Antimicrobial Effect of Simira ecuadorensis Extracts and Their Impact on Improving Shelf Life in Chicken and Fish Products.

The objective of this work was to evaluate the antimicrobial potential of different extracts of Simira ecuadorensis, a characteristic plant of Ecuador, and to validate its potential as a food preservative. Four extracts referred to as ethanol, ethanol-water (50:50 v/v), spray-dried, and freeze-dried were obtained under different processes. Initially, their antimicrobial activities were evaluated against a wide group of microorganisms consisting of 20 pathogenic and spoilage microbial strains found in foods through the agar diffusion method. Then, the extracts with the best yields and antimicrobial properties against microorganisms of greatest interest were selected to determine their effect on model foods preserved under normal commercial conditions through challenge tests. Spray-dried and ethanol-water extracts were tested for their ability to inhibit C. jejuni in chicken model products, where is a common pathogen and Shew. putrefaciens in fish model products as it is a spoilage microorganism frequently found in fish. One solid and one liquid were chosen as model foods: burger and broth, respectively. Campylobacter jejuni and Shewanella putrefaciens were effectively inhibited by the four extracts with minimum inhibitory concentration (MIC) of 80 mg/mL. Bacillus cereus, Yersinia enterocolitica, Clostridium perfringens, and Leuconostoc mesenteroides were also inhibited by ethanolic extract. The ethanol-water extract showed greater antimicrobial activity in fish products, whereas spray-dried extract had low growth inhibition of C. jejuni in chicken burgers; however, it was quite effective on C. jejuni in broth. The spray-dried extract significantly decreased the pH of the chicken burgers, while the ethanolic extract had a slight impact on the pH of the fish burgers. The presence of antibacterial effects revealed that the S. ecuadorensis extracts could be potentially used in food preservation and as a natural antimicrobial.

Genotyping, virulence genes and antimicrobial resistance of Campylobacter spp.isolated during two seasonal periods in Spanish poultry farms.

Campylobacter spp. are the leading causes of bacterial human gastroenteritis worldwide; being poultry farms the main source of infections. In order to obtain information on prevalence and diversity of Campylobacter-infected flocks in the North of Spain, fourteen farms were studied between autumn and spring in 2014 and 2015, respectively. Moreover, virulence genes involved in pathogenicity and antimicrobial resistance were investigated. A survey about preventive hygiene practices at farms was performed to determine the risky practices that could contribute to the presence of Campylobacter in this step of the poultry food chain. Testing the presence of Campylobacter spp. showed 43 % of the farms were positive during autumn, whereas only 31 % were positive in spring. A very high prevalence within-flock was observed (43.1 % to 88.6 %) and C. jejuni was the most prevalent species in both periods. Genotyping by pulsed field gel electrophoresis (PFGE) showed a high heterogeneity among farms (309 isolates clustered into 21 pulsotypes). Virulence genes were present in all C. jejuni isolates while cdtA and cdtC were absent in C. coli. On the contrary, the latter showed higher antimicrobial resistance than C. jejuni. This study suggests that environment might be one of the main sources for Campylobacter transmission, as water supply seemed to be a clear cause of the contamination in a specific farm. However, in other farms other environmental factors contributed to the contamination, confirming the multifactorial origin of Campylobacter colonization in broilers. Therefore, biosecurity measures in farms are crucial to reduce Campylobacter contamination, which may have important implications for human and animal health.

A mathematical modeling approach to the quantification of lactic acid bacteria in vacuum-packaged samples of cooked meat: Combining the TaqMan-based quantitative PCR method with the plate-count method.

The TaqMan-based quantitative Polymerase Chain Reaction (qPCR) method and the Plate Count (PC) method are both used in combination with primary and secondary mathematical modeling, to describe the growth curves of Leuconostoc mesenteroides and Weissella viridescens in vacuum-packaged meat products during storage under different isothermal conditions. Vacuum-Packaged Morcilla (VPM), a typical cooked blood sausage, is used as a representative meat product, with the aim of improving shelf-life prediction methods for those sorts of meat products. The standard curves constructed by qPCR showed good linearity between the cycle threshold (CT) and log10 CFU/g, demonstrating the high precision and the reproducible results of the qPCR method. The curves were used for the quantification of L. mesenteroides and W. viridescens in artificially inoculated VPM samples under isothermal storage (5, 8, 13 and 18 °C). Primally, both the qPCR and the PC methods were compared, and a linear regression analysis demonstrated a statistically significant linear correlation between the methods. Secondly, the Baranyi and Roberts model was fitted to the growth curve data to estimate the kinetic parameters of L. mesenteroides and W. viridescens under isothermal conditions, and secondary models were used to establish the dependence of the maximum specific growth rate on the temperature. The results proved that primary and secondary models were adequate for describing the growth curves of both methods in relation to both bacteria. In conclusion, the results of all the experiments proved that the qPCR method in combination with the PC method can be used to construct microbial growth kinetics and that primary and secondary mathematical modeling can be successfully applied to describe the growth of L. mesenteroides and W. viridescens in vacuum-packaged morcilla and, by extension, other cooked meat products with similar characteristics.

Characterization by culture-dependent and culture-independent methods of the bacterial population of suckling-lamb packaged in different atmospheres.

This study offers insight into the dynamics of bacterial populations in fresh cuts of suckling lamb under four different atmospheric conditions: air (A), and three Modified Atmosphere Packaging (MAP) environments, 15%O2/30%CO2/55%N2 (C, commercial), 70%O2/30%CO2 (O), and 15%O2/85%CO2 (H) for 18 days. Microbial analyses by both conventional methods and PCR-DGGE were performed. Controversial and surprising results emerged from comparing both methods in relation to the genus Pseudomonas. Thus, conventional methods detected the presence of high numbers of Pseudomonas colonies, although PCR-DGGE only detected this genus in air-packaged samples. PCR-DGGE detected higher microbial diversity in the control samples (A) than in the modified atmospheres (C, O, H), having atmosphere H the fewest number of species. Brochothrix thermosphacta, LAB (Carnobacterium divergens and Lactobacillus sakei), and Escherichia spp. were detected in all the atmospheres throughout storage. Moreover, previously undescribed bacteria from lamb meat such as Enterobacter hormaechei, Staphylococcus equorum and Jeotgalicoccus spp. were also isolated in this study by DGGE. Additionally, qPCR analysis was used to detect and characterize strains of Escherichia coli. Virulence genes (stx1, stx2 and eae) were detected throughout storage in 97% of the samples. A high CO2 atmosphere was the most effective packaging combination doubling storage time in comparison with commercial atmosphere.

Behaviour of non-stressed and stressed Listeria monocytogenes and Campylobacter jejuni cells on fresh chicken burger meat packaged under modified atmosphere and inoculated with protective culture.

Numerous investigations have provided evidence that chicken products are a source of Listeria monocytogenes and Campylobacter jejuni. Different strategies applied in final products are needed to prevent consumers' contamination. In this work, the combination of modified atmosphere packaging (MAP) and protective culture to control the growth of freeze stressed and non-stressed L. monocytogenes and C. jejuni on fresh chicken meat burger was studied. Meat burgers were inoculated with L. monocytogenes, C. jejuni and Leuconostoc pseudomesenteroides PCK 18, as protective strain against L. monocytogenes. Prior to the addition of the protective culture, half of the L. monocytogenes and C. jejuni - inoculated meat was frozen at -18°C for 48 h to subject cells to stress. Following the addition of the protective culture, meat burgers were packaged in air or MAP (50% CO(2)/50% O(2)) and stored under refrigeration conditions. L. monocytogenes counts were not reduced by the freezing temperature applied; however, the addition of Lc. pseudomesenteroides PCK 18 reduced its counts for 0.90 log cfu/g when chicken meat burgers were packaged under MAP. Furthermore, freezing stress was an effective strategy to reduce C. jejuni counts but only in combination with a high-O(2) MAP, it was completely eliminated. Chicken meat burgers' shelf-life under aerobic packaging conditions was reduced by the effect of freeze-thawing, while the use of MAP extended the product's shelf-life till 21days. Therefore, the combination of freezing, protective culture and MAP could extend the shelf-life and enhance the food safety of this kind of chicken products.

The influence of different preservation methods on spoilage bacteria populations inoculated in morcilla de Burgos during anaerobic cold storage.

Blood sausage is a widely consumed traditional product that would benefit from an extended shelf life. The two main spoilage bacteria in vacuum-packaged morcilla de Burgos are Weissella viridescens and Leuconostoc mesenteroides. This study examines the way in which three preservation treatments--organic acid salts (OAS), high-pressure processing (HPP) and pasteurization--influence these bacterial populations and their spoilage behaviour. HPP and pasteurization treatments were found to inhibit growth of the inoculated species and delay sensory spoilage of the product. In both treatments, L. mesenteroides was observed to have a longer recovery time; even so, once its growth started, it grew faster than W. viridescens. This longer recovery time might be due to metabolic modification following treatment, which would affect the production of metabolites such as acetic acid and some aldehydes. W. viridescens was the first strain to recover from the two treatments. It preserved its spoilage behaviour and even increased the production of certain compounds such as acetoin or ethanol. The extended product shelf life following HPP and pasteurization treatments might be due to a combination of various factors such as the fall in both microbial populations, as well as the delay in spoilage caused by damage to L. mesenteroides cells, as this strain is the fastest-acting, most intensive spoilage microorganism. It was observed that the addition of organic salts neither diminished nor delayed the growth of the two inoculated species. Nevertheless, the results also indicate that this treatment inhibits the metabolic activity of L. mesenteroides, resulting once again in an extended product shelf life.

Microbial, sensory and volatile changes during the anaerobic cold storage of morcilla de Burgos previously inoculated with Weissella viridescens and Leuconostoc mesenteroides.

Blood sausage, a widely consumed traditional product, would benefit from an increased commercial life. It is therefore pertinent to investigate the type, the evolution, and the behaviour of the Lactic Acid Bacteria responsible for their spoilage. This study aims to clarify the role played by Weissella viridescens and Leuconostoc mesenteroides, identified as their principal spoilage agents in vacuum-packaged morcilla de Burgos, through the study of microbiological, sensory, and volatile profile changes, following inoculation of the morcilla, both jointly and separately, with the two species. L. mesenteroides grew more rapidly and influenced the drop in pH, milky exudates and the sour smell, whereas W. viridescens influenced vacuum loss. With respect to volatile profiles, L. mesenteroides samples were richer in aldehydes (hexanal) and acids (acetic), on the contrary W. viridescens samples showed greater amounts of alcohols (ethanol) and ketones (acetoin and diacetyl). Both species inoculated together increased particular signs of morcilla spoilage.

Effectiveness of combined preservation methods to extend the shelf life of Morcilla de Burgos.

Morcilla de Burgos is the most famous blood sausage in Spain. However, while producers are interested in extending its shelf life, the consumer is increasingly demanding more natural food. This situation has led to the current search for new and mild preservation technologies. Two batches of four different products: control without any treatment, control with organic acid salts (CnOAS; a 3% mixture of potassium/sodium l-lactate), control with high hydrostatic pressure processing (CnHPP; 600MPa-10min), and a combination of both treatments (OAS+HPP), were carried out to evaluate any synergistic effect that occurs when combining OAS and HPP, and the influence of different preservative treatments on the spoilage bacterial population and their evolution. HPP (with or without addition of OAS) can be considered the most suitable method for preserving morcilla de Burgos as it does not produce negative changes in sensory attributes. No clear selective effect of different treatments on the composition of the spoilage bacteria was seen and similar spoilage patterns were observed independently of the preservation treatment used.

Spoilage of blood sausages morcilla de Burgos treated with high hydrostatic pressure.

In this study, the microbial ecology of the blood sausage morcilla de Burgos, subjected to high hydrostatic pressure treatment (HPP), was studied by culture-dependent and -independent methods. Morcilla de Burgos is the most traditional and famous blood sausage in Spain. The producers are interested in extending its shelf-life in order to expand their market and to reduce losses attributed to spoilage. Sausage batter prior to stuffing and blood sausages HPP treated or not (control) were analyzed at 0, 9, 14, 21, 28 and 35 days of storage at 4 degrees C. Lactic acid bacteria, Pseudomonas spp. and aerobic mesophilic bacteria were investigated by traditional plating. PCR-denaturing gradient gel electrophoresis (DGGE) was used to analyze the DNA and the RNA extracted directly from the blood sausages, as well as bulk cells of LAB and Pseudomonas spp. The results showed that HPP improved the shelf life of morcilla de Burgos to 28 days in comparison with control samples. The populations responsible for spoilage, namely LAB, remained lower in HPP treated samples when compared with the control samples. Only at 35 days of storage they reached values of 10(8) cfu/g, leading to the spoilage of the product. Although, HPP affected the LAB population, they were able to recover the injury provoked by the treatment. Lastly, HPP seemed to affect differently LAB species detected. While Leuconostoc mesenteroides was completely inactivated by HPP, Weissella viridescens was able to recover and carry out the typical spoilage of the product. Pseudomonas spp. remained under detection level (<10(2) CFU/g) after the HPP treatment.

Application of organic acid salts and high-pressure treatments to improve the preservation of blood sausage.

Blood sausages are traditional products in many parts of the world. In most cases, a very short shelf-life limits their consumption to the areas in which they are produced. In this work, different mild preservation methods were applied to Morcilla de Burgos, a Spanish blood sausage, consisting of a range of organic acid salts (OAS) and high-pressure processing (HPP), with the aim of increasing its shelf-life. In the first experiment, three batches of morcillas were produced using three different commercial OAS-PL (3% potassium lactate), PL+SL (3% potassium and sodium lactate) and PL+SD (2.5% potassium lactate and sodium diacetate)-together with a control batch and were stored under chill conditions (4 degrees C) for 35 days. In a further experiment, vacuum-packaged morcillas were treated at three different pressure levels-300, 500 and 600MPa-for 10min, and stored under chill conditions for 28 days. In both batches, a sensory difference test was performed on day 1 after treatment and the morcilla samples were subjected to microbiological and sensory analysis after each week in storage. The results suggest that, in both cases, an addition of PL+SL and the application of 600MPa for 10min increases the shelf-life of the morcillas by 15 days. Once again, it is evident that the initial opportunities for contamination play a very important role in improving the shelf-life of food products.

Microbiological and sensory changes in "Morcilla de Burgos" preserved in air, vacuum and modified atmosphere packaging.

"Morcilla de Burgos" is the most popular blood sausage in Spain. Traditionally, this product is distributed and sold without packaging in the local market. To extend its shelf-life and expand the market, different packaging methods have been employed and compared: "morcilla" stored in air (without packaging), in vacuum and in modified atmosphere packaging (MAP) using three different CO(2) concentrations 30%, 50% and 80% and balanced with N(2). Total viable count (TVC), psychrotrophs, lactic acid bacteria (LAB), pseudomonads, enterobacteria, moulds and yeasts, enterococci, Staphylococcus aureus, and sulfite reducing clostridia were analysed during storage at 4°C. Sulfite-reducing clostridia, pathogenic staphylococci, and enterococci were not detected in any sample. In air-stored "morcilla" a significant increase in all microbial groups was observed during storage. Pseudomonads were the predominant microorganisms reaching a population higher than 8 log cfu/g after 27 days of storage. On the other hand, a decrease in pH was noticed in MAP and in vacuum packaged "morcilla" (pH 4.73) during storage. At the same time, LAB becomes the predominant species in all these packaged samples. The rest of the microbiota did not grow during storage. In "morcilla" packed with 50% and 80% of CO(2), counts of pseudomonads and enterobacteria were lower than found in the vacuum packs. Sensory analysis showed that shelf-life of "morcilla" stored in air did not exceed 17 days, while samples packed under vacuum and with 30% CO(2) were acceptable until 22 days of storage. "Morcillas" packaged with 50% and 80% CO(2) were sensorially acceptable for 32 days.