotypic characters, which showed sausages are characterized by a long ripening period (30 typical fermentation profiles as described by Chamkha et to 45 days) at low temperatures (from 11 to 25 oC) and at al. (2001). Among all clostridia isolates, isobutiric acid was relative humidity of 60 to 90 %. This long processing time, not produced only by the isolates Te15(A)TSC, when compared with the short process applied in Te63(R)OPSP and Te16(R)OPSP. Higher amounts of Portuguese smoked sausages (1 to 3 days) at temperatures isovaleric acid were detected in Te11(R)TSC and below 10 ºC, could explain the lower values of pH promoted Te48(R)OPSP. Considering the fermentation profiles and through lactic fermentation from meat sugars and sugar the relationship of species based on the 16S rDNA- formulation ingredients (Sousa and Ribeiro, 1983). phylogenetic tree, clostridia species were divided into 6 LAB species have also been identified as major groups: Clostridium bifermentans, C. aminovalericum, C. spoilage populations of vacuum-packaged emulsion-type celerecrescens, C. sp., C. subterminale, and C. botulinum. sausages and other processed meats stored at refrigeration temperatures (Samelis et al., 2000; Borch et al., 1996; The late shelf life bacteria of chouriços type Alentejano Korkeala and Bjorkroth, 1997). Controlling the spoilage and Ribatejano, after 120 days at 20 ± 5 ºC in modified progress by LAB is difficult as these bacteria are atmosphere package (55 % N2 / 45 % CO2) psychotropic, microaerophilic, and resistant to nitrite, salt, The majority of the species isolated from type A and smoke (Franz and von Holy, 1996). sausage were assigned to the species Enterococcus Non lactic acid bacteria were characterized as faecium (34.3 %), Staphylococcus epidermidis (31.4 %), Bacillus and Clostridium species in both types of Bacillus cereus (11.4 %), Clostridium bifermentans (8.6 sausages and also as Staphylococcus epidermidis in %) and sporadic isolates of Lactobacillus casei, sausage type Alentejano. Identification of Bacillus strains Enterococcus solitarius, Paenibacillus sp., Clostridium isolated from Spanish fermented sausages during the sp. and Bacillus subtilis. In type R sausage the microflora manufacture and ripening performed by Encinas et al. included mainly Bacillus subtilis (30.9 %), Enterococcus (1996) revealed, in a total of 69 isolates, Bacillus subtilis faecium (16.7 %), Bacillus pumilus (11.9 %), Pediococcus (37), B. megaterium (22), B. pumilus (5), B. circulans (3) acidilactici (11.9 %), Clostridium spp. (9.5 %), Bacillus and 2 unidentified species. These authors, therefore, cereus (4.7 %) and Enterococcus faecalis, Bacillus considered Bacillus the second most important group licheniformis and Bacillus subtilis type A (4.8 %). of spoilage bacteria after the lactic acid bacteria. Also The lactic acid bacteria (LAB) species identified te Giffel et al. (1996) showed the high prevalence of (Pediococcus acidilactici, Lactobacillus casei, Bacillus species (Bacillus subtilis and Bacillus cereus) Enterococcus faecium and Enterococcus faecalis) are in meat products compared with raw meat (Konuma et recognized among the most frequently isolated LAB from al., 1988; Kramer and Gilbert, 1989) supporting the role dry sausages processed with different technologies played by meat additives as source of contamination of (Santos et al., 1998; Papamanoli et al., 2003; Hugas et al., these species. Pafumi (1986) appointed spices as the 2003; Devriese et al., 1995) and packaged in modified principal source of spore-forming bacteria in large atmosphere (Samelis et al., 2000; Pexara et al., 2002). The volumes of foods and, Trigo and Fraqueza (1998) persistence of enterococci during ripening of fermented discussed that mesophilic spores such as Bacillaceae sausages can be attributed to their wide range of growth species were dominant in beef dry casings. Also, temperatures and their high tolerance to salt (Hugas et al., previous studies on the microbiology of spices have 2003). It seems that in sausages, no hurdles are found for demonstrated that Bacillus cereus and Clostridium their growth, allowing them to coexist with lactobacilli as perfringens are the sporeformers most commonly found the dominant population. In fact, in sausages with less (Antai, 1988; Banerjee and Sarkar, 2003b; Kneifel and content of fermentable sugars and higher pH (> 5.0), Berger, 1994; Powers et al., 1976). enterococci become more competitive and also find better The presence of aerobic Bacillus related species conditions for survival and growth. Presence of in these MAP chouriços, where the air in the package was enterococci in fermented foods might also reflect higher replaced by a specific mixture of carbon dioxide and level of contamination or poorer curing process (Hugas et nitrogen (45 % CO2 and 55 % N2), can be explained al., 2003). In the present work, the pH of sausages (5.70 considering that oxygen is not always completely removed and 5.80 after production) indicates that no relevant lactic and may also permeate through the packaging material acid fermentation had taken place when compared with (Vermieren et al., 1999). The level of residual oxygen in other types of dry fermented sausages. In fact, Fanco et MAP packs may be due to factors such as oxygen al. (2002), González and Díez (2002), and Ferrer and Arboix permeability of the packaging material, the ability of the (1986), in several types of dry-cured sausages, reported food to trap air, poor heat sealing ability of the packaging pH values in the range of 4.42 - 4.50. These differences material causing air to leak in ineffective evacuation and/ may be related to the maturation period and the microbial or ineffective gas flushing (Smith et al., 1986), the storage

170 SOMENTA ©2006Matos 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).

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).

Organic acidse Gas

Molar ratio (%) (mM) 16S Identitya Number of Total Bpc Similard Morphology F A P IB B IV V IC C H L S H CO isolates (mM) 2 2 Bacillib Bacillus licheniformis [Te21(R)BP; Te17(R)OPSP] 2 1445-1491 98-99 Rod 36.5 9.6 2.7 0.0 0.0 0.3 0.0 0.0 0.0 0.2 0.0 85.6 1.8 0.0 35.3 Bacillus pumilus strain M1-9-1 [Te23(R)BP; Te68(R)BP; Te26(R)TGE; 4 1477-1497 98-99 Rod 6.8 17.7 26.7 1.5 0.8 2.4 0.0 0.0 0.0 1.2 0.0 40.0 9.6 0.0 9.8 Te29(R)TGE] Bacillus pumilus strain KL-052 [Te34(R)TGE] 1 1481 98 Rod 21.4 67.4 20.9 0.3 0.0 0.6 0.0 0.0 0.0 0.3 0.2 7.8 2.6 3.9 28.1 Bacillus cereus [Te27(R)TGE; Te72(R)TGE; Te28(A)TGE; Te30(A)TGE;

171 6 1475-1504 97-99 Rod 35.6 13.3 12.6 0.1 0.1 0.3 0.0 0.0 0.0 0.2 0.0 71.2 2.2 0.0 17.7 Te71(A)TGE; Te73(A)TGE] Bacillus subtilis type A [Te31(R)TGE; Te76(R)TGE] 2 1468-1495 98-99 Rod 39.6 3.7 3.3 0.2 0.0 0.4 0.0 0.1 0.0 0.2 0.2 90.4 1.6 0.0 13.6 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; 13 938-1501 97-99 Rod 5.9 20.0 32.9 1.7 0.8 2.7 0.0 0.0 0.0 1.4 0.0 29.6 10.8 0.0 11.4 Te84(R)TGE; Te85(R)TGE; Te87(R)TGE; Te89(R)TGE; Te90(R)TGE] Bacillus subtilis [Te36(A)TGE] 1 1458 97 Rod 18.0 52.2 30.0 0.5 0.0 0.8 0.0 0.2 0.0 0.5 0.7 11.8 3.3 1.3 10.9 Paenibacillus sp. [Te79(A)TGE] 1 1493 99 Rod 23.0 63.1 28.0 0.3 0.0 0.6 0.0 0.2 0.0 0.4 0.5 4.7 2.5 9.2 31.0 Clostridiab C. botulinum [Te11(R)TSC] 1 1379 98 Rod 57.2 1.5 70.8 0.6 4.9 10.2 8.8 0.0 0.0 0.1 0.0 2.1 1.1 0.0 36.6 et al. C. subterminale [Te48(R)OPSP] 1 1450 99 Rod 41.6 4.5 59.6 0.6 5.9 13.3 11.6 0.0 0.0 0.2 0.0 2.6 1.6 2.3 22.7 Clostridium sp. [Te15(A)TSC] 1 1344 98 Rod 33.8 33.5 59.6 0.3 0.0 0.5 0.0 0.0 0.0 0.2 0.0 3.9 1.9 14.2 49.7 : Spoilageandpathogenicbacteria... C. celerecrescens [Te63(R)OPSP] 1 1469 99 Rod 26.1 11.8 58.3 0.4 0.0 0.5 0.0 0.0 0.0 0.3 0.0 25.8 2.9 14.1 36.6 C. aminovalericum [Te16(R)OPSP] 1 1439 98 Rod 14.8 8.2 72.6 0.0 0.0 1.1 0.0 0.4 0.0 0.6 0.0 12.4 4.6 12.0 45.4 C. bifermentans [Te19(A)TSC; Te20(A)TSC; Te62(A)TSC] 3 1434-1441 98 Rod 58.1 15.6 62.3 4.7 3.0 0.5 0.2 0.1 10.5 0.1 0.0 1.9 1.1 5.9 38.7 Lactic acid bacteriab Pediococcus acidilactici [Te02(R)MRS; Te03(R)MRS; Te53(R)MRS; 5 519-1503 96-99 Coccus 63.3 1.6 13.2 0.1 0.0 0.2 0.0 0.0 0.0 0.1 0.0 83.5 1.1 0.0 18.6 Te56(R)MRS; Te57(R)MRS Lactobacillus sakei [Te52(A)MRS] 1 1482 97 Rod 68.8 1.6 14.3 0.1 0.0 0.2 0.0 0.0 0.0 0.1 0.0 82.6 1.0 0.0 18.8 Enterococcus faecalis [Te14(R)MRS; Te65(R)TSC] 2 1462-1492 98-99 Coccus 51,4 7.3 15.4 0.2 0.0 0.3 0.0 0.0 0.0 0.2 0.0 75.4 1.4 0.0 24.4 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; 18 1372-1500 97-99 Coccus 48.9 3.1 8.2 0.2 0.0 0.3 0.0 0.0 0.0 0.2 0.0 86.7 1.3 0.0 18.8 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] 1 1393 98 Coccus 35.9 23.6 22.8 0.3 0.0 0.4 0.0 0.0 0.0 0.2 0.0 50.7 1.8 0.0 21.9 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; 11 1494-1503 98-99 Coccus 47.6 5.3 11.3 0.3 0.0 0.4 0.1 0.0 0.0 0.3 0.0 81.0 1.6 0.0 27.9 Te66d(A)BP; Te66e(A)BP; Te66f(A)BP] Cienc. Tecnol. Aliment. 5(3) 165-174 (2006) ISSN 1135-8122 ©2006 SOMENTA

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 0.10 Te93 (R) EVA Te95 (A) EVA Enterococcus faecium CECT410T (AJ420800)

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

172 SOMENTA ©2006 Matos et al.: Spoilage and pathogenic bacteria... period and temperature (Smiddy et al., 2002). Smiddy et CONCLUSIONS al. (2002), in twenty-five packs of 13 processed cooked meats packaged in modified atmospheres of 70 % N2 and Although contributions from other bacteria than

30 % CO2, observed that oxygen was present in 88 % of those isolated can not be excluded, Enterococcus faecium packs 24 h after packaging. Also, studies conducted in (24.7 %), Bacillus subtilis (23.4 %), Staphylococcus vacuum-packaged Vienna sausages, with in-package epidermidis (14.3 %), B. cereus (7.8 %), Pediococcus pasteurization stored at 8 ºC for 128 days (Franz and von acidilactici (6.5 %) Bacillus pumilus (6.5 %), Clostridium Holy, 1996); and for 71 days (Dykes et al., 1996), showed bifermentans (3.9 %), Bacillus licheniformis, and that the bacterial populations during storage period were Enterococcus faecalis (2.6 %) were found in varieties predominantly LAB (Pediococcus species) and further, the Alentejano and Ribatejano of Portuguese chouriço after non-lactic acid bacteria were dominated by Bacillus 120 days at 20 ± 5 ºC in MAP. No pathogens strains of isolates as in this study. Enterobacteriaceae were found. Further studies must be The presence of staphylococci in Portuguese carried out to confirm the results obtained in this work in chouriço is in agreement with other authors whom also order to characterize the bacterial spoilage population identified Staphylococcus strains in different varieties of which might decrease shelf-life and compromise product spanish chorizo (García-Varona et al., 2000; Daporta Padín, safety 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 ACKNOWLEDGEMENT species involved in fermented sausage production as Staphylococcus xylosus. Daporta Padín (1988) and García- The authors wish to express their gratitude to the Varona et al. (2000) also identified species of S. technicians at Danish Institute of Agricultural Sciences, intermedius, S. saprophyticus, S. hominis, S. epidermidis Mona Dinesen, Karin Durup and Thomas Rebsdorf for and S. aureus in samples obtained from chorizo. Sausages excellent technical assistance. We are grateful to the with longer ripening times contain high numbers of Danish Institute of Agricultural Sciences (Denmark) and Micrococcaceae in the early stages of fermentation to the Foundation for Science and Technology (Portugal) (Cantoni and Comi, 1986) and, studies of the microbial for financial support. 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 REFERENCES et al., 1986). However, both microbial genera play a role in the fermentation of sausages as they contribute to color Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. 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