Microbial Ecology of the Soppressata of Vallo di Diano, a Traditional Dry Fermented Sausage from Southern Italy, and In Vitro and In Situ Selection of Autochthonous Starter Cultures

Enumeration and isolation of bacteria.Ten samples of dry fermented sausages, labeled A to L (Table 1), were purchased from different local factories in Campania (southern Italy). The pH and water activity (a_w) values for all of the samples were determined. Standard enumeration methods were used to determine the microbial populations at the end of ripening. Ten grams of each sample was transferred into a sterile Stomacher bag, diluted with 90 ml of Ringer solution (Oxoid), and treated with a Stomacher machine for 2 min. Serial decimal dilutions were made, and several microbial populations were targeted in duplicate as follows: (i) LAB on De Man, Rogosa, Sharpe (MRS) agar (Oxoid) incubated in anaerobic conditions at 30°C for 48 h, (ii) gram-positive CNC on mannitol salt agar (MSA; Oxoid) incubated at 30°C for 48 h, (iii) enterococci on Slanetz and Bartley medium (Oxoid) incubated at 37°C for 48 h, (iv) yeasts and molds on dichloran rose Bengal chloramphenicol (DRBC) agar base (Oxoid) supplemented with chloramphenicol selective supplement (Oxoid) incubated at 30°C for 72 h, (v) total enterobacteria on violet red bile glucose agar (Oxoid) incubated with a double layer at 30°C for 24 to 48 h, (vi) clostridia on tryptone-sulfite-neomycin agar (Sanofi Diagnostics Pasteur, Marnes, La Coquette, France) incubated in anaerobic conditions at 37°C for 72 h. Three to five colonies of LAB and staphylococci were randomly isolated from countable MRS and MSA plates, purified, and stored at −20°C in MRS and tryptone soy broth with 25% glycerol. Species-specific PCR assays performed by using two set of primers targeting xylulokinase (xylB) and 60-kDa heat shock protein genes (hsp60) of S. xylosus were used as previously reported (5) to identify the staphylococcal isolates belonging to the species S. xylosus. The isolated lactobacilli were selected for the absence of plasmid-mediated (tetracycline, erythromycin, oleandomycin, lincomycin, and clindamycin) antibiotic resistance according to the method of Charteris et al. (11) and for their capability to survive at low pH as previously described (42). Selected strains were identified by 16S rRNA gene sequencing as previously reported (4) (see Table 8). Both staphylococci and lactobacilli were also selected for their growth ability at pH 5.5 and at 14°C determined according to Casaburi et al. (9).

Collection of bulk cells.In order to investigate the cultivable microbiota of the sausages, bulk cells (22) were collected from LAB, gram-positive CNC, enterococci, and yeast cells by using the whole content of the countable dilution plates used for the enumeration of these microbial groups. In particular, for each microbial group, all colonies present on the specific growth medium were resuspended in 5 ml of tryptone soy broth (Oxoid), added to 20% glycerol, and stored at −20°C prior to further analysis.

DNA extraction.Ten grams of each soppressata sample was diluted with 90 ml of Ringer solution (Oxoid) and treated with a Stomacher machine for 2 min. Two milliliters of the solution obtained was centrifuged at 17,000 × g for 10 min at room temperature and washed once with 200 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). After washing, the solution was again centrifuged at 17,000 × g for 10 min at room temperature. The pellet was suspended in 100 μl of TE and used for DNA extraction with a Wizard DNA purification kit (Promega, Madison, WI) as previously described (22). After DNA precipitation with isopropanol (0.7 volume), the resulting pellet was washed with 70% ethanol. It was then dried and resuspended in 50 μl of DNA rehydration solution by incubation at 60°C for 45 min. Finally, 5 μl of RNase (10 mg/ml) was added and the solution was incubated at 37°C for 30 min. The DNA extracted was stored at −20°C. For DNA extraction from bulk cells, 500 μl of the bulk mixture was centrifuged at 17,000 × g for 10 min at room temperature. The pellet was suspended in 100 μl of TE and used for DNA extraction as described above. Finally, 500 μl of yeast bulk cells was centrifuged at 17,000 × g for 10 min at room temperature. The pellet was used for DNA extraction with InstaGene matrix (Bio-Rad Laboratories, Hercules, CA) by following the conditions described by the supplier.

PCR-denaturing gradient gel electrophoresis (DGGE) analysis.The primers V3f and V3r spanning the V3 region (positions 340 to 356 and 533 to 517, respectively) of the 16S rRNA gene (38) were used. A GC clamp was added to the forward primer according to the method of Muyzer et al. (38). Amplification was performed in a programmable heating incubator (MJ Research Inc., Madison, WI). Each mixture (final volume, 50 μl) contained 1 μl of template DNA (about 25 ng), each primer at a concentration of 0.2 μM, each deoxynucleoside triphosphate at a concentration of 0.25 mM, 2.5 mM MgCl₂, 5 μl of 10× PCR buffer (Invitrogen), and 2.5 U of Taq polymerase (Invitrogen). Template DNA was denatured at 94°C for 5 min, and a touchdown PCR was performed (38). The primers NL1 and LS2 (12) spanning the 5′-end region (positions 266 to 285 on the S. cerevisiae 26S rRNA gene (GenBank accession number M19229) were used to amplify DNA extracted from the yeast bulks. A GC clamp was added to primer NL1 (38). For the amplification a mixture (final volume, 50 μl) was prepared as described above. Reactions were run for 30 cycles: denaturation was at 95°C for 60 s, annealing was at 52°C for 45 s, and extension was at 72°C for 60 s. An initial denaturation at 95°C for 5 min and a final extension at 72°C for 7 min were used. The primers P1f and P2r spanning the V1 region (positions 41 to 60 and 130 to 111, respectively) of the 16S rRNA gene (32) were used to amplify DNA extracted from bulk cells collected from MRS agar. A GC clamp was added to the forward primer according to the method of Muyzer et al. (38). Each mixture (final volume, 50 μl) was prepared as described above. Template DNA was denatured at 94°C for 5 min, and a touchdown PCR was performed (38). Aliquots (3 μl) of all the PCR products were routinely checked on 2% (wt/vol) agarose gels.

PCR products were analyzed by DGGE by using a Dcode apparatus (Bio-Rad). Samples were applied to 8% (wt/vol) polyacrylamide gels in 1× Tris-acetate-EDTA buffer. Parallel electrophoresis experiments were performed at 60°C by using gels containing a 20%:50% and a 30%:50% urea-formamide denaturing gradient (100% corresponded to 7 M urea and 40% [wt/vol] formamide) for the separation of V3 (or D1) and V1 amplicons, respectively. The gels were analyzed by electrophoresis for 4 h at 200 V, stained with ethidium bromide for 5 min, and rinsed for 10 min in distilled water.

Sequencing of DGGE bands.Small pieces of selected DGGE fragments were punched from the gel with sterile pipette tips. The pieces were transferred into 20 μl of sterile water and incubated overnight at 4°C. Two milliliters of the eluted DNA was used for reamplification, and the PCR products were checked by DGGE; DNA amplified from the respective DGGE profile was used as a control. Only products that migrated as a single band and at the same position as the control were purified by using a QIAquick PCR purification kit (QIAGEN, Milan, Italy) and sequenced by use of primer V3r, P2r, or NL1. The DNA sequences were determined by the dideoxy chain termination method by using a DNA sequencing kit (Perkin-Elmer Cetus, Emeryville, CA) according to the manufacturer's instructions. Research to identify DNA similarity was performed using the GenBank and EMBL databases with the BLAST program to determine the closest known relatives of the partial 16S rRNA gene sequences obtained (1).

In vitro technological characterization of Staphylococcus and Lactobacillus strains.The strains belonging to the S. xylosus species were studied to evaluate catalase, nitrate reductase, proteolytic, lipolytic, and antioxidant activities as described in a previous work (9) in order to determine their potential for use as a starter for fermented sausage production.

Briefly, the nitrate reductase activity was evaluated spectrophotometrically according to Casaburi et al. (9) and lipolytic activity was assayed on pork fat (using concentrations of 10⁸ CFU ml⁻¹ of each culture and incubation at 14°C for 14 days) by titration and expressed as a percentage of oleic acid as previously described (36). In order to determine the proteolytic activity, 1 ml of cell suspension (10⁸ CFU ml⁻¹) in phosphate-buffered saline (Oxoid) was inoculated in 10 ml of sterile-meat sarcoplasmic (1.77 mg ml⁻¹) or myofibrillar (0.12 mg ml⁻¹) extracts and incubated at 14°C for 14 days. At time zero and after 14 days the cultures were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (33) on 12% polyacrylamide gels by use of a Mini-PROTEAN III cell (Bio-Rad) as electrophoresis equipment. The electrophoretograms were run with Precision Plus Protein standards (Bio-Rad) of known molecular weight, and the gels were subsequently stained for 1 h in a 0.1% (wt/vol) solution of brilliant blue R in methanol:acetic acid:water (40:10:50) and then destained for 2 h in the staining solution without brilliant blue. Sarcoplasmic and myofibrillar extracts were run as a control in the gel. Prior to loading, the myofibrillar extracts were first dialyzed (3500 MWCO unit with Spectra/Por 3 tubing; Spectrum Medical Industries Inc., Los Angeles, CA) in distilled water overnight, lyophilized, and then loaded at a concentration of 1.77 mg ml⁻¹ in sample buffer. The antioxidant properties of the strains were evaluated by determining the amount of malondialdehyde (MDA) produced in 10 ml of broth containing 1% tryptone, 0.5% yeast extract, 3% NaCl, and 4% (g/vol) of pork fat at pH 7.0 inoculated with each strain at a concentration of 10⁸ CFU ml⁻¹ after incubation for 14 days at 14°C with shaking. The MDA concentration was determined by using the thiobarbituric acid test described by Bolumar et al. (7). Briefly, 10 ml of broth was mixed with 10 mg of butyl hydroxide toluene and 20 ml of 5% trichloroacetic acid. The mixture was homogenized for 20 s and centrifuged at 9,000 × g for 10 min at 4°C, and the supernatant was filtered through Whatman paper. Four milliliters of thiobarbituric acid (0.02 M) was added to 4 ml of filtrate and incubated at 100°C for 1 h, and the mixture was then centrifuged at 9,000 × g for 5 min at 4°C. The supernatants obtained were spectrophotometrically read at 532 nm (7); the results as expressed as milligrams of MDA per gram of pork fat. The acidifying activity of the Lactobacillus strains was evaluated by measuring growth (according to the optical density at 600 nm) and pH every 24 h during growth in meat broth (0.1% glucose, 3% NaCl, 0.3% meat extract, 0.5% peptone, 250 ppm KNO₃, pH = 7.0) at 14°C for 14 days.

In situ technological characterization of Staphylococcus and Lactobacillus strains.The in situ characterization of the technological properties of the selected strains was performed by simulating laboratory-scale manufacture of soppressata of Vallo di Diano. A pork meat dough was prepared by mixing for 5 min the following ingredients: lean pork (76%), pork fat (4%), NaCl (2.8%), black pepper in grains (0.6%), white wine, and potassium nitrate (250 ppm). Aliquots (200 g) of dough were inoculated with cell suspensions of each Lactobacillus or Staphylococcus strain at a final concentration of 10⁷ CFU g⁻¹; the sausages were prepared in natural casings, dried at 18°C for 4 h at 86% RH, and then ripened for 14 days at 14°C and 68% RH. Every strain was assayed in duplicate tests; noninoculated sausages were prepared and ripened as a control. At time zero and after 14 days of ripening, sausage samples were taken for microbiological and chemical analyses. Microbial counts were performed on MRS agar under anaerobic conditions after 48 h at 30°C and for staphylococci on MSA after 48 h at 30°C. pH measurement was performed by using a Mettler-Toledo GmbH pH meter (model MP-120-B; Novate Milanese).

The nitrate reductase activity was determined spectrophotometrically according to the method of Baldini et al. (3). Briefly, nitrates and nitrites were extracted from meat and treated with Carrez reagents, and the colorimetric determination was performed at 540 nm after reduction of residual nitrates to nitrites on a cadmium sponge. The nitrate and nitrite content was expressed in milligrams per kilogram of meat. For the proteolytic activity analyses the sarcoplasmic and myofibrillar proteins were extracted according to the method described by Mauriello et al. (35) and the protein concentrations were determined using a Bio-Rad protein assay with bovine serum albumin as the standard. The extracts were diluted to a final concentration of 1.77 mg ml⁻¹, and SDS-PAGE was performed as described above. For the lipolytic activity, the total lipid content of the samples was determined after they had been thawed and minced. The extraction was performed using 5 g of minced meat according to the method of Folch et al. (25). The lipid phase was resuspended in ethanol:ethyl ether (1:2) and titrated with an alcoholic NaOH solution (0.02 N). The results are expressed as a percentage of oleic acid as previously described (36). A lipolysis index was also calculated as the percentage of oleic acid/percentage of total lipids according to the method of Cattaneo et al. (10). The determination of MDA as a marker of oxidation was performed as described above by use of 5 g of sausages.

Statistical analysis.Statistical analyses were performed using SPSS 11.5 software for Windows (SPSS, Chicago, IL). The analysis of variance (Duncan test) was applied to determine differences in means. All the values represent the mean values obtained from three independent assays for each trait.

Enumeration of microorganisms and pH and a_w measurements.The results obtained by the enumeration of microorganisms and determination of the pH and a_w values for the soppressata samples are shown in Table 1. All the samples had a pH far higher than 5.5 and had low a_w values. The LAB load was extremely heterogeneous, ranging from 7.4 × 10⁶ (sample F) to 3.2 × 10⁹ (sample B) CFU g⁻¹. Staphylococcal loads ranged from 2.4 × 10⁴ (sample D) to 3.4 × 10⁷ (sample A) CFU g⁻¹. Enterococcal loads ranged from 2.0 × 10³ (sample D) to 1.5 × 10⁷ (sample C) CFU g⁻¹. For samples E and G, enterococcal loads did not reach 10³ CFU g⁻¹. The numbers of yeasts and molds did not differ greatly among all samples, with values ranging from 2.0 × 10³ (sample D) to 7.2 × 10⁴ (sample A) CFU g⁻¹. The number of Enterobacteriaceae was high only in sample C (Table 1). An absence of clostridia was observed for all the soppressata samples.

DGGE profiles and species identification.The total DNA extracted from each soppressata sample (A to L) was employed in PCR assays using the primers V3f and V3r to obtain 200-bp fragments of the V3 region of the 16S rRNA gene, and the fragments were analyzed by DGGE. The results obtained by this analysis are shown in Fig. 1; multiple bands could be detected in most of the samples. All the bands were excised from the gel and reamplified prior to sequencing. The results of the identification are presented in Table 2. Members of the genus Lactobacillus and the genus Staphylococcus were predominant in the samples analyzed. In particular, Lactobacillus spp. were found in samples B, E, G, H, I, and L. The species group L. sakei/curvatus/graminis was found in samples B, E, I, and L, while L. plantarum occurred in samples G and H (Table 2). Members of the genus Staphylococcus were found in five samples (A, B, C, D, and H). S. succinus was found in samples C and D, while S. equorum was found in sample B (Table 2). Enterococcus spp. were detected in samples C and L and members of the genus Carnobacterium in samples E and F. Finally, Tetragenococcus halophilus was identified in sample I.

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FIG. 1.

DGGE profiles of the V3 region of the 16S rRNA gene obtained by PCR amplification of DNA extracted directly from the 10 soppressata samples A, B, C, D, E, F, G, H, I, and L. Identification of each band is presented in Table 2.

PCR-DGGE was also applied to PCR products from bulk cells collected from MRS agar, MSA, Slanetz and Bartley medium, and DRBC agar. Bulk cells were not identified when the bacterial load for a certain group was low or below the detection limit. DGGE profiles obtained by the analysis of the V3 region of the 16 rRNA gene of staphylococci from MSA are shown in Fig. 2; the closest relatives of the sequenced fragments are reported in Table 3. The species mainly detected in the eight bulk cells analyzed were S. xylosus, S. equorum, and S. succinus. Each species was present in five samples, and sample L was the only one containing all the three staphylococcal species. In particular, S. xylosus was found in samples A, B, C, I, and L; S. equorum in samples B, E, H, I, and L; and S. succinus in samples C, E, G, H, and L. DGGE profiles obtained by the analysis of the V3 region of the 16S rRNA gene of LAB are shown in Fig. 3; the closest relatives of the sequenced fragments are reported in Table 4.

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FIG. 2.

DGGE profiles of the V3 region of the 16S rRNA gene obtained by PCR amplification of DNA extracted from bulk cells collected from MSA for soppressata samples A, B, C, E, G, H, I, and L. Identification of each band is reported in Table 3.

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FIG. 3.

DGGE profiles of the V3 region of the 16S rRNA gene obtained by PCR amplification of DNA extracted from bulk cells collected from MRS for soppressata samples A, B, C, D, E, F, G, H, I, and L. Identification of each band is reported in Table 4.

Several bands of samples A, B, C, and D were identified as representing non-Lactobacillus species after sequencing. Almost all the detected bands belonging to the genus Lactobacillus could not be identified at the species level but were identified in groups of multiple species (Table 4). Therefore, to improve the study of the LAB population, the V1 region of the 16 rRNA gene was chosen for PCR-DGGE analysis of bulk cells from MRS agar (Fig. 4). The closest relatives of the sequenced fragments are reported in Table 5. The primer set used was suitable for obtaining good differentiation among Lactobacillus spp. without band comigration as already reported by Cocolin et al. (14). With the sole exception of sample A, species of Lactobacillus were found in all bulk cells collected from MRS agar. L. sakei was found in samples D, F, and H and L. curvatus in samples C and E, whereas both species were detected in samples B, G, L, and I (Fig. 4; Table 5). Sequence information for the DGGE bands obtained by analyzing the V3 region of the 16S rRNA gene of the microbial bulk cells collected from Slanetz and Bartley medium is reported in Table 6. The bands displayed in Fig. 5 were identified as representing Enterococcus faecalis (samples A, B, C, and D) and E. hirae (samples A and L). However, some fragments were identified as harboring Staphylococcus spp. (samples A, B, and H) or Lactobacillus spp. (sample L). Sequence information for the DGGE bands obtained by analyzing the 26S rRNA gene of the yeast communities from DRBC agar is reported in Table 7, and the corresponding DGGE profiles are shown in Fig. 6. One band was detected in all the samples analyzed and was identified as representing Debaryomyces hansenii. Candida zeylanoides was detected in samples B, C, and E, while Pichia guillermondii was detected only in sample H.

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FIG. 4.

DGGE profiles of the V1 region of the 16S rRNA gene obtained by PCR amplification of DNA extracted from bulk cells collected from MRS for soppressata samples A, B, C, D, E, F, G, H, I, and L. Identification of each band is reported in Table 5.

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FIG. 5.

DGGE profiles of the V3 region of the 16S rRNA gene obtained by PCR amplification of DNA extracted from bulk cells collected from Slanetz and Bartley medium for soppressata samples A, B, C, D, H, and L. Identification of each band is reported in Table 6.

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FIG. 6.

DGGE profiles of 26S 16S rRNA gene region obtained by PCR amplification of DNA extracted from bulk cells collected from DRBC agar base for soppressata samples A, B, C, E, G, and H. Identification of each band is reported in Table 7.

In vitro characterization of Staphylococcus and Lactobacillus strains.The results of the lipolytic activity analyses are shown in Table 8. Strong lipolytic activity was observed for the staphylococci, while weak lipolysis was observed for the lactobacilli. Indeed, the oleic acid values were between 7.05% and 31.02% for the S. xylosus strains whereas they ranged from 2.82% to 4.94% for the lactobacilli (Table 8). Moreover, no significant antioxidant activity was observed in the strains characterized; only L. sakei IVL42 was able to produce 4.23 mg MDA per g of fat, a concentration which was lower than the concentration of MDA found in the control (Table 8).

All the strains of S. xylosus tested in this study showed nitrate reductase activity when assayed in vitro; strains AVS5, IVS38, and IVS39 showed better activity than the others (results not shown).

The results of the analyses of proteolysis on sarcoplasmic proteins are shown in Fig. 7. Weak proteolytic activity was found for the lactobacilli except for the strain of L. sakei FVL26 that did not exhibit any activity on the sarcoplasmic proteins. The electrophoretic profiles showed digestion of the 14-kDa band for all the lactobacilli (Fig. 7A). The strongest proteolytic activity was shown by L. sakei IVL42 (Fig. 7A), L. sakei BVL6, and L. curvatus BVL7 and CVL12 (Fig. 7B), which showed a reduction in the 22.7- and 11.5-kDa bands and an increase in the 13-kDa band. The S. xylosus IVS39, LVS48, and IVS37 strains were able to hydrolyze the sarcoplasmic proteins after 14 days of incubation (Fig. 7C), showing a reduction in the 22.7-kDa band and digestion of the doublet of about 20 kDa. None of the staphylococci and lactobacilli showed proteolytic activity on the myofibrillar proteins in vitro, since the corresponding SDS-PAGE profiles were identical to that of the control (data not shown).

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FIG. 7.

SDS-PAGE of sarcoplasmic protein hydrolysis by different strains of Lactobacillus and S. xylosus. M, Precision Plus Protein standard; C₀, uninoculated control at time zero; C₁₄, uninoculated control after 14 days of incubation; a to p, samples containing different strains after 14 days of incubation. (A) a, L. sakei FVL26; b, L. sakei HVL36; c, L. curvatus IVL41; d, L. sakei IVL42. (B) e, L. sakei BVL6; f, L. curvatus BVL7; g, L. curvatus CVL12; h, L. sakei DVL17. (C) i, S. xylosus LVS49; l, S. xylosus AVS3; m, S. xylosus LVS48; n, S. xylosus IVS39; o, S. xylosus IVS38; p, S. xylosus IVS37.

In growth and acidification assays, all the lactobacilli showed slow growth at 14°C in meat broth; the highest effect on pH was shown by strains L. curvatus BVL7 and CVL12 and L. sakei DVL17 and HVL36, with a decrease in pH between 2.14 and 2.4 units compared to the control.

In situ characterization of Staphylococcus and Lactobacillus strains.After 14 days of ripening of meat at 14°C, viable counts showed an increase in the loads of lactobacilli ranging between 0.5 and 0.7 log cycles and an increase in 1 log cycle when strain L. sakei FVL26 was tested. The loads of staphylococci increased by about 1 log cycle in most cases (Table 9). The pH did not decrease during the first days of ripening, while it reached values of 5.53, 5.59, and 5.60 after 14 days when the meat had been inoculated with L. curvatus IVL41, DVL17, and FVL26, respectively (Table 9). Overall, the decrease in pH was registered after the first week of ripening.

The results of lipolytic activity analyses showed very weak lipolysis during meat ripening, with percent oleic acid values higher for lactobacilli than for staphylococci (Table 8). The lipolysis index confirmed the presence of better lipolytic activity by lactobacilli; the greatest rise in the lipolytic index was registered for the sausages inoculated with S. xylosus LVS49 and L. sakei FVL26, which showed increases of 1.42 and 1.38%, respectively, compared to the control strain results (data not shown).

After the 14 days of ripening the MDA concentrations were always higher than that seen with the control in the sausages inoculated with any of the Lactobacillus strains except for L. sakei DVL17 (Table 8). On the other hand, six of the nine sausages inoculated with S. xylosus showed MDA concentrations lower than the control results, with the lowest concentrations shown by strains IVS37 and IVS39. The results of the assessment of activities in vitro and in situ were found to be significantly different for both lipolytic activity (P < 0.01) and MDA determinations (P = 0.029) (Table 8).

After 14 days of ripening, the SDS-PAGE profiles of the sarcoplasmic proteins showed a reduction in the intensity of the 43.3- and 34.7-kDa bands in all the sausages, both inoculated and control (Fig. 8). The strains of S. xylosus assayed in situ did not exhibit proteolytic activity on the sarcoplasmic proteins, showing SDS-PAGE profiles identical to the control profiles (Fig. 8). The lactobacilli caused hydrolysis of the 14-kDa band; moreover, strains of L. sakei BVL6 and L. curvatus BVL7 caused a disappearance of the 34.7-kDa band and a reduction in the intensity of the 43.3-kDa band. As shown in Fig. 9, none of the staphylococci or lactobacilli was able to hydrolyze myofibrillar proteins during ripening of the fermented sausages.

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FIG. 8.

SDS-PAGE of sarcoplasmic proteins extracted from different sausages. M, Precision Plus Protein standard; C₀, uninoculated sausages (control) at time zero; C₁₄, uninoculated sausages (control) after 14 days of ripening; a to e, sausages inoculated with different strains after 14 days of ripening (a, S. xylosus IVS39; b, S. xylosus LVS48; c, L. sakei FVL26; d, L. sakei BVL6; e, L. curvatus BVL7).

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FIG. 9.

SDS-PAGE of myofibrillar proteins extracted from different sausages. M, Precision Plus Protein standard; C₀, uninoculated sausages (control) at time zero; C₁₄, uninoculated sausages (control) after 14 days of ripening; a to e, sausages inoculated with different strains after 14 days of ripening (a, L. curvatus BVL7; b, L. sakei BVL6; c, L. sakei FVL26; d, S. xylosus IVS39; e, S. xylosus LVS48; f, L. sakei HVL36).

The S. xylosus LVS48 strain showed the strongest nitrate reductase activity in situ, causing a reduction in the nitrate concentration, but did not exhibit nitrite reductase activity (Table 8). S. xylosus strains AVS3, AVS4, AVS5, IVS37, and IVS38 showed both nitrate and nitrite reductase activities, while strain IVS38 showed the best nitrite reductase activity, reducing the nitrite concentration by 53%.