Influence of starter cultures on the generation of antioxidant nitrogen compounds in Iberian dry‐fermented sausages

The purpose of this work was to investigate the influence of a starter culture on the generation of nitrogen compounds with antioxidant activity during the ripening of Iberian dry‐fermented sausages. Starters P200S34 (P. acidilactici M200 and S. vitulus RS34) and P198S34 (P. acidilactici MS198 and S. vitulus RS34) were used to make the Iberian dry‐fermented sausages ‘salchichón’ and chorizo; then, the physicochemical and microbial properties were determined during the ripening process. The oxygen radical absorbance capacity (ORAC) assay was employed to evaluate the antioxidant potential of the nitrogen extracts obtained during ripening of the sausages. This activity was correlated with the most relevant compounds detected by HPLC‐ESI‐MS in the final ripened extracts. Although a relevant part of the antioxidant activity was attributed to the predominant natural nitrogen fraction, the microbial population found in fermented sausages and the fermentation conditions significantly influenced the low molecular weight nitrogen profile and antioxidant activities. Inoculation with the starter culture P200S34 increased free amino acids and amines, such as methionine and tyramine, but other nitrogen compounds also increased the antioxidant activity of the low molecular weight nitrogen extracts. Thus, these starter cultures in Iberian sausages can contribute to delaying oxidative changes during storage.     

Effect of processing methods and starter culture (Staphylococcus xylosus and Pediococcus pentosaceus) on proteolytic changes in Turkish sausages (sucuk) during ripening and storage

Proteolytic changes in Turkish sausages (sucuk) produced by two methods (heat processing and traditional) were determined during processing and storage for 90 days. The sausages were produced with or without starter culture in both methods. A mixture of Staphylococcus xylosus and Pediococcus pentosaceus was used as starter culture for their acidic and proteolytic characteristics.The major changes in proteolytic characteristics of sucuk took place during the fermentation stage, with an increase in non-protein nitrogen (NPN) and a decrease in protein solubility. Proteolytic activity was observed in both starter-inoculated and non-inoculated (control) sausages during processing. Moreover, a slight increase in proteolytic activity was detected during storage in both starter-inoculated and control traditional sausages, and also in heat-processed sausages due to some heat-resistant proteolytic enzymes. Protein solubility was significantly affected by processing time and heat treatment. Sarcoplasmic and myofibrillar proteins were also affected by starter addition, fermentation, drying and heat processing. During fermentation, starter-inoculated and control sausages showed intense proteolysis in both the traditional and heat processing methods. After heating, intensive degradation of both sarcoplasmic and myofibrillar proteins due to denaturation was observed in heat-processed samples.     

Biochemical and sensory characteristics of traditional fermented sausages of Vallo di Diano (Southern Italy) as affected by the use of starter cultures

In this study, two strains of Staphylococcus xylosus isolated from traditional fermented sausages of Vallo di Diano (Southern Italy) were used in combination with an acidifying strain of Lactobacillus curvatus as starter culture for the production of fermented sausages. Two starter formulation were developed combining the proteolytic but not lipolytic (prt(+), lip(-)) S. xylosus CVS11 with the L. curvatus AVL3 (starter S1) and the S. xylosus FVS21 (prt(-), lip(+)) with the same strain of L. curvatus (starter S2). Proteolysis and lipolysis were observed during ripening by the increase in total free amino acids (FAA) and free fatty acids (FFA), respectively. Such activities were observed in both started and non started sausages (control). Moreover, the proteolytic and lipolytic activities were detected in products started by both formulations irrespective of the presence of such activities in the strains used. Therefore, it was not possible to conclude whether the effect of proteolysis and lipolysis during ripening of the started fermented sausages was due to the activity of the starter cultures or to the action of meat endogenous enzymes.     

Species diversity and metabolic impact of the microbiota are low in spontaneously acidified Belgian sausages with an added starter culture of Staphylococcus carnosus

Quality of fermented sausages is affected by acidifying lactic acid bacteria (LAB) and colour- and flavour-promoting coagulase-negative staphylococci (CNS), whether or not used as starter culture. Artisan fermented sausages are often perceived as superior to industrial variants, partially because of the specific microbiota due to spontaneous acidification, which may be considered as an artisan characteristic. Therefore, two kinds of spontaneously acidified Belgian sausages were prepared (Belgian-type salami and Boulogne sausage), but with addition of a Staphylococcus carnosus culture. The Belgian-type salami was made from pork and beef, whereas the Boulogne sausage contained pork and horse meat. In all cases, Lactobacillus sakei was the dominant LAB species present on the raw materials and during fermentation, whereas enterococci remained present in the background. Enterobacteriaceae vanished after fermentation. The CNS species diversity on the raw materials was large and differed between the pork, beef, and horse meat. Nevertheless, this species diversity was annihilated during fermentation by the added S. carnosus culture. The volatiles fraction was mainly composed of aldehydes that originated from lipid oxidation and spices-derived compounds. Aromatic compounds that are typically associated to CNS activity, such as end-products from the metabolism of branched-chain amino acids, were not present in the Belgian-type salami and only marginally present in the Boulogne sausage. In conclusion, spontaneous acidification of Belgian-type fermented sausages leads to dominance of L. sakei and is no guarantee for bacterial contribution to the aroma profile when S. carnosus is added as a starter culture.     

Characterization of Proteolytic Effect of Lactic Acid Bacteria Starter Cultures on Thai Fermented Sausages

The objective of this study was to investigate the effect of starter culture addition on proteolysis of Thai fermented sausages. Sausages inoculated with six different external starter cultures—Pediococcus pentosaceous, Pediococcus acidilactici, Weissella cibaria, Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus sakei—were compared with naturally fermented sausages. The results of microbiological analysis indicated that the dominance of lactic acid bacteria (LAB) could inhibit the growth of pathogens and spoilage. Proteolysis was observed during fermentation by the reduction of myofibrillar and sarcoplasmic proteins and the increase in nonprotein nitrogen (NPN) and total free amino acids. The highest increase in concentration of NPN and free amino acids was obtained from sausages inoculated with LAB. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed a similar pattern of proteolysis of sarcoplasmic proteins in all sausages, while that of the inoculated sausages with L. plantarum, L. pentsus, and L. sakei exhibited increased degradation of myofibrillar protein bands at 200 and 45 kDa.     

Use of Fourier transform infrared spectroscopy to evaluate the proteolytic activity of Yarrowia lipolytica and its contribution to cheese ripening

The interaction between tyrosine-decarboxylase and proteolytic activities of a Lactobacillus curvatus and Staphylococcus xylosus, respectively, on biogenic amine production during the ripening and the storage of dry fermented sausages was investigated. Water content, pH, proteolysis parameters, microbial counts, and biogenic amine contents were monitored in spontaneously and starter fermented sausages. The use of proteolytic staphylococci as starter resulted in a higher content of non-protein nitrogen and total free amino acids. Tyramine was the main amine produced in all batches. However, tyrosine-decarboxylase activity of the L. curvatus starter strain was weak and yielded lower amounts of tyramine than those produced by the wild mioroflora in the control batch. Association between tyramine production and proteolysis could only be established in a defectively dried batch. Putrescine and cadaverine accumulation was efficiently reduced in the starter-mediated fermentation, in agreement with the lower development of enterobacteria. Phenylethylamine and tryptamine were only detected in the spontaneously fermented sausages, while histamine, spermine and spermidine did not vary during the ripening. Biogenic amine levels and related parameters showed significant changes during the storage of dry sausages depending on the temperature and the batch. As a general rule, changes in the pH, proteolysis, microbial counts, and biogenic amine contents were stronger at 19 degrees C than at 4 degrees C. The results suggest that refrigeration would be advisable for preventing further accumulation of biogenic amines during the storage of dry fermented sausages.     

Utilisation possibility of Enterococcus faecalis isolates from neonate's faeces for production of fermented sausages as starter cultures

This study aimed at assessing the utilisation possibility of Enterococcus faecalis as starter cultures for production of fermented sausages. The E. faecalis isolates isolated from faeces samples of 5‐day‐old healthy newborn babies, identified by 16SrDNA sequencing and free from virulence determinants as proven by genomic sequencing, were used for fermented sausages production as starter culture. Six treatments (four inoculated with four different E. faecalis isolates, one with commercial starter culture and one control) were prepared. The inoculation with E. faecalis showed a stronger acidification compared with the control. The treatments with E. faecalis showed significantly higher flavour and acceptability scores in comparison with the control or commercial starter culture. Both of commercial starter culture and E. faecalis were positive with tyramine. Apart from the amine production, the inoculation with E. faecalis generally improved the acidifying activity and flavour of the products, suggesting their application potentials in fermented sausages production as starter cultures.     

Effect of proteolytic starter cultures of Staphylococcus spp. on biogenic amine formation during the ripening of dry fermented sausages

The effect of proteolytic starter cultures of Staphylococcus carnosus and Staphylococcus xylosus on biogenic amine production was examined during the fermentation process of dry sausages. Microbial counts (lactic acid bacteria, Micrococcaceae and Enterobactenaceae), pH, moisture and proteolysis-related parameters were also studied. The polyamines spermine and spermidine were the main amines found in the raw material and they only showed slight fluctuations during the fermentation. The four elaborated batches presented a significant (P < 0.001) formation of tyramine and putrescine. The main rate of amine production was during the first three days, when a sharp pH decrease and the development of lactic acid bacteria occurred. Sausages fermented with starters had lower amounts of tyramine than naturally fermented sausages (control), but differences in the Micrococcaceae counts were only significant during the first week of the ripening process. A slight formation of diaminopropane, cadaverine, agmatine, tryptamine and phenylethylamine was observed. The amounts of histamine were constant and remained below 0.5 mg/kg of dry matter, while serotonin, octopamine and dopamine were not detected. The sausages with Staphylococcus as starter culture showed strong proteolysis that was correlated with higher pH values than those of the control sausages. However, no positive correlation was found between the proteolysis index and biogenic amine production. Since proteolysis was stronger during the second half of the ripening process, the release of free amino acids as amine precursors occurred later than the early amine production.     

Fermentation of bighead carp (Aristichthys nobilis) surimi and the characteristics of fermented bighead carp surimi products

BACKGROUND: To improve the properties and functionality of bighead carp (Aristichthys nobilis) muscle, bighead carp surimi was inoculated with combinations of Saccharomyces cerevisiae, Lactobacillus and Monascus and fermented, and then bighead carp sausages were prepared. The characteristics of fermented bighead carp surimi and sausages were investigated. RESULTS: During the 24 h fermentation at 30 °C, bighead carp surimi inoculated with mixed starter cultures resulted in a rapid decrease of pH, and suppression in the growth of Enterobacteria. The bighead carp sausages exhibited better colour, taste, flavour and appearance than the control (P < 0.05). The changes in non‐protein nitrogen (NPN), free amino acid in the fermented bighead carp surimi and SDS–PAGE patterns indicated that severe hydrolysis of muscle protein had occurred during fermentation. The sausages inoculated with the mixed starter cultures gained higher scores for flavour and overall acceptability than the control. CONCLUSIONS: The characteristic of fermented bighead carp surimi and sausages can be significantly improved by fermentation with the combinations of yeast, Lactobacillus and Monascus. Copyright © 2008 Society of Chemical Industry     

Comparison of the effects of a commercial and an autochthonous Pediococcus acidilactici and Staphylococcus vitulus starter culture on the sensory and safety properties of a traditional Iberian dry‐fermented sausage “salchichón”

The effect of the addition of an autochthonous starter culture and commercial starter culture on the physico‐chemical and sensory characteristics of the dry‐fermented sausage ‘salchichon’ was investigated. Sausages were prepared with a mixture of Pediococcus acidilactici MC184 and Staphylococcus vitulus RS34 as the autochthonous starter culture (P184S34), ripened for 86 days, and compared with a commercial starter batch (CS1). Strains of acid lactic bacteria and Staphylococcus were identified in the samples along the ripening process which demonstrated that the inoculation of sausages assures their implantation during the ripening process. However, P184S34 starter culture showed a higher inhibitory effect on enterobacteria and coliform flora than the CS1 batches, guaranteeing a better quality hygienic sausages. Dry‐fermented sausages ripened with P184S34 showed higher amounts of NPN and volatile compounds derived from amino acid catabolism than the CS1 batches. Especially important was the result found in biogenic amines, since the P184S34 reduced their accumulation compared to the commercial starter batch. The inoculation of a decarboxylase‐negative autochthonous starter culture P184S34 reduced the biogenic amine accumulation guaranteeing safety and homogeneity of the products without producing a negative effect on the sensory characteristics of the traditional fermented sausages.     

Effects of branched-chain amino acids and Lactobacillus plantarum CGMCC18217 on volatiles formation and textural properties of dry-cured fermented sausage

This study aimed to evaluate the effect of branched-chain amino acids (BCAAs, leucine, isoleucine and valine) combined with Lactobacillus plantarum CGMCC18217 (L. plantarum) on the flavours and quality of fermented sausages. The parameters included pH, water activity, colour, texture, BCAAs metabolites and flavour compounds of fermented sausages in eight different groups (sausages with L. plantarum and individual BCAAs, that is L + Leu, L + Ile and L + Val groups; sausages with individual BCAAs, that is the Leu, Ile and Val groups; sausages only with L. plantarum assigned as the L group; and sausages with no L. plantarum and BCAAs assigned as the CK group). The results showed that the addition of BCAAs and L. plantarum significantly increased the hardness, cohesiveness and springiness of sausages. A total of sixty-nine flavour compounds were identified in fermented sausages. The content of methyl-branched alcohols, aldehydes, acids in the L. plantarum and BCAAs group significantly increased compared with the other groups (P < 0.05). Therefore, our data suggest the addition of L. plantarum and BCAAs potentially improved the texture of fermented sausages and formation of volatile flavours.     

Effect of Starter Culture Addition and Processing Method on Proteolysis and Texture Profile of Traditional Dry-Fermented Sausage Petrovská klobása

Proteolysis and texture profile of Petrovská klobása, made with addition of starter culture, were investigated during the ripening in different conditions (traditional versus industrial). Due to intensive degradation of myofibrillar proteins, significant increases of non-protein nitrogen and free amino acids nitrogen contents were registered in all samples. Similar proteolytic changes were found in both starter and non-starter sausages, but were more pronounced in samples ripened in industrial room. In relation to ripening conditions hardness and chewiness differed significantly, while starter culture had significant effect only on hardness of sausages processed in industrial room.     

Characterization of proteolysis during the ripening of semi-dry fermented sausages

The respective contribution of indigenous enzymes and enzymes from starter bacteria to proteolysis in fermented sausages were determined by comparing the proteolytic changes occurring in sausages resulting from the presence of a proteolytic strain of Staphylococcus carnosus, i.e. S. carnosus MC 1 to the proteolytic changes occurring in control sausages containing glucono-δ-lactone (GDL) and an antibiotic mixture. Proteolysis was quantified by assaying for non-protein nitrogen (NPN) and free amino acids. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed phase high performance liquid chromatography (RP-HPLC) were used to qualitatively assess the proteolytic changes in the sarcoplasmic and myofibrillar proteins as ripening progressed. The concentration of NPN and free amino acids increased in both sausages initially, but subsequently decreased towards the end of ripening in sausages inoculated with the starter culture. SDS-PAGE showed a similar pattern of proteolysis of sarcoplasmic proteins in both sausages, while of the two sausage types; the S. carnosus MC 1 inoculated sausages exhibited the most intense degradation of myofibrillar proteins, especially myosin and actin. RP-HPLC profiles of 2% trichloroacetic acid (TCA)-soluble peptides for the two sausage types were similar, with the production of numerous hydrophilic peptides. N-Terminal amino acid sequence analysis and sequence homology with proteins of known primary structure showed that six of the TCA-soluble peptides were released from the sarcoplasmic (myoglobin and creatine kinase) and myofibrillar (troponin-I, troponin-T and myosin light chain-2) proteins. In addition, the initial degradation of sarcoplasmic proteins was due to the activity of indigenous proteinases, while both indigenous and bacterial enzymes contributed to the initial degradation of myofibrillar proteins. Furthermore, indigenous enzymes were responsible for the release of TCA-soluble peptides, which, were further hydrolysed by bacterial enzymes.     

The use of commercial starter cultures in the production of Turkish semi-dry fermented sausages

 The effects of three different starter cultures (Pediococcus acidilactici, Staphylococcus xylosus plus P. pentosaceus, S. carnosus plus Lactobacillus pentosus) were evaluated during the production of Turkish semi-dry fermented sausages. Sausages were studied during the fermentation phase, after heat processing and after drying for 24 h and 72 h. Chemical and organoleptical results indicated that in the processing of these semi-dry sausages a starter culture of P. acidilactici should be used. The use of this culture significantly reduced the pH, increased the lactic acid content and percentage of total heme pigments converted to the cured pigment and improved the development of the sausages' characteristics, i.e. color, appearance, flavor and general acceptability. Received: 20 February 1998 / Revised version: 7 May 1998     

Nonprotein Nitrogen (NPN) and Free amino acid contents of dry, fermented and nonfermented sausages

Fermented and nonfermented sausages were analyzed for concentration changes in total and nonprotein nitrogen (NPN) and in free amino acid profiles during processing. Sausages fermented by Pediococcus pentaceous at 38°C and heated to 60°C had increases (p < 0·05) in total N and NPN contents and increases in concentration (≥5 mg per 100 g dry sample) for 14 of 20 free amino acids. Nonfermented sausages had no increase (p > 0·05) of NPN content and increases in only 6 of 20 free amino acids after heating to 60°C. When the effects of three starter cultures were compared, NPN concentrations after fermentation and heating and after drying were highest to lowest in the following order: P. pentaceous >P. acidilactici with M. varians >P. acidilactici. Variations of free amino acid patterns in the fermented, dried sausages were found which indicated differences in metabolic activity between the cultures. These variations did not (p > 0·05) affect sensory scores for the dried sausages. Overall, these results for rapidly fermented and mildly heated dry sausages were similar to results previously reported for 'European-style' dry sausages processed with prolonged low temperature fermentations.     

Proteomic and peptidomic insights on myofibrillar protein hydrolysis in a sausage model during fermentation with autochthonous starter cultures

The hydrolysis of myofibrillar proteins during fermentation of sausage models by an autochthonous starter culture was investigated. In order to provide a whole map of the generated products, proteomic and peptidomic were used and complemented with the amino acid profile. Beaker sausages (BS) were used as models which were inoculated or not with Lactobacillus curvatus CRL705 and Staphylococcus vitulinus GV318 as starter cultures. The hydrolysis of actin, myosin light chain 1/3 (MLC 1/3), myosin regulatory light chain-2 (MRLC-2) and myosin heavy chain (MHC) was evidenced by two-dimensional gel electrophoresis (2-DE). In addition, a total of 33 peptides arisen from troponin T, MRLC-2 and particularly from actin were identified by LC–MS/MS. These results showed that the starter culture significantly enhanced the proteolysis of the proteins named above, even when the endogenous enzymes induced a clear breakdown. L. curvatus CRL705 highly enriched both peptide pattern and amino acid concentrations. When the autochthonous starter culture was inoculated, although proteolysis was remarkably reinforced, a reduction in peptide and amino acid composition was observed. Regarding actin primary structure, three regions of this protein were highly susceptible to degradation by the starter culture. Additionally, the essential role of exopeptidases – from meat and bacteria – in diversity of actin peptides during fermentation was shown. This study improved the knowledge of the proteolysis of myofibrillar proteins and the involved enzymes, as well as, completed the previously reported degradation of sarcoplasmic proteins by the same autochthonous starter culture. The singular peptides and amino acids pattern generated might contribute to the uniqueness of produced fermented sausages while they may be used as quality markers.     

Effect of starter culture on proteolytic changes during processing of fermented beef sausages

Fermented beef sausages inoculated with four different starter cultures (Pediococcus acidilactici,Lactobacillus curvatus, Lactobacillus sake, or Streptomyces griseus) were evaluated for proteolysis during process stages (prefermentation, fermentation, drying and heating). Increases (p ? 0.05) in the nonprotein nitrogen (NPN) fraction were found at sequential stages of processing, while starter cultures had no major effects on NPN content. Concentrations of most free amino acids increased (p ? 0.05) during fermentation and drying, and culture effects were found for differences among concentrations of some individual free amino acids. From SDS–PAGE analysis of sarcoplasmic and myofibrillar protein fractions after fermentation and drying, myosin heavy and light chains, actin and troponin were degraded during processing. However, starter culture effects were absent from SDS–PAGE protein patterns.     

Proteolytic and lipolytic starter cultures and their effect on traditional fermented sausages ripening and sensory traits

In this study, three starter formulations including Lactobacillus curvatus and Staphylococcus xylosus strains selected in vitro on the basis of their lipolytic and proteolytic activities were employed for the manufacture of traditional fermented sausages of southern Italy. Microbial population, proteolysis, lipolysis, changes in free amino acids (FAA) and free fatty acids (FFA) and development of characteristic taste and flavor of the final product were investigated. Proteolysis and lipolysis were observed in sausages inoculated with proteolytic and lipolytic S. xylosus coupled with L. curvatus, while the sausage started with only S. xylosus without lactobacilli was identical to the non-inoculated control, indicating that the proteolysis could be due to both microbial activity and endogenous proteases activated by the decrease in pH. The statistical analysis applied to the instrumental and sensory data showed that there was an effect of the starter used on the characteristics of the sausage obtained. In particular, the control samples showed very close features different from the sausages obtained by adding starter cultures. Finally, analyzing the sensory parameters the sausages ripened without starter addition and those started without the L. curvatus AVL3 showed similar features indicating an influence of the presence of the lactobacilli on the final organoleptic quality of the sausages. An appropriate choice of a combination of strains in a starter formulation is fundamental to obtain products of the expected quality.     

Production of volatile compounds by Lactobacillus sakei from branched chain α-keto acids

Lactobacillus sakei belongs to the main flora of raw fermented sausages and is used as starter culture. Bacterial starter cultures can convert amino acids to α-keto acids by aminotransferases. These α-keto acids are the precursors of aroma active aldehydes, alcohols and carboxylic acids. In this study the formation of aldehydes, alcohols and carboxylic acids from leucine, isoleucine, valine and the corresponding α-keto acids are analysed in model fermentations with two different strains of L sakei. In the absence and upon addition of leucine, isoleucine and valine they produced 1 μg/ml 3-methylbutanoic, 0.2 μg/ml 2-methylbutanoic acid and 3 μg/ml 2-methylpropanoic acid, respectively. Upon addition of α-ketoisocaproic acid, α-keto-3-methyl-pentanoic acid or α-ketoisovaleric acid the amount of the corresponding carboxylic acid was increased to 40 μg/ml 3-methylbutanoic acid, 20 μg/ml 2-methylbutanoic acid and 35 μg/ml 2-methylpropanoic acid. The response patterns of the strains and amounts of carboxylic acids produced were similar. This behaviour was typical when compared with other strains of L. sakei and suggests general lack of transaminase activity and a limit in the transport of branched chain amino acids and their conversion to volatiles, some of which can contribute to the aroma of fermented sausages.     

Type of bacterial starter culture,aging and fermentation effects on some characteristics of inoculated beef sausages

Beef sausage mixes were inoculated with either Pediococcus acidilactici with Staphylococcus xylosus or P. acidilactici with S. carnosus, subdivided and then held for 0, 24, 48 or 72 h at 8–10 °C prior to fermentation. After aging (pre-fermentation holding), the mixes were fermented for 16 h ending at 41 °C. Moisture, protein and fat contents of all sausage mixes did not differ due to holding effects over all starter cultures. The pH of mixes followed the same pattern for all mixes, declining (p < 0.05) from approximately 5.8 to pH 5.2–5.3 at 72 h aging and to 4.4–4.5 after fermentation. Total acidity of the mixes followed an inverse pattern to pH, increasing (p < 0.05) after fermentation although there was no effect due to type of starter culture. Aging had no effect on nonprotein nitrogen (NPN) content as ΔNPN among all cultures. After fermentation, however, sausages held 72 h and inoculated with S. carnosus had higher NPN contents compared to P. acidilactici alone (p < 0.05) and with S. xylosus (p < 0.10). The same effects of starter cultures on changes in total amino acid concentration were observed. Concentrations of individual amino acids showed increases depending on pre-fermentation aging period (0 h versus 72 h) followed by fermentation.