In-house validation of a real-time PCR method for rapid detection of<Emphasis Type="Italic"> Salmonella</Emphasis> ssp. in food products

A real-time polymerase chain reaction (PCR) method for Salmonella ssp. detection in food samples has been developed and validated in-house. The specificity of the assay was confirmed by tests with 295 different Salmonella strains, including four strains of Salmonella bongori. When tested with extracted Salmonella DNA the lowest detected amount was found to be 5 fg, which is equivalent to approximately one genome copy. The detection limit was further determined by artificial contamination of minced meat with S. Typhimurium cells and of pastry with S. enteritidis using the most probable number approach for cell dose dilutions. It was calculated that even one colony forming unit of Salmonella was still detectable in 25 g food after enrichment culture for 18 h. An additional PCR system for internal positive control, which was included in each reaction and detected in parallel via another reporter fluorescence dye, has no negative impact on the sensitivity of the assay. The method was evaluated with 1,293 naturally contaminated food samples and compared to the conventional cultural method. Of 55 positive PCR samples, 45 were confirmed by the cultural method. The statistical comparison revealed a correlation of 99.2% for specificity, of 100% for sensitivity and of 99.2% for trueness. The results of the comparative analysis and the advantages of the real-time PCR method for detection of Salmonella ssp. under routine laboratory conditions are discussed.     

Extracts of plant cell cultures of <Emphasis Type="Italic">Lavandula vera</Emphasis> and <Emphasis Type="Italic">Rosa damascena</Emphasis> as sources of phenolic antioxidants for use in foods

Ethanolic extracts of plant cell cultures of lavender (Lavandula vera) and rose (Rosa damascena) have been examined as potential food antioxidants. The L. vera cell extract quenched the radicals Fremy’s salt, DPPH (2,2-diphenyl-1-picrylhydrazyl radical), and ABTS^·+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic) radical) more efficiently than the R. damascena extract. Also the L. vera extract inhibited lipid oxidation in a methyl linoleate emulsion more efficiently than the R. damascena extract. However, the L. vera extract had a prooxidative effect on the iron-based Fenton reaction in an aqueous model system. A similar effect was observed for pure rosmarinic acid, but not for the R. damascena extract. The addition of L. vera extract to minced chicken meat reduced lipid oxidation (measured as thiobarbituric acid reactive species) and the loss of α-tocopherol during cold storage after the meat was cooked. This suggests the antioxidative properties of L. vera extracts dominate in a real food system.     

Antitumor activities of liposome-incorporated aqueous extracts of <Emphasis Type="Italic">Anodonta </Emphasis><Emphasis Type="Italic">woodiana</Emphasis> (Lea, 1834)

To acquire the effectiveness of oral treatments, aqueous extracts of Anodonta woodiana (HB) were incorporated into liposome and its antitumor activities evaluated in vivo. The HB-loaded liposomes (HBL) were prepared at a mean size of 14.85 μm by reverse-phase evaporation method. Referring to the maximum tolerated doses test, mice with orally administrated HBL, at a 3 g/kg body weight dosage, showed no obvious acute toxic sign. Furthermore, the tumoricidal activities of HBL against C26 murine colon carcinoma, Lewis lung tumor, human QGY hepatic carcinoma and MKN-45 gastric tumor were examined, respectively. In contrast to free HB, HBL possessed remarkable antitumor activity. Simultaneously, the effect of HBL was observed in a dose-dependent manner. For C26 murine colon carcinoma and Lewis lung tumor, the inhibitory ratios of HBL were 54.36 and 51.97% at a dose of 400 mg/kg, respectively. The suppression of tumor growth and the reduction in body weight were more pronounced in human QGY hepatic carcinoma and MKN-45 gastric tumors inoculated mice by treatment of HBL during 30 days. All these promising results implied that liposome-incorporated aqueous extracts of Anodonta woodiana had a more potential application as a natural antitumor and immunomodulator formulation.     

Production of biogenic amines by<Emphasis Type="Italic"> Morganella morganii</Emphasis>,<Emphasis Type="Italic"> Klebsiella pneumoniae</Emphasis> and<Emphasis Type="Italic"> Hafnia alvei</Emphasis> using a rapid HPLC method

The histidine decarboxylating activity and production of biogenic amines by Morganella morganii (NCIMB, 10466), Klebsiella pneumoniae (NCIMB, 673) and Hafnia alvei (NCIMB, 11999) were investigated using a rapid HPLC method. Derivatisation of the bacterial samples was carried out using benzoyl chloride. A gradient elution system was used for analysis with a mixture of acetonitrile and HPLC grade water. Bacterial strains not only produce histamine in histidine-enriched broth but also the other biogenic amines. The chromatographic results show that bacterial strains are also capable of producing spermine and spermidine in histidine-enriched broth. Bacterial ammonia production by all three strains was clearly detected since ammonia is generated during the degradation of histidine. The study demonstrates that the highest histamine production was obtained by Morganella morganii, followed by Klebsiella pneumoniae, and the lowest with the Hafnia alvei. Therefore, Morganella morganii and Klebsiella pneumoniae have strong histidine decarboxylase activity since they are prolific histamine-forming bacteria     

Antioxidant effect of dittany (<Emphasis Type="Italic">Origanum dictamnus</Emphasis>) in pre-cooked chicken meat balls during chill-storage in comparison to rosemary (<Emphasis Type="Italic">Rosmarinus officinalis</Emphasis>)

Dittany (Origanus dictamnus L.) has been compared with rosemary (Rosmarinus officinalis L.) as an antioxidant in pre-cooked meat balls made from chicken breast and added 0.50% salt during chill storage for up to ten days packed in atmospheric air. For an addition of 0.10% of dried leaves, dittany yielded protection of the product against oxidation a little less efficiently but comparable to dried rosemary added at the same concentration. For addition of 0.050%, dittany was less efficient than rosemary, while dittany at this concentration protected vitamin E against degradation in the product during storage even better than rosemary.     

Modeling high pressure inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Listeria innocua</Emphasis> in whole milk

The survival curves of Escherichia coli and Listeria innocua inactivated by high hydrostatic pressure (HHP) were obtained at room temperature (∼22 °C) and at five pressure levels (400, 450, 500, 550 and 600 MPa) in whole milk. These curves were described by the Weibull model and parameters of this model were reduced from two to one with slight loss of goodness-of-fit. The logarithm of the time constant parameter (δ) of the reduced Weibull model was described with respect to high pressure (P). This approach can be used to define a z _p value analogous to the modeling of the classical D value (increase in pressure that results in one log unit decrease of δ values). The development of accurate survival models under high pressure, as presented here, can be very beneficial to food industry for designing, evaluating and optimizing HHP processes as a new preservation technology.     

Efficiency of pulse pressure treatment for inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Listeria innocua</Emphasis> in whole milk

Escherichia coli ATCC 11775 and Listeria innocua ATCC 33090 in whole milk were inactivated by single- and multi-pulsed (up to 10 pulses) high hydrostatic pressure (HHP) treatments. Both bacteria showed similar resistance to single- and multi-pulsed HHP. The efficiency of pulsed pressure treatment depends on the combination of holding time of each pulse and number of pulses. It was observed that multi-pulsed pressure treatment instead of traditional single-pulsed HHP could be used to pasteurize milk at a lower pressure level. Nevertheless, an optimization is necessary between the pulse holding time, number of pulses, and pressure levels to reach the desirable log-reduction of microorganisms compatible with industrial application. This study was partly presented in Joint 21st AIRAPT and 45th EHPRG International Conference on High Pressure Science and Technology held September 17–21, 2007 in Catania (Italy).     

Development of immunoliposome-based assay for the detection of <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium

This study was aimed to produce a polyclonal antibody against Salmonella Typhimurium and to develop a liposome-based immunoassay to validate its detection. Formalin-killed cells of S. Typhimurium were subcutaneously injected as an antigen into rabbit to produce antibody. The optimum production of rabbit anti-Salmonella immunoglobulin G (anti-Salmonella IgG) antibody was reached to the highest level on 8 week. Purification of rabbit anti-Salmonella IgG from immunized rabbit sera was accomplished using caprylic acid and ammonium sulfate precipitation method. The purified anti-Salmonella IgG was used to conjugate with liposome to prepare anti-Salmonella IgG-tagged liposome (immunoliposome). Finally, an improved immunoassay method was developed using the immunoliposome for the detection of S. Typhimurium. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 25 and 51 kDa, corresponding to a light and a heavy chain, respectively. The immunoliposome was successfully developed for the detection of S. Typhimurium in a 96-well microtiter plate, using purified anti-Salmonella IgG antibody. The improved technique was able to detect 10³ cells/mL of Salmonellas. In the future, even better detection may be achieved by using this immunoliposome with some modifications or other detection methods.     

Tyramine production of technological important strains of <Emphasis Type="Italic">Lactobacillus</Emphasis>, <Emphasis Type="Italic">Lactococcus</Emphasis> and <Emphasis Type="Italic">Streptococcus</Emphasis>

The aim of this paper was to study the biogenic amines (histamine, tyramine, putrescine, cadaverine, agmatine, spermine and spermidine) production of selected technological important lactic acid bacteria (strains of the genera Lactococcus, Lactobacillus and Streptococcus). Three methods (ion-exchange chromatography (IEC), PCR and cultivation method with pH indicator) were used. Within the 39 strains of lactic acid bacteria tested, the production of tyramine (formed by tyrosine decarboxylase) was detected in eight strains (3 strains of Lactococcus lactis subsp. lactis, three strains of Lactococcus lactis subsp. cremoris, 1 strain of Streptococcus thermophilus and 1 strain of Lactobacillus delbrueckii subsp. bulgaricus). The other tested biogenic amines were not detected. Cultivation in decarboxylation broth seems to be the least accurate method for the detection of biogenic amines due to enhanced risk of false-positive reactions. Therefore, in order to detect bacteria producing biogenic amines, the combination of PCR and chromatographic methods (e.g. IEC) can be recommended.     

Quantitative determination of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. in milk using a series piezoelectric quartz crystal sensor

A series piezoelectric quartz crystal (SPQC) sensor was developed for quantitative determination of Lactobacillus spp. populations in milk. When the electrodes were immersed in a reaction cell with bacterial inoculum, the change of frequency was caused by the impedance change of the microbial metabolism. A significant frequency decrease was found due to the coagulation of milk when the Lactobacillus spp. was cultivated in the media. The SPQC sensor system established in this study demonstrated the specificity and selectivity for detection of Lactobacillus spp. in milk sample. The calibration curve of detection time against density of Lactobacillus spp. shows a linear correlation coefficient (R ² = 0.8453) over the range of 10²–2.4 × 10⁵ CFU ml⁻¹. The detection time was influenced by the addition of peptone and glucose. The sensor exhibited rapid (within 4 h) and enabled real time monitoring of Lactobacillus spp. growth. This system is potentially applicable to detect Lactobacillus spp. concentration at local farm when a suitable temperature control device is adapted.     

Investigations of the natural microflora of poppy seeds (<Emphasis Type="Italic">Papaver somniferum</Emphasis>) and hazelnut kernels (<Emphasis Type="Italic">Corylus avellana</Emphasis>) including microbiological decomposition

Fatty seeds of Papaver somniferum and Corylus avellana undergo a rapid microbial degradation after being ground. Those bacteria and fungi which are mainly responsible for the microbial decay were identified, and the most important growth and death processes were documented using crucial indicator-organisms. Additionally, an aflatoxin-screening was carried out in order to assess the possible risk-potential of food intoxication. The acid value (indicator for free fatty acids) of poppy seeds and hazelnut kernels was determined during their fermentation in order to document the decomposition of triglycerides.In this study it could be proved that initially a natural decay of oil seeds is caused by bacteria, yeasts and mould fungi. After the bacteria died in the course of time, yeasts and mould fungi dominated the germ spectrum. Bacteria taking part in the degradation were identified as varieties of Staphylococcus xylosus, Enterobacteriaceae and coliforms. Yeasts were identified as Pichia burtonii, and the mould fungi are associated with the genus Alternaria.On account of the absence of the genus Aspergillus in the spectrum of mould fungi, no aflatoxin was produced.     

Antioxidant activity of <Emphasis Type="Italic">Dioscorea batatas</Emphasis> Decne glycoprotein

Dioscorea batatas Decne (DBD) has been traditionally used as folk medicine and health food in Korea. One glycoprotein was isolated from DBD and confirmed to have 30 kDa molecular weight. The DBD glycoprotein was tested its antioxidative activity and characterized in various chemical conditions. The DBD glycoprotein has the optimal free radical scavenging activities in acidic and neutral pH and up to 85 °C. In the M²⁺ ions (Ca²⁺, Mn²⁺, and Mg²⁺) in the presence of EDTA, the activities of DBD glycoprotein reduced, compared to DBD glycoprotein alone without metal ion. Interestingly, the results in this study indicated that the activities of DBD glycoprotein do not depend on the presence of EDTA. Interestingly, when DBD glycoprotein was treated with deactivation agents (pronase E or NaIO₄), scavenging activity of DBD glycoprotein was decreased. The anti-oxidative effects of DBD glycoprotein on hydroxyl radicals in cell-free system revealed, and the DBD glycoprotein has remarkable scavenging effects on hydroxyl radicals generated by G/GO. Furthermore, the results in this study showed that the DBD glycoprotein (200 μg/ml) significantly inhibits intracellular ROS amounts and protects from cytotoxicity in primary mouse splenocyte culture treated with GO (30 mU/ml). Therefore, we speculate that DBD glycoprotein has an antioxidative potential as one of natural antioxidants.     

Influence of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> on<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> growth in a fresh vegetable model system

The influence of a Lactobacillus plantarum (B₄) on the growth of Staphylococcus aureus (Sa₄) was verified by impedometric methods in a suitable model reproducing the characteristics of fresh vegetables. The inoculum size of the single strains and their growth temperature were varied according to a Central Composite Design. The results obtained via statistical analysis showed that the temperature affected the growth of both S. aureus and L. plantarum strains. The pathogenic strain, independently of its inoculum size, was inhibited by L. plantarum at all the tested temperatures. A proper combination of specific lactic acid bacteria and storage temperature should improve the safety of the vegetable products.     

Novel anticoagulant compound from fermented red alga <Emphasis Type="Italic">Pachymeniopsis elliptica</Emphasis>

Natural fermentation was tested as a method of releasing active compounds during screening for potential anticoagulant activity in three types of algae (Pachymeniopsis elliptica, Sargassum horneri, and Ulva pertusa). Freeze dried algae samples (2.5 g) were fermented by adding 75 g of sugar and 500 mL of water and thereafter kept at room temperature (25 °C) for 3 months. Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were measured every 2 weeks for 3 months to determine the optimum time for the highest activity. Fermented P. elliptica, (which had the highest activity) was subjected to anion exchange chromatography (DEAE-cellulose) and sepharose 4B gel permeation chromatography. The purified sample was analyzed by agarose-gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) to confirm the purification and to determine the molecular mass, respectively. The 360 μg/mL of purified compound (Mwt > 500,000 Da) had both APTT and PT activities (>1,000 s). However, at the concentrations of 180 μg/mL, purified compound and heparin showed 540 and >1,000 s APTT activity, respectively. Though, the purified compound of P. elliptica considered as a weaker anticoagulant than heparin, this purified anticoagulant polysaccharide could be considered as a good alternative source as an anticoagulant. Moreover, the technique of fermentation is an inexpensive and feasible, this purified anticoagulant polysaccharide compound could be used in pharmaceutical and biomedical industry. Further investigations need to be performed to determine the mechanism of this novel anticoagulant compound. The authors Prashani Mudika Ekanayake and Chamilani Nikapitiya contributed equally to this work.     

Effects of suni-bug (<Emphasis Type="Italic">Eurygaster</Emphasis> spp.) damage on size distribution of durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> L.) proteins

Wholemeal samples were obtained from five durum wheat cultivars at two different bug (Eurygaster spp.) damage levels (medium and high damage). The samples were incubated (60 and 120 min) and used in size exclusion high performance liquid chromatography (SE-HPLC) analyses. The results showed that the amount of larger polymeric protein (TP1) and smaller polymeric protein (TP2) obtained from total (sodium dodecyl sulfate soluble) proteins decreased significantly in the bug-damaged samples, while the amount of total larger monomeric proteins (TP3) increased. The polymeric/monomeric protein ratio of all cultivars decreased at 60 min of incubation with increasing damage level. For all cultivars, the ratio of unextractable polymeric protein (UPP%) significantly decreased at 60 min of incubation except cv. Diyarbakir. The results suggested that bug protease caused depolymerization and/or disaggregation of polymeric proteins to lower their average molecular size. The changes in protein structure as determined using SE-HPLC supported by the decreases in gluten content and gluten index values which decreased with suni-bug damage. Deteriorative effects of bug damage on durum wheat quality were found to be quite similar to those on bread wheats.     

Effects of process parameters on growth and metabolism of<Emphasis Type="Italic"> Lactobacillus sanfranciscensis</Emphasis> and<Emphasis Type="Italic"> Candida humilis</Emphasis> during rye sourdough fermentation

The effects of temperature, pH, inoculum level, and NaCl on the growth and metabolism of Lactobacillus sanfranciscensis and Candida humilis in rye sourdough were determined. The temperature optima for growth of C. humilis and L. sanfranciscensis were 28 and 32 °C, respectively. Yeast growth was inhibited at 35 °C. The pH did not affect yeast growth in the range 3.5–5.5, whereas growth of L. sanfranciscensis was inhibited at pH 4.0. A NaCl concentration of 4% (flour base) inhibited growth of L. sanfranciscensis but not C. humilis. The effects of the process parameters on the formation of lactate, acetate, ethanol, and CO₂ by the organisms were generally in agreement with their effects on growth. However, decreased formation of acetate by L. sanfranciscensis was observed at 35 °C although lactate and ethanol formation were not affected. In conclusion, the study provides a rationale for the stable persistence of L. sanfranciscensis and C. humilis in traditional sourdoughs and will facilitate the optimisation of sourdough fermentations in traditional and new applications.     

Development of a nucleic acid lateral flow immunoassay for simultaneous detection of <Emphasis Type="Italic">Listeria</Emphasis> spp. and <Emphasis Type="Italic">Listeria</Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> in food

We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food samples.     

Genetic typification by pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) of wild <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> wine strains

Genetic typification of 120 bacterial isolates of Lactobacillus plantarum and Oenococcus oeni from different Rioja musts and wines was performed by numerical analysis of pulsed-field gel electrophoresis (PFGE) patterns with endonuclease SfiI, and 46 of them were also studied by randomly amplified polymorphic DNA (RAPD)-PCR. A comparative study of both typification methods applied to L. plantarum and O. oeni oenological strains was performed. Bacterial species was determined both by biochemical identification methods and by specific PCR analysis. A wide variety of restriction digest patterns were detected by PFGE among L. plantarum strains (36 unrelated patterns and one closely related pattern, out of 48 isolates), as well as among O. oeni strains (18 unrelated patterns out of 72 isolates). PFGE was shown to be a suitable method for strain differentiation and to determine which strains are present in wine fermentations, with a discriminatory power to type L. plantarum and O. oeni strains higher than that of RAPD-PCR.     

Cross-linking of tyrosine-containing peptides by hydrogen peroxide-activated <Emphasis Type="Italic">Coprinus Cinereus</Emphasis> peroxidase

Hydrogen peroxide-activated Coprinus Cinereus peroxidase (CIP) can initiate polymerization of tyrosine-containing peptides via initial formation of an intermediate tyrosyl radical, which for the first time has been identified by spin trap electron spin resonance spectroscopy as located on carbon 1 in the aromatic ring, and subsequent formation of either dityrosine or isodityrosine bonds through a net elimination of two hydrogen atoms between peptides. The rate and degree of polymerization were found to depend on peptide size and the amino acid adjacent to tyrosine, as longer peptides and amino acids with bulky side groups were less reactive. In the forwarded hypothesis for the reaction mechanism upon peroxidase-initiated cross-linking of tyrosine-containing peptides and proteins, it is suggested that the polymerization takes place through a radical chain reaction. The polymerization reaction shows the potential of CIP as a protein structure-engineering tool to control functionality of proteinious food matrices or in biopolymer formation.