Development of a novel target-enriched multiplex PCR (Tem-PCR) assay for simultaneous detection of five foodborne pathogens

In the present study, a novel target-enriched multiplex PCR (Tem-PCR) assay was developed for simultaneous detection of Salmonella spp., Listeria (L.) monocytogenes, Staphylococcus (S.) aureus, Escherichia (E.) coli O157:H7 and Shigella spp.. DNA primers including universal primer and composite primer pairs were used in this Tem-PCR assay. Of which, the universal primer was linked to the 5'-end of each specific primer designed targeting Salmonella invA gene, Staphylococcus aureus femA gene, Shigella ipaH gene, Listeria monocytogenes hly gene and Escherichia coli O157:H7 rfbE gene, respectively, generating the composite primers. During PCR amplification, the composite primer pairs were employed to enrich target genes of different pathogens in initial cycles and the universal primer was employed to enrich the amplicons produced with the composite primers priming in later PCR cycles. Significantly, the Tem-PCR assay overcomes the amplification disparity resulting from primers competition observed in conventional multiplex PCR assay. With the evaluation of the Tem-PCR assay for the detection of these five foodborne pathogens, the assay showed high specificity to the target bacteria with an analytical detection limit of <2.0 × 10² CFU/mL for each, and practical detection capability, suggesting a novel multiplex PCR method for detecting foodborne pathogens.     

Assessment of polyphenolic profile and antibacterial activity of pomegranate peel (Punica granatum) flour obtained from co-product of juice extraction

The aim of this work were to determine the polyphenolic profile and the antibacterial properties of pomegranate peel flour (PPF), in view to its application in the food industry as potential antimicrobial “natural” agent. For thus, the antibacterial properties of PPF were tested against: Listeria monocytogenes, Listeria innocua, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Salmonella sp. bacterial strains. The polyphenolic profile was determined by HPLC. The Total Phenolic and Total Flavonoid content were also determined. The HPLC analysis of the PPF, showed a total of eight phenolic compounds. Punicagalin was the main component found in PPF (16.67 mg/g) followed by ellagic acid (0.15 mg/g). PPF had antibacterial activity against all bacteria tested with minimal inhibitory concentrations comprised between 20 and 50 mg/mL. The antimicrobial activity was in a decreasing order: Salmonella sp = E. coli = S. aureus = L. monocytogenes > P. aeruginosa > L. innocua. The antibacterial properties of pomegranate peel flour suggested that it is a promising antibacterial agent.     

Short communication: Fate of major foodborne pathogens and Bacillus cereus spores in sterilized and non-sterilized Korean turbid rice wine (Makgeolli)

The objective of this study was to examine the fate of foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus) and B. cereus spores in Korean turbid rice wine (Makgeolli). Samples of sterilized and non-sterilized turbid rice wine were inoculated with each of the vegetative bacteria or B. cereus spores at 3–4 log CFU/ml. The samples were stored at 5 °C or 22 °C, and bacterial survival was monitored over 28 days. Despite the harsh environment (alcohol content: 6–7% and pH: 3.43–3.98), long-term survival of pathogens was observed. Survival time was different depending on the type of beverage (pathogens survived longer in sterilized wine than in non-sterilized wine), cellular state (spores survived longer than vegetative cells), species (B. cereus survived longer than other species), and storage temperature (pathogens survived longer at 5 °C then at 22 °C). The number of B. cereus spores remained constant at both temperatures. The vegetative B. cereus population declined rapidly within 1 day, but then remained steady for up to 28 days (1.20–1.55 log CFU/ml in sterile wine). These results indicate that B. cereus formed spores that survived for a long time; therefore, it is possible that B. cereus may exist as spores in turbid rice wine. E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus survived for up to 28, 14, 14, and 14 days, respectively, in sterilized wine at 5 °C. Thus, the health implications of the long-term survival of pathogens in alcoholic beverages should be carefully considered. The results provide new information that may be useful in predicting the potential microbiological hazards associated with turbid rice wine.     

Extension of the shelf life of chilled hake (Merluccius merluccius) by a novel icing medium containing natural organic acids

Three combinations of two natural organic acids, citric acid (CA) and lactic acid (LA), in the form of novel flake icing systems were evaluated for the preservation of European hake (Merluccius merluccius), one of the most commercialised gadoid fish species in Europe. The flake icing systems were prepared with fresh water and 0.075%/0.050% (C-75 batch), 0.125%/0.050% (C-125 batch) and 0.175%/0.050% (C-175 batch) CA/LA, respectively. Aerobic mesophiles, psychrotrophs, proteolytic bacteria, anaerobes, Enterobacteriaceae, trimethylamine, lipid hydrolysis, primary, secondary and tertiary lipid oxidation events, and pH levels were evaluated in hake muscle in all batches during 13 d of chilled storage and the results were compared with sensory analyses. Significantly (p < 0.05) lower microbial counts were found in the C-175 batch for all five microbial groups investigated when compared with the control batch, with differences between batches of greater than 2 log CFU/g in the case of aerobes and anaerobes. The presence of organic acids in the icing systems also inhibited (p < 0.05) the increase in trimethylamine-nitrogen (TMA-N) content, with greater inhibition as the CA concentration in the ice increased. An inhibitory effect (p < 0.05) on fluorescent compound formation due to the presence of organic acids in the icing systems, which is indicative of tertiary lipid oxidation, was also observed at advanced storage times. These results correlated well with sensory analysis, which revealed that the shelf lives of the C-125 and C-175 batches were extended. The melting of the ice crystals containing the natural organic acid solutions, especially in the C-175 batch, exerted an antimicrobial washing effect that may explain the better protection of hake quality when stored in the proposed flake icing system.     

Inhibition of food poisoning and pathogenic bacteria by Lactobacillus plantarum strain 2.9 isolated from ben saalga, both in a culture medium and in food

The bacteriocinogenic strain Lactobacillus plantarum 2.9 isolated from the traditional pearl millet-based African fermented food ben saalga was tested for inhibition of food poisoning and pathogenic bacteria in MRS broth and in a malted millet flour slurry. In MRS broth, strain 2.9 completely eliminated Bacillus cereus, Escherichia coli O157:H7 and Salmonella enterica cells within 48 h incubation at 22–30 °C. A much lower inhibition was observed at 15 °C. The inhibitory effect of strain 2.9 on the above-mentioned target bacteria was corroborated in the malted millet flour slurry, reducing viable cell counts below detection levels after 8 h storage for B. cereus or after 24 h for S. enterica and 48 h for E. coli. An Enterobacter aerogenes strain was only moderately inhibited in the slurry. Results from the present study suggest that strain 2.9 could be used as starter culture to improve the microbiological safety of fermented ben saalga.     

Inhibition of spoilage and toxigenic Bacillus species in dough from wheat flour by the cyclic peptide enterocin AS-48

Enterocin AS-48 was tested against rope-forming Bacillus subtilis CECT 4002 and Bacillus licheniformis CECT 20, as well as on Bacillus cereus and Bacillus pumilus strains in broth and in experimental dough from wheat flour. Vegetative B. subtilis cells in liquid broth were rapidly inactivated by AS-48 (7 AU/ml). In wheat dough, higher bacteriocin concentrations of 14 and 23 AU/g were required for inactivation of B. subtilis vegetative cells and endospores activated to germinate, respectively. B. cereus LWL1 and B. licheniformis CECT 20 were inactivated by AS-48 (14 AU/g) in doughs stored at 22 °C much faster compared to doughs stored at 10 °C. Strains of Bacillus pumilus were partially inactivated in dough by bacteriocin addition (14 AU/g). Results from this study indicate that enterocin AS-48 can reduce the populations of spoilage and potentially-toxigenic bacilli in wheat dough, decreasing the risks for spoilage and food intoxication.     

应用GM（1,1）模型分析反射波振幅与频率特征

GM(1,1)模型的变化趋势不同,建模所用参数反映的物理机制也不同.本文应用这种灰色系统理论中的GM(1,1)模型,分析反射波的振幅与输车特征所反映的地质信息.并介绍了GM(1,1)模型的建模过程;给出了合成与实际数据的建模例子.合成数据试算结果表明:当地层厚度变化而岩性不变时,振幅与频率的GM(1,1)模型的变化趋势较接近;当地层厚度不变而岩性变化时,振幅与频率的GM(1,1)模型的变化趋势差异较大、应用这些特征,对SN地区实际反射波振幅和频率所反映的地质信息进行了分析.该方法具有建模简单、所建模型直观、便于分析的特点.     

Occurrence and infection dynamics of anisakid larvae in Scomber scombrus,Trachurus trachurus,Sardina pilchardus,and Engraulis encrasicolus from Tarragona (NE Spain)

Between February 1996 and January 2000, parasitological examination of Scomber scombrus (n = 447), Trachurus trachurus (n = 175), Sardina pilchardus (n = 160) and Engraulis encrasicolus (n = 153) from the fishing-ground of Tarragona (NE Spain) were made for anisakids. Three anisakid species (Anisakis larva type I sensu Berland, 1961, Contracaecum sp. and Hysterothylacium aduncum) were morphologically identified. The prevalence of Anisakis larva type I sensu Berland (1961) ranged from 11.0% (S. scombrus) and 2.6% (T. trachurus) to 0.0% (S. pilchardus and E. encrasicolus). The prevalence of H. aduncum in S. scombrus was 1.1%. The prevalence of Contracaecum sp. was 2.0% in S. scombrus, and 0.7% in T. trachurus respectively. There was a positive and significant correlation between the host length and the number of Anisakis larva type I sensu Berland, 1961 found in S. scombrus.     

Purification and partial characterization of M1-UVs300, a novel bacteriocin produced by Lactobacillus plantarum isolated from fermented sausage

A novel bacteriocin-M1-UVs300, which was produced by Lactobacillus plantarum M1-UVs300, was purified and characterized. Bacteriocin-M1-UVs300 was purified sequentially by an aqueous two-phase system (ATPS) and a Sephadex G-50 gel chromatography assay, combined with reverse phase high-performance liquid chromatography (RE-HPLC). The purification resulted in a 3221.63 IU ml⁻¹ titer with a 20.41- fold purification of the original activity. According to a Tricine-SDS-PAGE analysis, the molecular weight of the bacteriocin-M1-UVs300 was approximately 3.4 kDa. A quantitative analysis of the secondary structure of bacteriocin-M1-UVs300 results showed that it had a major β-sheet content of 52.43%, α-helix of 16.17%, β-turn of 15.27%, and a random coil of 16.12%. A partial sequence, GAKSKYGNVG-, was obtained by N-terminal amino acid sequence analysis. The antimicrobial spectrum of bacteriocin-M1-UVs300 exhibited activity against Gram-positive bacteria and Gram-negative bacteria. In addition, it was relatively heat-resistant, active over a range of pH 2–8, and sensitive to proteolytic enzymes, but it was not sensitive to α-amylase. The additives significantly increased the activity of bacteriocin-M1-UVs300 significantly. These findings indicated that bacteriocin-M1-UVs300, is a novel bacteriocin with a broad inhibitory spectrum, and that it had the potential to act as a natural preservative in the food industry.     

Purification and characterization of bacteriocin F1, a novel bacteriocin produced by Lactobacillus paracasei subsp. tolerans FX-6 from Tibetan kefir,a traditional fermented milk from Tibet,China

A bacteriocin-producing lactic acid bacteria, FX-6, was isolated from Tibetan kefir using the agar well diffusion assay. Strain FX-6 was identified as Lactobacillus paracasei subsp. tolerans on the basis of 16S rDNA sequence analyses. This bacteriocin, which was designated bacteriocin F1, was purified by a three-step purification procedure. The molecular mass of bacteriocin F1 was 2113.842 Da by MALDI-TOF–MS analyses. It was also discovered to have blocked N-termini, hampering analysis by Edman degradation. Bacteriocin F1 exhibited a wide range of antimicrobial activity, strong heat stability (20 min at 121 °C, or 60 min at 100 °C) and pH stability (pH 3.0–9.0). Following treatment by pepsin and trypsin, the antibacterial activity was partly reduced. Bacteriocin F1 is the first reported bacteriocin produced by L. paracasei subsp. tolerans which can not only inhibit fungi but also bacteria. The characterization of bacteriocin F1 suggested that it was a novel bacteriocin with potential research and application value in food industry.     

Control of Listeria monocytogenes on sliced bologna sausage using a novel bacteriocin,amysin, produced by Bacillus amyloliquefaciens isolated from Thai shrimp paste (Kapi)

Listeria monocytogenes is a foodborne pathogen causing listeriosis, a disease of great concern due to high fatality rates. Furthermore the bacterium has the ability to survive under stressful conditions. The objectives of this study were to characterize and evaluate the effectiveness of new antilisteria bacteriocin designated as amysin produced by Bacillus amyloliquefaciens strain SP-1-13LM isolated from Thai shrimp paste or “Kapi”. The partially purified bacteriocin or PPB containing amysin prepared from strain SP-1-13LM showed a broad range of inhibition including L. monocytogenes, Salmonella sp. and Shigella sp. It was found that the antimicrobial activity of PPB was heat stable up to 100 °C for 60 min and active within the pH range of 3–9. The activity of PPB disappeared when treated with proteinase K, α-chymotrypsin and trypsin demonstrating its proteinaceous nature. Tris-Tricine SDS-PAGE analysis revealed that amysin prepared from B. amyloliquefaciens strain SP-1-13LM had an apparent molecular weight of 5.2 kDa. PCR analysis revealed the presence of malonyl CoA transacylase (ituD), lichenysin synthetase C (lchAC) and surfactin synthetase (sfp) coding for iturin, lichenysin C and surfactin, respectively. The potent antilisterial effect of amysin on the growth of L. monocytogenes in sliced bologna sausage was observed during seven days of storage at 4 °C. The potential application of amysin, non-nisin bacteriocin, might be exploited as a safe food biopreservative for used with nisin to inhibit all foodborne pathogens and minimize the nisin-resistant problem of L. monocytogenes in the food industry.     

Inactivation of Anisakis pegreffii larvae in anchovies (Engraulis encrasicolus) by salting and quality assessment of finished product

There is an increasing reporting of Anisakiasis in humans at the world level and this disease has become a concern for the public health and for the fish and derived product trade. Humans acquire the infection by the ingestion of live larvae present in raw or almost raw (e.g., marinate, salted) fish products if the processing is insufficient to devitalize the worms. The aim of this study was to asses a dry salting process in killing Anisakis pegreffii larvae in naturally infected European anchovies (Engraulis encrasicolus) and to evaluate the quality assessment. The results show that a dry salting process with a salt concentration of 21% in all parts of the anchovy fillets devitalize A. pegreffii larvae in a 15 day period. The finished product showed a good panel acceptance and anchovies reached a good quality grade.     

Degradation of AFB1 in aqueous medium by electron beam irradiation: Kinetics,pathway and toxicology

The degradation study of aflatoxin B₁ (AFB₁) in aqueous medium was performed under electron beam irradiation (EBI) at various AFB₁ initial concentrations. It has been proven that the degradation of AFB₁ in the selected ranges of concentrations follows pseudo first-order reaction kinetics well (R² > 0.95). Five degradation products of AFB₁ in aqueous solution were identified by UPLC-Q-TOF/MS, and the possible degradation pathway was proposed. The Ames and cytotoxicity tests were employed to evaluate the toxicity of the AFB₁ degradation products in aqueous solution, and the results indicated that the mutagenicity and cytotoxicity of EBI treated samples decreased significantly compared with that of untreated samples, but were not completely disappeared. The study provided clues involving the application of EBI methods in AFB₁ decontamination.     

Purification and partial characterization of a novel bacteriocin produced by Lactobacillus casei TN-2 isolated from fermented camel milk (Shubat) of Xinjiang Uygur Autonomous region,China

Identification, characterization and assessment of novel bacteriocins for their potential use as biopreservatives continue to be highlighted in LAB research from past to present. A bacteriocin-producing Lactobacillus casei TN-2 strain was isolated from fermented camel milk (Shubat) of Xinjiang Uygur Autonomous region, China, after which the bacteriocin (designated as caseicin TN-2) was purified by performing ammonium sulfate precipitation, gel filtration, anion exchange chromatography and reversed-phase HPLC separation. According to MS spectrum, the molecular mass of caseicin TN-2 was 6352 Da, which was significantly different from previous reported bacteriocins produced by L. casei strains. Antibacterial activity of caseicin TN-2 was retained over a wide pH range and survived a heat treatment of 121 °C for 20 min. It was sensitive to proteases, such as trypsin and papain. Caseicin TN-2 exhibited a broad antimicrobial spectrum against Gram-positive and Gram-negative food-borne pathogenic strains including some antibiotic-resistant strains. It was found that the minimum inhibitory concentration of caseicin TN-2 to Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 was 2.5 mg/mL and 5 mg/mL respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were carried out to investigate the effect of caseicin TN-2 on the target cells, where it was demonstrated that the bacteriocidal mode of action was pore formation in the cytoplasmic membrane.     

С����ѭ��������(SACV)�о����������еĸ�ʴ����ʴ�����û���

��The SACV method described in this paper used forward and backward single potential sweeps of Ecor±30mv with scanning rates of 60,6 and 0.6mv/s. It has been used to study the corrosion behavior of iron in 1N and 10N HCl with and without the addition of formalde-hyde (0.001-1M). The I-V curves obtained usually exhibit hysterious loops, which can be explained by the condenser charging of double layer and the delay of diffusion and electrode surface process in relationship with the corrosion mechanisms of the studied systems. It has been shown that the method may be useful for quick and convenient monitoring of the corrosion rate as well as for giving information of the true corrotion mechanism.     

Inhibition of aflatoxin B1, B2, G1 and G2 production by Aspergillus parasiticus in nuts using yellow and oriental mustard flours

Aflatoxins (AFs) are naturally occurring mycotoxin compounds produced by several species of Aspergillus, as Aspergillus flavus and Aspergillus parasiticus and are mutagenic, teratogenic, and carcinogenic compounds that have been implicated as causative agents in human hepatic and extra hepatic carcinogenesis. In this study, the reduction of the AFs present in dried fruits (peanut, cashew, walnut, almond, hazelnut and pistachio), produced by A. parasiticus CECT 2681, by isothiocyanates (ITCs) generated by the enzymatic hydrolysis of the glucosinolates (GLCs) present in oriental and yellow mustard flours was evaluated. The AFs reduction activity through ITCs application in dried fruits was carried out using a model and food system experiments. The quantification of the AFs in the food products analyzed was carried out employing the technique of the liquid chromatography (LC) coupled to the mass spectrometry detection in tandem (MS/MS). The ITCs produced through GLCs hydrolysis reduced the A. parasiticus growth in the food products tested and in particular in the model system experiments the AFs B₁, B₂, G₁ and G₂ reduction ranged meanly from 83.1 to 87.2% using the oriental mustard flour, whereas employing the yellow flour the mean reduction observed ranged from 27.0 to 32.5%. In the food system experiments carried out employing only the oriental mustard flour the mean AFs reduction observed ranged from 88 to 89%.     

Gamma radiation as a phytosanitary treatment against larvae and pupae of Bactrocera dorsalis (Diptera: Tephritidae) in guava fruits

A low-dose gamma radiation phytosanitary treatment against the oriental fruit fly, Bactrocera dorsalis Hendel, was developed for guava fruits. The measure for efficacy of the treatment is preventing adult emergence from late third instars that were reared in the fruit of guava, Psidium guajava L. The dose–response tests with 1-, 2-, 3-, 7-d-old larvae in guava were initiated to determine the most tolerant stages, the late-aged third instars. No adult emerged from a total of 100,684 late-aged third instars irradiated at the dose of 97–116 Gy, resulting in an efficacy of 99.9970% at the 95% confidence level. The minimum dose for 100% preventing adult emergence from 2-, 5-, 7-d-old pupae (1800 pupae in each dose) reared in artificial diets was 100, 500, and 1750 Gy, respectively. Quality determinations on ‘Taiwan’ guavas were conducted at 1, 3 and 7 days after gamma radiation at doses of 200, 400, 600, 800, 1,200, 2000 and 6000 Gy. The guavas could tolerate radiation dose up to 600–800 Gy as there were no significant changes in organoleptic characteristics (≤800 Gy), the chemical and nutritional contents (sugar, sucrose, total sugar, titratable acid, vitamin C, and soluble solid) (≤600 Gy). Therefore, a dose of 116 Gy, which give the disinfestations efficacy of 99.9968% for the late-aged larvae in guavas and 100% mortality of 2-d-old pupae, is suggested as the minimum absorbed dose for phytosanitary irradiation treatment of B. dorsalis in fruits.     

Phthalate,adipate and sebacate residues by HRGC-MS in olive oils from Sicily and Molise (Italy)

Phthalate, adipate and sebacate esters contamination in olive oils produced in Sicily in the crop years 2006–2009 and in Molise in the crop years 2007–2008 was studied by HRGC-MS. The statistical elaboration of plasticizer data was performed to compare the contamination of the Sicilian samples with those of the samples produced in Molise in the same crop years and to compare the contamination of the Sicilian samples produced in three consecutive crop years. In all analyzed samples DEA, DiHP, DOP, and DEHS residues were always less than their LOQ. Furthermore in Sicilian olive oils were not found residues of DBA and DiDP, while DiBP, BBP and DEHP were found to be the phthalates most abundants. In Molise olive oils there were not residues of DiBA, DMP and DEP, while DiNP and DiDP are the most abundants. Among all plasticizers found only DEHP was observed stronger that its proposed limit by BNN (3 mg/kg) in 9.1% of Molise olive oils and in 27.27% of Sicilian samples of the year 2007–2008.Finally, plasticizer values found in this study were compared with those obtained earlier in olive oils produced in Sicily in the crop years 2002–2003. The results showed that the contamination has not decreased.     

Modeling the inactivation of Neosartorya fischeri ascospores in apple juice by high pressure,power ultrasound and thermal processing

Neosartorya fischeri is a mould that spoils acid foods and can produce mycotoxins. In this work, the efficacy of high pressure processing (HPP, 600 MPa) and power ultrasound (24 kHz, 0.33 W/mL) in combination with 75 °C for the inactivation of four week old N. fischeri ascospores in apple juice was investigated and compared with 75 °C thermal processing alone. The HPP-75 °C process was the most effective technique for inactivating N. fischeri spores, resulting in 3.3 log reductions after 10 min vs. no inactivation for thermosonication (TS) and thermal processing. Unexpectedly, activation shoulders were observed during the TS process. Then, the effect of different temperatures on the ascospore inactivation in apple juice by HPP-thermal, TS and thermal processing was investigated, and the log survivors vs. time were modeled. Faster inactivation was achieved at higher temperatures for all the technologies tested, indicating the significant role of temperature for the spore inactivation, alone or combined with other processes. The Weibull model described the spore inactivation better by 600 MPa HPP-thermal (50, 60, 75 °C) and thermal (85, 90 °C), whereas Lorentzian was more appropriate for the TS treatment (65, 70, 75 °C). In conclusion, HPP is the best food preservation technology due to higher spore inactivation in apple juice at the same temperature.