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.     

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

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

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

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

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.     

Safety improvement and preservation of typical sensory qualities of traditional dry fermented sausages using autochthonous starter cultures

Traditional dry fermented sausages are manufactured without addition of starter cultures in small-scale processing units, their fermentation relying on indigenous microflora. Characterisation and control of these specific bacteria are essential for the sensory quality and the safety of the sausages. The aim of this study was to develop an autochthonous starter culture that improves safety while preserving the typical sensory characteristics of traditional sausages. An autochthonous starter composed of Lactobacillus sakei, Staphylococcus equorum and Staphylococcus succinus isolated from a traditional fermented sausage was developed. These strains were tested for their susceptibility to antibiotics and their production of biogenic amines. This starter was evaluated in situ at the French traditional processing unit where the strains had been isolated. Effects of the autochthonous starter were assessed by analysing the microbial, physico-chemical, biochemical and sensory characteristics of the sausages. Inoculation with the chosen species was confirmed using known species-specific PCR assays for L. sakei and S. equorum and a species-specific PCR assay developed in this study for S. succinus. Strains were monitored by pulse-field gel electrophoresis typing. Addition of autochthonous microbial starter cultures improved safety compared with the traditional natural fermentation of sausages, by inhibiting the pathogen Listeria monocytogenes, decreasing the level of biogenic amines and by limiting fatty acid and cholesterol oxidation. Moreover, autochthonous starter did not affect the typical sensory quality of the traditional sausages. This is the first time to our knowledge that selection, development and validation in situ of autochthonous starter cultures have been carried out, and also the first time that S. equorum together with S. succinus have been used as starter cultures for meat fermentation. Use of autochthonous starter cultures is an effective tool for limiting the formation of unsafe compounds in traditional sausage while preserving their original and specific sensory quality.     

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.     

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.     

Metabolism of amino acids, dipeptides and tetrapeptides by Lactobacillus sakei

The microbial degradation of proteins, peptides and amino acids generates volatiles involved in the typical flavor of dry fermented sausage. The ability of three Lactobacillus sakei strains to form aroma compounds was investigated. Whole resting cells were fermented in phosphate buffer with equimolar amounts of substrates consisting of dipeptides, tetrapeptides and free amino acids, respectively.Dipeptides disappeared quickly from the solutions whereas tetrapeptides were only partially degraded. In both approaches the concentration of free amino acids increased in the reaction mixture but did not reach the equimolar amount of the initial substrates. When free amino acids were fed to the bacteria their levels decreased only slightly. Although peptides were more rapidly degraded and/or transported into the cells, free amino acids produced higher amounts of volatiles.It is suggested, that after transport into the cell peptides are only partially hydrolyzed to their amino acids, while the rest is metabolized via alternative metabolic pathways. The three L. sakei strains differed to some extend in their ability to metabolize the substrates to volatile compounds. In a few cases this was due to the position of the amino acids within the peptides. Compared to other starter cultures used for the production of dry fermented sausages, the metabolic impact of the L. sakei strains on the formation of volatiles was very low.     

The effect of starter cultures on proteolytic changes and amino acid content in fermented sausages

The main objective of this work was to examine the effects of using five types of commercial starter cultures in fermented sausages. During the fermentation stage, changes in proteolytic characteristics were observed in fermented sausages. Proteolytic activity was high in Ls_b + Sc:(Lactobacillus sakei + Staphylococcus carnosus) and Pp + Sx:(Pediococcus pentosaceus + Staphylococcus xylosus) starter-inoculated sausages during processing. Moreover, a slight increase in proteolytic activity was detected during storage in both these sausages. Sarcoplasmic and myofibrillar proteins were also affected by this starter culture addition, during the fermentation, ripening and intense proteolysis were observed in both the fermented sausages. The content of free amino acids was similar at the beginning of the fermentation stage for all the studied batches. However, the high differences in the content of free amino acids at the end of the process could be attributed to the starter culture activity.     

Lactic acid bacteria ecology of three traditional fermented sausages produced in the North of Italy as determined by molecular methods

In this study the bacterial biodiversity during the maturation process of three traditional sausages produced in the North of Italy (Salame bergamasco, Salame cremonese and Salame mantovano) was investigated by using culture-dependent and -independent methods. Eleven plants, in the three provinces considered here, were selected because starter cultures were never used in the production. The bacterial ecology, as determined by plate counts, was dominated by lactic acid bacteria (LAB), with minor contribution of coagulase negative cocci and yeasts. After molecular identification of 486 LAB strains, the species more frequently isolated were Lactobacillus sakei and Lactobacillus curvatus. This evidence was also confirmed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE). All the samples analyzed were characterized by the constant presence of L. sakei and L. curvatus bands. A richer biodiversity was only detected at the beginning of maturation. The results obtained by the molecular characterization of the L. sakei and L. curvatus and by the cluster analysis of the DGGE profiles highlighted a plant-specific population, rather than a geographic characterization of the products, underlining how the environmental and processing conditions are able to select specific microbiota responsible for the main transformations during the fermentation and ripening of the sausages.     

Strategies to reduce biogenic amine accumulation in traditional sausage manufacturing

Different strategies in the reduction of biogenic amine accumulation during the manufacture of five European traditional fermented sausages were studied concerning sausage formulation, the increase of sugar in the Italian salame abruzzese reduced the accumulation of cadaverine up to 43%. However, the addition of sugar in the French saucisson did not show a significant amine reduction. The inoculation of a decarboxylase-negative autochthonous starter culture reduced the biogenic amine accumulation in a different manner depending on the species and strain(s). The highest reduction was achieved by Lactobacillus sakei used in the Greek aeros thasou, resulting in a total putrescine reduction and a significant decrease in tyramine (62%) and histamine (71%). In Portuguese chouriços cadaverine reduction was only of 45% when a single strain of Staphylococcus equorum was inoculated, whereas a single strain of L. sakei or a mixture of S. equorum yielded a 75% and 89% of reduction, respectively. In Spanish fuet, a combination of L. sakei CTC6626 plus S. xylosus CTC6013 had only a very slight effect on tyramine reduction (19%) in Spanish fuet, whereas L. sakei CTC494 plus S. xylosus CTC6013 was capable to reduce tyraminogenesis by nearly 50%, suggesting that L. sakei CTC494 was the strain responsible for the additional tyramine reduction.     

Effect of autochthonous starter cultures isolated from Siahmazgi cheese on physicochemical,microbiological and volatile compound profiles and sensorial attributes of sucuk,a Turkish dry-fermented sausage

The effect of adding autochthonous starter cultures isolated from Siahmazgi cheese, on the physicochemical parameters and microbial counts of sucuk was investigated during the ripening period. SPME–GC/MS was used in volatile compound analysis and a trained group of panelists carried out sensory analysis of the final product. After preliminary screening, three strains of Lactobacillus plantarum, which possess desirable technological properties, were used to prepare three starter cultures: LBP7, LBP10 and LBP14. The addition of LBP7 and LBP14 starter cultures had a significant effect (P < 0.05) on lightness, leading to higher L values compared to control sausages during the ripening period. Both LBP7 and LBP14 sausages showed higher counts of lactic acid bacteria, lower growth of Enterobacteriaceae and Gram-positive catalase-positive cocci and greatly lowered the pH value compared to control sausages throughout the ripening process. At the end of the ripening process, lactic acid bacteria counts were affected (P < 0.05) by the addition of starter culture since higher counts were observed in sausages prepared with LBP7 (9.14 log CFU/g) and LBP14 (8.96 log CFU/g) batches. The decrease of water activity during the ripening of sausages was not affected by the various starters. The texture profiles of all sausages were similar except for LBP10, which showed lower hardness and gumminess during ripening. Under the conditions of the study, volatile compounds were mainly from spices, and no marked differences were found among inoculated sausages. However, sensory evaluation revealed that most of the sensory attributes were scored higher for inoculated sausages than for the control ones. Therefore, LBP7 and LBP14 could be promising candidates for inclusion as starter cultures for the manufacture of sucuk.     

Functional properties of Lactobacillus strains isolated from kimchi

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) from kimchi, a traditional Korean fermented vegetable product generally consumed raw as a side-dish with practically every meal.Twelve mild acid producing facultatively heterofermentative Lactobacillus strains were selected for their potential as starter cultures for fermentation of kimchi, and evaluated for their functional properties. Eleven strains were identified as Lactobacillus sakei and one as Lactobacillus plantarum. The strains identified as L. sakei differed in some physiological features; of particular interest was the fact that 9 of these strains produced L(+) lactic acid from glucose in presence of acetate.All strains were able to survive gastrointestinal conditions simulating stomach and duodenum passage. In addition, they showed higher adherence to HT-29 cells than Lactobacillus rhamnosus GG, a commercial probiotic strain used worldwide. These strains also showed antimicrobial activity against a number of food-borne pathogens. Their ability to lower cholesterol was demonstrated by BSH (bile salt hydrolytic) activity, and cholesterol assimilation tests in vitro. The results suggest the probiotic potential of these strains for use in kimchi fermentation.     

Prevalence and impact of single-strain starter cultures of lactic acid bacteria on metabolite formation in sourdough

Flavour of type II sourdoughs is influenced by the ingredients, processing conditions, and starter culture composition. It is, however, not fully clear to what extent different sourdough lactic acid bacteria (LAB) contribute to flavour. Therefore, two types of flour (rye and wheat) and different LAB starter culture strains were used to prepare sourdoughs, thereby leaving the yeast microbiota uncontrolled. All LAB starter culture strains tested were shown to be prevalent and to acidify the flour/water mixture to pH values between 3.1 and 3.9 after 24 h of fermentation. Multiple aldehydes, alcohols, ketones, and carboxylic acids were produced by the sourdough-associated microbiota throughout the fermentation period. Based on the organoleptic evaluation of breads produced with these sourdoughs, five LAB strains were selected to perform prolonged wheat and rye fermentations as to their capacity to result in an acidic (Lactobacillus fermentum IMDO 130101, Lactobacillus plantarum IMDO 130201, and Lactobacillus crustorum LMG 23699), buttermilk-like (Lactobacillus amylovorus DCE 471), or fruity flavour (Lactobacillus sakei CG1). Upon prolonged fermentation, higher metabolite concentrations were produced. For instance, L. sakei CG1 produced the highest amounts of 3-methyl-1-butanol, which was further converted into 3-methylbutyl acetate. The latter compound resulted in a fruity banana flavour after 48 h of fermentation, probably due to yeast interference. Rye fermentations resulted in sourdoughs richer in volatiles than wheat, including 3-methyl-1-butanol, 2-phenylethanol, and ethyl acetate.     

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

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

Microbiological and sensory quality of dry fermented sausages containing alginate-microencapsulated Lactobacillus reuteri

The probiotic bacterium Lactobacillus reuteri was added to dry sausage batter, without or after being microencapsulated in alginate using either extrusion or emulsion technology. Pediococcus pentosaceus and Staphylococcus carnosus were added at 7 log cfu/g as starter cultures for fermentation. The sausage batter was stuffed in 55 mm fibrous casings and fermented, with smoking, at 相似文献   

Microbiological,physicochemical and rheological properties of fermented soymilk produced with exopolysaccharide (EPS) producing lactic acid bacteria strains

Growth of Lactobacillus plantarum 70810, Lactobacillus rhamnosus 6005 and a commercial yogurt starter culture in soymilk was investigated in the present study. It was found that the fermented soymilk using L. plantarum 70810 had significantly higher viable cell counts, water holding capacity (WHC, 88.27%), apparent viscosity (1840.35 mPa s) and exopolysaccharide (EPS) amount (832.15 mg/L) than the other two starter cultures in soymilk. Direct observation of microstructure in fermented soymilk indicated that the network structures of EPS-protein could improve the texture of fermented soymilk. Considering that the beneficial effects of L. plantarum 70810 in fermented soymilk, volatile compounds in fermented soymilk were further identified. Then the growth and fermentation characteristics of L. plantarum 70810 including changes in viable cell counts, pH, titratable acidity, apparent viscosity and EPS production during storage were investigated. In comparison to original soymilk base, the concentrations of the characteristic flavor compounds for fermented soymilk using L. plantarum 70810 increased, whereas hexanal, 2-pentylfuran and 2-pentanone in relation to beany flavor of soymilk decreased. In addition, fermented soymilk using L. plantarum 70810 maintained high viable cell count (>10⁸ cfu/mL), apparent viscosity (966.43 mPa s) and amounts of EPS (635.49 mg/mL) during storage at 4 °C for 21 days.     

Identification of staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP

Pathogenic, spoilage, and technologically important microorganisms were monitored in 21 spontaneously fermented Swiss meat products manufactured with meat from wildlife or animals grown in natural habitat. Thereby, PCR-restriction fragment length polymorphism (RFLP) on rpoB and 16S rRNA gene sequences provided a powerful tool for fast and accurate identification of the main microbial population. Lactobacillus sakei and Lactobacillus curvatus dominated in fermented meat products followed by Staphylococcus species, which constituted 88.2% of all Gram-positive, catalase-positive cocci (GCC⁺) with cell counts varying from 2.6 to 7.0 log cfu/g during maturation. Staphylococcus equorum was prevalent in frequency and cell counts during maturation (18.0%; 5.0-7.3 log cfu/g) and in the end products (28.4%; 1.8-6.2 log cfu/g) implicating a new presumptive starter species for meat fermentation. Nine out of 14 end products indicated safety risks to consumers due to the high incidence of Staphylococcus saprophyticus or Staphylococcus epidermidis combined with cell counts of 7.4 and 4.9 log cfu/g, respectively. This fact was supported by the detection of Staphylococcus aureus and Enterobacteriaceae in ready-to-eat products strongly exceeding the tolerable limit of 2 log cfu/g. Spontaneously fermented meat products produced from wildlife or animals grown in natural habitats not only gave rise to hygienic and safety concerns but also provided new presumptive starter strains.     

Molecular characterization of Lactobacillus species isolated from naturally fermented sausages produced in Greece,Hungary and Italy

The ecology of the lactic acid bacteria (LAB) in naturally fermented sausages produced in three European countries was investigated with the purpose of defining the geographic distribution of specific populations responsible for the fermentation and maturation of the sausages. Three-hundred and fifty eight strains belonging to the LAB group were isolated and identified by PCR-DGGE and sequencing. Three species were common to all three countries studied and were the most numerous. Lactobacillus curvatus, L. plantarum and L. sakei strains were subjected to RAPD-PCR followed by cluster analysis to obtain a genetic characterization. The distributions reflected in almost all cases the provenience of the strains, thereby distinguishing Italian, Greek and Hungarian LAB populations. The application of molecular methods for the identification and characterization of bacterial populations isolated from naturally fermented products allows a better understanding of the ecology. As a consequence measures can be taken to protect the biodiversity characterizing a specific system.     

Microbial and Physicochemical Characterization of the Horse Meat in Fermented Sausage

In this study, 150 lactic acid bacterial strains, originating from horse meat, were subjected to a step-by-step screening and characterization to search for potential protective cultures to be used in the meat industry. Strains were first tested for their homofermentation and salt tolerance. Second, the antibacterial capacities towards Listeria monocytogenes were determined in an agar spot test. In total, 50% of the tested strains were inhibitory towards Listeria monocytogenes. However, only 12 strains produced a bacteriocin. Finally, three strains with strong bacteriocin activity were competitively evaluated by comparing their growth rate, acidifying character and lactic acid production at 15°C under anaerobic conditions in a liquid broth. All three strains had a fast growth rate with rapid acidification caused by the production of high levels of lactic acid. Based on this Lactobacillus sakei was used as starter culture for producing sausage horse meat. In this study, fermentations were followed analyzing the microbiological and physicochemical aspects of this product. The sausages were characterized based on microbial activity of lactic acid bacteria that resulted in a product with a final pH of about 4.56. No Listeria monocytogenes, Salmonella spp. nor Sulfite reducing clostridia were ever isolated from the raw materials or the fermented sausages during the maturation, reflecting the safety of this product. The final water activity of the product was 0.85. Lactobacillus sakei was efficient in reducing amine production since this strain caused a quick pH drop during sausage fermentation.