Spanish Dry Fermented Sausages Research Articles

Inactive dry yeast to improve the oxidative stability of Spanish dry-fermented sausage “salchichón”

The use of inactive dry yeasts (IDY) of Saccharomyces cerevisiae as a natural alternative to sodium ascorbate to inhibit the oxidation reactions of Spanish dry-fermented sausage “salchichón” was studied. Control sausages and those treated with different IDY concentrations were monitored and analyzed at 0, 7, 14, 21 and 28 d throughout the ripening process. The application of IDY (1.5 and 3 g/100 g) did not affect the physicochemical parameters and microbial counts over time. However, IDY addition achieved a more stable phenolic content, antioxidant activity of samples was increased, and a lower lipid and protein oxidation were observed compared to the use of sodium ascorbate. Likewise, sausages with 3 g/100 g IDY showed similar or lower quantities of volatile compounds related to lipid oxidation at the end of ripening than those elaborated with sodium ascorbate. From a sensory point of view, IDY addition provided bread/yeast and sweet notes to sausages. However, consumers did not find differences among samples in a hedonic ranking test. Therefore, IDY could be employed in “salchichón” as natural preservatives providing pleasant sensory attributes to the product.

Influence of an industrial dry-fermented sausage processing on ochratoxin A production by Penicillium nordicum

Dry-fermented sausages are prone to be colonised by Penicillium nordicum, which is one of the main ochratoxin A (OTA)-producing species. Its ability to produce this mycotoxin on dry-fermented sausages has been reported. However, the influence of the conditions of a traditional processing of a Spanish dry-fermented sausage and the intrinsic physicochemical parameters of this product such as water activity (aw) and pH on OTA production has not been studied yet. Thus, the aim of this study was to evaluate the influence of traditional processing (interaction of relative humidity (RH) x temperature x ripening days) on the evolution of pH and aw during maturation of dry-fermented sausage “salchichón” and its relationship with OTA synthesis by P. nordicum. The expression of otapks and otanps genes, both involved in the biosynthesis of the mycotoxin, was also assessed. For this, 27 raw sausages were inoculated with P. nordicum and ripened for 26 days in a drying chamber (3 days at 5 °C and 84% RH, 17 days at 12 °C and 84% RH, and 6 days at 12 °C and 80% RH). From results, although it seems that the pH slightly influenced on OTA biosynthesis, the aw had a great impact on this mycotoxin production. In fact, the two highest OTA concentrations found coincided with a dramatic rise of the aw value (0.92 aw) by day 18 of incubation when the RH of the drying chamber was still 84% and at the end of the incubation time when the aw decreased noticeably (0.87 aw). The expression of the otapks and otanps genes correlated with the OTA produced by P. nordicum. Results from this work confirm that the traditional processing of Spanish dry-fermented sausages favours itself OTA synthesis by P. nordicum. Our findings may help in informed decision-making in relation to RH/temperature of drying chambers and shortening of the ripening process. This may be then effectively incorporated into the hygienic production system in the framework of HACCP together with other measures including the use of Penicillium nalgiovense as protective culture or the monitoring of otapks gene expression, and aw during the processing of dry-fermented sausages. All these strategies together may put ochratoxigenic Penicillia at a disadvantage and minimise OTA contamination risks in dry-fermented sausages.

Different Lactobacillus populations dominate in “Chorizo de León” manufacturing performed in different production plants

“Chorizo de Léon” is a high-value Spanish dry fermented sausage traditionally manufactured without the use of starter cultures, owing to the activity of a house-specific autochthonous microbiota that naturally contaminates the meat from the environment, the equipment and the raw materials. Lactic acid bacteria (particularly Lactobacillus) and coagulase-negative cocci (mainly Staphylococcus) have been reported as the most important bacterial groups regarding the organoleptic and safety properties of the dry fermented sausages. In this study, samples from raw minced meat to final products were taken from five different producers and the microbial diversity was investigated by high-throughput sequencing of 16S rRNA gene amplicons. The diverse microbial composition observed during the first stages of “Chorizo de Léon” evolved during ripening to a microbiota mainly composed by Lactobacillus in the final product. Oligotyping performed on 16S rRNA gene sequences of Lactobacillus and Staphylococcus populations revealed sub-genus level diversity within the different manufacturers, likely responsible of the characteristic organoleptic properties of the products from different companies.

A peptidomic approach to study the contribution of added casein proteins to the peptide profile in Spanish dry-fermented sausages

Peptidomics is a necessary alternative in the analysis of naturally generated peptides in dry-fermented processing. The intense proteolysis occurred during the processing of dry-fermented sausages is due to the action of endopeptidases and exopeptidases from both, endogenous muscle origin and lactic acid bacteria (LAB) added in the starter. Sodium caseinate is frequently used as an additive in this type of products because of its emulsifying properties, and consequently influences the protein profile available during the proteolysis. In this study, a mass spectrometry approach has been used to determine the impact of added sodium caseinate in the final peptide profile as well as to analyse its possible influence in the presence of certain previously described casein-derived bioactive peptides.

Effect of different autochthonous starter cultures on the volatile compounds profile and sensory properties of Galician chorizo, a traditional Spanish dry fermented sausage

This study deals with the effect of different autochthonous starter cultures used in the manufacture of Galician chorizo, a traditional dry fermented sausage produced in the north-west of Spain, on its volatile compounds profile and sensory properties.SPME-GC/MS was used in volatile compounds analysis and a trained group of panellists performed sensory evaluation of final products. In addition, physicochemical parameters and microbial counts were performed throughout the ripening in order to follow and control the process conditions.The starter cultures studied, consisting of a mix of Lactobacillus sakei strain and a strain of Staphylococcus equorum, Staphylococcus epidermidis or Staphylococcus saprophyticus, had been previously isolated from traditional Galician fermented meat products and technologically characterized.According to the data obtained in this study, no significant differences were found among batches inoculated with different starter cultures. However, sensory evaluation revealed that overall acceptance was higher for inoculated batches than for the uninoculated control batch.In conclusion, this work reinforce the idea that using selected autochthonous starter strains in sausage manufacture makes possible the obtaining of a homogeneous production without renouncing the desired typical characteristic obtained in artisanal elaborations.

Technological conditions influence aminogenesis during spontaneous sausage fermentation

The influence of two manufacturing processes on biogenic amine formation during the manufacture of Spanish dry fermented sausages of different diameters (fuet and llonganissa) was evaluated to elucidate which conditions allow better control of the aminogenic activity of spontaneous microbiota. Technological conditions affected both the amounts and the qualitative profile of biogenic amine accumulated. The higher processing temperature and relative humidity in process A (simulating those applied in industrial manufacture) favoured aminogenesis, since biogenic amine accumulation was faster and higher than in sausages manufactured under the process B (close to those used in traditional practices). The major amine differed depending on the diameter of the sausages, tyramine being the major amine in fuet (2.5 cm diameter sausage), and putrescine in llonganissa (4.5 cm). Moreover, sausages of higher diameter (llonganissa) had higher biogenic amine contents compared with the thinnest sausages (fuet). Conditions would modulate biogenic amine accumulation not only due to its influence on development of the bacterial population but also on its aminogenic activity. From the biogenic amine point of view, when sausages are spontaneously fermented, traditional lower temperatures and relative humidities are more appropriate than those usually applied in industrial processes.

The effect of the pediocin PA-1 produced by Pediococcus acidilactici against Listeria monocytogenes and Clostridium perfringens in Spanish dry-fermented sausages and frankfurters

The inhibitory effects of the pediocin PA-1 in frankfurters and the Pediococcus acidilactici MCH14 pediocin-producing strain itself in Spanish dry-fermented sausages were evaluated. The experiments were carried out by in situ assays using Listeria monocytogenes and Clostridium perfringens as target bacteria strains. The inhibition of L. monocytogenes by the P. acidilactici MCH14 pediocin-producing strain was effective in Spanish dry-fermented sausages, being the counts of this pathogen reduced by 2 log cycles compared to the control. In frankfurters, the counts of L. monocytogenes, using 5000 bacteriocin units/ml (BU/ml) of the pediocin PA-1 produced by P. acidilactici MCH14, decreased by 2 and 0.6 log cycles after storage at 4 °C for 60 days and at 15 °C for 30 days, respectively, with respect to the control. Similar results were obtained for C. perfringens, with 5000 BU/ml the counts of this strain were reduced by 2 and 0.8 log cycles after storage at 10 °C for 60 days and at 15 °C for 30 days, respectively, with respect to the control. Both the pediocin PA-1 and the P. acidilactici MCH14 pediocin-producing strain could be a useful alternative for protection against these food-borne pathogens in meat products.

Volatile compounds in Spanish dry-fermented sausage ‘salchichón’ subjected to high pressure processing. Effect of the packaging material

The effect of high pressure treatment (400 MPa, 10 min at 12 °C) on the volatile profile of Spanish dry-fermented sausage ‘salchichón’, packaged with or without aluminium foil in a multilayer polymeric bag, was investigated. The analysis of the volatile fraction was carried out by dynamic headspace extraction coupled to gas chromatography–mass spectrometry. Pressure-treated samples showed significantly higher levels of alcohols, aldehydes and alkanes and lower levels of two methylketones as compared with control samples. An intense migration was observed of compounds from the plastic material into the product, mainly linear and branched-chain alkanes, alkenes and benzene compounds. Most of these migrating compounds were significantly more abundant in pressurized samples than in untreated samples.

Physico-chemical and microbiological profiles of “salchichón” (Spanish dry-fermented sausage) enriched with orange fiber

Spanish dry-fermented sausages with three concentrations (0, 1 and 2%) of orange fiber (juice industry by-product) were prepared and studied. Fermentation and dry-curing processes were followed by physico-chemical (pH and water activity), chemical (moisture, lactic acid and residual nitrite level) and microbiological (aerobic mesophilic bacteria, lactic acid bacteria, Enterobacteriaceae, Micrococcaceae and molds and yeasts counts) analysis. Sensory analyses were conducted on the finished products. Only residual nitrite level and counts of micrococcus were affected by fiber addition during fermentation. pH, water activity, residual nitrite level and counts of Micrococcaceae were affected by fiber addition during dry-curing. Orange fiber decreases residual nitrite levels and favours micrococcus growth. Both effects have a positive impact on sausage safety and quality. The reduction in residual nitrite level decreases the risk of nitrosamine formation. For all sensorial attributes evaluated sausages containing 1% fiber showed similar scores to controls.

Microbiological Profiles, pH, and Titratable Acidity of Chorizo and Salchichón (Two Spanish Dry Fermented Sausages) Manufactured with Ostrich, Deer, or Pork Meat

Microbial counts, pH, and titratable acidity were determined in 102 Spanish dry fermented sausages (chorizo and salchichón) made with ostrich, deer, or pork meat. Average microbial counts (log CFU per gram) varied from 5.46±0.24 to 8.25±0.80 (total viable counts), from 4.79±0.36 to 7.99±0.20 (psychrotrophs), from 0.00±0.00 to 0.99±1.10 (undetectable values were assumed to be zero) (Enterobacteriaceae), from 0.00±0.00 to 4.27±1.47 (enterococci), from 5.15±1.15 to 8.46±0.49 (lactic acid bacteria), from 3.08±0.44 to 6.59±1.76 (Micrococcaceae), from 2.27±1.53 to 5.11±1.81 (molds and yeasts), from 0.00±0.00 to 2.25±0.81 (pseudomonads), and from 0.00±0.00 to 2.78±0.46 (Brochothrix thermosphacta). Average pH and titratable acidity varied from 5.07±0.25 to 5.63±0.51 (pH units) and from 0.30±0.01 to 0.86±0.19 (% lactic acid). Both type of sausage (P < 0.05) and species of meat (P < 0.001) influenced microbial counts. Salchichón samples showed lower average values than chorizo samples for most microbial groups (significant for Enterobacteriaceae, lactic acid bacteria, and B. thermosphacta) and titratable acidity. Sausages made from pork showed the highest microbial loads for total viable counts, psychrotrophs, Enterobacteriaceae, enterococci, lactic acid bacteria, and yeasts and molds. Higher counts were observed only for pseudomonads in ostrich sausages. B. thermosphacta levels were similar for all species of meat. The highest average pH value was observed in sausages made from ostrich meat, and the lowest titratable acidity level was found in pork sausages.

Biogenic amine content in Spanish retail market meat products treated with protective atmosphere and high pressure

Biogenic amine content, microbial counts and pH were investigated in 20 kinds of Spanish retail meat products treated with mild preservation technologies such as high pressure (HP) and protective atmospheres. High levels of tyramine were found in non-packed (NP) and protective atmospheres packed (PAP) Spanish dry fermented sausage, “chorizo”. These are also associated with the total viable count and high levels of lactic acid bacteria. Lower levels of tyramine were observed in chorizo and cooked white sausage “butifarra” treated with HP. High concentrations of agmatine were detected in these pressurised products, mainly in chorizo. High levels of cadaverine, together with high levels of putrescine, and agmatine were observed in sliced cooked ham in PAP and in vacuum packed (VP) butifarra. High concentrations of spermidine and spermine were observed in the different products analysed.

Enterococcus faecium P21: a strain occurring naturally in dry-fermented sausages producing the class II bacteriocins enterocin A and enterocin B

Enterococcus faecium P21 isolated from a Spanish dry-fermented sausage shows a narrow antimicrobial activity against closely related lactic acid bacteria and strong antimicrobial activity against spoilage and foodborne Gram-positive pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens and Clostridium botulinum. The antimicrobial activity is produced during growth in MRS broth at 32°C; it is heat resistant (20 min at 80–100°C) and abolished by protease treatment, and withstands exposure to pH 2–11, freeze-thawing, lyophilization and long term storage at −20°C. Purification of the antimicrobial activity by ammonium sulphate precipitation, gel filtration, and cation-exchange, hydrophobic interaction and reverse-phase chromatographies showed that the broad antimicrobial spectrum exerted by E. faecium P21 was indeed due to two peptide bacteriocins. Studies on theirN -terminal amino acid sequences and PCR and DNA analyses revealed that these bacteriocins are identical to the class II bacteriocins enterocin A and enterocin B. The genetic organization of the enterocin A (EntA) operon in E. faecium P21 shows that the bacteriocin structural gene (entA), the immunity gene (entiA) and the induction peptide gene (entF) are clustered and colinearly arranged. Strikingly, this organization was structurally different to that reported for the EntA-producer E. faecium CTC492.

Analysis of volatile compounds by GC–MS of a dry fermented sausage: chorizo de Pamplona

The profile of volatile compounds of a typical Spanish dry fermented sausage, chorizo de Pamplona, has been analyzed by GC–MS, using a simultaneous distillation-extraction (SDE) system. Qualitative and quantitative differences were found in the volatile profiles obtained in the five analyzed commercial brands. One hundred and ninety-three different substances were isolated, the group of acids being the most important from a quantitative point of view in all brands, accounting at least for the 60% of the total area. Aldehydes, basically from lipid oxidation, contributed between 7.72 and 13.97% to the total amount. Acids and aldehydes were the chemical families that showed the lowest variability among brands. In contrast, esters showed the highest coefficient of variation among brands (111%), followed by phenols (82%) and terpenes (76%). The variability observed in these three families could be attributed respectively to the different starter cultures, smoking process and spices employed in their production. Butylated hydroxytoluene (added as an antioxidant, E-321) was the third most abundant compound in three of the five brands.

Characterization of chorizo de Pamplona: instrumental measurements of colour and texture

A study of standardization of chorizo de Pamplona by instrumental measurements of colour and texture was carried out in five different commercial brands of this typical Spanish dry fermented sausage. Parameters related to colour showed little variability among brands, especially in L ∗ and a ∗ values. The higher variability of b ∗ could be related to the different amounts of paprika used in the formulations. Low ranges of variation were found in texture parameters. Some observed differences, especially in cohesiveness were significantly correlated with the pH of the products. Multivariate analysis, based on colour and texture parameters, led to 2 principal components which explained 80.80% of the total variance. Brands 1 and 5 appeared to be the most similar in function of the first component, which was mainly defined by hardness followed by gumminess, a ∗ and b ∗.

Relationship between biogenic amine contents and the size of dry fermented sausages

Three trials were carried out to study the influence of the diameter on biogenic amine contents and related parameters (pH, humidity and proteolysis) in fermented sausages. The first trial was done on three groups of Spanish dry fermented sausages with different diameter. In the second, two sections (centre and edge) of salchichón sausages were examined. The last trial consisted in the study of the ripening of two batches of sausages fermented under the same conditions but with two different diameters. Biogenic amine contents varied among the different type of products as well as among the same type of samples. Generally, amine levels in the biggest diameter sausages were higher than in the thinnest sausages. Amine contents were higher in the central part of the sausages than in the edge. During the ripening, larger tyramine amounts were formed in sausages with the biggest diameter. Statistical correlations were found among the diameter, the pH, the proteolysis and some amines. The results of the three trials agree with the hypothesis that the diameter is a factor that may affect the formation of biogenic amines during sausage fermentation.

Temperature and pH conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin sakacin K.

Sakacin K is an antilisterial bacteriocin produced by Lactobacillus sake CTC 494, a strain isolated from Spanish dry fermented sausages. The biokinetics of cell growth and bacteriocin production of L. sake CTC 494 in vitro during laboratory fermentations were investigated by making use of MRS broth. The data obtained from the fermentations was used to set up a predictive model to describe the influence of the physical factors temperature and pH on microbial behavior. The model was validated successfully for all components. However, the specific bacteriocin production rate seemed to have an upper limit. Both cell growth and bacteriocin activity were very much influenced by changes in temperature and pH. The production of biomass was closely related to bacteriocin activity, indicating primary metabolite kinetics, but was not the only factor of importance. Acidity dramatically influenced both the production and the inactivation of sakacin K; the optimal pH for cell growth did not correspond to the pH for maximal sakacin K activity. Furthermore, cells grew well at 35 degrees C but no bacteriocin production could be detected at this temperature. L. sake CTC 494 shows special promise for implementation as a novel bacteriocin-producing sausage starter culture with antilisterial properties, considering the fact that the temperature and acidity conditions that prevail during the fermentation process of dry fermented sausages are optimal for the production of sakacin K.

Characterization of the headspace volatile compounds of selected Spanish dry fermented sausages

The volatile profile of three different Spanish dry fermented sausages (`salchichón', `fuet' and `chorizo') was studied using dynamic capillary gas–liquid chromatography/mass spectrometry (GC–MS) headspace analysis. Complex mixtures of esters, ketones, alcohols, aldehydes and terpenes characterized retail samples of each of these products. Esters were dominant components of the volatile compounds from one type of salchichón (salchichón 2), terpenes of the other type (salchichón 1) and of one type of fuet (fuet 2). Common to both types of fuet was a number of ketones and alcohols. `Chorizo' had similar high numbers of esters as `salchichón 2' but also had three sulphur-containing compounds which are known components of garlic and are used as an ingredient in chorizo.

Simultaneous addition of Palatase M and Protease P to a dry fermented sausage (Chorizo de Pamplona) elaboration: Effect over peptidic and Lipid fractions

The effect of the simultaneous addition of a lipase (Palatase M 200L Novo Nordisk A/S) and a protease (Protease P 31.000 Solvay Enzymes GMBH&CO.KG) to the manufacture of a Spanish dry fermented sausage (Chorizo de Pamplona) was studied. In relation to the free aminoacid fraction, significative increases in Glu, His, Lys, Ser, Ala, Pro, Val, Met, Ile, Leu and Phe were found. Smaller differences were observed in the aminoacids obtained from peptides. The addition of the lipase caused significant increases in palmitic, oleic and linolenic acids. Despite the observed changes, no differences were found in the sensory quality compared with the control, except for a slight softening.

Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria

The present work compares, under the same stated experimental conditions, the antimicrobial activity of crude and purified enterocin L50, pediocin PA-1, nisin A and lactocin S, produced by lactic acid bacteria (LAB) isolated from Spanish dry-fermented sausages. The bacteriocins were purified to homogeneity by ammonium sulphate precipitation, gel filtration (for lactocin S), and cation-exchange, hydrophobic-interaction, and reverse-phase-chromatography; high yields of pure bacteriocins were obtained. Minimal inhibitory concentration (MIC) of pure enterocin L50, pediocin PA-1, nisin A and lactocin S was determined against a broad spectrum of Gram-positive bacteria, including spoilage and foodborne pathogenic bacteria. The purified bacteriocins showed a broad antimicrobial spectrum similar to that exerted by crude bacteriocins. Enterocin L50 and pediocin PA-1 were very active againstListeria monocytogenes, which was quite resistant to nisin A and lactocin S. Enterocin L50 also displayed antimicrobial activity againstStaphylococcus aureus,Clostridium perfringensandClostridium botulinum. However, these pathogens were weakly inhibited, or not at all, by the other pure bacteriocins.

Comparative study of lactic acid bacteria house flora isolated in different varieties of `chorizo'

A total of 516 strains of lactic acid bacteria isolated from `chorizo' (a Spanish dry fermented sausage) were identified. The `chorizo' was from three zones of Castilla and León in Spain: Burgos, Segovia and Salamanca. Two factories were chosen in each zone and the samples were taken at three stages of ripening. L. sake was the most predominant species present (68.8%) followed by L. curvatus (16.47%) and Pediococcus sp. (8.52%). Different strains that do not belong to the above species were grouped as Lactobacillus sp. Group S1 comprising maltose and lactose negative L. sake was the main group present in all factories except in a factory in Segovia where group S3 comprising lactose positive L. sake and pediococci were the predominant ones. Group S1 increased during the ripening process in all six factories and it dominated in the ripened `chorizo' except in the mentioned factory in Segovia. In general strains of L. sake and L. curvatus which fermented maltose but not lactose were more dominant at the beginning and in the middle of the process, whereas, L. sake and L. curvatus which could ferment lactose, or lactose and maltose occurred in higher numbers in semi-ripened `chorizo' and in the final product. This indicates that strains which could ferment lactose were more competitive towards the end of the process. Strains from group S1 were the microorganisms responsible for the pH drop in most of the factories, giving the correct texture. As a result it would appear that a strain from this group would be most suitable for use as starter culture.