Controlled Release of Volatile Antimicrobial Compounds from Mesoporous Silica Nanocarriers for Active Food Packaging Applications.

Essential oils and their active components have been extensively reported in the literature for their efficient antimicrobial, antioxidant and antifungal properties. However, the sensitivity of these volatile compounds towards heat, oxygen and light limits their usage in real food packaging applications. The encapsulation of these compounds into inorganic nanocarriers, such as nanoclays, has been shown to prolong the release and protect the compounds from harsh processing conditions. Nevertheless, these systems have limited shelf stability, and the release is of limited control. Thus, this study presents a mesoporous silica nanocarrier with a high surface area and well-ordered protective pore structure for loading large amounts of natural active compounds (up to 500 mg/g). The presented loaded nanocarriers are shelf-stable with a very slow initial release which levels out at 50% retention of the encapsulated compounds after 2 months. By the addition of simulated drip-loss from chicken, the release of the compounds is activated and gives an antimicrobial effect, which is demonstrated on the foodborne spoilage bacteria Brochothrixthermosphacta and the potentially pathogenic bacteria Escherichia coli. When the release of the active compounds is activated, a ≥4-log reduction in the growth of B. thermosphacta and a 2-log reduction of E. coli is obtained, after only one hour of incubation. During the same one-hour incubation period the dry nanocarriers gave a negligible inhibitory effect. By using the proposed nanocarrier system, which is activated by the food product itself, increased availability of the natural antimicrobial compounds is expected, with a subsequent controlled antimicrobial effect.

Bioelectrical phase angle at diagnosis as a prognostic factor for survival in advanced head and neck cancer.

OBJECTIVE: Bioelectrical impedance analysis (BIA) is used to measure the patient's body composition, fat-free mass, phase angle (PA), and standardized phase angle (SPA), which are affected by malnutrition. Low values of PA and SPA have been found to be negative prognostic factors for survival in different types of cancer and other severe diseases. The aim of the current study was to investigate whether PA and SPA can be used to predict survival in head and neck (HN) cancer. METHODS: One hundred twenty-eight patients with advanced HN cancer treated in Western Sweden 2002-2006 were examined with BIA at diagnosis, and PA and SPA were calculated. Patients' age, gender, tumor site, TNM stage, and performance status were obtained, and weight, height, and BIA were measured. Survival up to 12 years was ascertained. RESULTS: The mean PA was 5.85° and the median was 5.91°. Lower PA and SPA values were significantly associated with shorter overall survival in univariate analyses, together with higher age, oral cancer, higher T class, worse performance status, more weight loss before diagnosis, lower: weight, height, BMI, and reactance. Age, performance status, T class, and PA were significant factors for the overall survival in the multivariable analysis. A PA cutoff value at 5.95° provided the best prediction of 5-year survival. CONCLUSIONS: PA and SPA at diagnosis are significant factors for survival in patients with advanced HN cancer. They are promising prognostic tools to use in treatment planning; further studies are needed.

Immunomodulatory oligonucleotides inhibit neutrophil migration by decreasing the surface expression of interleukin-8 and leukotriene B4 receptors.

Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin-8 (IL-8) and leukotriene B4 (LTB4 ) constitute key mediators by binding to the surface receptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll-like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down-regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL-8-induced and LTB4 -induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down-regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL-8 and LTB4 pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation.

Role of Lactobacillus reuteri cell and mucus-binding protein A (CmbA) in adhesion to intestinal epithelial cells and mucus in vitro.

Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA).

Microbes versus microbes: control of pathogens in the food chain.

Foodborne illness continues as a considerable threat to public health. Despite improved hygiene management systems and increased regulation, pathogenic bacteria still contaminate food, causing sporadic cases of illness and disease outbreaks worldwide. For many centuries, microbial antagonism has been used in food processing to improve food safety. An understanding of the mode of action of this microbial antagonism has been gained in recent years and potential applications in food and feed safety are now being explored. This review focuses on the potential opportunities presented, and the limitations, of using microbial antagonism as a biocontrol mechanism to reduce contamination along the food chain; including animal feed as its first link. © 2014 Society of Chemical Industry.

Establishment of a simple Lactobacillus plantarum cell consortium for cellulase-xylanase synergistic interactions.

Lactobacillus plantarum is an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including its ability to ferment both pentoses and hexoses, as well as its high acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent ability to deconstruct lignocellulosic substrates. As a first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding for a GH6 cellulase and a GH11 xylanase from a highly active cellulolytic bacterium into L. plantarum. For this purpose, we employed the recently developed pSIP vectors for efficient secretion of heterologous proteins. Both enzymes were secreted by L. plantarum at levels estimated at 0.33 nM and 3.3 nM, for the cellulase and xylanase, respectively, in culture at an optical density at 600 nm (OD600) of 1. Transformed cells demonstrated the ability to degrade individually either cellulose or xylan and wheat straw. When mixed together to form a two-strain cell-based consortium secreting both cellulase and xylanase, they exhibited synergistic activity in the overall release of soluble sugar from wheat straw. This result paves the way toward metabolic harnessing of L. plantarum for novel biorefining applications, such as production of ethanol and polylactic acid directly from plant biomass.

Screening for Antifungal Indigenous Lactobacilli Strains Isolated from Local Fermented Milk for Developing Bioprotective Fermentates and Coatings Based on Acid Whey Protein Concentrate for Fresh Cheese Quality Maintenance.

The demand for healthy foods without artificial food additives is constantly increasing. Hence, natural food preservation methods using bioprotective cultures could be an alternative to chemical preservatives. Thus, the main purpose of this work was to screen the indigenous lactobacilli isolated from fermented cow milk for their safety and antifungal activity to select the safe strain with the strongest fungicidal properties for the development of bioprotective acid whey protein concentrate (AWPC) based fermentates and their coatings intended for fresh cheese quality maintenance. Therefore, 12 lactobacilli strains were isolated and identified from raw fermented cow milk as protective cultures. The safety of the stains was determined by applying antibiotic susceptibility, haemolytic and enzymatic evaluation. Only one strain, Lacticaseibacillus paracasei A11, met all safety requirements and demonstrated a broad spectrum of antifungal activity in vitro. The strain was cultivated in AWPC for 48 h and grew well (biomass yield 8 log10 cfu mL-1). L. paracasei A11 AWPC fermentate was used as a vehicle for protective culture in the development of pectin-AWPC-based edible coating. Both the fermentate and coating were tested for their antimicrobial properties on fresh acid-curd cheese. Coating with L. paracasei A11 strain reduced yeast and mould counts by 1.0-1.5 log10 cfu mL-1 (p ≤ 0.001) during cheese storage (14 days), simultaneously preserving its flavour and prolonging the shelf life for six days.

Genome sequence of the naturally plasmid-free Lactobacillus plantarum strain NC8 (CCUG 61730).

Lactobacillus plantarum is a highly versatile lactic acid bacterium found in various ecological niches, such as fermented vegetable, meat, and dairy products and the gastrointestinal tract. We sequenced the genome of L. plantarum NC8, a naturally plasmid-free strain, which has been used as a model strain in many laboratories worldwide.

Microarray-based transcriptome of Listeria monocytogenes adapted to sublethal concentrations of acetic acid, lactic acid, and hydrochloric acid.

Listeria monocytogenes , an important foodborne pathogen, commonly encounters organic acids in food-related environments. The transcriptome of L. monocytogenes L502 was analyzed after adaptation to pH 5 in the presence of acetic acid, lactic acid, or hydrochloric acid (HCl) at 25 °C, representing a condition encountered in mildly acidic ready-to-eat food kept at room temperature. The acid-treated cells were compared with a reference culture with a pH of 6.7 at the time of RNA harvesting. The number of genes and magnitude of transcriptional responses were higher for the organic acids than for HCl. Protein coding genes described for low pH stress, energy transport and metabolism, virulence determinates, and acid tolerance response were commonly regulated in the 3 acid-stressed cultures. Interestingly, the transcriptional levels of histidine and cell wall biosynthetic operons were upregulated, indicating possible universal response against low pH stress in L. monocytogenes. The opuCABCD operon, coding proteins for compatible solutes transport, and the transcriptional regulator sigL were significantly induced in the organic acids, strongly suggesting key roles during organic acid stress. The present study revealed the complex transcriptional responses of L. monocytogenes towards food-related acidulants and opens the roadmap for more specific and in-depth future studies.

Heterologous Protein Production in Lactobacillus (plantarum) Using pSIP Vectors.

While lactobacilli are not generally regarded as efficient cell factories for heterologous proteins, these food-grade Gram-positive bacteria are attractive as expression hosts for medicinal proteins. Furthermore, tools have been developed not only to secrete the protein of interest, but also to anchor the protein to the cell membrane or the cell wall. Research efforts aimed at the production and surface display of complex vaccine proteins have shown that lactobacilli are capable of producing heterologous proteins that are otherwise difficult to produce in soluble form. Many recent studies on expressing a wide variety of proteins in lactobacilli have employed the pSIP vector system, which offers a wide range of possibilities for inducible expression, including various options for secretion and surface anchoring. The modular nature of the pSIP vectors allows for rapid screening of multiple expression strategies. This chapter describes the pSIP vector system and how it can be used to accomplish protein expression in lactobacilli.

Application of Edible Coating Based on Liquid Acid Whey Protein Concentrate with Indigenous Lactobacillus helveticus for Acid-Curd Cheese Quality Improvement.

Edible coatings as carriers for protective lactic acid bacteria (LAB) can enhance hygienic quality to dairy products. Thus, the aim of this study was to improve the quality of artisanal acid-curd cheese by applying liquid acid whey protein concentrate based edible coating with entrapped indigenous antimicrobial Lactobacillus helveticus MI-LH13. The edible fresh acid-curd cheese coating was composed of 100% (w/w) liquid acid whey protein concentrate (LAWPC), apple pectin, sunflower oil, and glycerol containing 6 log10 CFU/mL of strain biomass applied on cheese by dipping. The cheese samples were examined over 21 days of storage for changes of microbiological criteria (LAB, yeast and mould, coliform, enterobacteria, and lipolytic microorganism), physicochemical (pH, lactic acid, protein, fat, moisture content, and colour), rheological, and sensory properties. The coating significantly improved appearance and slowed down discolouration of cheese by preserving moisture during prolonged storage. The immobilisation of L. helveticus cells into the coating had no negative effect on their viability throughout 14 days of storage at 4 °C and 23 °C. The application of coating with immobilised cells on cheeses significantly decreased the counts of yeast up to 1 log10 CFU/g during 14 days (p < 0.05) of storage and suppressed growth of mould for 21 days resulting in improved flavour of curd cheese at the end of storage. These findings indicate that LAWPC-pectin formulation provided an excellent matrix to support L. helveticus cell viability. Acting as protective antimicrobial barrier in fresh cheeses, this bioactive coating can reduce microbial contamination after processing enabling the producers to extend the shelf life of this perishable product.

High stability of Stx2 phage in food and under food-processing conditions.

Bacteriophages (phages) carrying Shiga toxin genes constitute a major virulence attribute in enterohemorrhagic Escherichia coli (EHEC). Several EHEC outbreaks have been linked to food. The survival of such strains in different foods has received much attention, while the fate of the mobile Shiga toxin-converting phages (Stx phages) has been less studied. We have investigated the stability of an Stx phage in several food products and examined how storage, food processing, and disinfection influence the infectivity of phage particles. The study involved a recombinant Stx phage (Δstx::cat) of an E. coli O103:H25 strain from a Norwegian outbreak in 2006. Temperature, matrix, and time were factors of major importance for the stability of phage particles. Phages stored at cooling temperatures (4°C) showed a dramatic reduction in stability compared to those stored at room temperature. The importance of the matrix was evident at higher temperatures (60°C). Phages in ground beef were below the detection level when heated to 60°C for more than 10 min, while phages in broth exposed to the same heating conditions showed a 5-log-higher stability. The phages tolerated desiccation poorly but were infective for a substantial period of time in solutions. Under moist conditions, they also had a high ability to tolerate exposure to several disinfectants. In a dry-fermented sausage model, phages were shown to infect E. coli in situ. The results show that Stx phage particles can maintain their infectivity in foods and under food-processing conditions.

Global transcriptome response in Lactobacillus sakei during growth on ribose.

BACKGROUND: Lactobacillus sakei is valuable in the fermentation of meat products and exhibits properties that allow for better preservation of meat and fish. On these substrates, glucose and ribose are the main carbon sources available for growth. We used a whole-genome microarray based on the genome sequence of L. sakei strain 23K to investigate the global transcriptome response of three L. sakei strains when grown on ribose compared with glucose. RESULTS: The function of the common regulated genes was mostly related to carbohydrate metabolism and transport. Decreased transcription of genes encoding enzymes involved in glucose metabolism and the L-lactate dehydrogenase was observed, but most of the genes showing differential expression were up-regulated. Especially transcription of genes directly involved in ribose catabolism, the phosphoketolase pathway, and in alternative fates of pyruvate increased. Interestingly, the methylglyoxal synthase gene, which encodes an enzyme unique for L. sakei among lactobacilli, was up-regulated. Ribose catabolism seems closely linked with catabolism of nucleosides. The deoxyribonucleoside synthesis operon transcriptional regulator gene was strongly up-regulated, as well as two gene clusters involved in nucleoside catabolism. One of the clusters included a ribokinase gene. Moreover, hprK encoding the HPr kinase/phosphatase, which plays a major role in the regulation of carbon metabolism and sugar transport, was up-regulated, as were genes encoding the general PTS enzyme I and the mannose-specific enzyme II complex (EIIman). Putative catabolite-responsive element (cre) sites were found in proximity to the promoter of several genes and operons affected by the change of carbon source. This could indicate regulation by a catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR) mechanism, possibly with the EIIman being indirectly involved. CONCLUSIONS: Our data shows that the ribose uptake and catabolic machinery in L. sakei is highly regulated at the transcription level. A global regulation mechanism seems to permit a fine tuning of the expression of enzymes that control efficient exploitation of available carbon sources.

Inorganic Nanocarriers for Encapsulation of Natural Antimicrobial Compounds for Potential Food Packaging Application: A Comparative Study.

Design and development of novel inorganic nanocarriers for encapsulation of natural antimicrobial substances for food packaging applications have received great interest during the last years. Natural nanoclays are the most investigated nanocarriers and recently interest has also grown in the synthetically produced porous silica particles. However, these different carrier matrices have not been compared in terms of their loading capability and subsequent release. In this study, the feasibility of porous silica particles (with different pore structures and/or surface functionalities) and commercially available nanoclays were evaluated as encapsulation matrices. Two well-studied antimicrobial substances, thymol and curcumin, were chosen as volatile and non-volatile model compounds, respectively. The encapsulation efficiency, and the subsequent dispersibility and release, of these substances differed significantly among the nanocarriers. Encapsulation of the volatile compound highly depends on the inner surface area, i.e., the protective pore environment, and an optimal nanocarrier can protect the encapsulated thymol from volatilization. For the non-volatile compound, only the release rate and dispersibility are affected by the pore structure. Further, water-activated release of the volatile compound was demonstrated and exhibited good antimicrobial efficacy in the vapor phase against Staphylococcus aureus. This comparative study can provide a base for selecting the right nanocarrier aimed at a specific food packaging application. No nanocarrier can be considered as a universally applicable one.

Swedish National Multicenter Study on Head and Neck Cancer of Unknown Primary: Prognostic Factors and Impact of Treatment on Survival.

Introduction Head and neck cancer of unknown primary (HNCUP) is a rare condition whose prognostic factors that are significant for survival vary between studies. No randomized treatment study has been performed thus far, and the optimal treatment is not established. Objective The present study aimed to explore various prognostic factors and compare the two main treatments for HNCUP: neck dissection and (chemo) radiation vs primary (chemo) radiation. Methods A national multicenter study was performed with data from the Swedish Head and Neck Cancer Register (SweHNCR) and from the patients' medical records from 2008 to 2012. Results Two-hundred and sixty HNCUP patients were included. The tumors were HPV-positive in 80%. The overall 5-year survival rate of patients treated with curative intent was 71%. Age ( p < 0.001), performance status ( p = 0.036), and N stage ( p = 0.046) were significant factors for overall survival according to the multivariable analysis. Treatment with neck dissection and (chemo) radiation (122 patients) gave an overall 5-year survival of 73%, and treatment with primary (chemo) radiation (87 patients) gave an overall 5-year survival of 71%, with no significant difference in overall or disease-free survival between the 2 groups. Conclusions Age, performance status, and N stage were significant prognostic factors. Treatment with neck dissection and (chemo) radiation and primary (chemo) radiation gave similar survival outcomes. A randomized treatment study that includes quality of life is needed to establish the optimal treatment.

Intra- and interlaboratory performances of two commercial antimicrobial susceptibility testing methods for bifidobacteria and nonenterococcal lactic acid bacteria.

In a small-scale harmonization study involving nine laboratories in eight European countries, the intra- and interlaboratory performances of two commercially available systems, i.e., the VetMIC microplate system and Etest, for antimicrobial susceptibility testing of nonenterococcal lactic acid bacteria (NELAB) and bifidobacteria were analyzed. In addition, one laboratory also performed standard broth microdilution as a reference method. MICs of tetracycline, erythromycin, ampicillin, gentamicin, clindamycin, and streptomycin for the type strains of 25 species of NELAB and bifidobacteria and MICs of vancomycin for a selection of relevant taxa were determined. The previously described lactic acid bacterium susceptibility test medium (LSM) and related mixed-medium formulations, all including Iso-Sensitest broth as a basic component, were used as test media. The overall agreement of median MIC ranges +/- 1 log(2) dilution determined by the VetMIC and Etest methods with the median MICs determined by the reference method was very good for tetracycline, ampicillin, and streptomycin (92.3 to 100%) but low for erythromycin (19.5 to 30.7%) and clindamycin (50.0 to 80.8%). There was a consensus among the participating laboratories that VetMIC was preferred over Etest because of its lower cost, better growth support, and more uniform criteria for MIC end point reading. With the range for acceptable intralaboratory reproducibility being defined as the median MIC +/- 1 log(2) dilution, VetMIC results (with 69.2% of all data sets in the acceptable range) were shown to display greater reproducibility than Etest results (with 58.8% of all data sets in the acceptable range). Also at the interlaboratory level, the proportion of MIC values obtained with VetMIC that belonged to the complete agreement category (60.0%) was higher than the proportion of such values obtained with Etest (47.0%), which indicates a higher degree of interlaboratory reproducibility for the former method. Apart from some agent-specific effects, the majority of VetMIC and Etest replicate data sets were situated within a 1- to 2-log(2) dilution range, suggesting that the two methods can be considered to be equivalent for recognizing resistance phenotypes. This multicenter study has further validated the standard use of LSM and related mixed-medium formulations with commercially available systems and formed the basis for the ongoing development of the ISO 10932/IDF 223 standard for susceptibility testing of NELAB and bifidobacteria.

Primary metabolism in Lactobacillus sakei food isolates by proteomic analysis.

BACKGROUND: Lactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish. RESULTS: Proteins, the expression of which varied depending on the carbon source were identified, such as a ribokinase and a D-ribose pyranase directly involved in ribose catabolism, and enzymes involved in the phosphoketolase and glycolytic pathways. Expression of enzymes involved in pyruvate and glycerol/glycerolipid metabolism were also affected by the change of carbon source. Interestingly, a commercial starter culture and a protective culture strain down-regulated the glycolytic pathway more efficiently than the rest of the strains when grown on ribose. The overall two-dimensional gel electrophoresis (2-DE) protein expression pattern was similar for the different strains, though distinct differences were seen between the two subspecies (sakei and carnosus), and a variation of about 20% in the number of spots in the 2-DE gels was observed between strains. A strain isolated from fermented fish showed a higher expression of stress related proteins growing on both carbon sources. CONCLUSIONS: It is obvious from the data obtained in this study that the proteomic approach efficiently identifies differentially expressed proteins caused by the change of carbon source. Despite the basic similarity in the strains metabolic routes when they ferment glucose and ribose, there were also interesting differences. From the application point of view, an understanding of regulatory mechanisms, actions of catabolic enzymes and proteins, and preference of carbon source is of great importance.

A candidate probiotic with unfavourable effects in subjects with irritable bowel syndrome: a randomised controlled trial.

BACKGROUND: Some probiotics have shown efficacy for patients with irritable bowel syndrome (IBS). Lactobacillus (L.) plantarum MF1298 was found to have the best in vitro probiotic properties of 22 strains of lactobacilli. The aim of this study was to investigate the symptomatic effect of L. plantarum MF1298 in subjects with IBS. Primary outcome was treatment preference and secondary outcomes were number of weeks with satisfactory relief of symptoms and IBS sum score. METHODS: The design was a randomised double blind placebo-controlled crossover trial. 16 subjects with IBS underwent two three-week periods of daily intake of one capsule of 10(10) CFU L. plantarum MF 1298 or placebo separated by a four-week washout period. RESULTS: Thirteen participants (81%; 95% CI 57% to 93%; P = 0.012) preferred placebo to L. plantarum MF1298 treatment. The mean (SD) number of weeks with satisfactory relief of symptoms in the periods with L. plantarum MF1298 and placebo were 0.50 (0.89) and 1.44 (1.26), respectively (P = 0.006). IBS sum score was 6.44 (1.81) in the period with L. plantarum MF1298 treatment compared with 5.35 (1.77) in the period with placebo (P = 0.010). With a clinically significant difference in the IBS sum score of 2 in disfavour of active treatment, the number needed to harm was 3.7, 95% CI 2.3 to 10.9. CONCLUSIONS: This trial shows for the first time an unfavourable effect on symptoms in subjects with IBS after intake of a potential probiotic.

Growth Behavior of Listeria monocytogenes in a Traditional Norwegian Fermented Fish Product (Rakfisk), and Its Inhibition through Bacteriophage Addition.

Listeria monocytogenes may persist in food production environments and cause listeriosis. In Norway, a product of concern is the traditional and popular fermented fish product "rakfisk", which is made from freshwater salmonid fish by mild-salting and brine maturation at low temperatures for several months. It is eaten without any heat treatment, and L. monocytogenes, therefore, poses a potential hazard. We investigated the effect of salt and temperature on the growth of L. monocytogenes in rakfisk during the 91 days of maturation. The amounts of organic acids produced during fermentation were too low to inhibit growth of L. monocytogenes. Temperature was clearly the most important parameter for controlling L. monocytogenes. At 7 °C, approximately 2 log growth was observed during the first 14 days of fermentation, and the level of L. monocytogenes thereafter remained constant. At 4 °C, only a little growth potential of the pathogen was recorded. We also investigated the effect of the anti-Listeria bacteriophage P100 on rakfisk with added L. monocytogenes. The phage was introduced to the L. monocytogenes-inoculated fish before fermentation, and an average of 0.9 log reduction was observed throughout the fermentation period. This is the first study of L. monocytogenes behavior in rakfisk and points to possible measures for increasing the product safety.

Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1.

BACKGROUND: Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality. RESULTS: We have constructed a library of 76 Sec-type signal peptides from L. plantarum WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA. CONCLUSION: This is the first comprehensive experimental study showing that predicted SPs in the L. plantarum genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in L. plantarum. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.