Effect of reuterin,produced by Lactobacillus reuteri on the surface of sausages to inhibit the growth of Listeria monocytogenes and Salmonella spp.

Reuterin is a bacteriocin produced by some strains of Lactobacillus reuteri. The strain used in this study was isolated from raw milk from a dairy farm nearby Ankara. Beef sausage is a long years produced bratwurst style meat product in Turkey, as well as in some other countries in the Mediterranean region. Sausages are produced by raw meat; sometimes lactic starter cultures are added or spontaneous fermentation is employed. The production and storage conditions of the product promotes the growth of Listeria monocytogenes and Salmonella spp. Although nitrate is added as an antimicrobial substance against many pathogens, sometimes however nitrate application is not preventive enough on the surface because of the natural film around the sausages. Since most of the contaminations take place at post production steps, pathogenic growth is more effective on the surface of the sausages in refrigerated conditions. In this study, reuterin was applied to the surface of the sausages in order to prevent the growth of these two pathogens along with nitrate used as an additive in the product. Reuterin has inhibited the growth of L. monocytogenes considerably but not of Salmonella spp. on the surface of the sausages.     

Modelling contributes to the understanding of the different behaviour of bacteriocin-producing strains in a meat environment

Bacteriocins from lactic acid bacteria are potent antibacterial agents that enable the producer cells to selectively and efficiently inhibit a part of the competing background flora, including several spoilage bacteria and foodborne pathogens. The use of bacteriocinogenic lactic acid bacterium strains, either as starters or as protective cultures, is very limited in the food industry, possibly because many questions about the in situ functionality of such strains remain unanswered. A modelling approach was developed to study the functionality of lactic acid bacterium bacteriocin producers in a meat environment. The kinetic properties of three potential bacteriocin-producing starter cultures for sausage fermentation, namely Lactobacillus sakei CTC 494, Lactobacillus curvatus LTH 1174, and Lactobacillus amylovorus DCE 471 were compared in MRS, used as a meat simulation medium. Moreover, the application possibilities of the bacteriocin-producing Enterococcus faecium RZS C5 strain were evaluated. It turned out that growth and bacteriocin production by Lb. sakei CTC 494 and Lb. curvatus LTH 1174 are adapted to the meat environment, whereas Lb. amylovorus DCE 471 is kinetically not able to dominate the meat fermentation process. Furthermore, the use of bacteriocin-producing enterococci as functional cocultures seems to be attractive.     

Simulation of the effect of sausage ingredients and technology on the functionality of the bacteriocin-producing Lactobacillus sakei CTC 494 strain

Bacteriocin-producing lactic acid bacteria may be applied as novel functional starter cultures for sausage fermentation. In this way, safer and more standardised end products may be obtained. However, it is not clear how such strains behave under sausage fermentation conditions. In this study, the combined effects of typical sausage ingredients and process technology on the functionality of Lactobacillus sakei CTC 494 were simulated by a modelling approach. Under simulated sausage fermentation conditions, the strain was able to produce a considerable amount of bacteriocin. Model simulations indicate that sausage fermentation conditions of temperature and pH favour bacteriocin production, whereas salting and curing with sodium nitrite decrease growth and bacteriocin production. Sodium nitrite inhibits cell growth under its undissociated nitrous acid form, and its inhibitory effect seems to parallel lactic acid production. Whereas oxygen and magnesium levels did not influence bacterial functionality, manganese limitations severely decreased cell growth. Moreover, the presence of large amounts of fat, which is typical for a sausage environment, leads to an apparent bacteriocin inactivation, probably due to adsorption of the bacteriocin molecules from the water phase to the fat particles.     

Inhibition of Listeria monocytogenes in cold smoked salmon by addition of sakacin P and/or liveLactobacillus sakei cultures

Listeria monocytogenes is the causative agent of food-related listeriosis. It has the ability to grow in vacuum-packaged food at chiller temperatures and is relatively tolerant to salt and low pH, thus it is difficult to control its growth in food.Listeria monocytogenes was added to vacuum-packed cold smoked salmon together with known concentrations of the bacteriocin sakacin P or nisin and/or one of two isogenic strains of the lactic acid bacteriaLactobacillus sakei . The vacuum-packaged salmon samples were incubated at 10°C for 4 weeks. Of the isogenic L. sakei strains, one produced sakacin P and the other did not. Both sakacin P and nisin had an initial inhibiting effect on growth of L. monocytogenes. Addition of the sakacin P-producing or the non-producing L. sakei had a bacteriostatic effect on L. monocytogenes during the complete storage period. When the sakacin P-producing L. sakei culture was added to vacuum-packed cold smoked salmon together with sakacin P, a bacteriocidal effect on L. monocytogenes was observed.     

Effect of reuterin produced by Lactobacillus reuteri on the surface of sausages to inhibit the growth of Listeria monocytogenes and Salmonella spp

Reuterin is a bacteriocin produced by some strains of Lactobacillus reuteri. The strain used in this study was isolated from raw milk from a dairy farm nearby Ankara. Beef sausage is a long years produced bratwurst style meat product in Turkey, as well as in some other countries in the Mediterranean region. Sausages are produced by raw meat; sometimes lactic starter cultures are added or spontaneous fermentation is employed. The production and storage conditions of the product promotes the growth of Listeria monocytogenes and Salmonella spp. Although nitrate is added as an antimicrobial substance against many pathogens, sometimes however nitrate application is not preventive enough on the surface because of the natural film around the sausages. Since most of the contaminations take place at post production steps, pathogenic growth is more effective on the surface of the sausages in refrigerated conditions. In this study, reuterin was applied to the surface of the sausages in order to prevent the growth of these two pathogens along with nitrate used as an additive in the product. Reuterin has inhibited the growth of L. monocytogenes considerably but not of Salmonella spp. on the surface of the sausages.     

Effect of Extrinsic Parameters on the Production of a Bacteriocin by Lactobacillus buchneri,Isolated from Mexican Raw Sausages

Bacteriocins are antimicrobial peptides acting on certain pathogens and spoilage microorganisms. They have recently been considered as natural alternative to food preservatives. The objective of this work was to study the inhibition spectrum of a bacteriocin produced by Lactobacillus buchneri, isolated from a Mexican sausage, against Listeria monocytogenes and several lactic acid bacteria, and the influence of several extrinsic parameters on bacteriocin activity. Culture media (MRS or APT) had no significant effect on bacteriocin production (p < 0.060) although higher activity was observed in MRS (220 AU/mL). Conversely, temperature significant affected production (p > 0.001). Bacteriocin activity was also increased by 100% nitrogen atmosphere (180 AU/mL) as compared to N₂/CO₂ (50:50) (150 AU/mL) and 100% CO₂ (60 AU/mL). Maximum activity occurred at the end of the exponential growth phase. This bacteriocin inhibited some Listeria monocytogens strains and several lactic acid bacteria, mainly Lactobacillus sp.     

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

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

Effect of the lactic acid bacteria on the control of listerial activity and shelf life of smoked salmon scraps

The potential protective activity of lactic acid bacteria (LAB) from cold smoked salmon scraps was evaluated towards Listeria monocytogenes. Seventy‐three LAB strains were isolated and identified by biomolecular methods; Lactobacillus curvatus and Lactobacillus sakei prevailed. Three of the strains tested, identified as L. sakei, profile O, had a significant inhibitory activity against L. monocytogenes ATCC 19115 and clones DUP‐1042 and DUP‐18596. The evolution of microbial populations and chemical parameters were determined at time intervals to verify the shelf life. Listeria monocytogenes was isolated in half the packages also exceeding the legal limit. The shelf life of scraps was set at 30 days. Clonal characterisation of L. monocytogenes was performed by ribotyping. DUP‐1042, one of the human pathogen clones, was the most represented pattern. The results suggest further studies aimed at the selection of autochthonous nonspoilage LAB strains as bioprotective agents for cold smoked salmon.     

Lactic acid bacteria as protective cultures against Listeria spp. on cold-smoked salmon

Three bacteriocin producing (Bac⁺) strains of Lactobacillus sakei were used singly and in combination with each other as protective cultures to control the growth of listeria in cold-smoked salmon. Challenge experiments were conducted under practical conditions in a smokehouse. The surface of salmon sides was inoculated with 10⁴ cfu/g of Listeria innocua and 10⁷ cfu/g of Bac⁺ lactic acid bacteria as well as a L. sakei Bac⁻ control. After smoking the counts of listeria and lactic acid bacteria were determined at days 1 and 14. All Bac⁺ L. sakei strains reduced the counts of L. innocua by >2 log units. Strain LTH5754 was an isolate from cold-smoked salmon and achieved even a 5 log reduction of L. innocua within the storage period. In vitro experiments showed, that the Bac⁺ strains were also effective against L. monocytogenes (three strains tested) and L. ivanovii (1 strain). The pH as well the sensorial properties of the smoked salmon were not affected by the L. sakei inocula.     

Bacteriocin-producing lactobacilli in Spanish-style fermented sausages: characterization of bacteriocins

Three bacteriocins were studied. Sakacin K, a bacteriocin from Lactobacillus sake CTC494 was purified to homogeneity by a four-step system involving ammonium sulphate precipitation, binding to a cation exchanger, hydrophobic interaction and reverse phase chromatography in FPLC (fast performance liquid chromatography) system. The peptide sequence was determined by Edman degradation. The first 30 amino acid residues from the N-terminus of sakacin K were identical to those of curvacin A from Lactobacillus curvatus LTH1174 and sakacin A from Lactobacillus sakei Lb706. The structural gene of sakacin K in Lb. sake CTC494 was located on a 60 Kbp plasmid (as has been previously reported) by curvacin A in Lb. curvatus LTH1174 and sakacin A in Lb. sakei Lb706. Plantaricin D, a bacteriocin-like compound isolated from Lb. plantarum CTC305 was purified and sequenced using the same procedure as described for sakacin K. The first 15 amino acid residues of plantaricin D were identical to those obtained from the bacteriocin inducer peptide (termed plantaricin A) of the bacteriocinogenic system in Lb. plantarum C11. The structural gene of the plantaricin D peptide was located on a similar EcoRl chromosomal fragment when compared to plantaricin A in Lb. plantarum C11. These two isolated bacteriocin-like peptides (sakacin K and plantaricin D) were purified from two new different strains obtained from fermented meat, confirming the ecological importance of these substances. Lactobacillus sakei CTC372, the third bacteriocinogenic strain selected in this study from fermented sausages, produced a bacteriocin named sakacin T. The bacteriocin is not found free in the supernatant of the cells. It is active against Listeria monocytogenes and Staphylococcus aureus. The genetic determinant of this bacteriocin was localized in a 84·4 Kbp plasmid by conjugative transfer and curing assays.     

Safety and functional aspects of pre‐selected pediococci for probiotic use in Iberian dry‐fermented sausages

The purpose of this study was to investigate pre‐selected pediococci for potential probiotic use in Iberian dry‐fermented sausages. A total of twelve strains isolated from Iberian dry‐fermented sausages and pig faeces were evaluated according to safety and functional characteristics including biogenic amines and d ‐lactic acid production, antibiotic susceptibility, cell adhesion and antimicrobial activity against food‐borne pathogens. The strain P. acidilactici SP979 was able to establish itself and compete with enteropathogens such as Salmonella choleraesuis on the intestinal epithelium and an inhibition of such pathogenic bacteria as Listeria monocytogenes and Staphylococcus aureus in vitro. This strain was also considered safe to be used with regard to its void aminogenic potential, low d ‐lactic acid production and antibiotic resistance pattern; being identified as a potential probiotic meat starter culture suitable for manufacture of dry‐fermented Iberian sausages.     

Incidence and antibiotic susceptibility of bacteriocin‐producing lactic acid bacteria from dairy products

Screening for bacteriocin production by strains of lactic acid bacteria (LAB) from local dairy products in Iran resulted in the detection of 10 bacteriocin‐producing strains. Among 105 isolated, 10 bacteriocin producers were phenotypically and genotypically identified as Enterococcus spp. The antimicrobial compounds produced by these novel strains were inactivated by trypsin, proteinase k. These bacteriocins also were active in a wide range of pH and temperature values, and inhibited not only the closely related LAB, but also Listeria monocytogenes.     

Technological and safety properties of lactic acid bacteria isolated from Spanish dry-cured sausages

Technological and safety-related properties were analyzed in lactic acid bacteria isolated from Spanish dry-cured sausages in order to select them as starter cultures. In relation to technological properties, all the strains showed significative nitrate reductase activity; Lactobacillus plantarum, Lactobacillus paracasei and 52% of the Enterococcus faecium strains showed lipolytic activity and only Lactobacillus sakei strains (43%) were able to form biofilms. Related to safety aspects, E. faecium strains were the most resistant to antibiotics, whereas, L. sakei strains were the most sensitive. In relation to virulence factors, in the E. faecium strains analyzed, only the presence of efaA gene was detected. The analysis of biogenic amine production showed that most E. faecium strains and L. sakei Al-142 produced tyramine. In conclusion, L. paracasei Al-128 and L. sakei Al-143 strains possess the best properties to be selected as adequate and safe meat starter cultures.     

Safety and functional aspects of pre-selected lactobacilli for probiotic use in Iberian dry-fermented sausages

The purpose of this study was to investigate lactobacilli for potential probiotic use in Iberian dry-fermented sausages. A total of 18 strains, previously selected, isolated from different sources were evaluated according to safety and functional characteristics including biogenic amines and d-lactic acid production, antibiotic susceptibility, haemolysis, cell adhesion, and antimicrobial activity against food-borne pathogens. The strains Lactobacillus reuteri PL519, L. reuteri PL542, and Lactobacillus fermentum HL57 were able to establish their self on the intestinal epithelium, and an inhibition of such pathogenic bacteria as Listeria monocytogenesin vitro. These strains were also considered safe to be used with regard to their low aminogenic potential and d-lactic acid production, antibiotic resistance pattern and haemolytic activity. Thus, they were selected as a potential probiotic meat starter culture suitable for manufacture of dry-fermented Iberian sausages.     

Inhibition of Listeria monocytogenes and Escherichia coli O157:H7 in liquid broth medium and during processing of fermented sausage using autochthonous starter cultures

The antimicrobial effect of two autochthonous starter cultures of Lactobacillus sakei was evaluated in vitro (in liquid broth medium) and in situ assays. The inactivation of foodborne pathogens Listeria monocytogenes (serotype 4ab No 10) and Escherichia coli O157:H7 ATCC 43888 was investigated during the production of fermented sausage according to a typical Greek recipe using L. sakei strains as starter cultures. The inactivation kinetics were modeled using GInaFiT, a freeware tool to assess microbial survival curves. By the end of the ripening period, the inhibition of L. monocytogenes was significant in treatments with L. sakei 8416 and L. sakei 4413 compared to the control treatment. A 2.2-log reduction of the population of E. coli O157:H7 resulted from the autochthonous starter culture L. sakei 4413 during sausage processing. The use of the autochthonous starter cultures constitutes an additional improvement to the microbial safety by reducing foodborne pathogens.     

Assessment of technological attributes of autochthonous starter cultures in Turkish dry fermented sausage (sucuk)

The study aims to evaluate the technological properties of autochthonous strains (Lactobacillus sakei S15, Lactobacillus plantarum S24, L. plantarum S91, Pediococcus pentosaceus S128b and Staphylococcus carnosus G109) in Turkish dry fermented sausage (sucuk). After 24 h of fermentaPtion, all lactic acid bacteria strains reduced the pH to below 5.0, while the pH in the control group was above 5.3. The number of lactic acid bacteria strains reached 10⁸–10⁹ cfu g⁻¹ during fermentation. Staphylococcus carnosus G109 remained at the inoculation level of 10⁶ cfu g⁻¹ during ripening. Lactobacillus sakei S15 as mono-culture showed higher TBARS values compared to other strains. The control group had the lowest L* value and autochthonous strains caused no significant difference for a* value. According to principal component analysis results, most volatile compounds were positively correlated with the group containing only L. sakei S15.     

Microbial and physicochemical succession in fermented sausages produced with bacteriocinogenic culture of Lactobacillus sakei and semi-purified bacteriocin mesenterocin Y

The influence of the bacteriocinogenic culture Lactobacillus sakei (10⁵/g) and semi-purified bacteriocin mesenterocin Y (2560 AU/kg) on the safety and quality of traditional Croatian fermented sausages was investigated. The addition of Lb. sakei and/or mesenterocin Y reduced microbial counts (P < 0.05) in the final products. After 28 days of ripening, coagulase-negative cocci decreased 1.5–2.0 log, yeasts 1.2–1.4 log and enterococci 1.7–2.7 log. In the case of the addition of Lb. sakei, the lactic acid bacteria count was significantly (P < 0.05) higher at day 7 of ripening, and was accompanied by a lower pH and a higher amount of lactic acid (P < 0.05). In the final product the amount of acetic acid was significantly lower. More intensive proteolysis and an increase in ammonia content were found at the beginning of fermentation, and in the second phase of ripening in the control samples, respectively. The free fatty acid concentration was significantly lower during the entire ripening process compared to the control (P < 0.05). Semi-purified mesenterocin Y did not affect the sensory properties of the sausages, whilst the addition of Lb. sakei enhanced them.     

Natural and artificial casings as bacteriocin carriers for the biopreservation of meats products

The aim of the study was to determine if natural and artificial casings (ovine, porcine, bovine, collagen, and cellulose casings), serve as carriers for the application of the bacteriocin sakacin G produced by Lactobacillus curvatus ACU‐1. Mode of action of the cell‐free supernatant (CFS) containing the bacteriocin was also studied to determine whether Listeria monocytogenes inhibition could be tantamount to Listeria innocua one. Cylindrical vessels were filled with sterile meat emulsion and were wrapped with the different casings. Half the systems were treated with the CFS containing sakacin G, while the other half was taken as control systems. All systems were inoculated with L. innocua ATCC 33090. L. innocua was able to grow on all the studied casings. Collagen casing treated with CFS inhibited Listeria growth. A bacteriostatic effect was observed in the systems with porcine, ovine, bovine and cellulosic casings treated with CFS. The bacteriocin maintained its activity until the end of the trial. All casings were widely promising as antimicrobial application supports, being effective carriers to be used in a wide range of meat products.

Practical applications

Based on its advantageous characteristics, bacteriocins attracted considerable interest as natural food preservatives to extend shelf life and safety of meat and meat products. The application of sakacin G produced by Lactobacillus curvatus ACU‐1 on different natural and artificial casings would allow the preservation of a wide range of meat products that are stuffed into them, such as several types of sausages, wiener, salami, and mortadella, among others. The major advantage relies on its application, since it is not necessary to modify the production process or add extra wrappings to support the antimicrobial substance.     

Molecular and technological characterization of lactic acid bacteria from traditional fermented sausages of Basilicata region (Southern Italy)

Lactic acid bacteria (LAB) from traditional fermented sausages of the Basilicata region were investigated by ARDRA-PCR and RAPD-PCR for taxonomic identification at species and strain level and characterized on the basis of the growth and acidification at different temperatures, incubation times, levels of NaCl and KNO₂, hydrolysis of sarcoplasmatic and myofibrillar proteins and antimicrobial, peptide/amino acid release and nitrate reductase activities.Lactobacillus sakei was the predominant species (67%) followed by Pediococcus pentosaceus (16%), Leuconostoc carnosum (8%), Lactobacillus plantarum (4%), Lactobacillus brevis (2%) and Leuconostoc pseudomesenteroides (2%). The technological characterization revealed that most of the isolates had good acidifying and proteolytic properties. Moreover, Lb. sakei strains showed antimicrobial ability, while Leuconostoc strains the highest reduction of nitrates.This work was a preliminary study in the formulation of autochthonous starter cultures in order to standardize the production process of sausages, to preserve their typical organoleptic and sensory characteristics and to improve the quality of final product.     

Characterization and selection of autochthonous lactic acid bacteria isolated from traditional Iberian dry-fermented salchichón and chorizo sausages

ABSTRACT:  A total of 192 lactic acid bacteria were isolated from 2 types of naturally fermented dry sausages at 3 different stages of the ripening process in order to select the most suitable strains as starter cultures in dry-cured sausage manufacture according to their technological characteristics such as glucose fermentation, lactic and acetic acid production, and proteolytic, lipolytic, and antimicrobial activities. Identification of the isolates revealed that 31.2% were Pediococcus pentosaceus , 26.9% Lactococcus lactis , 18.6% Pediococcus acidilactici , 17% Lactobacillus brevis , and sporadic isolates of Leuconostoc mesenteroides , Lactobacillus plantarum , and Lactobacillus curvatus . Most of the strains did not produce gas from glucose and showed the capacity to produce lactic acid rapidly. Some 25% of the strains were able to degrade tributyrin (esterase activity), but none showed lipolytic activity against olive oil and pork fat. Only 3 strains of P. acidilactici showed weak proteolytic activity against myofibrillar or sarcoplasmic proteins. Also, the same strains showed antimicrobial activity against Listeria monocytogenes . Nine strains with the best properties were preselected and tested for biogenic amine production. The results showed that two of the strains, identified as P. acidilactici by polymerase chain reaction, had the potential to be further tested as starter cultures in pilot processing of Iberian sausages.