Evaluation of enrichment procedures for recovering Listeria monocytogenes from dairy products

Six different enrichment media and five selective plating media were compared for their suitability for the recovery of Listeria monocytogenes from dairy products. These included media used to test milk products by the U.S. Food and Drug Administration (FDA) and the U.S. Centers for Disease Control (CDC), and media developed by the U.S. Department of Agriculture (USDA) for testing meat and poultry products. Test samples included naturally contaminated goat's milk, cultured milk products and ice cream manufactured with L. monocytogenes, and unpasteurized milk inoculated with heat- and freeze-injured cells of L. monocytogenes. Generally, the media and two-stage enrichment protocol developed by the USDA, with plating of samples after two consecutive 24-h incubation periods, yielded better recoveries than all other enrichment media incubated for 24 h. A modified USDA procedure, incorporating nonselective pre-enrichment of samples by omitting acriflavine and nalidixic acid from the primary USDA enrichment broth, and transfer of a larger volume of the initial culture broth to the secondary enrichment media, significantly increased recoveries of low numbers of sublethally stressed L. monocytogenes. Prolonged incubation of samples in the FDA enrichment broth, for 7 days, did not consistently improve recoveries over the initial 24-h incubation time of the medium. The selective plating medium developed by the USDA, lithium chloride-phenylethanol-moxalactam agar, was the most effective plating agar for isolation of L. monocytogenes following enrichment of samples in any broth culture, and increased recoveries of L. monocytogenes by 19-40% compared with other selective agar media tested.     

Development of a method to quantify in vitro the synergistic activity of "natural" antimicrobials

Despite numerous papers being published on the use of hurdle technology to control food-borne pathogens or spoilage organisms, there is no commonly accepted methodology to quantify the level of synergistic activity. This paper describes a method to quantify in vitro the synergistic activity of antibacterial agents against bacteria. Initially, a microtiter plate growth assay was used to determine the inhibitory concentrations of four "natural" antimicrobials (nisin, lauricidin, totarol, and the lactoperoxidase system (LPS)) against a panel of eight bacteria. Using the same microtiter system, the impact of various combinations of antimicrobials was assessed. The degree of synergy was based on the analysis of three criteria: (1) increase in lag phase, (2) reduction in culture density after 24 h, (3) and residual viability at 24 h. Only the lactoperoxidase system was active against all the Gram-positive and Gram-negative bacteria tested. Nisin, lauricidin, and totarol were only effective against the Gram-positive bacteria. The method successfully identified three combinations (nisin-lauricidin, LPS-nisin, and LPS-lauricidin) previously reported to have synergistic activity and highlighted the synergistic activity of two novel combinations (nisin-totarol and LPS-totarol). The development of a quick and reliable method to identify and quantify synergistic activity is a useful screening tool to establish preservative techniques that could have potential antimicrobial synergy in food-based systems.     

Altered sensitivity to a quaternary ammonium sanitizer in stressed Listeria innocua

Chemical sanitizers are commonly used to inactivate Listeria monocytogenes and other Listeria species that persist in food-processing environments after cleaning. In this study, Listeria innocua cultures were exposed to acid, heat, cold, and starvation stress and then assessed for sensitivity to the quaternary ammonium compound cetrimide. Unstressed and stressed cultures were exposed to cetrimide for 3 min, neutralized, and plated on tryptic soy agar with yeast extract to determine the percentage of survivors. Relative to controls, L. innocua exposed to acid and starvation conditions was less sensitive to cetrimide, whereas heat and cold stress increased cetrimide sensitivity (P < 0.05). The diminished sensitivity of acid- and starvation-stressed L. innocua to cetrimide suggests that these stressors might increase the persistence of this organism within food-manufacturing facilities. In contrast, enhanced L. innocua sensitivity to cetrimide following heat and cold stress suggests that these interventions might increase sanitation efficacy.     

Modelling the competitive growth of Listeria monocytogenes and Listeria innocua in enrichment broths

The overgrowth of Listeria innocua in enrichment broths designed for the isolation of Listeria monocytogenes is believed to result from two factors: a selective growth advantage of L. innocua, and/or an inhibitory interspecies interaction. The generation times of 13 isolates of L. innocua and L. monocytogenes were determined in Brain Heart Infusion (BHI) and a variety of enrichment media. No significant differences were found in growth characteristics between either species in the various media, suggesting that the growth advantage of L. innocua in enrichment media was not as significant as previously described. Kinetic analysis of mixed cultures of L. monocytogenes and isolates of L. innocua producing a variety of inhibitory activities demonstrated the possibility of an inhibitory interaction between these two species resulting in the overgrowth of the enrichment culture with L. innocua. Modelling the evolution of the ratio between two populations in an enrichment process was used to analyze the impact of a selective growth advantage in L. innocua in an enrichment process for growth of L. monocytogenes. These findings support the widely held view that an overgrowth of L. innocua in the enrichment process can result from both a selective growth advantage as well as the production of inhibitory compounds. From a practical perspective, these interactions can result in an increase in false negatives.     

Development of a method to quantify sucrose in soybean grains

The sucrose content of soybean seeds affects the final flavor of soy-derived products. The aim of this work was to develop a simple, low-cost, spectrophotometric method for sucrose quantification in soybean seeds. To achieve this goal, we combined the action of invertase, an enzyme that hydrolyses sucrose into fructose and glucose, with glucose oxidase, an enzyme widely used for glucose quantification. This system was adapted to ELISA plates, making large-scale analyses possible at low cost, with potential application in routine analyses. To validate this method, sucrose content was determined in seeds of 14 soybean cultivars by this new method, as well as by HPLC and the enzymatic method of Stitt. The correlation coefficients were high and significant between the results obtained with the new method and the HPLC method (r = 0.9766) and the Stiff method (0.9461).     

Development of a system to quantify muscle fibre destructuration

The aim of this study was to set up a method to quantify the loss or modification of muscle fibre structure (destructuration). Thanks to histology and image analysis, we managed to set up a reliable method with an uncertainty of 3.2%. To express the results, a new indicator called MDI (Meat Destructuration Indicator), was calibrated according to mechanically recovered meat market practices. The MDI indicator was strongly correlated (0.95) with the sensory assessment of a European panel (126 professionals). The threshold between raw materials that could be labelled as “meat” in processed products and mechanically separated meat (MSM) has been set at 58.1%, according to the panellist’s judgements. Thus MDI system can be used to characterise meat raw materials from mechanical recovery system according to Regulation (EC) No. 853/2004. It can also be used to study relationships between manufacturing parameters and destructuration or between destructuration of raw materials and processed products characteristics.     

Development of a laboratory scale clean-in-place system to test the effectiveness of "natural" antimicrobials against dairy biofilms

A laboratory scale system, partially reproducing dairy plant conditions, was developed to quantify the effectiveness of chlorine and alternative sanitizers in reducing the number of viable bacteria attached to stainless steel surfaces. Stainless steel tubes fouled in a continuous flow reactor were exposed to a standard clean-in-place regime (water rinse, 1% sodium hydroxide at 70 degrees C for 10 min, water rinse, 0.8% nitric acid at 70 degrees C for 10 min, water rinse) followed by exposure to either chlorine (200 ppm) or combinations of nisin (500 ppm), lauricidin (100 ppm), and the lactoperoxidase system (LPS) (200 ppm) for 10 min or 2, 4, 8, 18, or 24 h. There was significant variation in the effectiveness of the alkaline-acid wash steps in reducing cell numbers (log reduction between 0 and 2). Following a 10-min treatment, none of the sanitizers significantly reduced the number of attached cells. Two hours of exposure to chlorine, nisin + the LPS, or lauricidin + the LPS achieved 2.8, 2.2, and 1.6 log reductions, respectively. Exposure times > 2 h did not further decrease the number of viable bacteria attached to the stainless steel. The effectiveness of combinations of nisin, lauricidin, and the LPS was similar to that of chlorine (P > 0.05), and these sanitizers could be used to decontaminate the surfaces of small-volume or critical hard-to-clean milk processing equipment.     

Evolution of Listeria populations in food samples undergoing enrichment culturing

The isolation of Listeria monocytogenes from food is carried out using a double enrichment. It is believed that the double enrichment can allow the overgrowth of Listeria innocua in samples where both species are present. In this study, we have evaluated the impact of overgrowth between Listeria species and strains during each step of the enrichment process. The effect of factors minimizing interactions between strains or phage inhibitory effects has also been estimated. In an artificially contaminated food undergoing enrichment, overgrowth could result from competitive interactions between Listeria spp. resulting from the production of bacteriocins and bacteriophage at high initial contamination levels (>10(4) cfu/g), but not at lower levels (50-100 cfu/g) as generally found in contaminated foods. At high levels of inoculation, the competitive effect could be reduced by solidification of the selective broths, to limit the diffusion of the inhibitors. Overgrowth resulting from differences in growth rate occurred independent of the initial contamination level. However, in naturally contaminated foods undergoing enrichment, there were no absolute correlations between growth rates or inhibitory profiles in terms of strain evolution during enrichment. In fact, Listeria strains which were predominant in the original sample in most cases remained the dominant strains at the end of the enrichment, although the relative proportion of any given strain could change significantly over the enrichment process. Additional factors which have yet to be identified impact on the evolution of Listeria in the two-step enrichment process. Analysis of strain evolution in eight naturally contaminated foods has indicated that the second enrichment step in Fraser broth can be reduced from 48 to 24 h without impacting on the recovery of L. monocytogenes. Our limited survey of naturally contaminated foods also demonstrated that maximum recovery of L. monocytogenes and other Listeria strains was found following 24 h incubation in 1/2 Fraser Broth. This finding suggests that it may be possible to shorten the current two-step isolation method further without reducing method sensitivity.     

Development of real-time PCR methods to quantify patulin-producing molds in food products

Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g⁻¹ per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain.     

Comparison of a cold enrichment and the FDA method for isolating Listeria monocytogenes and other Listeria spp. from ready-to-eat food on retail sale in the U.K

A cold enrichment and the modified FDA selective enrichment method were compared for their ability to detect Listeria monocytogenes and other Listeria species from various ready-to-eat foods on sale in the UK. Of 57 food samples examined using cold enrichment, five yielded L. monocytogenes, and two L. innocua. The FDA enrichment method yielded three samples positive for L. monocytogenes only. Foods examined included soft cheeses, fermented meat sausages, pates and salads.     

Development of a predictive model describing the growth of Listeria Monocytogenes in Kimbab

This study is to develop mathematical models to predict the growth of Listeria monocytogenes in kimbab as a function of storage temperature. Kimbab which was inoculated with L. monocytogenes were incubated at 4, 10, 15, and 30°C. The primary model showed a good fit (R²=0.9845 to 0.9967) to a Gompertz equation to obtain specific growth rates (SGR) and lag time (LT) at each temperature. The SGR of L. monocytogenes in the kimbab increased and LT decreased by increasing temperature. Secondary polynomial model was developed using PRISM general nonlinear analysis software for SGR and LT. The secondary models were 0.1479−(0.02457×Temp)+(0.001296 ×Temp²) for SGR and 312.8−(30.21×Temp)+(0.6654 ×Temp²) for LT. This secondary polynomial model was judged as appropriate based on the coefficient of determination (R² of the SGR and LT model=0.9995, 0.9556), the bias factor (B _f of the SGR and LT model=0.97, 0.94), and the accuracy factor (A _f of the SGR and LT model=1.10, 1.68). Reliable predictions of L. monocytogenes SGR and LT in kimbab were based on temperature.     

Development of an in-vitro mouth model to quantify salt release from gels

In the present study, an in-vitro mouth model to quantify salt release from food structures has been developed. In this instance biopolymer gels were used as model food systems. The model aimed to reproduce key phenomena occurring during oral processing, such as diffusion through the sample and compression. Salt release profiles from different gels (gelatin, gellan and alginate), under quiescent conditions and compression, were determined at temperatures of 25 and 37 °C. In-vitro results indicated that salt release is affected both by the type of gelling agent and by temperature. When melting took place, release occurred within seconds. However, when diffusion through the biopolymer matrix was the controlling parameter, the time scale was in the order of hours. It has been shown that compression of the gel only affects release when fractures occur. This is believed to be a consequence of increased surface area. Finally, a mathematical model has been compiled to predict release profiles when diffusion is the controlling mechanism.     

Development of a multiplex real-time PCR to quantify aflatoxin, ochratoxin A and patulin producing molds in foods

The antimicrobial activity of 8 Bacillus spp. and 2 Lysinibacillus spp. representing the predominant aerobic sporeformers during traditional maari fermentations, a traditional fermented baobab seeds product from Burkina Faso, was investigated. The antimicrobial activity was assessed against a total of 31 indicator organisms representing various Gram-negative and positive pathogens. The screening showed that 3 Bacillus subtilis strains (B3, B122 and B222) in particular had antimicrobial activity against some Gram-positive organisms and were selected for further studies. It was found that the antimicrobial substances produced were heat stable, in-sensitive to catalase, sensitive to protease and trypsin but resistant to the proteolytic action of papain and proteinase K and equally active at pH values ranging from 3 to 11. Bacteriocin secretion started in late exponential growth phase and maximum activity was detected during the stationary growth phase. The production of bacteriocin by B. subtilis B3, B122 and B222 was dependent on the aeration conditions. Maximum production of bacteriocin was observed under reduced aeration. Specific primers were used to screen isolates B3, B122 and B222 for genes involved in the synthesis of the bacteriocins subtilosin A, subtilin, sublancin and ericin. Amplicons of the expected sizes were detected for iywB, sboA, sboX, albA and spaS involved in the biosynthesis of subtilosin and subtilin, respectively. The translated nucleotide sequences had 100% identity to the YiwB, SboX and SboA amino acid sequences of the subtilosin A producing B. subtilis subsp. subtilis strain 168. Interestingly there was a 3 amino acid deletion at the N-terminal part of AlbA in B3, B122 and B222 that probably alters the activity of this enzyme. Analysis of the spaS gene sequences of B3, B122 and B222, encoding a subtilin precursor peptide, showed that the translated nucleotide sequence had 98% identity with the corresponding SpaS amino acid sequence of subtilin producing B. subtilis subsp. spizizenii strain ATCC6633.     

Implementation of a novel tool to quantify dough microstructure

A method was developed to quantify the microstructure of wheat dough proteins assessed by a confocal laser scanning microscope (CLSM) in combination with an image processing and analyzing tool. A variation of the water addition especially showed high significant (p<0.01) linear correlations with the branching index (r=-0.92). This branching index exhibited high significant correlation coefficients with the rheological measures complex shear modulus (r=0.88), creep compliance (r=-0.71) and relative elastic part (r=0.82). In summary the results submit a novel view on the microstructure of dough. The obtained visual structure of the dough via CLSM in combination with image processing and analyzing has proven to be a reliable and powerful tool for the acquisition and validation of dough protein microstructure. The high dependency of rheology from structural elements could be verified.     

Efficiency of four secondary enrichment protocols in differentiation and isolation of Listeria spp. and Listeria monocytogenes from smoked fish processing chains

Four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h: Impedimetric method: Listeria electrical detection medium) were studied for their ability to isolate Listeria spp. and Listeria monocytogenes from fish and environmental samples collected along the processing chain of cold-smoked fish. From all methods, Listeria spp. and L. monocytogenes were respectively present in 56 and 34 of 315 samples analysed. Fraser broth incubated for 48 h gave the fewest false negative Listeria spp. results [4/56; (7.1%)], but concurrently only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes, Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp. positive samples. Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes positive samples correctly and gave fewer false positive results. The overall conclusion was that more than one isolation method is needed to accurately estimate L. monocytogenes contamination rates.     

Development of a new reflectance technique to investigate the mechanism of emulsification

A new reflectance technique has been developed to measure the droplet size evolution during the process of emulsification in real-time. This has been used to investigate the dynamic behaviour of 50% oil-in-water emulsions. The performed experiments were designed in order to investigate the occurring droplet break-up and droplet coalescence phenomena individually, by carefully creating processing conditions where each of those events is dominant. The effect of emulsifier (Tween 20) concentration and different hydrodynamic conditions on the droplet break-up and coalescence phenomena and the emulsion droplet size evolution during processing were all investigated. The concentration of Tween 20 was shown to be a key parameter affecting the droplet size of the emulsion at the early stages of processing (within the first 3 min). However, during the later stages of processing, hydrodynamic conditions have a more pronounced effect on determining the final droplet size. Unlike droplet break-up, droplet coalescence rate decreases by intensifying the hydrodynamic conditions of the process as a consequence of the high capillary pressure of the smaller droplets being produced.     

Development of a surface plasmon resonance-based immunoassay for Listeria monocytogenes

A polyclonal antibody was produced against Internalin B (InlB)-enriched extract and used to develop an inhibition assay to detect Listeria monocytogenes cells in solution using surface plasmon resonance. The gene sequence encoding for the InlB protein was cloned into a Qiagen pQE-60 vector, expressed in Escherichia coli, and purified by immobilized metal affinity chromatography. Protein G-purified anti-InlB-enriched extract polyclonal antibody was incubated with various concentrations of L. monocytogenes cells and subsequently injected over a purified-recombinant InlB (rInlB)-immobilized CM5 sensor chip surface. A decrease in antibody binding response was observed with increasing L. monocytogenes cell concentrations. Intraday and interday assay variability studies were carried out to evaluate precision and reproducibility. The assay had a limit of detection of less than 2 x 10(5) cells per ml and could be successfully reproduced with coefficients of variation of between 2.5 and 7.7%.     

单核细胞增生李斯特菌荧光定量PCR检测试剂盒的研制

目的:建立一种用于单核细胞增生李斯特菌快速检测的荧光定量PCR试剂盒.方法:根据本课题组前期工作基础,针对单核细胞增生李斯特菌hly基因序列设计TaqMan探针和引物,建立荧光定量PCR检测方法,将反应试剂组装成试剂盒,并对该试剂盒的各项参数进行评价.结果:特异性试验表明,组装的试剂盒对8林单核细胞增生李斯特菌标准菌株的检测均呈阳性反应,对6株其它种的李斯特菌及13株食品(环境)中常见的非李斯特污染菌(如沙门氏菌、金黄色葡萄球菌)呈阴性反应.该试剂盒的基因组DNA检测灵敏度为3.94fg/反应(约合1.34CFU/反应).该试剂盒批内平均变异系数为2.2%,批间变异系数为1.9%,具有良好的重复性;同时稳定性强,反复冻融处理对其无明显影响;可在-20℃下避光保存1年.结论:该试剂盒具有特异性强、灵敏度高、重复性好、稳定性强等优点,可应用于单核细胞增生李斯特菌的快速检测.     

The inhibitory effect of natural microflora of food on growth of Listeria monocytogenes in enrichment broths

The aims of this study were to (i) compare the inhibitory effects of the natural microflora of different foods on the growth of Listeria monocytogenes during enrichment in selective and non-selective broths; (ii) to isolate and identify components of the microflora of the most inhibitory food; and (iii) to determine which of these components was most inhibitory to growth of L. monocytogenes in co-culture studies. Growth of an antibiotic-resistant marker strain of L. monocytogenes was examined during enrichment of a range of different foods in Tryptone Soya Broth (TSB), Half Fraser Broth (HFB) and Oxoid Novel Enrichment (ONE) Broth. Inhibition of L. monocytogenes was greatest in the presence of minced beef, salami and soft cheese and least with prepared fresh salad and chicken pâté. For any particular food the numbers of L. monocytogenes present after 24 h enrichment in different broths increased in the order: TSB, HFB and ONE Broth. Numbers of L. monocytogenes recovered after enrichment in TSB were inversely related to the initial aerobic plate count (APC) in the food but with only a moderate coefficient of determination (R²) of 0.51 implying that microbial numbers and the composition of the microflora both influenced the degree of inhibition of L. monocytogenes. In HFB and ONE Broth the relationship between APC and final L. monocytogenes counts was weaker. The microflora of TSB after 24 h enrichment of minced beef consisted of lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas spp., Enterobacteriaceae, and enterococci. In co-culture studies of L. monocytogenes with different components of the microflora in TSB, the lactic acid bacteria were the most inhibitory followed by the Enterobacteriaceae. The least inhibitory organisms were Pseudomonas sp., enterococci and B. thermosphacta. In HFB and ONE Broth the growth of Gram-negative organisms was inhibited but lactic acid bacteria still reached high numbers after 24 h. A more detailed study of the growth of low numbers of L. monocytogenes during enrichment of minced beef in TSB revealed that growth of L. monocytogenes ceased at a cell concentration of about 10² cfu/ml when lactic acid bacteria entered stationary phase. However in ONE Broth growth of lactic acid bacteria was slower than in TSB with a longer lag time allowing L. monocytogenes to achieve much higher numbers before lactic acid bacteria reached stationary phase. This work has identified the relative inhibitory effects of different components of a natural food microflora and shown that the ability of low numbers of L. monocytogenes to achieve high cell concentrations is highly dependent on the extent to which enrichment media are able to inhibit or delay growth of the more effective competitors.