Brown Macroalgae (Phaeophyceae): A Valuable Reservoir of Antimicrobial Compounds on Northern Coast of Spain.

The search for new sources of antimicrobial compounds has become an urgent need, due to the threat that the spread of bacterial resistance represents for global health and food safety. Brown macroalgae have been proposed as a great reservoir in the search for novel antimicrobial compounds. In this study, mid-polarity extracts were performed with a selection of 20 brown macroalgae species from northern Spain. The total polyphenol, carbohydrate and protein contents were quantified by spectrophotometry. The volatile organic compounds (VOCs) of whole macroalgae were also studied as a biomarker of their metabolic state in the representative species of the tested families by gas chromatography-mass spectrometry (GC-MS). The antimicrobial potential of the extracts was assessed by a disk diffusion assay against 20 target bacteria and further determinations of the minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) were performed by a microdilution assay for the active extracts. Ericaria selaginoides, Bifurcaria bifurcata and Dictyota dichotoma showed an antimicrobial effect against six Gram-positive strains: Bacillus cereus, Bacillus subtilis, Geobacillus stearothermophilus, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus haemolyticus. The phenolic content was generally higher in the extracts that showed antimicrobial activity, followed by carbohydrates and low contents of proteins. The results obtained in this study reveal the potential of brown macroalgae as a promising alternative source of antimicrobial compounds as functional ingredients for the application in industrial fields.

Pulsed Light Application for Campylobacter Control on Poultry Meat and Its Effect on Colour and Volatile Profile.

Campylobacter on poultry meat needs to be controlled to reduce the risk of infection caused by the consumption of chicken meat. Pulsed light (PL) application on poultry meat was studied to control Campylobacter spp. The effect of this technology was evaluated regarding poultry meat colour and volatile compound changes. Two breast sample groups were prepared: inoculated with Campylobacter (107 bacteria of Campylobacter jejuni strains) and not inoculated. Samples were submitted to PL, five pulses/s of 300 ms, 1 Hz, and 1 J/cm2 in the apparatus, PL Tecum unit (Claranor). A response surface experimental design was applied regarding the factors of voltage (1828 to 3000 W) and distance to the source UV lamp (2.6 to 5.4 cm). The binomial factorial treatment (voltage and distance) with PL induced different energy doses (fluence J/cm2) received by samples, 2.82 to 9.67 J/cm2. Poultry meat pulsed light treated had a significant decrease of Enterobacteriaceae counts. The treatments applied were unable to reduce 1 log Campylobacter cfu/g of poultry meat. The poultry meat PL treated became slightly light, redder, and yellower than those not treated. PL can decrease the proportion of aldehydes on total volatiles in meat, particularly on those associated with chicken-like, chicken skin-like, and sweet odour notes in fresh poultry meat. Further studies of PL with higher energy doses will be necessary to confirm if there are Campylobacter reductions and about poultry meat treated under storage to evaluate if volatile compounds can affect the flavour of PL-treated meat samples.

Enterocin A-based antimicrobial film exerted strong antilisterial activity in sliced dry-cured ham immediately and after 6 months at 8 °C.

To minimize the survival of Listeria monocytogenes on ready-to-eat (RTE)-products, active antimicrobial packaging based on polyvinyl alcohol films with Enterocin A or ethyl-lauroyl-arginate (LAE) have been designed and its antimicrobial activity assessed in vacuum-packed sliced dry-cured ham stored under refrigeration. The Enterocin A-based antimicrobial film exerted a strong antilisterial activity, causing an immediate reduction of L. monocytogenes counts of 1 log units compared with the control without antimicrobial. Besides, Enterocin A film enhanced (4-fold higher) the die-off rate along the 6 months of storage at 8 °C. The antilisterial effect of Enterocin A film applied on dry-cured ham complies with the performance criteria requirement of Alternative 1 of the US Listeria rule regarding the control of L. monocytogenes. Films made with LAE did not exert an immediate bactericidal effect but slightly increased the die-off rate of the pathogen and reduced its counts during the shelf life compared to the control batch.

Assessment of the bioprotective potential of lactic acid bacteria against Listeria monocytogenes on vacuum-packed cold-smoked salmon stored at 8 °C.

Smoked salmon is a highly appreciated delicatessen product. Nevertheless, this ready-to-eat (RTE) product is considered at risk for Listeria monocytogenes, due to both the prevalence and growth potential of this bacteria on the product. Biopreservation may be considered a mild and natural effective strategy for minimizing this risk. In this study, we evaluated the following three potential bioprotective lactic acid bacterial strains against L. monocytogenes in three smoked salmon types with different physicochemical characteristics, primarily fat, moisture, phenol and acid acetic content: two bacteriocin-like producers that were isolated from smoked salmon and identified as Lactobacillus curvatus and Carnobacterium maltaromaticum and a recognized bioprotective bacteriocin producer from meat origin, Lactobacillus sakei CTC494. L. sakei CTC494 inhibited the growth of L. monocytogenes after 21 days of storage at 8 °C in all the products tested, whereas L. curvatus CTC1742 only limited the growth of the pathogen (<2 log increase). The effectiveness of C. maltaromaticum CTC1741 was dependent on the product type; this strain limited the growth of the pathogen in only one smoked salmon type. These results suggest that the meat-borne starter culture, L. sakei CTC494, may potentially be used as a bioprotective culture to improve the food safety of cold-smoked salmon.

MLVA subtyping of Listeria monocytogenes isolates from meat products and meat processing plants.

Listeria monocytogenes is widely distributed in meat products and the meat-processing industry thus posing a risk to consumers. The aim of this study was to evaluate the suitability of the multilocus variable-number tandem-repeat analysis (MLVA) for use as a L. monocytogenes subtyping technique for surveillance and routine control in meat products and meat processing plants. A collection of 113 isolates (including control strains and isolates from meat products and meat processing plants) were subject to MLVA analysis using two different platforms for fragment sizing: 1.) ABI 3730xl DNA analyzer (Life Technologies) as the reference method and 2.) The QIAxcel Advanced System (Qiagen). Although discrepancies in fragment sizing were observed it was possible to standardize results in order to assign the same allele for a given fragment independently of the platform used for fragment sizing. A total of 27 different MLVA profiles were obtained considering all the isolates (N=113), 24 of them corresponding to the meat industry isolates (N=106). MLVA and multilocus sequence typing (MLST) results were compared and yielded Simpson's diversity indices of 0.907 and 0.872, respectively. The congruence between both typing methods was measured with the adjusted Wallace coefficient (AW). Using MLVA as the primary method, AW=0.946 suggested that MLVA can predict the sequence type with high accuracy. Given its discriminatory power and high throughput, MLVA could be considered a rapid, reliable, and high-throughput alternative to existing subtyping methods for surveillance and control of L. monocytogenes in the meat-processing industry.

Diversity and distribution of Listeria monocytogenes in meat processing plants.

Listeria monocytogenes is a major concern for the meat processing industry because many listeriosis outbreaks have been linked to meat product consumption. The aim of this study was to elucidate L. monocytogenes diversity and distribution across different Spanish meat processing plants. L. monocytogenes isolates (N = 106) collected from food contact surfaces of meat processing plants and meat products were serotyped and then characterised by multilocus sequence typing (MLST). The isolates were serotyped as 1/2a (36.8%), 1/2c (34%), 1/2b (17.9%) and 4b (11.3%). MLST identified ST9 as the most predominant allelic profile (33% of isolates) followed by ST121 (16%), both of which were detected from several processing plants and meat products sampled in different years, suggesting that those STs are highly adapted to the meat processing environment. Food contact surfaces during processing were established as an important source of L. monocytogenes in meat products because the same STs were obtained in isolates recovered from surfaces and products. L. monocytogenes was recovered after cleaning and disinfection procedures in two processing plants, highlighting the importance of thorough cleaning and disinfection procedures. Epidemic clone (EC) marker ECI was identified in 8.5%, ECIII was identified in 2.8%, and ECV was identified in 7.5% of the 106 isolates. Furthermore, a selection of presumably unrelated ST9 isolates was analysed by multi-virulence-locus sequence typing (MVLST). Most ST9 isolates had the same virulence type (VT11), confirming the clonal origin of ST9 isolates; however, one ST9 isolate was assigned to a new VT (VT95). Consequently, MLST is a reliable tool for identification of contamination routes and niches in processing plants, and MVLST clearly differentiates EC strains, which both contribute to the improvement of L. monocytogenes control programs in the meat industry.

Molecular basis of the behavior of hepatitis a virus exposed to high hydrostatic pressure.

Food-borne hepatitis A outbreaks may be prevented by subjecting foods at risk of virus contamination to moderate treatments of high hydrostatic pressure (HHP). A pretreatment promoting hepatitis A virus (HAV) capsid-folding changes enhances the virucidal effect of HHP, indicating that its efficacy depends on capsid conformation. HAV populations enriched in immature capsids (125S provirions) are more resistant to HHP, suggesting that mature capsids (150S virions) are more susceptible to this treatment. In addition, the monoclonal antibody (MAb) K24F2 epitope contained in the immunodominant site is a key factor for the resistance to HHP. Changes in capsid folding inducing a loss of recognition by MAb K24F2 render more susceptible conformations independently of the origin of such changes. Accordingly, codon usage-associated folding changes and changes stimulated by pH-dependent breathings, provided they confer a loss of recognition by MAb K24F2, induce a higher susceptibility to HHP. In conclusion, the resistance of HAV to HHP treatments may be explained by a low proportion of 150S particles combined with a good accessibility of the epitope contained in the immunodominant site close to the 5-fold axis.

The potential probiotic Lactobacillus rhamnosus CTC1679 survives the passage through the gastrointestinal tract and its use as starter culture results in safe nutritionally enhanced fermented sausages.

The human-derived potential probiotic strain Lactobacillus rhamnosus CTC1679 was used as a starter culture in reduced fat and sodium low-acid fermented sausages (fuets) to assess its ability to survive through the gastrointestinal tract (GIT) in a human intervention study consisting of 5 healthy volunteers who consumed 25 g fuet a day for 21 days. Faecal samples were analysed during and after consumption. L. rhamnosus CTC1679 produced a transient colonisation of the human GIT and persisted during the ingestion period of fuet containing L. rhamnosus CTC1679 at levels ca. 8log CFU/g. After 3 days of non-consumption, the strain was still recovered in the faeces of all the volunteers. To evaluate the safety of the nutritionally enhanced manufactured fuets, a challenge test was designed in a separately manufactured batch. L. rhamnosus CTC1679 was able to grow, survive and dominate (levels ca. 10(8) CFU/g) the endogenous lactic acid bacteria (LAB), prevented the growth of Listeria monocytogenes throughout the whole ripening process of the fuets and eliminated Salmonella. After 35 days of storage at 4 °C, L. monocytogenes was not detected, achieving absence in 25 g of the product. The application of high hydrostatic pressure (HHP) treatment (600 MPa for 5 min) at the end of ripening (day 14) produced an immediate reduction of L. monocytogenes to levels <1log CFU/g. After 35 days of storage at 4 °C the pathogen was not detected. Thus, the strain L. rhamnosus CTC1679 is a suitable starter culture for producing safe potentially probiotic fermented sausages.

Nutritionally enhanced fermented sausages as a vehicle for potential probiotic lactobacilli delivery.

The suitability of three potential probiotic lactobacilli strains (Lactobacillus casei CTC1677, L. casei CTC1678 and Lactobacillus rhamnosus CTC1679), previously isolated from infants' faeces and characterized, and three commercial probiotic strains (Lactobacillus plantarum 299v, L. rhamnosus GG and L. casei Shirota) was assessed during the manufacture of low-acid fermented sausages (fuets) with reduced Na(+) and fat content. The inoculated strains were successfully monitored by RAPD-PCR during the process. L. rhamnosus CTC1679 was the only strain able to grow and dominate (levels ca. 10(8)CFU/g) the endogenous lactic acid bacteria population in two independent trials, throughout the ripening process. Thus, fuet containing L. rhamnosus CTC1679 as a starter culture could be a suitable vehicle for putative probiotic bacteria delivery. All the final products recorded a satisfactory overall sensory quality without any noticeable off-flavour, and with the characteristic sensory properties of low-acid fermented sausages.

Characterization of lactic acid bacteria isolated from infant faeces as potential probiotic starter cultures for fermented sausages.

A total of 109 lactic acid bacteria isolated from infant faeces were identified by partial 16S rRNA, cpn60 and/or pheS sequencing. Lactobacillus was the most prevalent genus, representing 48% of the isolates followed by Enterococcus (38%). Lactobacillus gasseri (21%) and Enterococcus faecalis (38%) were the main species detected. A further selection of potential probiotic starter cultures for fermented sausages focused on Lactobacillus as the most technologically relevant genus in this type of product. Lactobacilli strains were evaluated for their ability to grow in vitro in the processing conditions of fermented sausages and for their functional and safety properties, including antagonistic activity against foodborne pathogens, survival from gastrointestinal tract conditions (acidity, bile and pancreatin), tyramine production, antibiotic susceptibility and aggregation capacity. The best strains according to the results obtained were Lactobacillus casei/paracasei CTC1677, L. casei/paracasei CTC1678, Lactobacillus rhamnosus CTC1679, L. gasseri CTC1700, L. gasseri CTC1704, Lactobacillus fermentum CTC1693. Those strains were further assayed as starter cultures in model sausages. L. casei/paracasei CTC1677, L. casei/paracasei CTC1678 and L. rhamnosus CTC1679 were able to lead the fermentation and dominate (levels ca. 10(8) CFU/g) the endogenous lactic acid bacteria, confirming their suitability as probiotic starter cultures.

Assessment of safe enterococci as bioprotective cultures in low-acid fermented sausages combined with high hydrostatic pressure.

Three bacteriocinogenic, non-aminogenic and non-virulent Enterococcus strains (Enterococcus faecium CTC8005, Enterococcus devriesei CTC8006 and Enterococcus casseliflavus CTC8003) were used as starter cultures in low-acid fermented sausages to assess their competitiveness and their bioprotective potential against Listeria monocytogenes and Staphylococcus aureus. The inoculated strains were successfully monitored by RAPD-PCR. All the strains were able to grow, survive and dominate the endogenous enterococci population and avoided the growth of Enterobacteriaceae. E. devriesei CTC8006 and E. faecium CTC8005 particularly inhibited the growth of L. monocytogenes during the whole ripening process. S. aureus was not affected by the inoculated bacteriocinogenic enterococci strains. The application of high hydrostatic pressure (HHP) treatment (600 MPa for 5 min) at the end of ripening (day 21) produced an immediate reduction in the counts of Enterobacteriaceae to levels <1 log cfu/g and promoted a decrease of 1-log unit in the counts of S. aureus. E. faecium CTC8005, which reduced the counts of L. monocytogenes ca. 2 log cfu/g immediately after stuffing and in combination with HHP treatment promoted a further reduction of 1 log cfu/g in the pathogen counts. The combination of E. faecium CTC8005 and HHP was the most efficient antilisterial approach.

High hydrostatic pressure and biopreservation of dry-cured ham to meet the Food Safety Objectives for Listeria monocytogenes.

This work aimed to evaluate the effect of nisin application (biopreservation) combined with high hydrostatic pressure processing (HHP) on the behavior of Listeria monocytogenes CTC1034 intentionally inoculated (at ca. 10(7)cells/g) onto the surface of ready-to-eat (RTE) sliced dry-cured ham. Two types of dry-cured ham, which had different water activities and fat contents were studied (a(w) of 0.92 and 14.25% fat and a(w) of 0.88 and 33.26% fat). Three batches were prepared for each type of product: (C) control, without nisin; (N) nisin directly applied (200 AU/cm(2)) and (F) nisin applied through active packaging, polyvinyl alcohol films with 200 AU/cm(2). Half of the samples were pressurized at 600 MPa for 5min. Counts of L. monocytogenes were periodically monitored throughout 60 days of storage at 8°C. The physico-chemical characteristics of the products enabled the survival of L. monocytogenes, but it was significantly reduced by the presence of nisin. The effect of biopreservation was greater when applied directly to the surface and in the product with lower water activity in comparison with the active packaging and the high water activity products, respectively. The immediate inactivation of L. monocytogenes by HHP ranged from 1.82 to 3.85 Log units, depending on the type of dry-cured ham. The lower the water activity, the less was the inactivation induced by HHP, both immediately and during storage. The reduction of L. monocytogenes immediately after HHP and during storage was more evident in batches with nisin applied directly to the surface of the product. The pathogen was not detected in some samples from day 5 of storage in the product with higher water activity. The effect of nisin applied through active packaging was lower than the direct application. The results of the present study indicated that HHP, as post-processing listericidal treatment, is more effective (both immediately and long term) than the use of nisin as an antimicrobial measure. However, the both hurdles combined (i.e. biopreservation and HHP) provided a wider margin of safety in the control of L. monocytogenes during the storage of RTE cured meat products.

Prevalence of Salmonella spp. and Listeria monocytogenes at small-scale spanish factories producing traditional fermented sausages.

The manufacturing of fermented sausages is subject to natural contamination processes that can potentially carry foodborne pathogens along the process chain and result in contamination of the final product. The aim of this study was to evaluate the occurrence of Salmonella spp. and Listeria monocytogenes at different sampling points during the manufacturing process of fuet, a type of traditional fermented sausage, at 10 small-scale Spanish factories. The presence of both pathogens was studied in the raw materials (19 casings and 19 meat batters), the final products (19 fermented sausages), and the factory equipment (12 mincing, 12 mixing, and 19 stuffing machines, 19 cutting tables, 11 knives, and 12 cold rooms) by using classical microbiological techniques and real-time PCR. Salmonella was not detected in the equipment analyzed or in the final products, but it was detected in the raw materials (23.7% of samples). L. monocytogenes showed higher incidence than Salmonella and was detected in the equipment (11.8% of samples), the raw materials (28.9%), and the final products (15.8%), confirming its ubiquity throughout the manufacturing process of fermented sausages. Five factories were further investigated to study the changes in the distribution of pathogens in the fuet production process over a period of either 2 or 3 years. There was considerable variation in the incidence of both pathogens at different sampling periods, and there was no relation between seasonal variations or geographic location of the factories.

Inactivation and recovery of Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus after high hydrostatic pressure treatments up to 900 MPa.

High hydrostatic pressure (HP) processing is used in the food industry to enhance the safety and extend the shelf-life of food. Although a drastic decrease in microbial viability is achieved immediately after the application of HP treatments, under favorable conditions the injured bacteria can recover. The present study evaluated the inactivation and recovery of five strains of Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus subjected to pressures of 400, 600, and 900 MPa under stressing and non-stressing conditions in a complex medium. Treatments at 400 and 600 MPa were found to greatly affect the viability of L. monocytogenes and S. enterica, but only a treatment of 5 min at 900 MPa decreased the levels of the three pathogens to below the detection limit (8-9 log units reduction). After HP treatment, not only the baroresistant S. aureus but also several replicates of L. monocytogenes and S. enterica strains recovered during subsequent incubation under favorable conditions. However, when HP was combined with low pH and nitrite but not with NaCl or lactate, the viability of pressurized S. aureus cells progressively decreased. As pathogenic bacteria can recover even after the application of very high pressure levels, the combination of HP with other hurdles for microbial growth, either intrinsically present in the food product or extrinsically applied, may be needed to guarantee the efficacy of technologies aimed at pathogen reduction and shelf-life extension.

Inactivation and recovery of Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus after high hydrostatic pressure treatments up to 900 MPa

High hydrostatic pressure (HP) processing is used in the food industry to enhance the safety and extend the shelf-life of food. Although a drastic decrease in microbial viability is achieved immediately after the application of HP treatments, under favorable conditions the injured bacteria can recover. The present study evaluated the inactivation and recovery of five strains of Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus subjected to pressures of 400, 600, and 900 MPa under stressing and non-stressing conditions in a complex medium. Treatments at 400 and 600 MPa were found to greatly affect the viability of L. monocytogenes and S. enterica, but only a treatment of 5 min at 900 MPa decreased the levels of the three pathogens to below the detection limit (8-9 log units reduction). After HP treatment, not only the baroresistant S. aureus but also several replicates of L. monocytogenes and S. enterica strains recovered during subsequent incubation under favorable conditions. However, when HP was combined with low pH and nitrite but not with NaCl or lactate, the viability of pressurized S. aureus cells progressively decreased. As pathogenic bacteria can recover even after the application of very high pressure levels, the combination of HP with other hurdles for microbial growth, either intrinsically present in the food product or extrinsically applied, may be needed to guarantee the efficacy of technologies aimed at pathogen reduction and shelf-life extension (AU)

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Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages.

The single and combined effects of enterocin AS-48 and high hydrostatic pressure (HHP) on Listeria monocytogenes, Salmonellaenterica, and Staphylococcus aureus was investigated in fuet (a low acid fermented sausage) during ripening and storage at 7 degrees C or at room temperature. AS-48 (148 AU g(-1)) caused a drastic 5.5 log cfu g(-1) decrease in L. monocytogenes (P<0.001) and a significant (P<0.01) inhibition (1.79 logs) for Salmonella at the end of ripening (10 d). After pressurization (400 MPa) and storage Listeria counts remained below 5 cfu g(-1) in all fuets containing AS-48 (pressurized or not). HHP alone had no anti-Listeria effect. HHP treatment significantly reduced Salmonella counts, with lowest levels in pressurized fuets with AS-48. S. aureus showed similar growth for all treatments and storage conditions. These results indicate that AS-48 can be applied alone to control L. monocytogenes and combined with HHP treatment to control Salmonella in fuets.

Physical performance of biodegradable films intended for antimicrobial food packaging.

Antimicrobial films were prepared by including enterocins to alginate, polyvinyl alcohol (PVOH), and zein films. The physical performance of the films was assessed by measuring color, microstructure (SEM), water vapor permeability (WVP), and tensile properties. All studied biopolymers showed poor WVP and limited tensile properties. PVOH showed the best performance exhibiting the lowest WVP values, higher tensile properties, and flexibility among studied biopolymers. SEM of antimicrobial films showed increased presence of voids and pores as a consequence of enterocin addition. However, changes in microstructure did not disturb WVP of films. Moreover, enterocin-containing films showed slight improvement compared to control films. Addition of enterocins to PVOH films had a plasticizing effect, by reducing its tensile strength and increasing the strain at break. The presence of enterocins had an important effect on tensile properties of zein films by significantly reducing its brittleness. Addition of enterocins, thus, proved not to disturb the physical performance of studied biopolymers. Development of new antimicrobial biodegradable packaging materials may contribute to improving food safety while reducing environmental impact derived from packaging waste. Practical Application: Development of new antimicrobial biodegradable packaging materials may contribute to improving food safety while reducing environmental impact derived from packaging waste.

Modeling the combined effects of enterocins A and B, lactate, and EDTA on the growth of Salmonella at different temperatures.

The effects of enterocins A and B (produced by Enterococcus faecium CTC492), lactate, and ethylenediaminetetraacetic acid (EDTA) on the growth of Salmonella were modeled together with temperature using the response surface methodology. Six serovars of Salmonella enterica were inoculated (ca. 10(3) cells/ml) in brain-heart infusion broth with different levels of the studied factors and then incubated at different temperatures. The results showed that while Salmonella growth was affected by all the factors, temperature was the most important factor influencing the time to detection of the pathogen. All factors, including temperature, showed significant two-way interactions. The presence of enterocins A and B, lactate, and EDTA had an inhibitory effect that was enhanced at suboptimal temperatures for growth, thus delaying the time to detection. Moderate-low concentrations of lactate and EDTA increased the inhibitory effect of enterocins A and B. The effectiveness of these bacteriocins was not further enhanced by high concentrations of lactate (>3.6%) or EDTA (>200 mg/l). The mathematical model obtained from these analyses provides a useful tool to assess the effects of natural antimicrobials and their interactions with other growth-related factors on the growth response of Salmonella. The results can be applied to determine the most effective combination of hurdles to be used in the preservation of food products.

Identification of Enterococcus species by melting curve analysis of restriction fragments.

A new method for the identification of Enterococcus species has been developed. It combines PCR amplification of sodA gene and 16S-23S intergenic spacer region with restriction enzyme digestion followed by a melting curve analysis of the restriction fragments (MCARF). All strains analyzed were correctly identified by MCARF. This method was proved to be a reliable enterococcal identification tool.