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Technological and microbiological evaluation of traditional processes as modified for the industrial manufacturing of dry fermented sausage in Greece
Abstract
The technology and microbiological quality of dry fermented sausage was compared during processing at three (A, B and C) industrial meat plants. In plants A and B salamis having final pHs below 5.0 and characterised by strong acidic taste and poor aroma. were produced even in the absence of starter cultures. In plant C, moderately acidified (final pH 5.0-5.2) sausages with excellent sensory characteristics were produced regardless of the greater increase in lactic acid bacteria, The microbial composition of the sausages in plant C was more diverse, with Micrococcaceae constituting a significant part of it throughout processing, Salmonella spp. were absent from all batches after stuffing, while pseudomonads. enterobacteria, coagulase-positive staphylococci, sulfite-reducing clostridia and aerobic spore-formers were maintained at normal levels and were practically eliminated during ripening. Listeria monocytogenes and other Listeria spp. contaminated most batches during formulation, but were not detected in the finished products. Enterococci were significantly present in plants A and C during the whole process. It is concluded that traditional processes for making salami lead to a microbiologically safe product when applied under industrial manufacturing conditions. but should be closely monitored to give a less acidified and delicately flavoured final product.
... The total viable mesophilic counts showed an average value of 8.65 log CFU/g. This average value was of the same order of those observed by Dolazo, Steinhof, Pfeiffer, and Ring (1998) in Galician chorizo, by Garcı´a Fonta´n, Lorenzo, Parada, Franco, and Carballo (2007) in Androlla, and by Domı´nguez, Gutie´rrez, Lo´pez, Seco, and Zumalaca´rregui (1989) at the end of the ripening process of the Chorizo of Leo´n, around a log unit higher than those observed by Gonza´lez and Dı´ez (2002) in Chorizo, by Serrano Moreno (1979) in Salchicho´n and by Samelis, Metaxopoulos, Vlassi, and Pappa (1998) and Metaxopoulos, Samelis, and Papadelli (2001) in Greek traditional salami, and around two log units higher than those observed by Lizaso, Chasco, and Beriain (1999) in Salchicho´n at the end of the ripening process. ...
... In agreement with the results obtained by other authors in previous studies carried out on other raw-cured sausages (Dolazo et al., 1998;Domı´nguez et al., 1989;Garcı´a Fonta´n et al., 2007;Gonza´lez & Dı´ez, 2002;Lizaso et al., 1999;Metaxopoulos et al., 2001;Samelis et al., 1998), the lactic acid bacteria were the main microbial group, which is logical in a product that experiences a true lactic fermentation. The average counts of lactic acid bacteria observed in our study (8.87 log CFU/g) basically coincide with those observed in different varieties of Chorizo (Dolazo et al., 1998;Domı´nguez et al., 1989;Gonza´lez & Dı´ez, 2002) and in Androlla (Garcı´a Fonta´n et al., 2007) and they are slightly higher (0.5-1 log units) than those observed in Greek salami (Metaxopoulos et al., 2001;Samelis et al., 1998), Naples-type salami (Coppola, Mauriello, Aponte, Moschetti, & Villani, 2000) and Salchicho´n (Lizaso et al., 1999). ...
... In agreement with the results obtained by other authors in previous studies carried out on other raw-cured sausages (Dolazo et al., 1998;Domı´nguez et al., 1989;Garcı´a Fonta´n et al., 2007;Gonza´lez & Dı´ez, 2002;Lizaso et al., 1999;Metaxopoulos et al., 2001;Samelis et al., 1998), the lactic acid bacteria were the main microbial group, which is logical in a product that experiences a true lactic fermentation. The average counts of lactic acid bacteria observed in our study (8.87 log CFU/g) basically coincide with those observed in different varieties of Chorizo (Dolazo et al., 1998;Domı´nguez et al., 1989;Gonza´lez & Dı´ez, 2002) and in Androlla (Garcı´a Fonta´n et al., 2007) and they are slightly higher (0.5-1 log units) than those observed in Greek salami (Metaxopoulos et al., 2001;Samelis et al., 1998), Naples-type salami (Coppola, Mauriello, Aponte, Moschetti, & Villani, 2000) and Salchicho´n (Lizaso et al., 1999). Serrano Moreno (1979) observed lower values (on average 6.76 log CFU/g) at the end of the ripening process of Salchicho´n. ...
Counts of total aerobic mesophilic flora on Standard Plate Count (SPC) agar, lactic acid bacteria on de Man Rogosa Sharpe (MRS) agar, salt tolerant flora on SPC agar +7.5% NaCl, and Enterobacteriaceae on Violet Red Bile Glucose (VRBG) agar, and some physico-chemical parameters (Total solids and NaCl contents and pH and aw values) were determined in 15 units of Botillo, a traditional dry-fermented sausage made in the NW of Spain.In general, high counts of all the investigated microbial groups were observed, with average values of 8.65 log Colony Forming Units (CFU)/g for the total aerobic mesophilic flora, 8.87 log CFU/g for the lactic acid bacteria, 6.56 log CFU/g for the salt tolerant flora, and 2.98 log CFU/g for the Enterobacteriaceae.After the end of the incubation period, from MRS agar, SPC agar +7.5% NaCl, and VRBG agar, 10 colonies were randomly taken from each Botillo unit and from each culture medium. A total of 150 strains per culture medium were then identified using classical methods.Among the isolates from MRS agar, Lactobacillus sakei predominated (23.3% of the isolates), followed by Lactobacillus alimentarius (17.3%), Lactobacillus curvatus (15.3%), Lactobacillus plantarum (12%) and Lactobacillus farciminis (10%). Of the 150 isolates obtained from SPC agar +7.5% NaCl, only 34 strains (22.6% of the isolates) belonged to the Staphylococcaceae or Micrococcaceae families. Among the Staphylococcaceae, Staphylococcus saprophyticus (8.6% of the isolates) was the main species, followed by Staphylococcus xylosus (4%), Staphylococcus lentus (2%) and Staphylococcus cohnii cohnii (1.3%); Staphylococcus epidermidis, Staphylococcus sciuri and Staphylococcus capitis were isolated in very low proportions. The Micrococcaceae strains isolated, belonging to the Micrococcus genus, could not be identified at species level. Of the 140 isolates obtained from VRBG agar, Hafnia alvei (32.14% of the isolates) was the main species, followed by Serratia liquefaciens (12.85%), Klebsiella oxytoca (5.71%) and Enterobacter cloacae (3.71%); only one isolate was identified as Salmonella (Salmonella arizonae) and one as Escherichia coli.
... LAB and total aerobic microbes are the fastest growing during the production of sausages. From the initial counts of 10 2 -10 3 cfu/g, they reach values of 10 7 -10 8 cfu/g in the first three days of fermentation (Sanz et al., 1988;Samelis et al., 1993Samelis et al., , 1998Rebecchi et al., 1998;Cocolin et al., 2001a,c;Metaxopoulos et al., 2001;Papamanoli et al., 2002Papamanoli et al., , 2003Aymerich et al., 2003;Mauriello et al., 2004;Drosinos et al., 2005), and this situation establishes both in the core and in the external layers of the sausage (Coppola et al., 2000). Their counts remain quite stable in number throughout the ripening period. ...
... Yeasts only in few cases were found to be relevant to sausage fermentation. Samelis et al. (1993) determined a stable number of 10 5 cfu/g in all stages of production, and this data was confirmed by Metaxopoulos et al. (2001). Coppola et al. (2000) found yeasts to be a predominant flora, together with LAB and CNC, in Naples-type salami, with Debaryomyces spp., described as a proteolytic agent during fermentation of sausages (Santos et al., 2001), as the main representative. ...
... In some studies, after an increase to 10 5 cfu/g up to 20 days of fermentation, a reduction in the numbers was observed, leading to a final count of about 10 2 cfu/g (Cocolin et al., 2001a,c;Aymerich et al., 2003;Papamanoli et al., 2003). On the other hand, several papers reported a stable population of enterococci of about 10 5 -10 6 cfu/g at the end of the fermentation (Rebecchi et al., 1998;Samelis et al., 1998;Metaxopoulos et al., 2001;Comi et al., 2005;Rantsiou et al., 2005c), becoming an important population possibly influencing the final organoleptic characteristics of the product. Since enterococci are able to produce ammonia and other amines, they possibly contribute to the final flavour of the product. ...
The microflora of different types of fermented sausages has been defined by isolation and biochemical identification of the microorganisms commonly found in these products. It is generally agreed that the main microbial groups involved in such products are lactic acid bacteria and coagulase-negative cocci. In addition, and depending on the product, other groups may play a role, such as yeasts and enterococci. Since it appears that the types of microbial groups, or even the specific strains of a given microbial group, that dominate the fermentation, significantly affect the organoleptic profile of the final product, there is an increasing interest in the description of the microbiota that are found in different fermented sausages. More recently, new tools, based on molecular methods, allowing fast and unequivocal identification of strains, isolated from fermented sausages, became available. These methods have been successfully applied and, in general, biochemical and molecular identification compared well. However, new information comes to light when molecular methods are applied to DNA and/or RNA extracted directly from sausages. This approach eliminates problems related to traditional isolation. This review deals with the recent findings and results of the application of molecular methods, in a culture-dependent and culture-independent manner, on the study of the microflora of fermented sausages.
... Cured (dry fermented) sausages made with ostrich and deer meats have recently appeared in the Spanish market and are being sold alongside traditional pork and beef products. The microbiological characteristics of fermented dry sausages have been extensively studied (9,14,16,19,23,25,30). However, these studies focus on sausages manufactured with pork and beef meats. ...
... The low Enterobacteriaceae counts found in the present study agree with findings in Greek fermented sausages (14,23,30) and in Italian fermented salami at the end of the ripening period (5,19) and suggest the strong competitive effect that lactic acid bacteria (LAB) have on other endogenous flora (9, 16, 19, 25). The chemical compounds (antibiotic-like substances) excreted by LAB and the decrease in pH as a result of their high acidifying capability may partially explain the reduction and disappearance of some microbial groups (Enterobacteriaceae, enterococci, and pseudomonads) in fermented meat products. ...
... Gram-positive, catalase-positive cocci are necessary for the reduction of nitrate to nitrite; they also enhance color stability and prevent rancidity by consuming oxygen. By their catalase activity, they destroy peroxides, and they release various aromatic substances because of their intense lipolytic and proteolytic activities (14,19,23). Microbiological analysis showed that counts of these bacteria in deMan Rogosa Sharpe (MRS) agar constituted the major microflora of both chorizo and salchichón sausages (average, 7.10 Ϯ 1.64 and 6.53 Ϯ 1.24, respectively; P Ͻ 0.05). ...
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. Salchich6n 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.
... The total viable mesophilic counts showed an average value of 8.99 log cfu/g. This value was similar to those observed by Dolazo et al. (1998) in Galician chorizo and by Domı´nguez et al. (1989) at the end of the ripening process of the chorizo of Leo´n, around a log unit higher than those observed by Gonza´lez and Dı´ez (2002) in chorizo, by Serrano Moreno (1979) in salchicho´n and by Metaxopoulos et al. (2001) and Samelis et al. (1998) in Greek traditional salami, and around two log units higher than those observed by Lizaso et al. (1999) in salchicho´n at the end of the ripening process. In agreement with the results obtained by other authors in previous studies carried out on other raw-cured sausages (Domı´nguez et al., 1989;Dolazo et al., 1998;Samelis et al., 1998;Lizaso et al., 1999;Metaxopoulos et al., 2001;Gonza´lez and Dı´ez, 2002), the lactic acid bacteria were the major microbial group. ...
... This value was similar to those observed by Dolazo et al. (1998) in Galician chorizo and by Domı´nguez et al. (1989) at the end of the ripening process of the chorizo of Leo´n, around a log unit higher than those observed by Gonza´lez and Dı´ez (2002) in chorizo, by Serrano Moreno (1979) in salchicho´n and by Metaxopoulos et al. (2001) and Samelis et al. (1998) in Greek traditional salami, and around two log units higher than those observed by Lizaso et al. (1999) in salchicho´n at the end of the ripening process. In agreement with the results obtained by other authors in previous studies carried out on other raw-cured sausages (Domı´nguez et al., 1989;Dolazo et al., 1998;Samelis et al., 1998;Lizaso et al., 1999;Metaxopoulos et al., 2001;Gonza´lez and Dı´ez, 2002), the lactic acid bacteria were the major microbial group. The average counts of lactic acid bacteria observed in our study (9.11 log cfu/g) basically coincide with those observed in different varieties of chorizo (Domı´nguez et al.,1989;Dolazo et al., 1998;Gonza´lez and Dı´ez, 2002) and they are lightly higher (around one log unit) to those observed in Greek salami (Samelis et al., 1998;Metaxopoulos et al., 2001), Italian salami (Coppola et al., 2000) and salchicho´n (Lizaso et al., 1999). ...
... In agreement with the results obtained by other authors in previous studies carried out on other raw-cured sausages (Domı´nguez et al., 1989;Dolazo et al., 1998;Samelis et al., 1998;Lizaso et al., 1999;Metaxopoulos et al., 2001;Gonza´lez and Dı´ez, 2002), the lactic acid bacteria were the major microbial group. The average counts of lactic acid bacteria observed in our study (9.11 log cfu/g) basically coincide with those observed in different varieties of chorizo (Domı´nguez et al.,1989;Dolazo et al., 1998;Gonza´lez and Dı´ez, 2002) and they are lightly higher (around one log unit) to those observed in Greek salami (Samelis et al., 1998;Metaxopoulos et al., 2001), Italian salami (Coppola et al., 2000) and salchicho´n (Lizaso et al., 1999). Serrano Moreno (1979) observed lower values (on average 6.76 log cfu/g) at the end of the ripening process of salchicho´n. ...
Counts of total aerobic mesophilic microflora, lactic acid bacteria, salt-tolerant microflora, Enterobacteriaceae, enterococci, moulds and yeasts, and staphylococci, and some physico-chemical parameters (total solids, NaCl and nitrate contents and pH and aw values) were determined in 20 units of "androlla", a traditional dry-fermented sausage made in the NW of Spain. In general, high counts of all the investigated microbial groups were observed, with average values of 8.99 +/- 0.46 log cfu/g for the total aerobic mesophilic microflora, 9.11 +/- 0.16 log cfu/g for the lactic acid bacteria, 6.87 +/- 0.68 log cfu/g for the salt-tolerant microflora, 2.80+/-1.85 log cfu/g for the Enterobacteriaceae, 3.25 +/- 1.86 log cfu/g for the enterococci, 4.30 +/- 1.73 log cfu/g for the moulds and yeasts, and 3.62 +/- 0.60 log cfu/g for the staphylococci. From MRS agar, SPC agar + 7.5% NaCl, VRBG agar, and KAA agar, 10 colonies were randomly taken from each androlla unit and from each culture medium. A total of 200 strains per culture medium were then identified using the classical methods. Among the isolates from MRS agar, Lactobacillus sakei predominated, followed by Lactobacillus curvatus, Lactobacillus alimentarius and Lactobacillus plantarum. Of the 200 isolates obtained from SPC agar + 7.5% NaCl, only 56 strains belonged to the Staphylococcaceae or Micrococcaceae families. Among the Staphylococcaceae, Staphylococcus xylosus was the main species, followed by Staph. epidermidis; Staph. equorum, Staph. capitis and Staph. saprophyticus were isolated in very low proportions. Among the Micrococcaceae, Micrococcus luteus predominated, followed by Micrococcus lylae, Kocuria varians and Kocuria kristinae. Of the 150 isolates obtained from VRBG agar, Hafnia alvei was the main species, followed by Serratia liquefaciens and Enterobacter amnigenus; six isolates were identified as Salmonella. Among the 190 isolates obtained from KAA agar, 122 were considered enterococci; 20 isolates were identified as Enterococcus faecium, one as Enterococcus faecalis and 101 as Enterococcus inter faecalis-faecium.
... Natural fermented sausages have a long tradition in the South European countries, and extensive studies have been performed on the characterization of traditional sausages produced in Greece, Italy and Spain (Metaxopoulos et al., 2001;Samelis et al., 1994Samelis et al., , 1998Cantoni et al., 1967;Comi et al., 1992Comi et al., , 2005Coppola et al., 1998;Parente et al., 2001;García-Varona et al., 2000;Garriga et al., 1996;Hugas et al., 1993;Rodríguez et al., 1994;Santos et al., 1998). ...
... LAB strains were previously isolated from three different plants in Greece, named plants B, E and T, where fermented sausages are produced without the use of starter cultures (Metaxopoulos et al., 2001). They were isolated from MRS plates (Oxoid, Basingstoke, Hampshire, UK) and after preliminary screening using the Gram staining and the catalase test, they were stored at − 80°C in MRS broth supplemented with 30% glycerol before being subjected to molecular analysis. ...
... The plants selected are carrying out only natural fermentations, and the use of starter cultures is not considered in the production procedures. Three-hundred and thirty eight strains, isolated in a previous study (Metaxopoulos et al., 2001), were first subjected to molecular identification by means of species-specific PCR and 16S rDNA sequencing. ...
In this study, our purpose was to molecular characterize Lactobacillus strains isolated from naturally fermented sausages, produced in three different processing plants in continental Greece, in order to investigate the differences of strains coming from different producing areas. Three-hundred and thirty eight strains were isolated throughout the fermentation periods on MRS. They were identified by species-specific PCR and sequencing of partial 16S rRNA gene. The results obtained highlighted that the main populations involved in the fermentations studied belonged to the species Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus. However, for each of the fermentations, the percentage of isolation for the three main species was different. Molecular characterization of these strains was performed by RAPD-PCR with primer M13 and cluster analysis was applied to define relations and degrees of similarities between strains. This analysis led to the understanding that some strains were plant-specific, whereas others shared a degree of homology independently of the provenience. This evidence is highlighting the capability of the strains to adapt to specific production condition, becoming the main responsible for the transformations, thereby influencing the final characteristics of the sausages.
... Selective enumeration of Staphylococcus aureus was carried out on spread plates of Baird-Parker Agar Media (MM043, HiMedia), with appropriate addition of Egg Yolk Tellurite Emulsion (FD046, HiMedia) 11 . Salmonella-Shigella Agar (M108, HiMedia) was used for the detection of Salmonella and Shigella and Listeria identification agar base (M1064, HiMedia) with Listeria selective supplement (FD 061, HiMedia) for Listeria in the samples following the standard method of Metaxopolous et al. 11 Most Probable Number (MPN) counts of coliforms was determined as described by Harrigan 12 . ...
... Selective enumeration of Staphylococcus aureus was carried out on spread plates of Baird-Parker Agar Media (MM043, HiMedia), with appropriate addition of Egg Yolk Tellurite Emulsion (FD046, HiMedia) 11 . Salmonella-Shigella Agar (M108, HiMedia) was used for the detection of Salmonella and Shigella and Listeria identification agar base (M1064, HiMedia) with Listeria selective supplement (FD 061, HiMedia) for Listeria in the samples following the standard method of Metaxopolous et al. 11 Most Probable Number (MPN) counts of coliforms was determined as described by Harrigan 12 . The water samples were tested for MPN which included presumptive, confirmatory and completed. ...
The Hazard Analysis Critical Control Points (HACCP) system basically applies on food processing to identify specific hazards and measures for their control to ensure the safety of foods. Kinema is a naturally fermented soybean food of the Eastern Himalayas. The present study on HACCP of kinema revealed that marketed kinema has higher microbial load as compared to the one prepared under laboratory condition and home-made kinema. Furthermore, the Critical Control Point (CCP) was checked during kinema preparation in both traditionally prepared method and kinema prepared under laboratory condition. HACCP model for optimised production of kinema has been proposed. © 2012 The Council of Scientific and Industrial Research, New Delhi. All rights reserved.
... After that, their number decreases due to acidification performed by LAB Papamanoli et al. 2003;Drosinos et al. 2005). Fermented sausages are usually free of sulphite-reducing clostridia and coagulase-positive staphylococci (CPS) Rantsiou and Cocolin 2006), but in some studies suspected colonies of Staphylococcus aureus were isolated from the meat mixture (Rebecchi et al. 1998;Metaxopoulos et al. 2001). With the aim of evaluating the safety aspect of the fermented sausages, several studies have investigated the presence of Listeria monocytogenes and Salmonella spp. ...
... during the different stages of the production Rantsiou et al. 2005). Listeria monocytogenes can contaminate the fresh sausage mixture, but it is generally undetectable at the end of the fermentation process (Samelis et al. 1998;Metaxopoulos et al. 2001;Drosinos et al. 2005). ...
Two traditional sausage products (“salsiccia” and “salame”) processed from the raw meat of the Black Sicilian swine “Suino Nero dei Nebrodi” were microbiologically investigated during the manufacturing and ripening stages. Both products were dominated by lactic acid bacteria (LAB), especially rod-shaped types. The concentration of enterococci was consistent in salame. Coagulase-negative cocci increased slower than LAB. Yeasts showed an increasing trend during the ripening of both products. Enterobacteriaceae were counted at a constant level of about 105 CFU/g in both products, while pseudomonads diminished during ripening. Coagulase-positive staphylococci, Listeria monocytogenes and Salmonella spp. were not detected at the end of the ripening process. Characterisation of LAB at the strain and species level revealed that Lactococcus lactis was found only in the meat mixture, while Lactobacillus sakei and various enterococci persisted during the monitoring period. Some LAB strains isolated from sausages were also identified on the surface of the factory equipment. Two strains (Lactobacillus sakei SS106A and Enterococcus faecalis SS91) were characterised by their anti-Listeria properties due to bacteriocin-like inhibitory substance production. A multiple strain starter composed of Lactobacillus sakei and enterococci has been proposed to maintain the typical characteristics of the two fermented meat products microbiologically investigated in this study.
... The sausages had high initial pH values (pH > 6.0) ( Table 2) and this is related to the use of frozen pork, which generally had higher pH than fresh pork and beef meats (Metaxopoulos, Samelis, & Papadelli, 2001). Also, the higher initial pH in batch 1 was due to the use of pork and beef meats with higher pH value (data not shown). ...
... Nitrite depletion was observed in all batches. Salt concentration increased and moisture content decreased due to sausage dehydration (Table 2) (Metaxopoulos et al., 2001). ...
The microbial flora of naturally fermented sausages was studied. Lactic acid bacteria were the dominant species at the end of fermentation in all 3 batches (ca. 10(8) cfu g(-1)). Enterobacteria, Pseudomonas, yeasts and aerobic spore-formers decreased during fermentation and the ripening process and were below the detection limit in the end product. Enterococci exceeded 10(4)-10(5) cfu g(-1) during fermentation and remained constant at this level during ripening. Gram-positive, catalase-positive cocci exceeded 10(5) cfu g(-1), except for batch 1, during the first days of fermentation and then decreased until the end of ripening (10(2)-10(4) cfu g(-1)). No pathogenic staphylococci, sulfite reducing clostridia or Salmonella spp. were detected. Listeria spp. occurred in the first days of fermentation but were eliminated by the end of whole process in all batches. Identification showed that the majority of lactobacilli isolated from MRS agar strains were assigned to the species of Lactobacillus plantarum and Lb. plantarum/pentosus. All the isolated strains from the mannitol salt agar belonged to the genus of Staphylococcus. The predominant species were Staphylococcus saprophyticus, Staphylococcus xylosus and Staphylococcus simulans. The tests used to characterize the lactic acid bacteria and staphylococci as well as their distribution on the three batches were also discussed.
... After incubation for 72 hours at 37 o C. The Petri plates were flooded with Lugols Iodine solution (2gm iodine, 2gm ammonium sulfate, and 300ml distilled water) for 1 minute and the diameter of the clear zone and colony was measured. The amylolytic activity was expressed as the ratio of clear zone diameter to colony diameter [14]. ...
... The ratio between LAB and GCC ϩ was highly balanced in the slightly fermented sausages analyzed, and a correlation between the LAB/GCC ϩ ratio and pH could be established. It has been reported that sausages with a predominance of LAB are tangier and less flavorful (46,50,56), while sausages with higher numbers of GCC ϩ contain more aromatic compounds (25). ...
Detection of six species of lactic acid bacteria and six species of gram-positive catalase-positive cocci from low-acid fermented sausages (fuets and chorizos) was assessed by species-specific PCR. Without enrichment, Lactobacillus sakei and Lactobacillus curvatus were detected in 11.8% of the samples, and Lactobacillus plantarum and Staphylococcus xylosus were detected in 17.6%. Enriched samples allowed the detection of L. sakei and S. xylosus in all of the samples (100%) and of Enterococcus faecium in 11.8% of the sausages. The percentages of L. curvatus, L. plantarum, Staphylococcus carnosus, and Staphylococcus epidermidis varied depending on the sausage type. L. curvatus was detected in 80% of fuets and in 57% of chorizos. L. plantarum was found in 50% of fuets and 100% of chorizos. S. epidermidis was detected in only 11.8% of fuets, and S. carnosus was detected in only 5.9% of chorizos. Lactococcus lactis, Staphylococcus warneri, and Staphylococcus simulans were not detected in any sausage type. From a microbiological point of view, 70.6% of the samples could be considered of high quality, as they had low counts of Enterobacteriaceae and did not contain any of the food-borne pathogens assayed.
... As one of the most common pathogens in hospital infections, E. faecalis leads to various opportunistic infections [9][10][11][12][13] such as urinary tract infections, bacteremia, prosthetic joint infections, abdominal-pelvic infections, endocarditis [14][15][16], periodontal disease, and endodontic infections [17,18]. Despite its pathogenicity, E. faecalis is also a probiotic and is often found as part of the autochthonous flora in a variety of fermented foods, namely cheeses and sausages [19][20][21][22][23]. ...
Enterococcus faecalis (E. faecalis) is a Gram-positive, facultative anaerobic bacterium that is highly adaptable to its environment. In humans, it can cause serious infections with biofilm formation. With increasing attention on its health threat, prevention and control of biofilm formation in E. faecalis have been observed. Many factors including polysaccharides as well as autolysis, proteases, and eDNA regulate biofilm formation. Those contributors are regulated by several important regulatory systems involving the two-component signal transduction system (TCS) for its adaptation to the environment. Highly conserved WalRK as one of 17 TCSs is the only essential TCS in E. faecalis. In addition to biofilm formation, various metabolisms, including cell wall construction, drug resistance, as well as interactions among regulatory systems and resistance to the host immune system, can be modulated by the WalRK system. Therefore, WalRK has been identified as a key target for E. faecalis infection control. In the present review, the regulation of WalRK on E. faecalis pathogenesis and associated therapeutic strategies are demonstrated.
... Fleming et al. (1995), Perez-Diaz et al., 2017Mustard E. durans, Lb. alimentarius, Lb. brevis, Lb. casei, Lb. coryniformis, Lb. farciminis, Lb. fermentum, Lb. pentosus, Lb. plantarum, Lb. versmoldensis, Ln. citreum, Ln. mesenteroides, Ln. pseudomesenteroides, Pd. pentosaceus, W. cibaria, W. paramesenteroides Tamang et al. (2005, Chen et al. (2006), Chao et al. (2009), Nguyen et al. (2013), Goswami et al. (2017 Leek C. maltaromaticum, Lb. brevis, Lb. crustorum, Lb. hammessii/parabrevis, Lb. nodensis, Lb. parabrevis, Lb. plantarum, Lb. sakei, Lb. sakei/curvatus, Lc. lactis, Lc. raffinolactis, Ln. gasicomitatum, Ln. gelidum, Ln. kimchii, Ln. in Greece. In that case, the fermentation step takes place at a temperature and RH that decrease gradually from 18-24 o C and 60-94 per cent RH to 11-19 o C and 60-90 per cent RH within five to seven days (Coppola et al., 1998;Metaxopoulos et al., 2001;Papamanoli et al., 2003;Comi et al., 2005;Greco et al., 2005;Drosinos et al., , 2007. The second step may as well be omitted and the whole procedure may be carried out in two steps, as in the case of chorizo, salchichon or the Naplestype salami (Lizaso et al., 1999;Garcia-Varona et al., 2000;Mauriello et al., 2004). ...
... However, during processing there was not an increase in this microbiological parameter in any of the analyzed cases, mainly due to the decrease in the a w until values around 0.78 at the end of maturation but also by the slight decrease in pH, and the presence of NaCl and nitrites. These data are consistent with those observed in other dry-cured fermented sausages [46][47][48]. ...
In the present work, the effect of processing of dry-cured fermented sausage “salchichón” spiked with the selected Lactobacillus sakei 205 was challenge-tested with low and high levels of L. monocytogenes. The evolution of the natural microbial population throughout the “salchichón” ripening was also evaluated. For this, a total of 150 “salchichón” were elaborated and divided into six equal cases which were inoculated with different levels of L. monocytogenes, and L. sakei 205. Afterwards, sausages were ripened for 90 days according to a typical industrial process. Moisture content (%) and water activity (aw) decreased throughout the ripening up to values around 26% and 0.78, respectively. No differences for moisture content, aw, pH, NaCl and nitrite concentration were observed between the analyzed cases. Lactic acid bacteria counts in the L. sakei 205 inoculated cases were always higher than 6 log CFU g−1 during ripening. Enterobacteriaceae counts were reduced during ripening until non-detectable levels at the end of processing. Reductions in L. monocytogenes counts ranged from 1.6 to 2.2 log CFU g−1; therefore, the processing of “salchichón” itself did not allow the growth of this pathogen. Reduction in L. monocytogenes was significantly higher in the cases inoculated with L. sakei 205.
... This kind of product is characterized by low acidity and the final pH is of about 5.3 to 6.2 (Aymerich, Martín, Garriga, & Hugas, 2003). Greece, Italy and Spain are the countries where more efforts have been dedicated to the study of this traditional product (Comi, Citterio, Manzano, & Cantoni, 1992;Coppola, Giagnacovo, Iorizzo, & Grazia, 1998;García-Varona, Santos, Jaime, & Rovira, 2000;Garriga et al., 1996;Hugas et al., 1993;Metaxopoulos, Samelis, & Papadelli, 2001;Parente, Griego, & Crudele, 2001;Rodríguez et al., 1994;Samelis, Maurogenakis, & Metaxopoulos, 1994;Samelis, Metaxopoulos, Vlassi, & Pappa, 1998;Santos, González-Fernández, Jaime, & Rovira, 1998). Moreover, molecular techniques have been applied either to directly profile the main populations involved in the production of Italian fermented sausages (Cocolin, Manzano, Aggio, et al., 2001) or to identify isolated LAB and nonpathogenic staphylococci from artisanal low-acid sausages produced in Spain (Aymerich et al., 2003). ...
... Thus, the initial yeast-mold population in the batter is usually less than 5 log cfu/g (Samelis et al. 1993, Osei Abunyewa et al. 2000, Metaxopoulos et al. 2001, Drosinos et al. 2005, Aquilanti et al. 2007). During fermentation their population may remain stable, increase or diminish depending on product formulation and technology applied ; in any case the population may hardly exceed 6 log cfu/g. ...
... Similar to traditional sausages made with pork meat and fat, goose sausage ripening is based on microbial and tissue enzymes (Comi et al., 2005(Comi et al., , 2000Coppola et al., 1998). CNCPC and lactic acid bacteria (LAB) are the main microorganisms responsible for ripening (Talon et al., 2007;Iacumin et al., 2006;Metaxopoulos et al., 2001;Garcia-Verona et al., 2000). Although these bacteria are normally present in salt and both pork and goose meat, they are often intentionally added to fat and meat mixtures for sausages as microbial starters to ensure a consistent aroma and flavor, to improve quality and to reduce the length of the curing period Comi et al., 2005Comi et al., , 2000Tjener et al., 2003;Luongo et al., 2001). ...
The aim of this work was to determine the microorganisms present and to investigate their metabolites that cause spoilage of many goose sausages produced in Friuli, a northeast region of Italy. The defect was observed by sensorial analysis using the “needle probing” technique; the spoiled sausages were unsafe and not marketable. Despite the addition of starter, the microorganisms, particularly enterococci and Enterobacteriaceae, grew during ripening and produced a large amount of biogenic amines; therefore, these sausages represented a risk to consumers. The production of those compounds was confirmed in vitro. Furthermore, a second cause of spoilage was attributed to moulds that grew during ripening; the fungi grew between the meat and casing, producing a large amount of total volatile nitrogen, and consequently an ammonia smell was present either in the ripening area or in the sausages. This is the first description of this type of defect in goose sausages.
... Violet red bile glucose agar w/o lactose (M581, HiMedia) was used for detection of enterobacteriaceae in samples [12], Salmo-nellaeShigella agar (M108, HiMedia) was used for the detection of Salmonella and Shigella, Listeria identification agar base (M1064, HiMedia) with Listeria selective supplement (FD061, HiMedia) for Listeria [13], Cetrimide agar (MM024, HiMedia) was used for the detection of Pseudomonas spp. [14], and Kaper's medium (M1169, HiMedia) was used for the detection of Aeromonas spp. ...
Background: Consumption of ethnic street foods is very popular in both Gangtok and Nainital in India, both being popular tourist resorts in the Indian Himalayas. Common ethnic street foods of Gangtok are samosa, kachori, puchkka, alu chop, vegetable momo, pork momo, alu-cheura, vegetable chowmein, jhal-muri, and sya-faley. Common ethnic street foods of Nainital are samosa, kachori, pani puri, alu tikki, vegetable momo, mutton momo, bread chop, vegetable chowmein, jhal-muri, and vegetable pakoda.
Methods: A microbiological analysis was conducted to determine bacteria, including pathogenic bacteria, in the food samples. Enterotoxins were also determined.
Results: Lactococcus lactis, Lactobacillus plantarum, Lactobacillus brevis, Enterococcus faecium, Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus cereus, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Salmonella enteritica, Staphylococcus aureus, Staphylococcus epidermidis, and Shigella flexneri were isolated from 233 samples of different street foods collected from Gangtok and Nainital. The dominant contaminant bacteria were enterobacteriaceae followed by Staphylococcus spp. and B. cereus in the food samples tested. Only a few street foods tested positive for toxin production. Salmonella toxins and Staphylococcus enterotoxins were not detected in the street foods tested.
Conclusion: The risks associated with street foods may be controlled by educating vendors about hygienic conditions. In conclusion, street foods are important ethnic foods sold in popular tourist spots in India for marginal local vendors.
... Enterococci are used as probiotics and in food fermentation processes, where they contribute to the flavour and aroma of cheeses. The ability to produce bacteriocins effective against pathogenic bacteria, especially Listeria, might be a powerful tool to protect the products from spoiling: for instance, raw milk cheeses, traditional cheeses with an improved aroma, or medium-pH fermented sausages (Bover-Cid et al. 2001;Gardini et al. 2001;Lauková et al. 2001;Metaxopoulos et al. 2001;Giraffa 2002;Fontán et al. 2007;Choho et al. 2008). Because enterococci are so common in the gastrointestinal tract of many animals, the contamination of raw milk or meat is often unavoidable (Huys et al. 2004). ...
We determined the prevalence and antibiotic resistance of enterococci isolated from raw foods of animal origin. All samples were positive for enterococci. The lowest count of enterococci was found in pork (2.00 log CFU/cm2), while bryndza cheese contained the highest count (4.99 log CFU/g). Among the 349 Enterococcus isolates, 49% were E. faecalis, 29% E. faecium, and 13% Enterococcus spp. Tetracycline and gentamicin resistance was the most common. We found the highest tetracycline resistance levels (91%) in isolates from poultry samples. The isolates from the poultry samples also displayed multidrug resistance to all antibiotics tested. The most common vancomycin-resistant species in poultry and milk was E. faecalis. In contrast, the pork samples contained vancomycin-resistant E. faecium isolates. It is interesting to note that vancomycin resistance in the pork and poultry samples was found only in combination with either four (28%) or all five (14%) of the tested antibiotics. Our results suggest that raw products of animal origin are possible reservoirs of multi-antibiotic resistant enterococci in the food chain.
... Occurrence of Listeria sp in about 80% of 45 retailed sausage samples (from three types) indicated the absence of hygienic conditions during manufacturing and absence of exacting quality control in many factories. These results are in the same trend of Metaxopoulos et al. (2001). On the other hand, fermented sausage had the lowest percentage of Listeria sp. ...
Forty five of retailed sausage samples (including fresh, frozen and fermented sausage) were collected from local and central markets of Qalubia, Cairo and Giza Governorates and analyzed for some microbial groups enumeration, pathogens detection and chemical characteristics. Data showed that, all sausage types contained coliform microorganisms where frozen sausage had coliform counts over the permissible limits followed by fresh and fermented sausage. On the other hand, fermented sausage had the highest records of lactic acid bacteria, proteolytic and lipolytic bacteria. All samples were positive for presence of Staph. aureus especially fresh sausage which recorded the highest mean counts. Listeria monocytogenes, E. coli O157:H7 and Salmonella spp. were also presented at higher percentage in fresh sausage followed by frozen and fermented ones. Concerning chemical analysis, frozen sausage had the highest values of sodium nitrite and total volatile nitrogen while, the highest mean values of thiobarbaturic acid was observed in fermented one. Furthermore, antimicrobial activity of eight essential oils of spices and two strains of lactic acid bacteria against four strains of foodborne pathogens isolated and identified from the previous three types of sausage was studied. Maximum mean values of inhibition zones of spices against the tested pathogens were obtained by marjoram followed by cumin and mint essential oils. In addition, all tested pathogens were inhibited by either Lactococcus lactis or Lactobacillus plantarum and their mixture which gave the highest mean values of inhibition zones.
... Samples were also tested for pathogenic bacteria using selective media Bacillus cereus agar base (M833, HiMedia, India) for B. cereus, violet red bile glucose agar w/o lactose (M581, HiMedia, India) for enterobacteriaceae (Han, Beumer, Rombouts, & Nout, 2001). Salmonella-Shigella agar (M108, HiMedia, India) was used for the detection of Salmonella and Shigella and Listeria identification agar base (M1064, HiMedia, India) with Listeria selective supplement (FD 061, HiMedia, India) for Listeria in the samples following the standard method of Metaxopoulos, Samelis, & Papadelli (2001). ...
Ethnic people of the Himalayan regions of India, Nepal, Bhutan and China (Tibet) prepare various types of indigenous meat products using their traditional knowledge. Some of these ethnic meat products such as sausages and dried or smoked meats are sold in local markets contributing to local economy. There is no literature on traditional processing of indigenous meat products of the Himalayas. The paper is aimed to document the traditional knowledge of the ethnic Himalayan people on preparation of various traditionally processed sausages and meat products such as kargyong, kheuri, satchu, suka ko masu, chilu, chartayshya, gemma and arjia.
... The results of the analysis indicated possible failures in processing by detection of aerobic mesophilic, Enterobacteriaceae and Salmonella spp. Similar reports of aerobic mesophilic bacteria and Enterobacteriaceae in sausage have been documented (Metaxopoulos et al. 2001;Fontán et al. 2007a, b). ...
The production of fresh pork sausages inoculated with different multi-resistant bacterial strains, isolated from sausage commercially purchased from Brazil, was conducted to evaluate microbial inhibition by lactic acid. The isolates were identified through 16S rDNA region sequencing and the bacteria with antimicrobial multi-resistance profiles were selected. The minimum bactericidal concentration was determined using lactic and acetic acids. Samples were contaminated by Salmonella Typhi, Salmonella Typhimurium, Salmonella Paratyphi, Escherichia coli and high counts of aerobic mesophilic bacteria. Antibiotic resistance was detected in 21 of the 27 analyzed isolates (six Salmonella spp. and 21 E. coli). In the minimum bactericidal concentration test, lactic acid was more effective than acetic acid in the reduction of the bacterial population. The food matrix inoculated with two bacterial strains (E. coli, Salmonella spp.) was treated with lactic acid at the concentrations of 1.5 and 4.0 M and showed no significant effect on the bacterial population.
Fresh sausages are potential vehicles for pathogenic microorganisms, which are frequently associated with foodborne diseases. Organic acids have been used to improve the microbiological quality of food products including meat. Bacteria were isolated from fresh pork sausages and antibiotic resistance and susceptibility of lactic and acetic acids in vitro of the identified strains were tested. The best results were used to test the functionality in fresh pork sausages inoculated with Escherichia coli and Salmonella spp. These results may be applied by local producers of fresh pork sausages.
... This kind of product is characterized by low acidity and the final pH is of about 5.3 to 6.2 (Aymerich, Martín, Garriga, & Hugas, 2003 ). Greece, Italy and Spain are the countries where more efforts have been dedicated to the study of this traditional product (Comi, Citterio, Manzano, & Cantoni, 1992; Coppola, Giagnacovo, Iorizzo, & Grazia, 1998; García-Varona, Santos, Jaime, & Rovira, 2000; Garriga et al., 1996; Hugas et al., 1993; Metaxopoulos, Samelis, & Papadelli, 2001; Parente, Griego, & Crudele, 2001; Rodríguez et al., 1994; Samelis, Maurogenakis, & Metaxopoulos, 1994; Samelis, Metaxopoulos, Vlassi, & Pappa, 1998; Santos, González-Fernández, Jaime, & Rovira, 1998 ). Moreover , molecular techniques have been applied either to directly profile the main populations involved in the production of Italian fermented sausages (Cocolin, Manzano, Aggio, et al., 2001) or to identify isolated LAB and nonpathogenic staphylococci from artisanal low-acid sausages produced in Spain (Aymerich et al., 2003). ...
In the Friuli Venezia Giulia region, in the North East of Italy, a traditional fermented sausage is produced without the use of microbial starters. It is characterized at the end of the ripening period by accentuated acidity, slight sourness and elastic, semi-hard consistency. In this study, three fermentations, carried out in different seasons (winter, spring and summer) were followed analyzing the microbiological, physicochemical and sensory aspects of this product. The sausages were characterized by an important microbial activity of lactic acid bacteria and micro/staphylococci that resulted in a product with a final pH of about 5.6-5.7. An interesting aspect was the high number of fecal enterococci that can play an important role in the definition of the organoleptic profile of the final product. No Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus were ever isolated from the raw materials or the fermented sausages during the maturation, underlining the safety of this product. The final water activity of the product was 0.91-0.92. One hundred and fifty lactic acid bacteria were isolated and identified by molecular methods to understand which species were more predominant in the product. Lactobacillus curvatus and Lactobacillus sakei were the most numerous (54 and 64 strains isolated, respectively) and they were the only species common to all three fermentations. A cluster analysis of the profiles obtained from these strains after RAPD-PCR highlighted a population distribution that was fermentation-specific.
... Colonies of all microorganisms were selected randomly and purity of the isolates was checked by streaking again on fresh agar plates of the isolation media and sub-culturing in the corresponding broths, followed by microscopic examinations. Isolates were preserved in their respective isolation media supplemented with 15% (v/v) glycerol, at À20 C. Samples were also tested for the presence of pathogenic bacteria using Bacillus cereus agar base (M833, HiMedia,) for B. cereus; Violet Red Bile Glucose agar without lactose (M581, HiMedia) for Enterobacteriaceae (Han et al., 2001); Salmonella-Shigella Agar (M108, HiMedia) for the detection of Salmonella; and Shigella, and Listeria identification agar base (M1064, HiMedia) with Listeria selective supplement (FD 061, HiMedia) for Listeria following the standard method of Metaxopoulos et al. (2001). ...
We used culture- and molecular-biology-based methods to investigate the diversity of lactic acid bacteria (LAB) in the ethnic chevon (goat) meat products chartayshya, jamma and arjia of the Western Himalayas. In six chartayshya, six jamma and four arjia samples, LAB were the predominant microbial component involved in the fermentation of these samples, and the total LAB population in arjia (7.8 ± 0.1 log cfu g(-1); mean ± SD) was significantly higher (P < 0.05) than in chartayshya (6.9 ± 0.1 log cfu g(-1)) and jamma (7.5 ± 0.1 log cfu g(-1)). We identified 53 LAB samples by 16S rRNA and phenylalanyl-tRNA synthase (pheS) genes sequencing. The LAB isolates were identified as Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Leuconostoc citreum, Leuconostoc mesenteroides, Pediococcus pentosaceus, and Weissella cibaria. These results revealed that there is a high level of diversity of LAB in the Himalayan ethnic preserved meat products.
... Colonies of all microorganisms were selected randomly and purity of the isolates was checked by streaking again on fresh agar plates of the isolation media and sub-culturing in the corresponding broths, followed by microscopic examinations. Isolates were preserved in their respective isolation media supplemented with 15% (v/v) glycerol, at À20 C. Samples were also tested for the presence of pathogenic bacteria using Bacillus cereus agar base (M833, HiMedia,) for B. cereus; Violet Red Bile Glucose agar without lactose (M581, HiMedia) for Enterobacteriaceae (Han et al., 2001); Salmonella-Shigella Agar (M108, HiMedia) for the detection of Salmonella; and Shigella, and Listeria identification agar base (M1064, HiMedia) with Listeria selective supplement (FD 061, HiMedia) for Listeria following the standard method of Metaxopoulos et al. (2001). ...
Native microorganisms from some ethnic meat products of the Eastern Himalayas such as lang kargyong, yak kargyong, faak kargyong, lang satchu, yak satchu and suka ko masu were isolated and characterized. The bacterial isolates included Lactobacillus sake, Lactobacillus curvatus, Lactobacillus divergens, Lactobacillus carnis, Lactobacillus sanfrancisco, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus brevis, Enterococcus faecium, Leuconostoc mesenteroides, Pediococcus pentosaceous, Bacillus subtilis, Bacillus mycoides, Bacillus thuringiensis, Bacillus lentus and Bacillus licheniformis, Micrococcus and Staphylococcus. Yeast isolates included Debaryomyces hansenii, Debaryomyces polymorphus, Debaryomyces pseudopolymorphus, Pichia burtonii, Pichia anomala, Candida famata and the mould Rhizopus was also identified. Many of the LAB isolates demonstrated some antimicrobial activity, enzymatic activity and a few showed a high degree of hydrophobicity. None of the strains produced biogenic amines.
... During fermentation, the contaminating enterococci may survive and multiply. In natural fermented sausages, Metaxopoulos et al. (2001) found 10 2-10 3 cfu g À 1 of enterococci contaminating the sausage batter in three different manufacturing plants in Greece. In two out of the three plants, enterococci grew during fermentation and ripening to 10 4 cfu g À 1 after 28 days of processing while in the third plant no growth was observed during the first week and no recovery was possible until after the end of the process. ...
The presence of enterococci in meat fermentation is a constant as reported in the literature. Despite the concern about pathogenicity of enterococci, recent studies point out that food and meat enterococci, especially Enterococcus faecium have a much lower pathogenicity potential than clinical strains. Enterococci possess a competitive advantage over other microbiota in meat fermentations, and many enterococci isolated from sausages have the ability to produce enterocins harbouring antimicrobial activity against pathogens and spoilage microorganisms of meat concern. The application of enterocins producing enterococci or their purified metabolites, as extra hurdles for preservation in sausage fermentation and in sliced-vacuum packed cooked meat products can be beneficial, preventing the outgrowth of Listeria monocytogenes and slime-producing lactic acid bacteria. Enterocins and bacteriocinogenic enterococci hold considerable promise as alternatives to traditional chemical preservatives and they could be exploited for the control of emergent pathogens in meat products. Their inhibitory effect can be increased when used in conjunction with particular physical and chemical processes, but current regulation is hampering the application of purified bacteriocins.
... In our study, the formulation of the sausages mix as well as the technology applied for their manufacture did not differ from the ones already described by Metaxopoulos et al. (2001), Papamanoli et al. (2002) and Drosinos et al. (2005a) and thus the absence of significant differences in the physico-chemical and microbiological characteristics is rightfully justified. Table 4 Physiological and biochemical tests used for the identification of staphylococci isolated from naturally fermented sausages of southern Greece 19 (50) Percentage of positive strains is given in parenthesis. ...
The physicochemical and microbiological characteristics of spontaneously fermented sausages made by two medium-sized enterprises (MSE) located in southern Greece have been studied. A total of 300 lactic acid bacteria and 300 staphylococcal strains have been isolated and identified by their physiological characteristics. Lactobacillus plantarum strains were found to dominate the lactic acid bacteria microbiota in most of the cases with L. sakei strains prevailing in some of them and L. rhamnosus strains occasionally accompanying the dominant lactic acid bacteria microbiota. On the other hand, S. saprophyticus strains were found to dominate the staphylococcal microbiota in all spontaneously fermented sausages with of S. simulans, S. xylosus, S. gallinarum and S. cohnii cohnii strains being sporadically present. Following the identification, an evaluation of their technological properties, namely proteolytic and lipolytic capacities as well as production of biogenic amines and antimicrobial compounds, took place. None of the lactic acid bacteria and staphylococci was found to possess lipolytic activity whereas a total of 6 lactic acid bacteria and 51 staphylococci strains were found to be able to hydrolyse either the sarcoplasic, myofibrillar or both protein fractions. Furthermore, only one L. sakei strain and 185 staphylococci strains were found to possess decarboxylase activity against lysine, tyrosine, ornithine or histidine. Finally none of the staphylococcal microbiota and 3 lactic acid bacteria strains were found to be able to produce antimicrobial compounds of proteinaceous nature against Listeria monocytogenes.
Recently, during the ripening of goose sausage, a defect consisting of ammonia and vinegar smell was noticed. The producer of the craft facility, located in Lombardia, a Northern region of Italy, asked us to identify the cause of that defect. Therefore, this study aimed to identify the potential responsible agents for the spoilage of this lot of goose sausages. Spoilage was first detected by sensory analysis using the “needle probing” technique; however, the spoiled sausages were not marketable due to the high ammonia and vinegar smell. The added starter culture did not limit or inhibit the spoilage microorganisms, which were represented by Levilactobacillus brevis, the predominant species, and by Enterococcus faecalis and E. faecium. These microorganisms grew during ripening and produced a large amount of biogenic amines, which could represent a risk for consumers. Furthermore, Lev. brevis, being a heterofermentative lactic acid bacteria (LAB), also produced ethanol, acetic acid, and a variation in the sausage colour. The production of biogenic amines was confirmed in vitro. Furthermore, as observed in a previous study, the second cause of spoilage can be attributed to moulds which grew during ripening; both the isolated strains, Penicillium nalgiovense, added as a starter culture, and P. lanosocoeruleum, present as an environmental contaminant, grew between the meat and casing, producing a large amount of total volatile nitrogen, responsible for the ammonia smell perceived in the ripening area and in the sausages. This is the first description of Levilactobacillus brevis predominance in spoiled goose sausage.
The aim of this work was to investigate the microorganisms and their metabolites responsible for the spoilage of goose sausages produced in a small facility in Lombardy, a northern region of Italy. Spoilage was first detected by sensorial analysis using the “needle probing” technique; however, the spoiled sausages were not marketable due to the high ammonia and vinegar smell. The added starter culture did not limit or inhibit the spoilage microorganisms, which were represented by Levilactobacillus brevis, the predominant species, and by Enterococcus faecalis and E. faecium. These microorganisms grew during ripening and produced a large amount of biogenic amines, which could represent a risk for consumers. Furthermore, Lev. brevis, being a heterofermentant, also produced ethanol, acetic acid and a variation in the sausage colour. The production of biogenic amines was confirmed in vitro. Furthermore, as observed in a previous work, a second cause of spoilage can be attributed to moulds that grew during ripening; both the isolated strains, Penicillium nalgiovense, added as starter culture, and P. lanoso-coeruleum, present as environmental contaminant, grew between the meat and casing, producing a large amount of total volatile nitrogen, responsible for the ammonia smell perceived either in the ripening area or in the sausages. This is the first description of Levilactobacillus brevis predominance in spoiled goose sausage.
This study investigated the effect of gamma irradiation on shelf life of smoked guinea fowl meat stored at refrigerated temperature. Dressed guinea fowl meats were smoked, packaged and gamma irradiated at 2.5, 5.0 and 7.5 kGy with a dose rate of 0.74 kGy h⁻¹ and compared with a control (un-irradiated). Physicochemical, sensory and microbial properties of the treated meats were determined over refrigerated storage period using appropriate procedures. Titratable acidity (TA) and acid value (AV) decreased significantly (p ≤ 0.05) with gamma irradiation, but increased with storage. pH of the meat samples were however within the neutral range which was numerically insignificant to affect quality characteristics of the processed meat. Bacterial isolates of natural microbiota identified on the basis of their mass spectra of protein profiles on the smoked guinea fowl meat were Staphylococcus aureus, Serratia marcescens and Enterobacter cloacae. The total bacterial counts (7.23 log10 cfu/g) decreased with increasing doses of gamma irradiation and storage (1.29 log10 cfu/g). Irradiation had highly significant effects (p ≤ 0.05) on the reduction of microbial population. Irradiation had no significant effects (p > 0.05) on the sensory attributes (aroma, colour, tenderness and taste) of the smoked guinea fowl meat; but the taste of the irradiated meat samples was influenced at the end of the seven weeks refrigerated storage period. Gamma irradiation has a potential to enhance shelf life of smoked guinea fowl meat at a refrigeration condition.
Forty five of retailed sausage samples (including fresh, frozen and fermented sausage) were collected from local and central markets of Qalubia, Cairo and Giza Governorates and analyzed for some microbial groups enumeration, pathogens detection and chemical characteristics. Data showed that, all sausage types contained coliform microorganisms where frozen sausage had coliform counts over the permissible limits followed by fresh and fermented sausage. On the other hand, fermented sausage had the highest records of lactic acid bacteria, proteolytic and lipolytic bacteria. All samples were positive for presence of Staph. aureus especially fresh sausage which recorded the highest mean counts. Listeria monocytogenes, E. coli O157:H7 and Salmonella spp. were also presented at higher percentage in fresh sausage followed by frozen and fermented ones. Concerning chemical analysis, frozen sausage had the highest values of sodium nitrite and total volatile nitrogen while, the highest mean values of thiobarbaturic acid was observed in fermented one. Furthermore, antimicrobial activity of eight essential oils of spices and two strains of lactic acid bacteria against four strains of foodborne pathogens isolated and identified from the previous three types of sausage was studied. Maximum mean values of inhibition zones of spices against the tested pathogens were obtained by marjoram followed by cumin and mint essential oils. In addition, all tested pathogens were inhibited by either Lactococcus lactis or Lactobacillus plantarum and their mixture which gave the highest mean values of inhibition zones.
Fresh pork has a varied microflora depending on the sources of contamination. To avoid spoilage and to increase the shelf life, the food technology offers different preservation procedures. Because of the distinct features of the environment after different preservation methods are used and the differing sensitivities of the distinct microbial groups to these preservation systems, the microbial species present and their proportions vary notably in the different pork products. In this chapter, the microbial ecology of pork and various pork products is summarized.
The fermentation of sausages is a complex process involving different species of lactic acid bacteria, coagulase-negative cocci, and yeasts. The molecular techniques developed to date allow for a fast and precise identification of strains; the use of culture-independent methods applied at the DNA and RNA level permits problems related to traditional isolation to be overcome and, together with culture-dependent methods, allows for the definition of microbial dynamics during fermentation. Furthermore, the application of techniques for the characterization of isolates allows the study of intraspecies biodiversity. In this light, this Chapter aims to describe the findings of the application of molecular methods in order to provide a better comprehension of the ecology of sausage fermentation.
The aim of the paper was to find the origin of odorous compounds in traditional Friuli sausages produced by a small factory in the Udine-San Daniele area. The sensorial alteration (mephitic type) of dry salami was due to the pork meat with a pH over 6.2 (DFD - Dark, Firm, Dry type) and to an anomalous fermentation of Lactic acid bacteria/enterococci, CNC (Coagulase negative cocci), and enterobacteriaceae. These bacteria have produced hydrogen sulphur and metanthiol, which are responsible for the mephitic aroma of the sausages.
Probiotic food products are a fast growing area. Although probiotic strains are currently used in dairy products, their commercial application in fermented meat products is not yet common. The aim of this study was to assess the competitiveness of two probiotic Lactobacillus strains (Lactobacillus plantarum 299V and Lactobacillus rhamnosus GG) during the manufacture of Spanish fermented sausages and their effect on the hygienic and sensory qualities of the final products. The inoculated strains were successfully monitored by Randomly Amplified Polymorphic DNA (RAPD)-PCR. Both strains prevented the growth of Enterobacteriaceae throughout the entire ripening process. L. rhamnosus GG and L plantarum 299V at high inoculum (ca. 10(7) CFU/g) produced a sharp decrease of pH values and low growth of Gram-positive Catalase-positive Cocci (GCC+), leading to a negative effect on the sensory attributes evaluated. Nevertheless, L plantarum 299V inoculated at 10(5) CFU/g achieved and maintained high counts until the end of ripening and storage (ca.10(8) CFU/g), co-dominating (60%) with the endogenous microbiota, producing functional sausages with a satisfactory overall sensory quality. No major differences in physico-chemical parameters or sensory attributes were recorded when compared to spontaneously fermented sausages, thus adding further value to this type of meat product as a probiotic vehicle.
We have performed a transcriptomic in vivo study with Enterococcus faecalis OG1RF in the intestine of living mice to identify novel latent and adaptive fitness determinants within E. faecalis. From 2,658 genes that are present in E. faecalis strain OG1RF, 124 genes were identified as significantly differentially expressed within the intestinal tract of living mice as compared to exponential growth in BHI broth. The groups of significantly up- or down-regulated genes consisted of 94 and 30 genes, respectively, for which 46 and 18 a clear annotation to a functionally described protein was found. These included genes involved in energy metabolism (e.g., dhaK and glpK pathway), transport and binding mechanisms (e.g., phosphoenolpyruvate carbohydrate PTS) as well as fatty acid metabolism (fab genes). The novel putative fitness determinants found in this work may be helpful for future studies of E. faecalis adaptation to the intestinal tract, which is also a prerequisite for infection in a compromised or inflamed host.
Ekung, eup and hirring are some common indigenous fermented bamboo products of Northeast India. We have isolated, characterized, and identified the predominant lactic acid bacteria (LAB) from 44 samples of ekung, eup, and hirring and studied their technological properties. The phenotypic characterizations of LAB isolates were based on physiological, biochemical tests and API kits, and were identified as Lactobacillus plantarum, L. brevis, L. casei, L. fermentum, Lactococcus lactis, and Tetragenococcus halophilus. Technological properties of LAB such as acidifying capacity, antimicrobial activities, degradation of phytic acid and oligosaccharides, bile-salt tolerance, enzymatic activities, biogenic amines production, and degree of hydrophobicity also were studied. This study showed that strains of LAB played important roles by their functional properties related to acidifying capacity, degradation of antinutritive factors, tolerance to bile-salt, wide enzymatic activities, and nonproducers of biogenic amines. Understanding the biological and biochemical basis of indigenous knowledge of the ethnic people of Northeast India for production of nonperishable bamboo shoots by lactic acid fermentation has merit. It helps to develop both low-cost functional foods, and understand the functionality of microbial diversity. Some of the LAB strains possess functional properties, which render them interesting candidates for use as LAB starter cultures.
Enterococcus (E.) faecalis is found as commensal in healthy humans, in a variety of fermented foods. It can serve as probiotic but also as pathogen causing endocarditis, bacteremia and urinary tract infections. We have employed a proteomic study with E. faecalis strain OG1RF under different growth conditions and in contact to mouse intestinal cells to identify novel latent and adaptive fitness determinants. These relate to changes in catabolic pathways (BudA), protein biosynthesis (AsnS), cellular surface biosynthesis (RmlA) and regulatory mechanisms (OmpR). This knowledge can be used to derive novel evidence-based targets, which can be used to further elucidate gene expression changes enhancing pathogenicity or fitness in a commensal strain and possibly delineate this species into groups of higher and lower risk for applications in a food or a medical context versus improved treatment strategies of the so far hard to cure diseases.
Selroti is an ethnic fermented rice food of the Himalayas. A total of 125 samples of selroti batters were collected from different villages and markets of the Himalayas. The microbial population of selroti batters showed that lactic acid bacteria (LAB) were present in viable numbers above 108 cfu/g, followed by yeasts at 105 cfu/g. LAB Leuconostoc mesenteroides, Enterococcus faecium, Pediococcus pentosaceus and Lactobacillus curvatus and yeasts Saccharomyces cerevisiae, Saccharomyces kluyveri, Debaryomyces hansenii, Pichia burtonii, and Zygosaccharomyces rouxii were identified. The most prevalent LAB and yeasts in selroti batters were Leuc. mesenteroids (42.9%) and S. cerevisiae (35.6%). Molds and pathogenic bacteria were not detected. It was observed that seasons affect the development and prevalence of microorganisms in the fermented batters. LAB and yeast strains were screened for their acidifying and coagulating capacity, and it was found that most of the LAB strains acidified with lowering of pH up to 4.3. These strains showed a wide spectrum of enzymatic profiles in commercial API-zym kits. All strains of LAB showed antimicrobial activities under the applied condition. The nutritional value of fermented batters was found to be increased. This is the first report on selroti concerning its microbiology and nutritional value.
The main objectives of fermentation and ripening are to achieve a desired and characteristic color and flavor, to develop firmness, and to inhibit pathogenic and spoilage bacteria. The intrinsic factors of the sausage mix and the final water activity and pH exert a natural selectivity for the development of desired salt-tolerant microflora. While a few species seem to be able to dominate at the end of sausage ripening, biodiversity at the strain level inside the predominant species has mainly been reported to exploit the potential of molecular methods. Although typical preparations based on indigenous microflora still exist, the use of starter cultures is increasing, as these guarantee safety, standardization of product properties, and a shorter ripening period. Relevant safety aspects such as bacteriocin production, absence of amino acid-decarboxylase activity, and a nontoxicogenic and nontransferable antibiotic resistance profile are important to the evaluation of potential strains.
The effects of sage extract, applied alone or combined with sodium isoascorbate, on water activity (Aw), pH and microbiological and sensory qualities of turkey meatballs were studied. The samples were vacuum packed and stored at 3C for 15 days. It was found that the additives had a positive effect on the quality of turkey meatballs. Aw and pH values of samples with mixture extract of sage and sodium isoascorbate were lower than those of control samples. Sage extract combined with sodium isoascorbate reduced the counts of mesophilic bacteria and coliforms in raw meatballs more effectively than sage extract applied alone. During cold storage, both sage extract and sage extract with sodium isoascorbate mixture inhibited the growth of coliforms and sulfate-reducing Clostridium sp. The products with additives were characterized by a better sensory quality than the control samples. Samples with mixture of sage extract and sodium isoascorbate showed a higher overall desirability because of lower intensity of taste and aroma of spices.
Microbiological and sensory changes are the main causes of quality decrease of cooked poultry products during cold storage. The addition of sage extract and mixture of sage extract and sodium isoascorbate inhibited the growth of coliforms and sulfate-reducing Clostridium species in turkey meatballs. The samples with both additives had a better sensory quality compared to samples without additives. According to my results, sage extract alone or in combination with sodium isoascorbate could be used to extend the shelf life of turkey meat products during refrigerate storage.
The natural fermentation of Sausages is a complex microbial process in which the main participants are represented by lactic
acid bacteria (LAB) and coagulase-negative cocci (CNC). The microflora of different types of fermented Sausages has been defined
by using traditional microbiological methods based on isolation and biochemical identification. Since it appears that the
types of microbial groups, or even the specific strains of a given microbial group, that dominate the fermentation, significantly
affect the organoleptic profile of the final product, there is an increasing interest in the description of the microbiota
that are found in different fermented Sausages. Recently new tools, based on molecular methods, allowing fast and unequivocal
identification of strains, isolated from fermented Sausages, became available. These methods have been successfully applied
and, in general, biochemical and molecular identification compared well. However, new information comes to light when molecular
methods are applied to DNA and/or RNA extracted directly from Sausages. This approach eliminates problems related to traditional
isolation. This chapter deals with the recent findings and results of the application of molecular methods, in a culture-dependent
and culture-independent manner, on the study of the microflora of fermented Sausages.
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.
In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent
and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well
as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant
counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection
limit (>100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns
and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The
bacterial ecology was mainly characterized by the stable presence of Lactobacillus curvatus and Lactobacillus sakei, but Lactobacillus paracasei was also repeatedly detected. An important piece of evidence was the presence of Lactococcus garvieae, which clearly contributed in two fermentations. Several species of Staphylococcus were also detected. Regarding other bacterial groups, Bacillus sp., Ruminococcus sp., and Macrococcus caseolyticus were also identified at the beginning of the transformations. In addition, yeast species belonging to Debaryomyces hansenii, several Candida species, and Willopsis saturnus were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness
of the fermentation processes studied.
Coagulase-negative cocci (CNC) ecology in naturally fermented sausages from Friuli Venezia Giulia region, in the North East of Italy, was investigated. A total of 617 CNC strains, isolated from three different plants during the fermentation process, were identified by traditional methods (biochemical tests) and molecular methods based on species specific PCR, PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and sequencing of the V3 region of the 16S rRNA gene. The identification, by using biochemical tests, was not successful for 130 strains. Moreover, incongruent results were observed comparing the traditional with the molecular identifications. The same species of CNC were found in all three processing plants, but their contribution to the fermentations was different. In two plants Staphylococcus xylosus was the main species involved in fermentation process, while in the third the maturation was carried out equally by three species: S. xylosus, Staphylococcus warneri and Staphylococcus pasteuri.
Listeria monocytogenes NCTC10527 was examined with respect to its nonthermal inactivation kinetics in fermented sausages from four European countries: Serbia-Montenegro, Hungary, Croatia, and Bosnia-Herzegovina. The goal was to quantify the effect of fermentation and ripening conditions on L. monocytogenes with the simultaneous presence or absence of bacteriocin-producing lactic acid bacteria (i.e., Lactobacillus sakei). Different models were used to fit the experimental data and to calculate the kinetic parameters. The best model was chosen based on statistical comparisons. The Baranyi model was selected because it fitted the data better in most (73%) of the cases. The results from the challenge experiments and the subsequent statistical analysis indicated that relative to the control condition the addition of L. sakei strains reduced the time required for a 4-log reduction of L. monocytogenes (t(4D)). In contrast, the addition of the bacteriocins mesenterocin Y and sakacin P decreased the t(4D) values for only the Serbian product. A case study for risk assessment also was conducted. The data of initial population and t(4D) collected from all countries were described by a single distribution function. Storage temperature, packaging method, pH, and water activity of the final products were used to calculate the inactivation of L. monocytogenes that might occur during storage of the final product (U.S. Department of Agriculture Pathogen Modeling Program version 7.0). Simulation results indicated that the addition of L. sakei strains significantly decreased the simulated L. monocytogenes concentration of ready-to-eat fermented sausages at the time of consumption.
Three strains of Staphylococcus aureus (S-6, 137 and 472) were inoculated, in duplicate, into Italian-style dry salami made with finished product as starter and processed under commercial manufacturing conditions. Five levels of S. aureus ranging from 2.2 × 102 - 1.8 × 107 cells/g were used. A fourth strain (264) was inoculated at a level of 105 cells/g. All strains of S. aureus grew at every level of inoculation, but the amount of growth was dependent on inoculum size. Strains S-6 and 472 increased in number by 1.2 - 2.9 logs (mean 2.14) at inoculum levels of 2.3 × 102 - 2.5 × 103 cells/g, and by 2.1 - 3.2 logs (mean 2.66) at inoculum levels of 3.7 × 104 - 6.6 × 105 cells/g. Strain 137 was very sensitive to salami environment and only increased by 0.47 - 1.86 logs (mean 1.23) even at the greatest inoculum level. Strain 264 increased in numbers by 1.5 logs in the presence of 5 × 105 inoculated lactobacilli/g and by 2.5 logs in the presence of 6 × 104 naturally occurring lactic acid bacteria. Staphylococci occurring naturally in salami mix were unable to grow to levels greater than 2 × 104 cells at any time during processing of experimental sausages. Thermonuclease was detected only in salamis inoculated with strains S-6 and 472 at initial levels of greater than 3.7 × 104 cells/g and only when growth reached levels greater than 107 cells/g. No enterotoxin was detected in any of the inoculated samples. Development of regression equations allowed description of the growth of inoculated S. aureus in the salami during manufacturing as affected by a number of variables.
The hydrolytic changes in the lipid fraction and the changes in microbial growth during the natural fermentation and ripening of Greek dry sausages were studied. Two batches were manufactured under industrial conditions, without the addition of starter cultures. The results of the microbiological analyses indicated that, among the micro-organisms well known for their lipolytic activity, micrococci were predominant during the whole fermentation-ripening process. It was shown that the fatty acid composition of the hydrolysed lipid fraction did not remain constant with time. Analysis of the experimental results indicated that, given the length of the aliphatic chain (16 or 18 carbon atoms), the more unsaturated fatty acids were hydrolysed in preference to the saturated homologues. On the other hand, fatty acids having 16 carbon atoms were hydrolysed in preference to the iso-unsaturated ones having 18 carbon atoms.
In recent years a number of food poisoning episodes due to the consumption of fermented meat product were reported by the National Center for Disease Control (NCDC). These episodes, though small in number, took the fermented meat industry by surprise, inasmuch as safety problems connected with such products had not been reported for years. A critical look in this area of meat technology indicated that some of the production was based on tradition rather than science. The making of many fermented meats has remained for long an art, with 'trade secrets' in the industry handed down from generation to generation and processing methods guarded jealously. As a result, knowledge of microbiologic and chemical changes taking place during the fermentation and knowledge of the impact of the overall manufacturing practices on the safety of some fermented meats remained limited. The purpose of this report is to review the current state of knowledge in these areas and to recommend actions for improvement.
The present paper examined the presence of Listeria spp. in the environment of domestic, retail and industrial refrigerators. From 136 household refrigerators, 136 surface samples were taken from the walls or shelves, and 125 from cheese compartments. Only two refrigerators harboured L. monocytogenes. From 228 food store refrigerators, 335 samples were taken. Of these, 118 were in in contact with cheeses, 69 with sausages, 21 with cheese and sausages, 20 with miscellaneous products and 107 from refrigerator handles. Listeria spp. and L. monocytogenes were found in 3.1% and 1.7%, of the samples respectively. Listeria spp. was not detected in any of the nine dairy plant refrigerators examined. Listeria monocytogenes and L. innocua were found in 4.5 and 36.4%, respectively, of the 22 refrigerators inside meat processing plants, with only one of 22 refrigerators handles being positive for L. monocytogenes. Temperature distribution in the refrigerators was also investigated. Fifty five per cent of the 136 domestic and 32% of the 228 retail store refrigerators had temperatures of greater than or equal to 9 degrees C. The range of refrigeration temperatures of the industrial refrigerators was 0-2 degrees C for meat plants and 2-7 degrees C for dairy plants. No correlation of any kind could be established between the prevalence of Listeria spp. and the temperature of the various refrigerators due to the low number of positive samples.
Dichloran 18% glycerol (DG18) agar was originally developed to enumerate xerophilic foodborne moulds. However, some laboratories are using DG18 agar as a general medium to enumerate foodborne moulds and yeasts. A collaborative study, with the participation of seven laboratories, was undertaken to compare DG18 agar with dichloran rose bengal chloramphenicol (DRBC) agar, tryptone glucose yeast extract chloramphenicol (TGYC) agar, and plate count agar supplemented with chloramphenicol (PCAC) for enumerating 14 species of common food spoilage yeasts. Comparison of the mean values of populations of all yeasts recovered on each medium revealed no significant differences among DRBC agar, PCAC, and TGYC agar, while each of these media supported the development of significantly (P < or = 0.05) higher numbers of colonies than DG18 agar. However, differences were only 0.08 to 0.10 log10 cfu/ml, making the practical significance questionable. The overall coefficient of variation (CV) for within laboratory repeatability was 1.71%, while the CV for reproducibility of counts obtained among laboratories was 6.96%. Compared to DRBC agar, TGYC agar, and PCAC, yeast colonies were smaller on DG18 agar. Growth of Brettanomyces anomalus, Cryptococcus albidus, and Rhodotorula mucilaginosa was particularly retarded or inhibited on DG18 agar. Based on the performance of media in supporting colony development and ease of counting colonies, the use of DG18 agar as a general enumeration medium for foodborne yeasts cannot be recommended.
The microbiological changes in relation to changes in pH, moisture, salt and nitrite content of four batches of Creek country-style sausage during manufacture followed by storage at 3 degrees and 12 degrees C in air were studied. Results showed that certain processing stages of the traditional manufacturing method, such as aging of the meat and drying of the sausages by ventilation followed by smoking at 25 degrees-40 degrees C, favoured lactic acid bacteria (LAB) to exceed 10(8) cfu/g and acidify the product. However, the growth rate of LAB and, accordingly, the acidification rate varied significantly among batches due to differences in the initial LAB numbers and possible variations in the prevalent types of LAB. In samples where LAB failed to predominate during manufacture, Gram-negative bacteria increased during storage at 12 degrees C, but not at 3 degrees C. Staphylococci grew to a level of 10(6) cfu/g in samples stored at 12 degrees C, however, Staphylococcus aureus remained below 10(3) cfu/g in all samples. In most samples, yeasts increased to a level of 10(7) cfu/g with prolonged storage and caused an increase in pH, mainly at 12 degrees C. Yeasts formed of odours associated with spoilage of sausages. The shelf-life of samples stored at 3 degrees C was at least 6 weeks. At 12 degrees C samples spoiled in less than 2 weeks, as spoilage was accelerated by surface-growing moulds. It was concluded that chilling increased the shelf-life of country-style sausage stored in air at least four-fold.
The fate of Listeria monocytogenes was followed during the manufacture of uncooked German, American and Italian-style sausages. In the German-style sausages prepared with starter culture, levels of L. monocytogenes decreased about 2- to 3-logs after the fermentation and smoking, and a further 1- to 2-logs during the drying period. Similar results were observed with the starter culture prepared American-style sausages, where close to a 5-log reduction in levels of L. monocytogenes was observed after fermentation and smoking.With the Italian-style sausages not produced with starter culture, numbers of L. monocytogenes increased slightly during the fermentation period, remained constant in number during drying, and then decreased slightly during a 4 week holding period at 4°C.Sausages prepared with starter culture under good manufacturing practices should present little or no risk to the consumer. However, sausages made without starter culture could posssibly pose some risk to the consumer. With these products, extra attention should be paid to good manufacturing practices and additional safety measures or 'hurdles' should be considered to provide an extra measure of safety.
The microbial flora associated with the natural fermentation and ripening of five batches of Greek dry salami was enumerated and characterized over time. Micrococci-staphylococci increased at a level of 107 cfu g-1 during early fermentation. By day 4, lactic acid bacteria outnumbered Micrococcaceae in all batches, since they exceeded — with the exception of batch III — 108 cfu g-1. Yeasts remained below 106 cfu g-1 during the whole process, but tended to increase at a late ripening stage. Almost all colonies grown on mannitol salt agar were catalase positive cocci. Nitrate-reducing micrococci were progressively replaced by less acid-sensitive staphylococci. A high proportion (62·5%) of 112 Staphylococcus isolates grew weakly under anaerobic conditions. Typical staphylococci were less capable of reducing nitrate. Yeast populations mainly comprised Debaryomyces strains. Characterization of 348 lactic isolates indicated that the salami microflora was dominated by homofermentative lactobacilli (61·4%) and leuconostoc-like bacteria (24·9%). Most lactobacilli (51·4%) belonged to the formerly-called `atypical' meat streptobacteria. Typical streptobacteria (10·0%), heterofermentative lactobacilli (10·0%) and homofermentative cocci (3·1%) were mainly isolated during the first days of fermentation and failed to compete with the other two groups. The microbial interactions as reflected by the distribution and succession of different general/subgenera in each batch and their possible effect on physicochemical and sensory characteristics of the sausages were discussed.
In an attempt to determine the composition and origin of the spoilage flora of refrigerated vacuum-packed cooked ham, the changes in microbial numbers and types were followed along the processing line. Results revealed Lactobacillus sake and Leuconostoc mesenteroides ssp. mesenteroides as the major causative agents of spoilage of sliced ham stored at 4 °C and 12 °C, due to recontamination in the cutting room. On the contrary, the progressive deterioration of whole ham under the same storage conditions was associated with a non-identifiable group of leuconostoc-like bacteria. Except for lactic acid bacteria, no other organism grew in vacuum packs of either sliced or whole ham. Although atypical leuconostocs could not be detected among isolates recovered from freshly produced whole ham, they appeared to survive cooking and proliferate during storage. Neither these organisms however, nor Lact. sake and Leuc. mesenteroides were important in curing and tumbling as carnobacteria, mainly Carnobacterium divergens, and Brochothrix thermosphacta dominated at this stage. A progressive inversion of the ham microflora from mostly Gram-negative at the beginning of processing to highly Gram-positive prior to cooking was noted. Listeria monocytogenes cross-contaminated ham during tumbling. However, the pathogen was always absent from the vacuum-packed product provided that heating to a core temperature of 70 °C occurred and recontamination during slicing and packing was prevented. The percentage distribution of different species of lactic acid bacteria as well as the uncommon phenotypic characteristics of some strains were discussed.
The occurrence and distribution of listeriae in a meat processing plant was studied to determine the major sources and routes of contamination. Listeria monocytogenes and other Listeria spp. were isolated from 51% and 49% of samples of frozen raw meat taken from several incoming lots. Turkey necks and breasts, pork trimmings and lard were the principal sources of initial contamination. As a consequence, listeriae colonized certain processing sites where raw materials were handled and hygienic conditions were not strict. Mainly tumbled meats were contaminated heavily during tumbling as the need to operate tumblers continuously did not enable their proper cleaning and disinfection on a daily basis. Also the use of mechanically deboned turkey-neck meat in cooked sausages raised contamination at a pre-cooking stage. Listeriae survived in tumbled meats cooked in boilers at core temperatures below 70°C, and in country-style sausages heated to 65–68°C. In contrast, listeriae were killed in oven-cooked tumbled meats and emulsion-type sausages heated to 72–75°C, and in fully ripened salamis. Heat survivors appeared to be the main cause of post-process contamination as spreading of listeriae in the cutting room was restricted to processing lines where precontaminated meat products were handled. The possible reasons leading to heat survival of listeriae and the measures taken to control the problem were discussed.
The main fermented meat products are fermented sausages in which lactic acid bacteria (LAB) are the essential agents of the ripening process. During indigenous fermentations Lactobacillus curvatus and L. sake are the dominating LAB. Their application as starter organisms ensures the dominance of the starter during the whole ripening process. The suppression of the competing fortuitous LAB depends on the quality of the raw materials and on technological factors. The physiological properties of lactic starters do not suffice to ensure a sensory quality which can be found in traditionally produced dry fermented sausages. Additional activities required are present in micrococci and yeasts which, therefore, are further components of starter culture preparations. Some strains of meat-borne lactobacilli exhibit the essential activities like nitrate reductase, nitrite reductase, catalase, lipase, and protease, respectively. To create the optimal starter cultures composed of lactobacilli, these activities have to be studied and optimized in strains of high competitiveness in the fermenting substrate.
This paper provides general information on the fermentation of meat, the types and manufacture of the resulting products, the microorganisms involved and the factors affecting microbial activity. Subsequently, recent developments in the following three main research fields are reviewed: (i) Instrumental control of meat fermentation: Sensors for continuous measurement of fermentation parameters such as pH, water activity and weight loss of fermenting meats have been developed, making the on-line control of the fermentation climate feasible. This could lead to a marked reduction in fermentation time and costs without affecting product quality. (ii) Selection of antagonistic lactic starter cultures: Such cultures, ideally, would kill rather than inhibit pathogens, not only in fermented meats sensu stricto but possibly also in non-fermented products such as sliced perishable meats. (iii) Role of microorganisms in flavour development: A better understanding of the effect of microorganisms (in particular Micrococcaceae and their ability to scavenge oxygen, destroy peroxides and hydrolyze lipids and proteins), meat enzymes and non-enzymic reactions on aroma and flavour of fermented meat is required to maintain a large diversity of fermented meats and to improve their sensory quality and shelf life.
Foods preserved by combined methods remain stable and safe even without refrigeration, and are high in sensory and nutritive properties due to the gentle processes applied. The concept is gaining ground in industrialized as well as in developing countries. Several topics will be discussed briefly: (1) water activity, (2) hurdle effect, (3) hurdle technology (4) shelf stable products, (5) intermediate moisture foods, and (6) perspectives.
Bacterial starter cultures, consisting of lactic acid bacteria (Lactobacillus, Pediococcus) and staphylococci (Staphlylococcus carnosus. S. saprophyticus, S. warneri), have an important effect on the pH, lactate, acetate and free fatty acid contents of sausages. Sausages made with L. sake had the lowest pH whereas no change of pH was noticed in the controls and in the sausages inoculated with P. acidilactici. Inoculation of S. saprophyticus led to sausages with a high acetate content. Lipolysis occurred not only in inoculated samples but also in the controls, but it was the highest in the sausages inoculated with S. warneri.
Sausages with and without Staphylococcus xylosus were manufactured with four replicates. Antibiotics and a fungicide to inhibit growth of naturally occuring microorganisms were added to the control sausages. The volatile compounds from the sausages were collected and identified by gas chromatography/mass spectrometry, the effluent evaluated by sniffing and the sausages assessed by a trained panel. The study showed that sausages fermented with Staphylococcus xylosus contained several fragrant esters that were not found in control sausages without microbial growth. Control sausages had an unpleasant, rancid odour compared to sausages with Staphylococcus xylosus, indicating that the esterase activity of Staphylococcus xylosus or other microorganisms is very important in order to obtain the proper fermented sausage aroma. Although sausages with Staphylococcus xylosus contained the highest amounts of free fatty acids, it seemed to be of no importance to the aroma development. It is therefore questionable whether lipolytic activity of starter cultures has any influence on sausage flavour.
Starter cultures applied in meat technology may contain moulds, yeasts and bacteria. Moulds have been selected with the primary intension to exclude any potential of mycotoxin production. Their sensory effects and the requirements of technology are also criteria for suitable starters. Their genetic potential can now be modified by genetic engineering. Yeasts are applied for sensory reasons mainly. The species in use are only fermentative and do not enhance nitrate reduction. Bacterial components of starters consist of micrococci, staphylococci, lactic acid bacteria (LAB) and, with minor importance, Streptomyces griseus or Aeromonas sp. For selection of the appropriate organisms and for ensuring optimum performance in the fermentation process, their technological, ecological, physiological and genetic properties should be well known. Most knowledge is available for LAB, which represent the most important group of starter organisms, since they are involve in the fermentation of all types of products and contribute to each single aim of the fermentation process. The study of the properties of the organisms may also contribute to reduction of potential hygienic risks not only in the classical fields of starter application but also in new fields where they may be employed as protective cultures that inhibit the growth of food pathogens or even spoilage organisms. Strains with an improved potential to reduce these risks and to exhibit further useful properties can be obtained by means of genetic engineering. Examples for successfully modified meat specific bacteria are Staphylococcus carnosus and lactobacilli L. curvatus and L. sake.
Microbiological developments during industrial meat fermentations (salami), made with and without commercial starter cultures, were followed at two factories in Germany and Italy. In the German product microbial growth was evident only for the first 48 h, followed by a gradual decline in numbers of most micro-organisms. The pH fell from 5.8 to 4.8 in the 28 d required for production. In Italy a similar situation was seen, except that a second period of bacterial growth began around 15 d, coincident with the appearance of intentional surface mould growth which reversed the pH fall, the final pH being 6.2. The German starter culture was a mixture of Lactobacillus plantarum and Staphylococcus carnosus, whereas in Italy only Staph. carnosus was used. The strain of Lact. plantarum used did not grow in the German product whereas the Staph. carnosus grew well in both products to form a substantial proportion of the final microflora.
Commercial cultures used in Canada for the manufacture of Italian dry sausage were examined to determine their microbial composition and suitability for low temperature (less than or equal to 20 degrees C) meat fermentations. Temperature optima in both laboratory media and commercial meat mixtures were generally too high to allow these cultures to be of substantial advantage in this application. In addition, media used currently for the enumeration of streptococci and related organisms from fermented meat products were found to be inadequately specific and often required confirmatory inspection of colonies by conventional phase contrast microscopy. Streptococci were isolated from Italian dry sausage manufactured commercially with and without added starter cultures. Streptococci persisted in sausages produced by both techniques with slightly higher numbers present in starter-acidulated sausages. About 55.5% of the 312 streptococci studied were enterococci (Lancefield's Group D). Streptococci were found in several samples of commercial starter cultures but it was felt that elevated ripening temperatures used for sausage manufactured by the starter-mediated process and meat handling practices were more important factors influencing streptococci recovery from sausage material.
Microbiological changes occurring during the commercial manufacture of Italian dry sausages (Genoa and salametti) were studied in two urban Canadian centres over a 5 month period. A comparison was made between 6 plants which used bacterial starter cultures and 4 plants where more traditional processes (without starters) were used. A total of 600 samples of raw, fermented and finished products were tested for the presence of coliforms, salmonellae, staphylococci, streptococci, the rate of pH reduction and final water activity (aW). Numbers of total bacteria peaked earlier and were significantly higher in sausages at the fermentation stage produced with starter cultures than in those traditionally manufactured. This corresponded with a more rapid drop in pH of the starter-inoculated products. Staphylococci and streptococci were significantly higher in starter-fermented Genoa sausages at the fermentation stage, but no significant differences were seen in the microbiological content or aW of mature finished sausages manufactured by the two different techniques. Of 128 randomly chosen isolates of coagulase-positive staphylococci, 34.4% were enterotoxin producers and 80% of these produced type A toxin. Enterotoxigenic staphylococci were found in 2 different samples of finished salametti and one sample of finished Genoa made with starter cultures and in one sample of finished Genoa made without added culture. Total numbers of staphylococci in these samples were not greater than 500/g. No correlation between the method of manufacture and presence of enterotoxigenic staphylococci could be made. Five subsamples from one lot of raw Genoa were the only samples positive for Salmonella during this study. Results indicated that low temperature traditional fermentations can yield products which are as safe as those produced by the higher temperature starter-controlled process. One of the most important elements in the traditional process was believed to be the selection and use of raw materials of the highest possible quality.
A total of 348 lactic acid bacteria isolated from five batches of naturally fermented dry salami at various stages of ripening were characterised. The majority of the strains were assigned to two main phylogenetic groups of species: (i) the psychrotrophic, formerly called atypical, meat streptobacteria (169 strains) and (ii) a new genus Weissella (120), which was recently proposed (Collins et al., 1993) to include Leuconostoc paramesenteroides and some other closely related species. Meat streptobacteria were identified as Lactobacillus curvatus (88 strains) and L. sake (76), whereas 5 strains were indistinguishable and, thus designated L. sake/curvatus. Non-psychrotrophic streptobacteria were also isolated and identified as L. plantarum (34 strains), L. farciminis (10), L. coryniformis (1) and L. casei subsp. pseudoplantarum (1). The majority of the Weissella strains (86) were leuconostoc-like bacteria; four of them were identified as W. viridescens, 11 belonged to the newly described W. hellenica (Collins et al., 1993), another 11 resembled W. paramesenteroides, whereas 60 isolates were not classified to any species. The latter group comprised strains that produced D(L)-lactate. The remaining Weissella were gas-forming, arginine-positive rods assigned to W. minor (31) and W. halotolerans (3). Other species identified were Enterococcus faecium (10), Leuconostoc mesenteroides (1), L. brevis (1) and Pediococcus sp. (1). The main criteria used to distinguish between above species as well as their distribution on the five salami batches in relation to their succession with time and suitability as starters were discussed.
The characterization of 254 strains of lactobacilli isolated from dry spontaneously fermented sausages from 15 different producers at two different stages of ripening time is reported. The species identified were Lactobacillus sake 55%, L. curvatus 26%, L. bavaricus 11% and L. plantarum 8%. The main criteria for the identification of isolates to species level were: production of lactic acid isomers, presence of mDpm acid in cell walls, deamination of arginine and fermentation of mannitol and melibiose. The composition of the populations of lactobacilli were the same for the two stages of ripening. The deamination of arginine was tested in aerobic and anaerobic cultures and in different media by checking the production of ammonia and detecting the production of citrulline. In 94% of strains tested both methods gave identical results. In two L. sake strains arginine catabolism was dependent on culture media; for two other L. sake strains the deamination of arginine only occurred when oxygen was scarce.
Growth of Penicillium chrysogenum, Aspergillus flavus, Cladosporium cladosporioides and Alternaria alternata at their respective optimum temperatures was studied in Potato Dextrose Agar (PDA) medium at different water activities (a(w)) adjusted with glycerol. The growth rate (mu) was expressed as the increase in colony radius per unit of time. This paper extends the model that showed the relationship between temperature and bacterial growth rate developed by Rosso et al. [J. Theor. Biol. 162 (1993) 447] to describe the influence of a(w) on fungal development. An excellent correlation between the experimental data and the model predictions was obtained, the regression coefficients (r2) were greater than 0.990, with the exception of that for A. flavus (r2 = 0.982). In addition, the use of such a model allows predictions of the cardinal water activities: a(wmin), a(wopt) and a(wmax). The estimation of the minimum water activity (a(wmin)) was in accordance with data literature for all the moulds considered here, but seemed to be slightly underestimated for P. chrysogenum and A. flavus when compared to our experimental values. The estimations of the optimal water activity (a(wopt)) and the optimal growth rate (muopt) were in excellent agreement to the experimental results for the four moulds. Through this example, it is suggested that the same approach for modelling can be used for various microorganisms (e.g. bacteria and moulds), and different environmental parameters (e.g. temperature and water activity).
The effect of starter culture and chemical acidulation on the growth and enterotoxigenesis of Staphylococcus aureus strain S-6 in Italian dry salami under commercial manufacturing conditions was studied. The experimental design included two levels of S. aureus (10 and 10/g), three levels of starter culture (0, 10, and 10/g), three levels of initial pH (pH(0)) (6.1, 5.5, and 4.8), two manufacturing plants, and three replications. S. aureus growth in the salami was affected significantly (P < 0.005) by pH(0), initial levels of S. aureus (staph(0)) and lactic acid bacteria (LAB(0)), day of fermentation, and by the interactions of pH(0) x day, pH(0) x LAB(0), LAB(0) x staph(0), pH(0) x staph(0), and pH(0) x location of fermentation. In general, the lower the pH(0) and the higher the LAB(0), the greater the inhibition of S. aureus. The LAB levels during the fermentation were affected significantly (P < 0.005) by pH(0), LAB(0), day of fermentation, location, LAB(0) x pH(0), and LAB(0) x day. Derived regression equations related level of S. aureus and LAB at any day of fermentation to a number of microbiological and chemical variables. Close similarity of observed and predicted levels of S. aureus and LAB growth demonstrated the usefulness of the experimental approach in evaluating the safety of a process. No detectable enterotoxin or thermonuclease was found at any stage of processing even when S. aureus reached levels of 10/g of salami.