The Presence of Salt and a Curing Agent Reduces Bacteriocin Production by Lactobacillus sakei CTC 494, a Potential Starter Culture for Sausage Fermentation

The specific conditions in the batter of raw fermented sausages may reduce the efficiency of bacteriocin-producing starter cultures. In this work, using in vitro fermentation, we found that sodium chloride and sodium nitrite interfere with the growth of Lactobacillus sakei CTC 494, an organism which produces the antilisterial bacteriocin sakacin K. Because sakacin K production follows primary metabolite kinetics, a decrease in cell formation resulted in a decrease in sakacin K production as well. Sodium chloride dramatically influenced bacteriocin production by decreasing both biomass production and specific bacteriocin production. Sodium nitrite, however, had no effect on specific bacteriocin production and decreased bacteriocin production only because of its effect on cell growth. Moreover, sodium nitrite enhanced the toxic effect of lactic acid on bacterial growth.     

Sequencing and expression analysis of sakacin genes in Lactobacillus curvatus strains

In this study, we focused our investigation on two strains of Lactobacillus curvatus, L442 and LTH1174, which are able to produce bacteriocins. L. curvatus LTH1174 is widely studied for its capability to produce curvacin A, while L. curvatus L442 was isolated from traditional Greek fermented sausages and was shown to possess a strong inhibitory activity toward Listeria monocytogenes. By polymerase chain reaction, we were able to target in both strains the genes for the production of sakacin P and sakacin Q, sppA and sppQ, respectively, both encoded chromosomally. While sppA was found to be conserved when compared with other sakacin P genes, sppQ showed a deletion of about 15 nucleotides when aligned with sequences obtained from Lactobacillus sakei. This difference did not affect the activity of sakacin Q as determined by testing sensitive strains. Expression analysis highlighted that sakacin P was expressed in L. curvatus L442 but not in L. curvatus LTH1174. Curing experiments were performed on L. curvatus LTH1174 to study the effect of the megaplasmid, present in this strain. In the plasmid-cured strain, expression of the sppA gene was detected. sppQ was expressed in both plasmid-cured and wild-type L. curvatus LTH1174, although expression was higher in the plasmid-cured strain.     

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

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

Cloning and heterologous expression of a bacteriocin sakacin P from <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sakacin P, a bacteriocin from Lactobacillus sakei, shows strong activity against food-borne pathogens such as Listeria monocytogenes. In L. sakei, the structural gene (sppA) encoding sakacin P is controlled by a strict regulatory mechanism, and the quantity of secreted sakacin P is limited. In this study, the sppA gene was synthesized by splicing overlap extension PCR and cloned into Escherichia coli. After the induction with isopropyl-β-d-thiogalactopyranoside, the recombinant sakacin P was successfully expressed. The collected cells were sonicated, and the activity was detected by agar diffusion method. The results also showed that the low-temperature induction can improve the activity of sakacin P.     

Inhibition of Listeria in dry fermented sausages by the bacteriocinogenic Lactobacillus sake CTC494

Lactobacillus sake CTC494 isolated from a naturally fermented sausage, produced an antibacterial agent active against selected strains of Listeria monocytogenes and L. innocua. The agent was bacteriolytic against L. monocytogenes and sensitive to proteolytic enzymes; it was identified as a bacteriocin and was designated as sakacin K. The ability of Lact. sake CTC494 to inhibit the growth of listeria, compared to a bacteriocinogenic negative control strain, was examined in a model sausage system and in dry fermented sausages. In dry fermented sausages Lact. sake CTC494 was able not only to suppress the growth of listeria but to diminish their number by 1.25 log compared to the non-bacteriocinogenic control strain. Thus, Lact. sake CTC494 has proved to be a good starter culture providing good organoleptical and sensorial qualities to the product and can be employed as a bioprotective starter culture in fermented meat products.     

Characterization of a New Bacteriocin Operon in Sakacin P-Producing Lactobacillus sakei, Showing Strong Translational Coupling between the Bacteriocin and Immunity Genes

Previous studies of genes involved in the production of sakacin P by Lactobacillus sakei Lb674 revealed the presence of an inducible promoter downstream of the known spp gene clusters. We show here that this promoter drives the expression of an operon consisting of a bacteriocin gene (sppQ), a cognate immunity gene (spiQ), another gene with an unknown function (orf4), and a pseudoimmunity gene containing a frameshift mutation (orf5). The leader peptide of the new one-peptide bacteriocin sakacin Q contains consensus elements that are typical for so-called “double-glycine” leader peptides. The mature bacteriocin shows weak similarity to the BrcA peptide of the two-peptide bacteriocin brochocin C. Sakacin Q has an antimicrobial spectrum that differs from that of sakacin P, thus expanding the antimicrobial properties of the producer strain. The genes encoding sakacin Q and its cognate immunity protein showed strong translational coupling, which was investigated in detail by analyzing the properties of a series of β-glucuronidase fusions. Our results provide experimental evidence that production of the bacteriocin and production of the cognate immunity protein are tightly coregulated at the translational level.     

Characterisation of an Antilisterial Bacteriocin Produced by Lactobacillus sakei CWBI-B1365 Isolated from Raw Poultry Meat and Determination of Factors Controlling its Production

Amongst 101 lactic acid bacteria isolated from meat and fish samples, strain CWBI-B1365, identified as Lactobacillus sakei, was found to produce the subclass IIa bacteriocin sakacin G. Partial sequencing of the gene involved in the biosynthetic pathways revealed an unusual gene organisation in that the accessory gene associated with bacteriocin transport did not occur immediately downstream of the gene encoding an ABC transporter, but upstream of the putative immunity gene and encoded on the opposite DNA strand. Sakacin G production was strongly regulated by pH, temperature and the carbon sources used in the growth medium, as well as the concentration of carbon and nitrogen sources. The condition of pH 5.5 and the temperature of 25°C appeared to be optimal for bacteriocin production. The use of sucrose during culturing and the fed batch addition of sucrose and meat extract greatly enhanced bacteriocin production.     

The presence of salt and a curing agent reduces bacteriocin production by Lactobacillus sakei CTC 494, a potential starter culture for sausage fermentation

The specific conditions in the batter of raw fermented sausages may reduce the efficiency of bacteriocin-producing starter cultures. In this work, using in vitro fermentation, we found that sodium chloride and sodium nitrite interfere with the growth of Lactobacillus sakei CTC 494, an organism which produces the antilisterial bacteriocin sakacin K. Because sakacin K production follows primary metabolite kinetics, a decrease in cell formation resulted in a decrease in sakacin K production as well. Sodium chloride dramatically influenced bacteriocin production by decreasing both biomass production and specific bacteriocin production. Sodium nitrite, however, had no effect on specific bacteriocin production and decreased bacteriocin production only because of its effect on cell growth. Moreover, sodium nitrite enhanced the toxic effect of lactic acid on bacterial growth.     

Anti‐listerial activity of bacteriocin‐producing Lactobacillus curvatus CWBI‐B28 and Lactobacillus sakei CWBI‐B1365 on raw beef and poultry meat

Aim: The study aimed to evaluate the effect of the bacteriocins produced by Lactobacillus sakei CWBI‐B1365 and Lactobacillus curvatus CWBI‐B28 on the growth and survival of Listeria monocytogenes in raw beef and poultry meat. Methods and Results: The sakacin P and sakacin G structural genes were identified in Lact. curvatus CWBI‐B28 and Lact. sakei CWBI‐B1365 using PCR amplification, respectively. The effect of the two bacteriocinogenic strains either alone or together, and that of the nonbacteriocin‐producing strain Lact. sakei LMG17302, on the growth of L. monocytogenes was evaluated in beef and poultry meat. In raw beef, the pathogenic bacteria were inhibited by the bacteriocinogenic strains. The bacteriocinogenic strains had no activity in raw chicken meat when inoculated separately, while they showed a clear anti‐Listeria effect when applied together. Conclusion: Sakacin G producing Lact. sakei and sakacin P producing Lact. curvatus may be applied in raw beef to inhibit L. monocytogenes. In poultry meat, the inhibition of L. monocytogenes could only be achieved by a combined application of these bacteriocin‐producing strains. Significance and Impact of the Study: In some meat products, the combined application of different class IIa bacteriocin producing lactic acid bacterium can enhance the anti‐listerial activity.     

Influence of autochthonous starter cultures on microbial dynamics and chemical-physical features of traditional fermented sausages of Basilicata region

In this study, a combination of a Lactobacillus sakei strain and a Staphylococcus equorum strain was used as autochthonous starter for an experimental production of Basilicata fermented sausages. The influence of starter addition on the safety and quality parameters and microbiological and chemical-physical properties of the products was investigated. Microbial counts of safety indicators were lower in the samples with the addition of starter culture, and, at the end of ripening, Enterobacteriaceae and Gram negative bacteria were detected only in samples made without the starter addition. The addition of starter led to a final product with better microbiological and chemical-physical features, and a positive effect on flavor and aroma compounds formation by a good proteolytic and lipolytic activities. The use of autochthonous starter cultures allows to obtain products with the organoleptic characteristics expected and steady in time and to standardize the production process, improving the safety and quality, but preserving the essential character of the product.     

Induction of bacteriocin production in Lactobacillus sake by a secreted peptide.

Lactobacillus sake LTH673 is known to produce a bacteriocin called sakacin P. Production of and immunity to sakacin P were found to depend on the presence of a protease-sensitive component that is produced by L. sake LTH673 itself. This component (called inducing factor [IF]) was purified from culture supernatants and shown to be a basic, nonbacteriocin peptide consisting of 19 amino acids, which in principle is capable of forming a highly amphiphilic helical structure. Circular dichroism studies showed that IF indeed could adopt a helical structure, but only in membrane-mimicking environments. Both purified IF and chemically synthesized IF induced expression of the structural gene for sakacin P and concomitant secretion of the gene product. In addition, IF induced its own production and immunity to sakacin P and related bacteriocins. These results indicate that bacteriocin production by L. sake LTH673 is controlled by an autoinduction pathway in which IF may function as a cell density signal.     

Two Different Tetracycline Resistance Mechanisms, Plasmid-Carried tet(L) and Chromosomally Located Transposon-Associated tet(M), Coexist in Lactobacillus sakei Rits 9

Lactobacillus sakei is extensively used as functional starter culture in fermented meat products. One of the safety criteria of a starter culture is the absence of potentially transferable antibiotic resistance determinants. However, tetracycline-resistant L. sakei strains have already been observed. In this paper, we show that tetracycline resistance in L. sakei Rits 9, a strain isolated from Italian Sola cheese made from raw milk, is mediated by a transposon-associated tet(M) gene coding for a ribosomal protection protein and a plasmid-carried tet(L) gene coding for a tetracycline efflux pump. pLS55, the 5-kb plasmid carrying the tet(L) gene, is highly similar to the pMA67 plasmid recently described for Paenibacillus larvae, a species pathogenic to honeybees. pLS55 could be transferred by electroporation into the laboratory strain L. sakei 23K. While the L. sakei 23K transformant containing pLS55 displayed an intermediate tetracycline resistance level (MIC, <32 μg/ml), L. sakei Rits 9, containing both tetracycline-resistant determinants, had a MIC of <256 μg/ml, suggesting that Tet L and Tet M confer different levels of resistance in L. sakei. Remarkably, in the absence of tetracycline, a basal expression of both genes was detected for L. sakei Rits 9. In addition, subinhibitory concentrations of tetracycline affected the expression patterns of tet(M) and tet(L) in different ways: the expression of tet(M) was induced only at high tetracycline concentrations, whereas the expression of tet(L) was up-regulated at lower concentrations. This is the first time that two different mechanisms conferring resistance to tetracycline are characterized for the same strain of a lactic acid bacterium.     

High-level gene expression in Lactobacillus plantarum using a pheromone-regulated bacteriocin promoter

AIMS: To use promoters and regulatory genes involved in the production of the bacteriocin sakacin P to obtain high-level regulated gene expression in Lactobacillus plantarum. METHODS AND RESULTS: In a plasmid containing all three operons naturally involved in sakacin P production, the genes encoding sakacin P and its immunity protein were replaced by the aminopeptidase N gene from Lactococcus lactis (pepN) or the beta-glucuronidase gene from Escherichia coli (gusA). The new genes were precisely fused to the start codon of the sakacin P gene and the stop codon of the immunity gene. This set-up permitted regulated (external pheromone controlled) overexpression of both reporter genes in L. plantarum NC8. For PepN, production levels amounted to as much as 40% of total cellular protein. CONCLUSIONS: Promoters and regulatory genes involved in production of sakacin P are suitable for establishing inducible high-level gene expression in L. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a system for controllable gene expression in lactobacilli, giving some of the highest expression levels reported so far in this genus.     

Microbial Ecology of the Soppressata of Vallo di Diano, a Traditional Dry Fermented Sausage from Southern Italy, and In Vitro and In Situ Selection of Autochthonous Starter Cultures

The microbial ecology of “soppressata of Vallo di Diano,” a traditional dry fermented sausage from southern Italy, was studied by using both culture-dependent and culture-independent approaches. The ripened fermented sausages were characterized by high microbial loads of both staphylococci and lactobacilli. Using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the variable V3 and V1 regions of the 16S rRNA gene and direct DNA sequencing, it was possible to identify Staphylococcus xylosus, S. succinus, and S. equorum among the staphylococci and Lactobacillus sakei and L. curvatus within the lactobacilli. Moreover, Debaryomyces hansenii was the main yeast species found by targeting the yeast 26S rRNA gene by PCR-DGGE. Selected strains of S. xylosus, L. sakei, and L. curvatus were characterized for their technological properties in the ripening conditions of the fermented sausages so as to select an autochthonous starter formulation. The selection included the determination of nitrate reductase, lipolytic, and antioxidant activity and proteolysis with myofibrillar and sarcoplasmic protein fractions. Such properties were evaluated in both in vitro and in situ assays; the latter were performed by using each strain as a starter in the laboratory-scale manufacture of soppressata of Vallo di Diano and by monitoring the microbiological and chemical changes at the end of ripening. The results show differences between the in vitro and in situ selection results and indicate that in situ evaluation of the technological performance of specific strains is better suited to selecting autochthonous starter cultures for fermented-meat products than in vitro evaluation.     

Influence of complex nutrients, temperature and pH on bacteriocin production by Lactobacillus sakei CCUG 42687

The effects of process conditions and growth kinetics on the production of the bacteriocin sakacin P by Lactobacillus sakei CCUG 42687 have been studied in pH-controlled fermentations. The fermentations could be divided into phases based on the growth kinetics, phase one being a short period of exponential growth, and three subsequent ones being phases of with decreasing specific growth rate. Sakacin P production was maximal at 20 °C. At higher temperatures (25–30 °C) the production ceased at lower cell masses, when less glucose was consumed, resulting in much lower sakacin P concentrations. With similar media and pH, the maximum sakacin P concentration at 20 °C was seven times higher than that at 30 °C. The growth rate increased with increasing concentrations of yeast extract, and the maximum concentration and specific production rate of sakacin P increased concomitantly. Increasing tryptone concentrations also had a positive influence upon sakacin P production, though the effect was significantly lower than that of yeast extract. The maximum sakacin P concentration obtained in this study was 20.5 mg l⁻¹. On the basis of the growth and production kinetics, possible metabolic regulation of bacteriocin synthesis is discussed, e.g. the effects of availability of essential amino acids, other nutrients, and energy. Received: 7 June 1999 / Received revision: 15 September 1999 / Accepted: 17 September 1999     

Autochthonous fungi are central components in microbial community structure in raw fermented sausages

Raw meat sausage represents a unique ecological niche rich in nutrients for microbial consumption, making it particularly vulnerable to microbial spoilage. Starter cultures are applied to improve product stability and safety as well as flavour characteristics. However, the influence of starter cultures on microbial community assembly and succession throughout the fermentation process is largely unknown. In particular the effect on the fungal community has not yet been explored. We evaluate the microbiological status of four different raw meat sausages using high-throughput 16S rRNA gene and ITS2 gene sequencing. The objective was to study temporal changes of microbial composition during the fermentation process and to identify potential keystone species that play an important role within the microbial community. Our results suggest that fungi assigned to the species Debaryomyces hansenii and Alternaria alternata play a key role in microbial community dynamics during fermentation. In addition, bacteria related to the starter culture Lactobacillus sakei and the spoilage-associated genera Acinetobacter, Pseudomonas and Psychrobacter are central components of the microbial ecosystem in raw fermented sausages. Elucidating the exact role and interactions of these microorganisms has the potential to have direct impacts on the quality and safety of fermented foods.     

Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.     

Lyophilized preparations of bacteriocinogenic Lactobacillus curvatus and Lactococcus lactis subsp. lactis as potential protective adjuncts to control Listeria monocytogenes in dry-fermented sausages

AIM: Study of the effectiveness of in situ bacteriocin production by lactic acid bacteria (LAB) to control Listeria monocytogenes in dry-fermented sausages. METHODS AND RESULTS: Two bacteriocin-producing strains: Lactococcus lactis subsp. lactis LMG21206 and Lactobacillus curvatus LBPE were grown in a pilot scale fermentor and lyophilized to be directly used in dry sausage fermentation. A commercial starter culture (Bel'meat SL-25) not inhibitory to L. monocytogenes (Bac- starter) was mixed (1 : 1) with each of the two lyophilized bacteriocin-producing strains to obtain starters active against the pathogen (Bac+ starter). Anti-Listeria effectiveness of the Bac+ starters was studied in dry-fermented sausages. The meat batter was experimentally contaminated with a mixture of four different strains of L. monocytogenes (10(2)-10(3) CFU g(-1)). The results showed that L. monocytogenes did not grow in any of the contaminated batches, but no significant decrease (P > 0.05) was observed either in the positive control (no added starter culture) or in samples fermented with the Bac- starter culture during the fermentation period and up to 15 days of drying. When the Bac+ starter contained Lb. curvatus LBPE, cell counts of L. monocytogenes decreased to below the detectable limit (<10 CFU g(-1)) after 4 h of fermentation and no survivors could be recovered by enrichment beyond day 8 of drying. When the Bac+ starter culture containing Lc. lactis LMG21206 was used, a decrease in Listeria counts to below the detectable limit was achieved after 15 days of drying. CONCLUSIONS: The bacteriocin-producing strains studied may be used as adjunct cultures for sausage fermentations to control the occurrence and survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Addition of the Bac+ strains, especially the Lb. curvatus strain would provide an additional hurdle to enhance the control of L. monocytogenes in fermented meat products.