Antilisterial activity and stability of nanovesicle-encapsulated antimicrobial peptide P34 in milk

Bacillus sp. P34, a strain isolated from aquatic environments of Brazilian Amazon basin, produces a bacteriocin-like substance (BLS) which was encapsulated in nanovesicles prepared from partially purified soy lecithin. The efficiency of free and encapsulated BLS P34 to control the development of L. monocytogenes and maintenance of antimicrobial activity was assessed over time in milk. The antimicrobial activity of free and encapsulated BLS P34 decreased approximately 50% after 4 days of storage (<4 °C) in skim and whole milk. After this period there was not significant loss of activity up to 21 days. The viable counts of Listeria monocytogenes in skim and whole milk containing 3200 AU/ml of free or encapsulated BLS P34 were always lower than those observed in controls without bacteriocin at both 30 °C and 7 °C. At 1600 AU/ml concentration, free and encapsulated BLS P34 were inhibitory to L. monocytogenes in skim milk, when compared with the control at 7 days. Nanovesicle-encapsulated and free BLS P34 shows potential use as biopreservative for application in milk-derived products.     

Characterization of a novel bacteriocin produced by Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish

A novel bacteriocin, sakacin LSJ618, produced by the strain Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish, was studied. L. sakei LSJ618 was identified by both phenotypical and physiological tests combined with 16S rDNA sequence analysis. Sakacin LSJ618 is sensitive to hydrolytic enzymes including lipase, is stable between pH 2-8, and is heat resistant (30 min at 121 °C). Sakacin LSJ618 exhibits inhibitory activity against food-spoiling bacteria and food-borne pathogens, including the Gram-positive Listeria monocytogenes, Staphylococcus aureus, Sarcina sp., Micrococcus luteus, and the Gram-negative Proteus sp. and Escherichia coli, but not against most of the lactic acid bacteria tested. Maximal production of bacteriocin was reached in the late stationary phase, and inhibitory activity declined within 26 h. The mode of action of sakacin LSJ618 was determined to be bactericidal, as evidenced by its action upon Micrococcus tetragenus. After partial purification by ammonium sulfate precipitation and Sephadex G-25 chromatography, the molecular weight of sakacin LSJ618 was determined to be 5.2 kDa by Tricine-SDS-PAGE. The identified properties of sakacin LSJ618 indicate that it is a novel bacteriocin with potential application as a biopreservative in the food industry.     

Induction of plantaricin MG under co-culture with certain lactic acid bacterial strains and identification of LuxS mediated quorum sensing system in Lactobacillus plantarum KLDS1.0391

A bacteriocin named plantaricin MG was produced by Lactobacillus plantarum KLDS1.0391 which was isolated from “jiaoke”, a traditional, naturally fermented cream from Inner Mongolia in China. Bacteriocin production was increased significantly when L. plantarum KLDS1.0391 co-cultured with certain lactic acid bacteria (LAB) strains. Among 76 strains belonging to the genera Lactobacillus, Lactococcus, Leuconostoc and Streptococcus, four strains namely Lactobacillus helveticus KLDS1.9207, Enterococcus faecium KLDS4.0352, Lactobacillus reuteri KLDS1.0737 and Enterococcus faecalis KLDS4.0313 were shown to induce bacteriocin production of L. plantarum KLDS1.0391. Cell numbers of L. plantarum KLDS1.0391 were greatly enhanced when co-cultured with four bacteriocin-inducing strains. Bacteriocin production was not induced by autoclaved cultures and cell-free supernatants (CFS) of inducing strains, indicating that living cells of inducing strains might be necessary for enhancement of bacteriocin production. The presence of plnD and luxS genes was detected by polymerase chain reaction (PCR), and the existence of plNC8HK gene was detected by single oligonucleotide nested PCR (SON-PCR).     

Inhibition of Listeria monocytogenes in vitro and in goat milk by liposomal nanovesicles containing bacteriocins produced by Lactobacillus sakei subsp. sakei 2a

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacteria isolated from a Brazilian meat product, capable to inhibit Listeria monocytogenes in vitro and in foods. In this study, bacteriocin produced by this strain were encapsulated in phosphatidylcholine (FC) and 1,2-dioleoyloxy-3-trimethylammonium-propane (DOTAP) liposomes, separately and in combination, were characterized and evaluated for activity against L. monocytogenes in vitro and in experimentally contaminated UHT goat milk, during storage at 7 °C for 14 days and 30 °C for 24 h. The FC and DOTAP/FC (3:1) nanovesicles containing bacteriocins (12.800 AU mL⁻¹) presented zeta potential of −1.54 and + 38.13 mV, Entrapment Efficiency of 80.0% and 94.1%, and diameter of 91.19 and 81.49 nm, respectively. DOTAP/FC nanovesicle presented excellent stability, and maintained the same physicochemical characteristics over 28 days. Both free and encapsulated bacteriocins controlled L. monocytogenes growth in BHI medium and goat milk stored at 30 °C for at least 8 h, in a similar pattern. After 24 h in BHI medium, bacteriocins encapsulated in FC nanovesicles were more effective (p < 0.05) than free bacteriocins. However, in goat milk, no significant differences (p ≥ 0.05) were observed for the two types of nanovesicles. At 7 °C, both free and encapsulated bacteriocins retarded the L. monocytogenes growth, and after 5 days, counts were 5 log lower than in the controls, both in BHI and in goat milk. Encapsulation of bacteriocin in FC and DOTAP/FC nanovesicles did not affect the antimicrobial activity, but the advantages of their application for control of L. monocytogenes in goat milk based dairy products, when compared to free bacteriocins, remain unclear and more studies are needed.     

Plantaricin MG active against Gram-negative bacteria produced by Lactobacillus plantarum KLDS1.0391 isolated from “Jiaoke”, a traditional fermented cream from China

A bacteriocin named plantaricin MG produced by Lactobacillus plantarum KLDS1.0391 which was isolated from “Jiaoke”, a traditional, naturally fermented cream from Inner Mongolia in China is reported in this article. Plantaricin MG was purified by ammonium sulfate precipitation followed by sequential gel filtration chromatography and reverse-phase chromatography. Mass spectrometry analysis showed the mass of plantaricin MG to be approximately 2180 Da. The bacteriocin showed a broad inhibitory activity against Gram-positive and Gram-negative bacteria including Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium and Escherichia coli. The bacteriocin was extremely heat-stable (30 min at 121 °C) and remained active after incubation at pH 2.0–10.0. It was found to be sensitive to proteolytic enzymes (pepsin, trypsin, papain, α-chymotrypsin, proteinase K, Neutrase and Alcalase). The mode of action of plantaricin MG was identified as bactericidal.     

Contamination of UHT milk by aflatoxin M1 in Iran

In this study the levels of aflatoxin M₁ (AFM₁) in UHT milk samples were determined in May, August and November and February. Two hundred and ten UHT milk samples were obtained from supermarkets in Tehran, Iran. The occurrence and concentration range of AFM₁ in the samples were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA) method. AFM₁ was found in 116 (55.2%) of 210 UHT milk samples examined. The levels of AFM₁ in 70 (33.3%) samples were higher than the maximum tolerance limit (0.05 μg/l) accepted by some European countries while none of the samples exceeded the prescribed limit of US regulations. The highest mean concentration of AFM₁ was recorded in February (0.087 μg/l). The lowest mean concentration of AFM₁ was recorded in August (0.021 μg/l). Statistical evaluation showed that there were significant difference (P < 0.01) between the mean concentrations of AFM₁ of UHT milk samples taken in February with May and August. AFM₁ contents of milk samples taken in February were not higher than UHT milk samples taken in November (P < 0.01). The AFM₁ incidence of exceeding legal limit in UHT milk samples (33.3%) was relatively much higher than some other countries. It was therefore concluded that, the levels of AFM₁ in UHT milk samples consumed in Iran were high and seemed to pose a threat to public health.     

Control of milk pH reduces biofilm formation of Bacillus licheniformis and Lactobacillus paracasei on stainless steel

Microbial biofilms present in dairy farms may contaminate milk during milk harvest and transfer diseases from the environment to cows. In order to reduce biofilm formation with respect to the role of pH, a study involving the control of milk pH during long-term biofilm formation of Bacillus licheniformis NBRC 12195 and Lactobacillus paracasei subsp. paracasei NBRC 15889 on stainless steel coupons in different dilutions of skim milk (0.1%, 1.0% and 5.0%) was conducted. During long incubation at 30 °C, pH decreased due to bacterial development in unadjusted samples. In pH-adjusted samples, pH was kept at around 7.0 by the addition of sterile sodium hydroxide. Biofilms formed on stainless steel coupons were daily stained by 0.1% Crystal Violet solution and assessed by the evaluation of optical density. The bacterial count of the suspensions showed that the control of pH enhanced the growth of bacteria in free-floating form. In contrast, optical densities of biofilms formed in the pH-adjusted samples were significantly lower than in the pH-unadjusted samples in all of three skim milk dilutions. Comparison of maximum OD values of adhered cells at different nutrient levels also implicated that for both tested strains, thicker biofilms were formed in milk dilutions at higher nutrient levels. These results suggested that, control of milk pH and milk residue level could significantly reduce biofilm formation of the tested bacteria.     

Technological characterization of Lactobacillus isolates from traditional Manchego cheese for potential use as adjunct starter cultures

Thirty Lactobacillus isolates from artisanal Manchego cheeses, 10 belonging to Lactobacillus plantarum and 20 belonging to Lactobacillus paracasei subsp. paracasei, were examined for potential use as adjunct cultures. Isolates were screened for acidifying and amino-biogenic capacity, for proteolytic, lipolytic, autolytic and aminopeptidase activities, and for sensory characteristics in UHT coagulated milk. Most of the lactobacilli displayed high aminopeptidase and autolytic activities and low acidifying activity. Only one isolate was able to produce tyramine. When growing on milk 53.3% of the Lb. paracasei subsp. paracasei isolates were able to produce pleasant flavours. Strains PBL210 (Lb. plantarum) and PBL213 and PBL226 (Lb. paracasei subsp. paracasei) presented the best technological characteristics, and also were still viable and with unaltered properties after freeze-drying and storage for 12 months. For these reasons, these strains could be good candidates for inclusion as adjuncts in a starter culture to manufacture industrial Manchego cheese.     

Potential of Escherichia coli as a surrogate indicator for postchill broiler carcasses with high Campylobacter counts

Surrogating Campylobacter contamination level in broiler carcasses with other bacterial indicators, used to evaluate the hygienic status of the slaughterline operations, might be stimulation to the broiler meat industry to improve control of Campylobacter during slaughter. Theoretically, Escherichia coli might have some practical merits as a potential indicator for carcasses contaminated with Campylobacter. This study investigates the correlation between the counts of E. coli and Campylobacter in 231 postchill broiler carcasses. The impact of setting a process hygiene target based on E. coli counts on reducing the frequency of carcasses contaminated with Campylobacter at level of ≥3 log₁₀ CFU/g was also investigated. Almost half (48.9% (46/94)) of the carcasses with enumerable Campylobacter (≥1 log₁₀ CFU/g) had E. coli counts between 3 and 4 log₁₀ CFU/g. In addition, 54.8% (17/31) of the carcasses contaminated with Campylobacter of ≥3 log₁₀ CFU/g were correlated with E. coli count range of ≥3 & <4 log₁₀ CFU/g. A theoretical scenario assuming that hygiene and processing measures could allow achieving a target for E. coli that not exceeding 3 log₁₀ CFU/g showed a parallel impact on Campylobacter contamination in broiler carcasses. In such scenario, the overall number of Campylobacter-positive carcasses could be dropped from 40.6% to 12.5%; in addition, 80.6% (25/31) of the carcasses contaminated with Campylobacter of ≥3 log₁₀ CFU/g could be eliminated. Findings from this study reveal that a hygiene target based on E. coli count could be used as an indirect sanitary tool for reducing the level of Campylobacter contamination in postchill broiler carcasses.     

Direct detection of Salmonella without pre-enrichment in milk, ice-cream and fruit juice by PCR against hilA gene

A novel PCR based assay was devised to specifically detect contamination of any Salmonella serovar in milk, fruit juice and ice-cream without pre-enrichment. This method utilizes primers against hilA gene which is conserved in all Salmonella serovars and absent from the close relatives of Salmonella. An optimized protocol, in terms time and money, is provided for the reduction of PCR contaminants from milk, ice-cream and juice through the use of routine laboratory chemicals. The simplicity, efficiency (time taken 3-4 h) and sensitivity (to about 5-10 CFU/ml) of this technique confers a unique advantage over other previously used time consuming detection techniques. This technique does not involve pre-enrichment of the samples or extensive sample processing, which was a pre-requisite in most of the other reported studies. Hence, this assay can be ideal for adoption, after further fine tuning, by food quality control for timely detection of Salmonella contamination as well as other food-borne pathogens (with species specific primers) in food especially milk, ice-cream and fruit juice.     

Effect of Hibiscus sabdariffa extract and Nigella sativa oil on the growth and aflatoxin B1 production of Aspergillus flavus and Aspergillus parasiticus strains

This study was undertaken to evaluate the inhibitory effect of Hibiscus sabdariffa calyx extract at concentrations of 5, 7.5, 10 and 12.5 g/100 ml and Nigella sativa oil at concentrations of 1, 2 and 3 ml/100 ml on the growth and aflatoxin B₁ production by Aspergillus parasiticus (CBS 921.7) and Aspergillus flavus (SQU 21) strains. The inhibition of aflatoxin B₁ production by the different concentrations of H. sabdariffa calyx ranged between 91.5-97.9% and 87.1-93.3% for A. flavus and A. parasiticus strains, respectively, whereas the inhibition by different concentrations of N. sativa oil ranged between 47.9 and 58.3% for A. flavus and 32-48% for A. parasiticus strains. The different concentrations of H. sabdariffa calyx and N. sativa oil had no significant effect on the growth of either Aspergillus species. Neither H. sabdariffa calyx nor N. sativa oil detoxified pure aqueous aflatoxin. Our results suggest that H. sabdariffa calyx and N. sativa oil extracted from seeds had metabolic effects on aflatoxin biosynthesis pathway of both Aspergillus species and can be used as an effective biocontrol and non-toxic biopreservatives in food industry against aflatoxin contamination.     

The sanitizing action of essential oil-based solutions against Salmonella enterica serotype Enteritidis S64 biofilm formation on AISI 304 stainless steel

Bacterial adhesion and biofilm formation by Salmonella enterica serovar Enteritidis is a serious concern in the food processing industry; organism persistence in biofilms represents a continual source of contamination. Due to unsuccessful disinfection processes and emerging resistance, conventional control methods are rapidly becoming ineffective, necessitating the development of new control strategies. The following study evaluated the anti-biofilm effect of disinfectant solutions formulated with essential oils (EOs) of peppermint (Mentha piperita) and lemongrass (Cymbopogon citratus) against biofilm formation by S. enterica serotype Enteritidis S64 on stainless steel surface AISI 304 (#4) after 10, 20 and 40 min of contact. A minimum inhibitory concentration (MIC) of 7.8 μL/mL was found for both EOs and disinfectant solutions were formulated based on these MIC values. Ten minutes of sanitizing solution contact significantly reduced (p ≤ 0.05) adhered bacterial populations for both EOs tested. After 20 and 40 min of treatment, cell counts were not detected. Thus, M. piperita and C. citratus EOs can be considered convenient, quality alternatives to the application of conventional sanitizing agents in the food industry; further, use of these EOs addresses the increasing consumer demand for natural products.     

Application of ozone sprays as a strategy to improve the microbial safety and quality of salmon fillets

Ozone research investigating the efficacy of spray application mechanisms for balancing microbial safety with chemical quality attributes of high lipid content fish is lacking. The objectives of this study were to investigate the influence of aqueous spray treatments of 1 mg/L and 1.5 mg/L ozone on the microbial and chemical quality indices of Atlantic salmon fillets inoculated with Listeria innocua as a surrogate species replacing the pathogen Listeria monocytogenes. In order to simulate industry processing parameters, number of passes under spray nozzles (1, 2, and 3) was also investigated.Listeria counts resulting from treatment with three passes of 1 mg/L ozone sprays were significantly lower (p ≤ 0.05) than non-ozonated controls on days 0, 3, 6, and 10 of refrigerated storage at 4 °C. At both ozone concentrations, Listeria counts were significantly influenced (p ≤ 0.05) by the number of passes under spray nozzles with increasing passes resulting in increasing reductions. The residual antimicrobial effectiveness of ozone at both concentrations against aerobic bacterial populations on treated samples was limited to <6 days storage at 4 °C. Lipid oxidation analyses indicate that ozone concentration did not significantly affect (p ≤ 0.05) TBARS but did influence headspace propanal with 1 mg/L ozone treatments yielding approximately 30% higher propanal values than spray treatments of 1.5 mg/L ozone. Both TBARS and propanal levels were significantly influenced (p ≤ 0.05) by storage time, yet the number of spray passes did not significantly affect either measure of lipid oxidation. Our results indicate that ozone sprays at concentrations up to 1.5 mg/L are effective in reducing initial counts of aerobic bacterial populations and in significantly reducing (p ≤ 0.05) counts of L. innocua without causing significant increases in lipid oxidation levels in farmed Atlantic salmon fillets during refrigerated storage at 4 °C.     

Safety aspects, genetic diversity and technological characterisation of wild-type Streptococcus thermophilus strains isolated from north Italian traditional cheeses

Antibiotic susceptibility, antimicrobial activity, genotypic and technological properties of 52 Streptococcus thermophilus isolates, collected from four north Italian traditional cheeses, was investigated. RAPD-PCR, was used to study genetic variability and distinguish closely related strains; the results showed a high degree of heterogeneity among isolates. With regard to technological properties, after 6 h of incubation in milk 25% of the streptococcal strains could be defined as fast acid producers, while after 24 h the majority of isolates (79%) displayed only weak acidification activity. Reduction activity was generally low; in fact, none of these S. thermophilus strains showed a Eh < −102 mV). All the studied S. thermophilus were susceptible to ciprofloxacin, levofloxacin, penicillin G, ampicillin, mupirocin, nitrofurantoin, quinupristin/dalfopristin and rifampicin. Nine isolates were classified as resistant to tetracycline, 6 to streptomycin, 5 to oxacillin, 3 to erytromycin, 3 to vancomycin and only one to chloramphenicol. PCR-based detection did not identify any of the common genetic determinants for vancomycin (vanA, vanB, vanC1, vanC2, vanC3, vanD, vanE, vanG) or erythromycin (ermB and ermC). The genetic basis of the tetracycline resistance phenotype in these strains was linked to tetS-tetL genes (8 isolates) or the tetM gene (1 isolate), and the integrase element int of the Tn916/Tn1545 family of transposons was negative. Four strains were able to produce antimicrobial compounds against Clostridium tyrobutyricum. The study provides new evidence of the resistance of S. thermophilus to antimicrobial agents, confirming the importance of including an accurate safety assessment of phenotypic/biotechnological data when carrying out strain selection for dairy applications.     

Isolation, identification and monitoring of contaminant bacteria in Iranian Kefir type drink by 16S rDNA sequencing

Iranian Kefir type drink (IKTD) is a highly consumed, traditional Iranian, fermented milk product. To improve monitoring procedures for food safety 32 industrial Kefir type drinks from 4 brands and 8 different production dates as well as 32 samples from pasteurized milk of the same Kefir manufacturers and air of the production sites were analyzed for contaminations. 16S rDNA extraction from Kefir samples as well as 16S rDNA obtained from samples incubated on Columbia agar were analyzed using PCR/DGGE, cloning, sequencing and phylogenetic classification. Already DGGE analysis indicated contaminations including Bacillus strains. Subsequently analysis of cultured clones indicated contaminations with Bacillus sp. including Bacillus cereus, Bacillus thuringiensis and Paenibacillus sp. in 9 (28%) from all analyzed samples. Also 38% of pasteurized milk samples were contaminated with B. cereus. The average count of B. cereus was 74 ± 19 cfu/ml. B. cereus and B. thuringiensis were found as contaminant bacteria in the air of the all manufacturing sites. These results suggest that milk is one of the most important sources of contamination with Bacillus sp., especially B. cereus for Kefir products in Iran. But bacterial contamination in Kefir samples might also originate from the air of the production sites. 16S rDNA analysis accelerates monitoring strategies.     

Inhibition of Listeria monocytogenes by pomegranate (Punica granatum) peel extract in meat paté at different temperatures

Natural antimicrobials are being more and more considered as alternative approach for controlling growth of microorganisms in food. The objective of this study was to evaluate the pomegranate extract’s (PE) potential to be used as a natural preservative in ready to eat meats. Listeria monocytogenes was the main target. In a preliminary assessment with the disk diffusion method PE showed inhibitory effect against all five tested species, in the following order of increasing sensitivity: L. monocytogenes, Bacillus subtilis, Bacillus cereus, Escherichia coli and Staphylococcus aureus.No viable cells of L. monocytogenes were detected after incubation in BHI broth in presence of 7.5% v/v of the liquid PE (or 24.7 mg dry PE/ml). This concentration was considered as the Minimal Bactericidal Concentration (MBC) of the tested PE. Two pure components commonly found in PE, namely gallic and ellagic acids were also tested in BHI broth, however they did not show considerable inhibition of L. monocytogenes. PE in a concentration equal to the measured MBC was tested against L. monocytogenes in meat paté at different temperatures. At 4 °C during 46 days the extract inhibited the growth in meat paté by 4.1 log CFU/g compared to the control, which had reached log 9.2 CFU/g already on the 18th day. Inhibition was less pronounced at higher temperatures. The results indicate that the PE has a potential to be used as a natural preservative in meat products.     

Antioxidant activity of essential oil and aqueous extract of Pelargonium graveolens L’Her

Pelargonium graveolens L’Her is an aromatic, rose-scented herb which is indigenous to various parts of southern Africa, and nowadays cultivated worldwide. This work presents the first phytochemical investigation of P. graveolens cultivated in Bosnia. The volatile profile of odorous parts of the plant was analysed by GC/MS. More than eighty compounds were identified in essential oils obtained from the leaves and stems, representing 92.3% and 96.3% in total, respectively. The major compounds in essential oils were oxygenated monoterpenes (64.3-74.2%), with geraniol (27.5-50.2%) and citronellol (14.2-19.0%) as the main representatives. The content of phenolic compounds in corresponding hydrosols as 34.88 ± 2.00 mg GAE/g in leaves and 102.44 ± 1.63 mg GAE/g in stems including flavonoid compounds of 32.35 ± 0.81 mg GAE/g to 101.87 ± 1.03 mg GAE/g. The radical-scavenging activity was measured by the DPPH method and IC₅₀ values ranged from 0.19 ± 0.05 mg/mL (stems) to 0.39 ± 0.04 mg/mL (leaves) for hydrosols, and from 63.70 ± 1.56 mg/mL (leaves) to 64.88 ± 1.12 mg/mL (stems) for essential oils.     

Influence of sunflower oil based nanoemulsion (AUSN-4) on the shelf life and quality of Indo-Pacific king mackerel (Scomberomorus guttatus) steaks stored at 20 °C

The influence of nanoemulsion (AUSN-4) on the microbiological, proximal, chemical, and sensory qualities of Indo-Pacific king mackerel (Scomberomorus guttatus) steaks stored at 20 °C was studied for a time period of 72 h. AUSN-4 treatment showed initial reduction (P > 0.05) in the heterotrophic, H₂S and lactic acid bacterial populations in 12 h, followed by a gradual increase in their respective populations. Irrespective of treatments, reduction in total carbohydrate, protein, and fat contents were observed in all samples with an increase in storage time (h), with AUSN-4 treated steaks having the lowest reduction. AUSN-4 treatment significantly (P < 0.05) decreased the values of chemical indicators of spoilage throughout the storage period. Organoleptic evaluation revealed that AUSN-4 treated steaks showed an extension of shelf life of 48 h, when compared with control and antibiotic treated samples, respectively. Based on the results obtained in our present study we conclude that sunflower oil based nanoemulsion preservative technique is able to extend the shelf life and maintain the quality of S. guttatus steaks during storage.     

Detection of Norovirus and Feline Calicivirus in spiked molluscs subjected to heat treatments

Aim of the study was the evaluation of the effect of heat treatment in shellfish experimentally contaminated with human Norovirus (NoV). Feline Calicivirus (FCV), often used as surrogate for human NoV, was examined in parallel to test for virus infectivity after treatment. The experiments were performed subjecting suspensions and spiked mussels to heat treatment (60 °C and 80 °C) for various times. Analysis by rRT-PCR showed a limited reduction of NoV (less than 1 log RT-PCR units ml⁻¹) and FCV (0.3-1.2 log TCID₅₀ ml⁻¹) both in virus suspensions and in spiked mussels, with the exception of NoV suspensions treated at 80 °C (reduction of 3 log RT-PCR units ml⁻¹). Cell culture assay showed that the infectious FCV in suspensions decreased of 4 log after 3 min of treatment while a lower reduction (2 log) was obtained in spiked samples treated for 15 min. Data showed that mussel matrix plays a protective role against heat inactivation of viruses. Despite the efficacy of rRT-PCR for viral nucleic acid detection, its inability to provide information on virus infectivity, represents a limit to the evaluation of product safety. Caution should therefore be used in the evaluation of efficacy of heat treatments on virus-contaminated foods and when the judgment of food product safety is based exclusively on rRT-PCR results.     

Adherence kinetics, resistance to benzalkonium chloride and microscopic analysis of mixed biofilms formed by Listeria monocytogenes and Pseudomonas putida

Comparison between the resistance to BAC and the microscopic structure between mixed-species biofilms formed by different strains of Listeria monocytogenes and Pseudomonas putida CECT 845 under different scenarios and that obtained by the corresponding monospecies L. monocytogenes biofilm was carried out. The association of P. putida with L. monocytogenes quickens biofilm formation and increases significantly (p < 0.05) the BAC-resistance of the biofilm after 4 days of incubation at 25 °C respecting to that formed by monospecies biofilms. According with the adherence profiles of P. putida, two different patterns of association between both species (A and B) were identified, being type A pattern found in the mixed biofilms much more resistant to BAC. After 11 days of incubation, a destructuration of mixed biofilms occurred in all experimental assays, being in 2 out of 5 experimental cases (4032 and BAC-adapted 5873 on polypropylene) accompanied by a sharp decrease in the number of adhered cells. Microscopic analyses demonstrated that complex three-dimensional microscopic structure showed the highest resistance to BAC (4032-SS). Obtained results clearly highlight that to improve disinfection protocols for assuring food safety, it is necessary to mimick those bacterial association that occur in nature.