Biogenic Amines in Wines and Pomegranate Molasses—A Non-Ionic Micellar Electrokinetic Chromatography Assay with Laser-Induced Fluorescence Detection

Biogenic amines in wine samples and pomegranate molasses were detected and quantified by a nonionic micellar electrokinetic chromatography method coupled to laser-induced fluorescence detection. The method provides a satisfactory and fast separation of seven biogenic amines and matrix peaks in food samples in less than 9 min. Detection limits are between 0.42 and 1.26 nM, and precisions were lower than 3% RSD for peak areas. The recovery values were between 93% and 104%, depending on the food matrix. The method is sensitive and rapid and widely applicable for the determination of biogenic amines in wine samples and fruit molasses.     

High-throughput detection of spore contamination in food packages and food powders using tiered approach of ATP bioluminescence and real-time PCR

A rapid and quantitative method for detection of Bacillus spores in food/non-alcoholic beverage packages and food powders has been developed using filtration-based ATP bioluminescence and real-time PCR, targeting the sporulation gene (spo0A). In combination with heat activation, the presence and amount of viable bacterial spores (i.e., Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus thuringiensis) was determined within 20 min through ATP signal amplifications. The detection limits of heat activation-ATP bioluminescence assay for B. amyloliquefaciens and B. licheniformis spores on food packages were 1.4 × 10² and 1.0 × 10³ CFU/cm², respectively. In contaminated food powders, B. thuringiensis spores could be detected by the ATP assay within the range of 7.9 × 10⁰ to 3.2 × 10⁴ CFU/mg powder while the PCR detection limit was 614 CFU/mg. Linear relationships between luminescent signal (RLU/mg) and plate count (CFU/mg) were found. The same sample after heat activation-ATP assay could be directly used for real-time PCR as a streamlined detection to confirm the identity of Bacillus spores in food packages and food powders even though some bacterial DNA loss was observed. This tiered approach, filtration-based one-tube ATP luminescence method as a rapid, viable screening and using real-time PCR as confirmation, could serve as a high-throughput tool for the detection of Bacillus spores in the food and beverage industry.     

The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 10² CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism.     

Survey of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products by commercial real-time PCR kits

Real-time PCR (RTiPCR) assays including enrichment stage were evaluated for the rapid detection of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products using molecular beacon probes available as commercial kits (WARNEX Genevision, Canada & AES Chemunex detection system, France). The accuracy of the assays was evaluated analyzing 1032 naturally contaminated food samples in combination to the conventional cultural methods. Presence/absence testing of the above pathogens was performed in 25 g samples of each product. In case of L. monocytogenes of 39 positive RTiPCR samples, 37 were confirmed by the cultural method (based on McNemar's test the difference between the two methods is insignificant). The highest incidence of L. monocytogenes in food products was found in desserts and the second highest in frozen pastries. None of the samples were cultural positive but negative in the RTiPCR test. One among the 343 investigated samples was positive for Salmonella spp. by RTiPCR and the cultural method. Out of 333 samples analyzed for E. coli O157:H7 no positive sample was detected. RTiPCR-based methods proved to be powerful tools for fast, sensitive and accurate pathogen detection in raw food ingredients and ready-to-eat products.     

Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in food products

We developed a rapid and reliable technique for simultaneous detection of Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes that can be used in food products. Magnetic nano-beads (MNBs) based immunomagnetic separation (IMS) was used to separate the target bacterial cells while multiplex PCR (mPCR) was used to amplify the target genes. To detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the DNA detection from dead cells. The results showed the detection limit of IMS-PMA-mPCR assay was about 10² CFU/ml (1.2 × 10² CFU/ml for S. Typhimurium, 4.0 × 10² CFU/ml for E. coli O157:H7 and 5.4 × 10² CFU/ml for L. monocytogenes) in pure culture and 10³ CFU/g (5.1 × 10³ CFU/g for S. Typhimurium, 7.5 × 10³ CFU/g for E. coli O157:H7 and 8.4 × 10³ CFU/g for L. monocytogenes) in spiking food products samples (lettuce, tomato and ground beef). This report has demonstrated for the first time, the effective use of rapid and reliable IMS combined with PMA treatment and mPCR assay for simultaneous detection of viable S. Typhimurium, E. coli O157:H7 and L. monocytogenes in spiked food samples. It is anticipated that the present approach will be applicable to simultaneous detection of the three target microorganisms for practical use.     

Determination of biogenic amines in foods using ultra-performance liquid chromatography (UPLC)

A rapid ultra-performance liquid chromatographic (UPLC) method for the determination of biogenic amines (putrescine, cadaverine, spermidine, spermine, phenylethylamine, histamine, tyramine and tryptamine) in selected food samples is described. The eight biogenic amines, which are the most important to be determined in food samples, were derivatized with dansyl chloride prior to UPLC separation. The dansylated amines were separated on an Agilent Zorbax Eclipse XDB – C18 column (50 × 4.6 mm ID, 1.8 μm) using gradient elution with a binary system of acetonitrile–water, a flow rate of 1.0 ml/min and UV detection at 225 nm. The analysis is very fast, all amines are well resolved and are eluted from the column in less than 6 min. The average repeatability of the method ranged between 1.02% and 2.14%. Limits of detection (LODs) for considered amines ranged between 0.032 and 0.098 μg/l; calibration curves showed very good linearity (r = 0.9994–1.0000). The method was applied to the analysis of amines in pork, beef, chicken and fish meat, cheese and edible mushrooms.     

Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to-eat meat samples

Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica are the most common foodborne bacterial pathogens and are responsible for many outbreaks. Therefore, multiplex detection of these three using a single assay platform is highly desirable. The objective was to develop and optimize a fiber optic sensor for simultaneous detection of these three from food. The streptavidin coated optical waveguides were immobilized with biotinylated polyclonal antibodies and exposed to the bacterial suspensions or enriched food samples for 2 h. Pathogens were detected after reacting with Alexa-Fluor 647-labeled monoclonal antibodies. Ready-to-eat beef, chicken and turkey meats were inoculated with each pathogen (∼100 cfu/25 g), enriched in SEL (Salmonella, E. coli, Listeria), a multipathogen selective enrichment broth for 18 h and tested with the biosensor. The biosensor was able to detect each pathogen, individually or in a mixture with very little cross-reactivity. The limit of detection for the sensor was ∼10³ cfu/ml for all three pathogens. Furthermore, the biosensor successfully detected each pathogen, grown in a mixture from enriched meat samples under 24 h. The pathogen presence was further verified by PCR and immunofluorescence assay. The multiplex fiber optic sensor shows promise for detection of the three pathogens if present in the same sample eliminating the use of multiple single pathogen detection platforms.     

Monitoring of Escherichia coli O157:H7 in food samples using lectin based surface plasmon resonance biosensor

A novel surface plasmon resonance (SPR) biosensor using lectin as bioreceptor was developed for the rapid detection of Escherichia coli (E. coli) O157:H7. The selective interaction of lectins with carbohydrate components from bacterial cells surface was used as the recognition principle for the detection. Five types of lectins from Triticum vulgaris, Canavailia ensiformis, Ulex europaeus, Arachis hypogaea, and Maackia amurensis, were employed to evaluate the selectivity of the approach for binding E. coli O157:H7 effectively. A detection limit of 3 × 10³ cfu mL^?1 was obtained for determination of E. coli O157:H7 when used the lectin from T. vulgaris as the binding molecule. Furthermore, the proposed biosensor was used to detect E. coli O157:H7 in real food samples. Results showed that the lectin based SPR biosensor was sensitive, reliable and effective for detection of E. coli O157:H7, which hold a great promise in food safety analysis.     

Simple,rapid and valid thin layer chromatographic method for determining biogenic amines in foods

A simple, rapid and valid thin layer chromatographic (TLC) method for determining biogenic amines in foods is established. Biogenic amines were extracted from foods with 5% TCA, the extract was washed by ethyl ether, the washed extract was dried and the dansyl derivatives were prepared. Separation of the dansylated amines was achieved on silica gel TLC plates by multiple development technique, and quantification was performed by densitometry at 254 nm. The response of the densitometer was linear and highly correlated with the amounts of dansylamines. The calculated determination coefficient (r²) ranged from 0.997 to 0.970. The sensitivity of the method was 45, 174, 703, 101, 132, 88, 515 and 61 for TRY, PUT, CAD, SPD, HIS, SPM, TYR and PHE, respectively. The relative standard deviation (RSD) of the proposed method (repeatability) ranged from 0.39 to 6.36% for TYR (80 ng) and PHE (20 ng), respectively. The accuracy of the proposed method ranged from 86.2 to 93.2% for PHE and HIS, respectively, and there was no significant differences (p<0.05) between the recoveries obtained for all kinds of food analyzed. The limit of detection of the proposed method was found to be 10ng for TRY, SPM and PHE, and 5 ng for the other biogenic amines tested. ©     

Antibacterial activities and membrane permeability actions of glycinin basic peptide against Escherichia coli

The objective of this study was to determine the antibacterial characteristics of glycinin basic peptide (GBP) and its effects on the cell membrane of Escherichia coli (E. coli). The antibacterial activities of GBP increased with increasing GBP concentrations and treatment times. Atomic force microscope analysis showed that GBP damaged the morphology of E. coli cells. GBP significantly (p < 0.05) increased the permeability of the outer membrane of E. coli cells treated with 80 μg/ml GBP, thereby enhancing the sensitivity of E. coli cells to erythromycin and rifampicin. Moreover, O-nitrophenyl-β-D-galactopyranoside (ONPG) entered into bacterial cells and immediately reacted with β-galactosidase in the cells due to the destruction of the inner membrane of E. coli. The damage to the bacterial membrane caused by GBP resulted in Ca^2 +, K⁺, and Mg^2 + leakage from the cells. SDS–PAGE of the membrane proteins further demonstrated that GBP significantly destroyed the cell membrane and promoted the extraction of membrane proteins in the presence of Triton X-114.Industrial relevanceIn recent years, chemical synthetic preservatives have aroused wide public concern because of various negative effects on human health. Novel natural food preservatives have received increasing interest from the food industry and researchers. The results of this study indicate that the antibacterial actions of GBP against E. coli occurred via interacting with destroying the bacterial membrane structure. Therefore, GBP may be a potential natural food preservative used in the food industry.     

Effect of food residues on efficiency of surfactant disinfectants against food related pathogens adhered on polystyrene and ceramic surfaces

The adhesion of pathogenic bacteria on food contact surfaces increases the risk of cross-contamination in the food industry. However, food-borne disease introduced by the production process can be mitigated by surfactant use. This study investigates the effect of food residues (milk, beef gravy and tuna gravy) on the bactericidal efficiency of benzalkonium chloride (BAC) and alkyldiaminoethylglycine hydrochloride (AGH). The test was conducted on pathogenic bacteria (Escherichia coli O26, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and B. cereus spores) dried and adhered to the surfaces of polystyrene and ceramic dishes at room temperature for 1.5 h. The protein and lipid rich food residues protected the bacterial cells from dehydration and from the adverse effects of disinfectants, although bacterial numbers were decreased after drying and the surfaces were clearly sterilized after disinfectant treatment at typical concentrations (0.5 mg/ml–2.0 mg/ml) for 10 min. Following general and proper washing processes, the bactericidal effect of the disinfectants became clearly visible. These results indicate that applying a proper washing process prior to disinfectant treatment can prevent cross-contamination.     

Efficacy of plant essential oils as antimicrobial agents against Listeria monocytogenes in hotdogs

The purpose of this study was to evaluate the antimicrobial activity of different plant essential oils against Listeria monocytogenes, both in vitro and in a food system. Essential oils of thyme, clove, and pimenta were found to be most effective, based on disc diffusion experiments. Thyme and clove proved to be highly effective against L. monocytogenes in peptone water (1 g/l) and reduced the bacterial population below detection limits at concentrations of 1 ml/l. Experiments were also carried out in hotdogs of different fat content (zero-, low-, and full-fat) to evaluate the antimicrobial activity of essential oils against L. monocytogenes. Thyme essential oil reduced bacterial populations significantly (P⩽0.05) at 1 ml/l level in zero- and low-fat hotdogs, but not in full-fat hotdogs. At 10 ml/l level it reduced the bacterial population >1.3 log₁₀ cfu/g in zero-fat hotdogs, but was less effective in low- and full-fat hotdogs. Clove essential oil also exhibited antimicrobial activity at 1 ml/l in all hotdogs, and was more effective than thyme at 5 ml/l. However, increasing concentration to 10 ml/l did not result in significant (P⩽0.05) reduction of bacterial population. It is concluded that efficacy of essential oils was reduced in a food system due to interaction with food components.     

Correlates of food choice in unemployed young people: The role of demographic factors,self-efficacy,food involvement,food poverty and physical activity

Associations between socio-demographic and psychological factors and food choice patterns were explored in unemployed young people who constitute a vulnerable group at risk of poor dietary health. Volunteers (N = 168), male (n = 97) and female (n = 71), aged 15–25 years were recruited through United Kingdom (UK) community-based organisations serving young people not in education training or employment (NEET). Survey questionnaire enquired on food poverty, physical activity and measured responses to the Food Involvement Scale (FIS), Food Self-Efficacy Scale (FSS) and a 19-item Food Frequency Questionnaire (FFQ). A path analysis was undertaken to explore associations between age, gender, food poverty, age at leaving school, food self-efficacy (FS-E), food involvement (FI) (kitchen; uninvolved; enjoyment), physical activity and the four food choice patterns (junk food; healthy; fast food; high fat). FS-E was strong in the model and increased with age. FS-E was positively associated with more frequent choice of healthy food and less frequent junk or high fat food (having controlled for age, gender and age at leaving school). FI (kitchen and enjoyment) increased with age. Higher FI (kitchen) was associated with less frequent junk food and fast food choice. Being uninvolved with food was associated with more frequent fast food choice. Those who left school after the age of 16 years reported more frequent physical activity. Of the indirect effects, younger individuals had lower FI (kitchen) which led to frequent junk and fast food choice. Females who were older had higher FI (enjoyment) which led to less frequent fast food choice. Those who had left school before the age of 16 had low food involvement (uninvolved) which led to frequent junk food choice. Multiple indices implied that data were a good fit to the model which indicated a need to enhance food self-efficacy and encourage food involvement in order to improve dietary health among these disadvantaged young people.     

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Occurrence of biogenic amines in Doubanjiang and Tofu

This study was performed to analyze biogenic amine contents and other parameters in doubanjiang and tofu. Through this study, it was found that doubanjiang contained considerably large amounts of most biogenic amines (>30 mg/kg of β-phenylethylamine in particular), and tofu had a relatively high level of spermidine (>20 mg/ kg). Therefore, the amounts of biogenic amines in the foods seem to be occasionally beyond the safe level for human consumption. Meanwhile, the biogenic amine contents in doubanjiang showed a good relationship with salt content (R²=0.89). The spermidine content in tofu samples was closely related to that in soybean, the raw material of tofu. There also appeared to be a good relationship (R²=0.82) between the biogenic amine contents and total plate counts in doubanjiang, but not in tofu. Most strains from the foods were capable of producing biogenic amines, and the identification revealed that bacterial ability to produce biogenic amines was determined at the level of strains rather than species. Taken together, it seems that biogenic amine contents in doubanjiang are mainly affected by fermentation processes, whereas those in tofu are primarily affected by raw materials.     

Use of small diameter column particles to enhance HPLC determination of histamine and other biogenic amines in seafood

Pre-column and post-column HPLC derivatization methods were modified and evaluated for the identification and quantification of nine biogenic amines in seafood. Two HPLC methods with column particles of 1.8 μm or 3 μm in diameter were modified and compared to classical methods using 5 μm column particles. Both pre-column derivatization with dansyl chloride and post-column derivatization with O-phthalaldehyde were studied. The HPLC methods were compared with respect to the time of elution, eluent consumption, backpressure as well as separation, sensitivity, recovery and repeatability for determination of biogenic amines in lean canned tuna and fatty frozen herring. The modified methods using smaller column particles of 1.8 μm or 3 μm allowed biogenic amines to be separated and quantified faster (23-59%) and with less eluent consumption (59-62%) than classical HPLC methods. Backpressures were below 170 bar and this allowed the use of classical HPLC systems rather than dedicated and costly ultra-high-pressure liquid chromatography (UHPLC) equipment. Biogenic amine separation, sensitivity, recovery and repeatability for the modified methods were similar to or performed better than for the classical HPLC methods. Due to reduced analyses time and eluent consumption the modified HPLC methods would improve biogenic amine analysis.     

Removal of meat biofilms from surfaces by ultrasounds combined with enzymes and/or a chelating agent

A curved ultrasonic transducer was devised to standardise biofilm removal for hygiene testing in internal or curved food contact surfaces. Meat biofilms made with Escherichia coli and Staphylococcus aureus on stainless steel sheets were studied. Ultrasounds (10 s at 40 kHz) alone failed to completely remove biofilms: 49 ± 5% and 39 ± 5% recovery rates were obtained for E. coli and S. aureus biofilms, respectively. A combined treatment, which involved the application of ultrasounds to EDTA and/or in enzymes solutions, allowed to remove up to 75 ± 4% and 100 ± 15% of E. coli and S. aureus biofilms, respectively. This application was in agreement with an industrial control i.e. a combined treatment: ultrasound generation in enzymes preparation restricted to an active chamber area with a fast and good reproducible recovery.Industrial relevanceThe biofilm phenomenon has been under intensive research for several years in food industry. A curved ultrasonic transducer was devised to standardise biofilm removal for hygiene testing in internal or curved food contact surfaces. This apparatus uses the mechanical effects of ultrasonic cavitation produced at 40 KHz (10 s) for the non-destructive detection of biofilms in food processing equipment. We report the utilisation of a combined treatment, which involved the application of ultrasounds to EDTA and/or in enzymes solutions on meat biofilms made with E. coli and S. aureus on stainless steel sheets. This application was in agreement with an industrial control i.e. a combined treatment: ultrasound generation in EDTA and/or enzymes preparation restricted to an active chamber area with a fast and good reproducible recovery.     

Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni

A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log₁₀/300 μl C. jejuni cells, one log₁₀ better (lower) than that of IMS-qPCR (2.1 log₁₀ CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log₁₀ CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.     

Effects of different freezing treatments on physicochemical responses and microbial characteristics of Japanese sea bass (Lateolabrax japonicas) fillets during refrigerated storage

In order to establish an effective freezing method for quality control, the present research evaluated the effects of the different freezing treatments on the quality of Japanese sea bass (Lateolabrax japonicas) over a period of 20 days storage at 0 ± 1 °C. Fish pH value, total volatile basic nitrogen (TVB-N), K-value, trimethylamine nitrogen (TMA-N), drip loss, hardness, color, biogenic amines, microbiological characteristics were measured. Sea bass fillets were stored at −18 °C (T1), −55 °C for 24 h and then −18 °C (T2), −55 °C (T3) for 3 months prior to refrigerated storage. T2 showed lower TVB-N, pH value, biogenic amines and drip loss than T3 and T1 did. Significant lower value of bacterial loads, b* value and hardness were observed in T1, T2 and T3 than those of control group. No significant differences were observed among T1, T2 and T3 for TMA-N and a* value. The study demonstrated that Japanese sea bass fillets treated with −55 °C for 24 h and then −18 °C up to 3 months maintain better quality during refrigerated storage.     

Biofilm formation of Salmonella Typhimurium on stainless steel and acrylic surfaces as affected by temperature and pH level

The effect of temperature (28, 37 and 42 °C) and pH (6 and 7) on the biofilm formation capability of Salmonella Typhimurium on stainless steel and acrylic was investigated. The rate of biofilm formation increased with increasing temperature and pH, while the number of attached cells after 240 h decreased with increasing temperature and was not different between pH 6 and 7. The surface hydrophobicity of bacterial cells was not significantly (p > 0.05) different among tested conditions. Electron-donating/accepting properties changed with pH and temperature, although these changes did not correlate with the ability to form biofilms under respective conditions. Attachment of S. Typhimurium showed a preference for stainless steel compared to acrylic surfaces under all conditions tested. The results suggest that salmonellae were less adherent to acrylic than to stainless steel surfaces; thus, acrylic-type surfaces should be considered for use in the food industry over stainless steel where applicable. The rate of biofilm formation increased at higher temperatures and pH levels within the tested ranges. Hurdle technology using lower temperatures reduced pH may help delay biofilm formation on food contact surfaces contaminated with S. Typhimurium.