New Active Packaging Based on Biopolymeric Mixture Added with Bacteriocin as Active Compound.

The objective of this work was to develop a chitosan/agar-agar bioplastic film incorporated with bacteriocin that presents active potential when used as food packaging. The formulation of the film solution was determined from an experimental design, through the optimization using the desirability function. After establishing the concentrations of the biopolymers and the plasticizer, the purified bacteriocin extract of Lactobacillus sakei was added, which acts as an antibacterial agent. The films were characterized through physical, chemical, mechanical, barrier, and microbiological analyses. The mechanical properties and water vapor permeability were not altered by the addition of the extract. The swelling property decreased with the addition of the extract and the solubility increased, however, the film remained intact when in contact with the food, thus allowing an efficient barrier. Visible light protection was improved by increased opacity and antibacterial capacity was effective. When used as Minas Frescal cream cheese packaging, it contributed to the increase of microbiological stability, showing a reduction of 2.62 log UFC/g, contributing a gradual release of the active compound into the food during the storage time. The film had an active capacity that could be used as a barrier to the food, allowing it to be safely packaged.

Studies on host-foodborne bacteria in intestinal three-dimensional cell culture model indicate possible mechanisms of interaction.

Spheroids of intestinal cells (Caco-2) were used to evaluate the adhesion/invasion ability of Listeria monocytogenes (pathogen) and Lactobacillus sakei 1 (potential probiotic). Besides, transcriptomic analyses of Caco-2 cells in three dimensional cultures were done, with the aim of revealing possible host-foodborne bacteria interactions. Result of adhesion assay for L. monocytogenes in Caco-2 spheroids was 22.86 ± 0.33%, but it was stimulated in acidic pH (4.5) and by the presence of 2% sucrose (respectively, 32.56 ± 1.35% and 33.25 ± 1.26%). Conversely, the invasion rate of L. monocytogenes was lower at pH 4.5, in comparison with non-stressed controls (18.89 ± 1.05% and 58.65 ± 0.30%, respectively). L. sakei 1 adhered to Caco-2 tridimensional cell culture (27.30 ± 2.64%), with no invasiveness. There were 19 and 21 genes down and upregulated, respectively, in tridimensional Caco-2 cells, upon infection with L. monocytogenes, which involved immunity, apoptosis; cytoprotective responses, cell signalling-regulatory pathways. It was evidenced despite activation or deactivation of several pathways in intestinal cells to counteract infection, the pathogen was able to hijack many host defense mechanisms. On the other hand, the probiotic candidate L. sakei 1 was correlated with decreased transcription of two genes in Caco-2 cells, though it stimulated the expression of 14 others, with diverse roles in immunity, apoptosis, cytoprotective response and cell signalling-regulatory pathways. Our data suggest the use of tridimensional cell culture to mimic the intestinal epithelium is a good model for gathering broad information on the putative mechanisms of interaction between host and bacteria of importance for food safety, which can serve as a basis for further in-depth investigation.

Avaliação de diferentes culturas starters na elaboração de salame tipo italiano / Evaluation of different starters cultures in the obtaining of Italian-type sausage

O Salame Tipo Italiano produzido no Brasil é predominantemente obtido de carne suína, com maturação aproximada de 30 dias, atingindo pH em torno de 5,4. O uso das bactérias do gênero Lactobacillus como culturas iniciadoras propicia o processo de fermentação e a obtenção de produtos uniformes e seguros, com redução do tempo de maturação devido à rápida formação de ácido lático, obtendo-se melhores características sensoriais, químicas e microbiológicas. Os estafilococos coagulase-negativos (comumente em produtos cárneos fermentados Staphylococcus xylosus e S. carnosus)possuem vantagens tecnológicas, como atividade de nitrito e nitrato redutase, consumo de oxigênio e atividade de catalase que melhoram a estabilidade de cor e diminuem o desenvolvimento de rancidez no produto, além de contribuírem para a geração de sabor devido à capacidade proteolítica e lipolítica. Portanto, este trabalho objetivou estudar a influência de diferentes culturas starters (S. xylosus, S. carnosus, L. sakei e L. plantarum)combinadascom diferentes concentrações de substrato de glicose (40,5 e 99,5%) na fabricação do Salame Tipo Italiano, bem como a influência da concentração destes no desempenho das culturas starters. Durante o processamento e shelf life, os salames foram avaliados quanto às características físico-químicas, microbiológicas e sensoriais. Em todas as amostras de Salame Tipo Italiano houve comportamento similar em relação aos parâmetros físico-químicos (umidade, proteína e gordura). Não houve crescimento de micro-organismos indesejáveis nas amostras analisadas após 60 dias de shelf life. Na análise sensorial, a amostra com menor concentração de substrato (40,5%), utilizando as culturas S. carnosus e L. sakei, foi a que recebeu as melhores notas e melhor aceitação pelos degustadores, além de apresentar valores de nitritos e nitratos de acordo com a legislação.(AU)

The Italian-Type Sausage produced in Brazil is predominantly obtained from swine meat, with maturation approximately 30 days, reaching pH around 5.3. The use of Lactobacillus bacteria as starter cultures provide fermentation processes and products more uniform and safe, reducing the maturation time due to the rapid formation of lactic acid, obtaining products with better sensorial, chemical and microbiological characteristics. Coagulase-negative staphylococci (commonly in S. xylosus and S. carnosus fermented meat products) have technological advantages such as nitrite and nitrate redutase activity, oxygen uptake and catalase activity that improve color stability and decrease the development of rancidity in the skin, contribute to the generation of flavor due to the proteolytic and lipolytic capacity. This work aimed to study the influence of different starters cultures (S. xylosus, S. carnosus, L. sakei and L. plantarum) combined with different concentrations of glucose substrate (40.5 and 99.5%) in properties of Italian-Type Sausage, as well as the influence of their concentration on starter performance. During the processing and shelf life the sausages were evaluated in relation to physic-chemical, microbiological and sensorial characteristics. In all samples of Italian-type Sausages have similar behavior in relation to physical-chemical parameters (moisture, protein, and fat). There was no growth of undesirable micro-organisms in the samples after 60 days of shelf life. By the sensorial analysis, the sample with low concentration of substrate (40.5%) and with S. carnosus and L. sakei cultures was the one that received the best grades and better acceptance by the tasters, and presented values of nitrites and nitrites according to the legislation.(AU)

Avaliação de diferentes culturas starters na elaboração de salame tipo italiano / Evaluation of different starters cultures in the obtaining of Italian-type sausage

O Salame Tipo Italiano produzido no Brasil é predominantemente obtido de carne suína, com maturação aproximada de 30 dias, atingindo pH em torno de 5,4. O uso das bactérias do gênero Lactobacillus como culturas iniciadoras propicia o processo de fermentação e a obtenção de produtos uniformes e seguros, com redução do tempo de maturação devido à rápida formação de ácido lático, obtendo-se melhores características sensoriais, químicas e microbiológicas. Os estafilococos coagulase-negativos (comumente em produtos cárneos fermentados Staphylococcus xylosus e S. carnosus)possuem vantagens tecnológicas, como atividade de nitrito e nitrato redutase, consumo de oxigênio e atividade de catalase que melhoram a estabilidade de cor e diminuem o desenvolvimento de rancidez no produto, além de contribuírem para a geração de sabor devido à capacidade proteolítica e lipolítica. Portanto, este trabalho objetivou estudar a influência de diferentes culturas starters (S. xylosus, S. carnosus, L. sakei e L. plantarum)combinadascom diferentes concentrações de substrato de glicose (40,5 e 99,5%) na fabricação do Salame Tipo Italiano, bem como a influência da concentração destes no desempenho das culturas starters. Durante o processamento e shelf life, os salames foram avaliados quanto às características físico-químicas, microbiológicas e sensoriais. Em todas as amostras de Salame Tipo Italiano houve comportamento similar em relação aos parâmetros físico-químicos (umidade, proteína e gordura). Não houve crescimento de micro-organismos indesejáveis nas amostras analisadas após 60 dias de shelf life. Na análise sensorial, a amostra com menor concentração de substrato (40,5%), utilizando as culturas S. carnosus e L. sakei, foi a que recebeu as melhores notas e melhor aceitação pelos degustadores, além de apresentar valores de nitritos e nitratos de acordo com a legislação.

The Italian-Type Sausage produced in Brazil is predominantly obtained from swine meat, with maturation approximately 30 days, reaching pH around 5.3. The use of Lactobacillus bacteria as starter cultures provide fermentation processes and products more uniform and safe, reducing the maturation time due to the rapid formation of lactic acid, obtaining products with better sensorial, chemical and microbiological characteristics. Coagulase-negative staphylococci (commonly in S. xylosus and S. carnosus fermented meat products) have technological advantages such as nitrite and nitrate redutase activity, oxygen uptake and catalase activity that improve color stability and decrease the development of rancidity in the skin, contribute to the generation of flavor due to the proteolytic and lipolytic capacity. This work aimed to study the influence of different starters cultures (S. xylosus, S. carnosus, L. sakei and L. plantarum) combined with different concentrations of glucose substrate (40.5 and 99.5%) in properties of Italian-Type Sausage, as well as the influence of their concentration on starter performance. During the processing and shelf life the sausages were evaluated in relation to physic-chemical, microbiological and sensorial characteristics. In all samples of Italian-type Sausages have similar behavior in relation to physical-chemical parameters (moisture, protein, and fat). There was no growth of undesirable micro-organisms in the samples after 60 days of shelf life. By the sensorial analysis, the sample with low concentration of substrate (40.5%) and with S. carnosus and L. sakei cultures was the one that received the best grades and better acceptance by the tasters, and presented values of nitrites and nitrites according to the legislation.

Characterization of multiple antilisterial peptides produced by sakacin P-producing Lactobacillus sakei subsp. sakei 2a.

Antimicrobial compounds produced by lactic acid bacteria can be explored as natural food biopreservatives. In a previous report, the main antimicrobial compounds produced by the Brazilian meat isolate Lactobacillus sakei subsp. sakei 2a, i.e., bacteriocin sakacin P and two ribosomal peptides (P2 and P3) active against Listeria monocytogenes, were described. In this study, we report the spectrum of activity, molecular mass, structural identity and mechanism of action of additional six antilisterial peptides produced by Lb. sakei 2a, detected in a 24 h-culture in MRS broth submitted to acid treatment (pH 1.5) and proper fractionation and purification steps for obtention of free and cell-bound proteins. The six peptides presented similarity to different ribosomal proteins of Lb. sakei subsp sakei 23K and the molecular masses varied from 4.6 to 11.0 kDa. All peptides were capable to increase the efflux of ATP and decrease the membrane potential in Listeria monocytogenes. The activity of a pool of the obtained antilisterial compounds [enriched active fraction (EAF)] against Listeria monocytogenes in a food model (meat gravy) during refrigerated storage (4 °C) for 10 days was also tested and results indicated that the populations of L. monocytogenes in the food model containing the acid extract remained lower than those at time 0-day, evidencing that the acid extract of a culture of Lb. sakei 2a is a good technological alternative for the control of growth of L. monocytogenes in foods.

Antimicrobial activity of whey protein films supplemented with Lactobacillus sakei cell-free supernatant on fresh beef.

The aim of this work was to evaluate the antimicrobial activity of whey protein isolate (WPI) films supplemented with Lactobacillus sakei NRRL B-1917 cell-free supernatant on beef inoculated with Escherichia coli ATCC 25922 or Listeria monocytogenes Scott A; additionally, sensory evaluation was performed on wrapped beef cubes. Supernatant concentrates were obtained from Lb. sakei cultures in MRS broth after centrifugation, filtering, and freeze-drying. Films were prepared with WPI (3% w/w), alginate (0.625% w/w), rehydrated supernatant (18 mg/ml), and glycerol. Films were used to wrap beef cubes inoculated with ≈103 CFU/g E. coli or L. monocytogenes. Sensory evaluation was carried out on grilled beef wrapped or not with the studied antimicrobial films. During refrigerated storage, antimicrobial films reduced 1.4 log10 CFU/g of L. monocytogenes after 120 h, while E. coli decreased 2.3 log10 CFU/g after 36 h. Grilled beef wrapped with antimicrobial film was well accepted by panelists, besides scores evidenced no significant differences (p > 0.05) between wrapped and unwrapped beef.

Microbial ecology involved in the ripening of naturally fermented llama meat sausages. A focus on lactobacilli diversity.

Llama represents for the Andean regions a valid alternative to bovine and pork meat and thanks to the high proteins and low fat content; it can constitute a good product for the novel food market. In this study, culture-dependent and independent methods were applied to investigate the microbial ecology of naturally fermented llama sausages produced in Northwest Argentina. Two different production technologies of llama sausage were investigated: a pilot-plant scale (P) and an artisanal one (A). Results obtained by High-Throughput Sequencing (HTS) of 16S rRNA amplicons showed that the production technologies influenced the development of microbial communities with a different composition throughout the entire fermentation process. Both sequencing and microbiological counts demonstrated that Lactic Acid Bacteria (LAB) contributed largely to the dominant microbiota. When a total of 230 isolates were approached by RAPD-PCR, presumptive LAB strains from P production exhibited an initial variability in RAPD fingerprints switching to a single profile at the final of ripening, while A production revealed a more heterogeneous RAPD pattern during the whole fermentation process. The constant presence of Lactobacillus sakei along the fermentation in both productions was revealed by HTS and confirmed by species-specific PCR from isolated strains. The technological characterization of Lb. sakei isolates evidenced their ability to grow at 15°C, pH4.5 and 5% NaCl (95%). Most strains hydrolyzed myofibrillar and sarcoplasmic proteins. Bacteriocins encoding genes and antimicrobial resistance were found in 35% and 42.5% of the strains, respectively. An appropriate choice of a combination of autochthonous strains in a starter formulation is fundamental to improve and standardize llama sausages safety and quality.

Control of Listeria monocytogenes biofilms on industrial surfaces by the bacteriocin-producing Lactobacillus sakei CRL1862.

The effect of the bacteriocin-producing Lactobacillus sakei CRL1862 and its bacteriocin in the control of Listeria biofilm formation on industrial surfaces at 10°C was investigated. A screening among different Listeria species was performed allowing selecting L. monocytogenes FBUNT for its use as a biofilm producer on stainless steel (SS) and polytetrafluoroe-thylene (PTFE) surfaces. Three conditions were simulated to evaluate the ability of the bacteriocinogenic strain to displace, exclude and compete pathogen biofilm formation. Lactobacillus sakei CRL1862 effectively inhibited biofilm formation by L. monocytogenes FBUNT through the three assayed mechanisms, pathogen inhibition being more efficient on PTFE than on SS surface. Moreover, co-culture of L. monocytogenes FBUNT with the bacteriocin-producer displayed the highest efficacy reducing the pathogen by 5.54 ± 0.12 and 4.52 ± 0.01 on PTFE and SS, respectively. Industrially, the pre-treatment with L. sakei CRL1862 or its bacteriocin (exclusion) constitutes the most realistic way to prevent pathogen biofilm settlement. The use of bacteriocins and/or the bacteriocin-producer strain represents a safe and environmentally-friendly sanitation method to mitigate post-processing food contamination.

In vitro modelling of simultaneous interactions of Listeria monocytogenes, Lactobacillus sakei, and Staphylococcus carnosus.

The Jameson effect model describes suppression of microorganism growth as being dependent on non-specific competition. This model was developed for simultaneous growth in a liquid medium and microbial interactions between Listeria monocytogenes, Lactobacillus sakei, and Staphylococcus carnosus with addition of NaNO2 to mimic the manufacturing process of salami for 48 h at 21°C and then for 14 days at 17°C. L. monocytogenes in the presence of L. sakei was inhibited by 2.120 log CFU/mL in the presence of NaNO2, and 1.146 log CFU/mL without NaNO2. Inhibition of L. monocytogenes cocultured with S. carnosus was 2.248 log CFU/mL at 48 h, but after 336 h was not significantly (p>0.05) different from L. monocytogenes in mono-culture. The interspecific competition parameter (ß) <1 indicated that the prevailing competition in co-cultures was intraspecific. Differentiation between 2 bacterial species interactions can be applied for use in starter cultures with pathogenic flora.

Optimization of growth and bacteriocin production by Lactobacillus sakei subsp. sakei2a.

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacterium isolated from Brazilian pork sausage, capable of inhibiting the growth of microbial pathogens, mainly Listeria monocytogenes. In order to optimize bacteriocin production for industrial applications, this study evaluated the effect of supplementation of MRS broth with glucose, Tween 20, Tween 80, sodium citrate, potassium chloride and cysteine, and effect of the initial pH and temperature of incubation of the medium on production of bacteriocins by L. sakei 2a. Adding glucose and Tween 20 to the medium, an initial pH of 5.0 or 5.5, and incubation temperatures of 25 °C or 30 °C resulted to the highest bacteriocin yields. Thus, a 2(4) factorial design with the four variables was performed, and statistical analysis showed that it was an adequate model (R (2) = 0.8296). In the studied range, the four parameters significantly influenced bacteriocin production, with the maximum yield produced at an initial pH between 5.5 and 7.0, a temperature between 25 and 30 °C and supplementation of the MRS broth with glucose from 3.25 to 6.0 g L(-1) and Tween 20 from 0.575 to 1.15% (v/v). Response Surface Methodology analysis indicated that the highest bacteriocin production (12800 AU mL(-1)) occurred in the MRS broth supplemented with 5.5 g L(-1) glucose and 1.05% Tween 20 at an initial pH of 6.28 and an incubation temperature of 25 °C. The amount of bacteriocin produced in commercial MRS broths under the same conditions was only 5600AU mL(-1).

Optimization of growth and bacteriocin production by Lactobacillus sakei subsp. sakei2a

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacterium isolated from Brazilian pork sausage, capable of inhibiting the growth of microbial pathogens, mainly Listeria monocytogenes. In order to optimize bacteriocin production for industrial applications, this study evaluated the effect of supplementation of MRS broth with glucose, Tween 20, Tween 80, sodium citrate, potassium chloride and cysteine, and effect of the initial pH and temperature of incubation of the medium on production of bacteriocins by L. sakei 2a. Adding glucose and Tween 20 to the medium, an initial pH of 5.0 or 5.5, and incubation temperatures of 25 °C or 30 °C resulted to the highest bacteriocin yields. Thus, a 2⁴ factorial design with the four variables was performed, and statistical analysis showed that it was an adequate model (R² = 0.8296). In the studied range, the four parameters significantly influenced bacteriocin production, with the maximum yield produced at an initial pH between 5.5 and 7.0, a temperature between 25 and 30 °C and supplementation of the MRS broth with glucose from 3.25 to 6.0 g L⁻¹ and Tween 20 from 0.575 to 1.15% (v/v). Response Surface Methodology analysis indicated that the highest bacteriocin production (12800 AU mL⁻¹) occurred in the MRS broth supplemented with 5.5 g L⁻¹ glucose and 1.05% Tween 20 at an initial pH of 6.28 and an incubation temperature of 25 °C. The amount of bacteriocin produced in commercial MRS broths under the same conditions was only 5600AU mL⁻¹..(AU)

Optimization of growth and bacteriocin production by Lactobacillus sakei subsp. sakei2a

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacterium isolated from Brazilian pork sausage, capable of inhibiting the growth of microbial pathogens, mainly Listeria monocytogenes. In order to optimize bacteriocin production for industrial applications, this study evaluated the effect of supplementation of MRS broth with glucose, Tween 20, Tween 80, sodium citrate, potassium chloride and cysteine, and effect of the initial pH and temperature of incubation of the medium on production of bacteriocins by L. sakei 2a. Adding glucose and Tween 20 to the medium, an initial pH of 5.0 or 5.5, and incubation temperatures of 25 °C or 30 °C resulted to the highest bacteriocin yields. Thus, a 2⁴ factorial design with the four variables was performed, and statistical analysis showed that it was an adequate model (R² = 0.8296). In the studied range, the four parameters significantly influenced bacteriocin production, with the maximum yield produced at an initial pH between 5.5 and 7.0, a temperature between 25 and 30 °C and supplementation of the MRS broth with glucose from 3.25 to 6.0 g L⁻¹ and Tween 20 from 0.575 to 1.15% (v/v). Response Surface Methodology analysis indicated that the highest bacteriocin production (12800 AU mL⁻¹) occurred in the MRS broth supplemented with 5.5 g L⁻¹ glucose and 1.05% Tween 20 at an initial pH of 6.28 and an incubation temperature of 25 °C. The amount of bacteriocin produced in commercial MRS broths under the same conditions was only 5600AU mL⁻¹.

Characterization of the spoilage lactic acid bacteria in "sliced vacuum-packed cooked ham".

The lactic acid bacteria are involved with food fermentation and in such cases with food spoilage. Considering the need to reduce the lactic acid bacteria growth in meat products, the aim of this work was to enumerated and investigated the lactic acid bacteria present on sliced vacuum-packed cooked ham stored at 4 °C and 8 °C for 45 days by phenotypic and molecular techniques. The quantification showed that the lactic acid bacteria were present from the first day with mean count of 1.98 log cfu/g for the four batches analyzed. The lactic acid bacteria grew rapidly on the samples, and plate counts around 7.59 log cfu/g and 8.25 log cfu/g were detected after 45 days of storage at 4 °C and 8 °C, respectively; storage temperatures studied showed significant influence on the microorganism in study growth. The predominant lactic acid bacteria associated with the spoilage samples at one day of storage includes Lactobacillus sp., the phenotypic overlap Leuconostoc / Weissella sp. and Enterococcus sp. At 45 days of storage at 4 and 8 °C the mainly specie was Lactobacillus curvatus , following by Lactobacillus sakei and Leuconostoc mesentereoides ; the Enterococcus sp. was not present in the samples.

Characterization of the spoilage lactic acid bacteria in “sliced vacuum-packed cooked ham”

The lactic acid bacteria are involved with food fermentation and in such cases with food spoilage. Considering the need to reduce the lactic acid bacteria growth in meat products, the aim of this work was to enumerated and investigated the lactic acid bacteria present on sliced vacuum-packed cooked ham stored at 4 °C and 8 °C for 45 days by phenotypic and molecular techniques. The quantification showed that the lactic acid bacteria were present from the first day with mean count of 1.98 log cfu/g for the four batches analyzed. The lactic acid bacteria grew rapidly on the samples, and plate counts around 7.59 log cfu/g and 8.25 log cfu/g were detected after 45 days of storage at 4 °C and 8 °C, respectively; storage temperatures studied showed significant influence on the microorganism in study growth. The predominant lactic acid bacteria associated with the spoilage samples at one day of storage includes Lactobacillus sp., the phenotypic overlap Leuconostoc/Weissella sp. and Enterococcus sp. At 45 days of storage at 4 and 8 °C the mainly specie was Lactobacillus curvatus, following by Lactobacillus sakei and Leuconostoc mesentereoides; the Enterococcus sp. was not present in the samples.

Characterization of the spoilage lactic acid bacteria in “sliced vacuum-packed cooked ham”

The lactic acid bacteria are involved with food fermentation and in such cases with food spoilage. Considering the need to reduce the lactic acid bacteria growth in meat products, the aim of this work was to enumerated and investigated the lactic acid bacteria present on sliced vacuum-packed cooked ham stored at 4 °C and 8 °C for 45 days by phenotypic and molecular techniques. The quantification showed that the lactic acid bacteria were present from the first day with mean count of 1.98 log cfu/g for the four batches analyzed. The lactic acid bacteria grew rapidly on the samples, and plate counts around 7.59 log cfu/g and 8.25 log cfu/g were detected after 45 days of storage at 4 °C and 8 °C, respectively; storage temperatures studied showed significant influence on the microorganism in study growth. The predominant lactic acid bacteria associated with the spoilage samples at one day of storage includes Lactobacillus sp., the phenotypic overlap Leuconostoc/Weissella sp. and Enterococcus sp. At 45 days of storage at 4 and 8 °C the mainly specie was Lactobacillus curvatus, following by Lactobacillus sakei and Leuconostoc mesentereoides; the Enterococcus sp. was not present in the samples.(AU)

Dietary administration of microalgae alone or supplemented with Lactobacillus sakei affects immune response and intestinal morphology of Pacific red snapper (Lutjanus peru).

The aim of this study was to evaluate the effect of dietary microalgae Navicula sp single or in combination with Lactobacillus sakei on growth performance, humoral immune parameters and intestinal morphology in Pacific red snapper, Lutjanus peru. The experimental fish were grouped into four treatment diets which were a control diet (commercial diet, Control), silage microalgae Navicula sp plus L. sakei (10(6) CFU g(-1), Navicula + L. sakei), lyophilized microalgae (Navicula) and L. sakei (10(6) CFU g(-1), L. sakei). The blood and intestine samples were collected on week 4 and 8. The weight gain showed an additive effect of Navicula + L. sakei at 8 weeks of treatment compared with fish fed control diet. Overall, physiological parameters such as total protein and hemoglobin were increased in fish fed with Navicula and L. sakei diets at 4 and 8 weeks of feeding assay, respectively. There was a significant improvement in immune parameters, principally in myeloperoxidase, lysozyme, total antiproteases activities and IgM in fish fed with Navicula + L. sakei and L. sakei diets at 4 or 8 weeks of treatments. Serum antioxidant capabilities revealed significant increase in phosphatase alkaline, esterase, protease, superoxide dismutase and catalase in groups which received diet supplemented with Navicula + L. sakei and L. sakei diets. Finally, light microscopy observations revealed no effect of experimental diets on microvilli height. Curiously, the presence of vacuoles inside the enterocytes was significant higher in the intestine of L. sakei group after four or six weeks of feeding. Elevated intraepithelial leucocyte levels and melanomacrophages centers were observed in fish fed Navicula or control diets at any time of the experiment. To conclude, the results of the present study demonstrate that the fish that were fed with Navicula + L. sakei or L. sakei diets yielded significantly better immune status and antioxidant capabilities.

Purification and characterization of the bacteriocin produced by Lactobacillus sakei MBSa1 isolated from Brazilian salami.

AIMS: The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. METHODS AND RESULTS: Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-ß, X, P, G and Q). CONCLUSIONS: In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.

Evaluation of bacteriocin-producing Lactobacillus sakei 1 against Listeria monocytogenes 1/2a growth and haemolytic activity

Bacteriocin-producing Lactobacillus sakei 1 was cultivated in Brain-Heart Infusion broth (24 h at 25ºC). The culture supernatant was neutralized, filter sterilized and used to test the activity of bacteriocin against Listeria monocytogenes 1/2a, at 8ºC and 15ºC. Non-bacteriocinogenic Lactobacillus sakei ATCC 15521 was used as a negative control. L. monocytogenes 1/2a was inoculated in culture supernatant medium from L. sakei 1 and L. sakei ATCC 15521 and the listerial populations were determined after 0, 5 and 10 days. The bacteriocin production was quantified as arbitrary units per mL (AU/mL) using agar antagonism test. Additionally, to investigate if L. monocytogenes virulence pattern could be changed after bactericion exposure, the ability of L. monocytogenes to cause haemolysis in sheep red blood cells was determined, before and after exposure to bacteriocin at 8ºC. In the presence of the antimicrobial peptide, at 8ºC, L. monocytogenes population decreased, but growth of resistant cells was observed. At 15ºC, there was no difference between test and control. Furthermore, the haemolytic activity of L. monocytogenes 1/2a was not altered by exposure to L. sakei 1 bacteriocin, which suggests no change in its virulence pattern.

Lactobacillus sakei 1 produtor de bacteriocina foi cultivado em caldo Infusão Cérebro-Coração por 24h a 25ºC. O sobrenadante da cultura foi neutralizado, esterilizado por filtração e usado para testar a atividade da bacteriocina frente a Listeria monocytogenes 1/2a, a 8ºC e 15ºC. Lactobacillus sakei ATCC 15521 não bacteriocinogênico, foi utilizado como controle negativo. L. monocytogenes 1/2a foi inoculada no sobrenadante da cultura de L.sakei 1 e L. sakei ATCC 15521 e as populações listeriais foram determinadas após 0, 5 e 10 dias. A produção de bacteriocina foi quantificada como unidades arbitrárias por mL (UA/mL), utilizando-se o teste de antagonismo em ágar. Adicionalmente, para investigar se o padrão de virulência de L. monocytogenes poderia ter mudado após exposição à bacteriocina, foi avaliada a habilidade de L. monocytogenes em causar hemólise em hemáceas de carneiro, antes e após exposição à bacteriocina a 8ºC. Na presença do peptídeo antimicrobiano, a 8ºC, a população de L. monocytogenes foi reduzida, mas o desenvolvimento de células resistentes foi observado. A 15ºC, não houve diferença entre os grupos controle e teste. Além disso, a atividade hemolítica de L. monocytogenes 1/2a não foi alterada pela exposição à bacteriocina de L. sakei, 1/2a não foi alterada pela exposição à bacteriocina de L. sakei, o que sugere que não houve mudança em seu padrão de virulência.