A multiplex PCR assay for fraud identification of deer products

Attempts were made to established one-step multiplex PCR assay for the fraud identification of the mostly used species in deer products (bovine, ovine, porcine and poultry). Primers were selected from published papers or designed in the well-conserved region of tRNA-Val and 16S rRNA mitochondrial genes after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 124, 183, 225 and 290 bp length for bovine, poultry, ovine and porcine, respectively. The detection limit was 1 ng for porcine and ovine primers, 5 ng for poultry primers and 0.5 ng for bovine primers. The results demonstrated that fraud phenomena are very epidemic in the deer products, especially heart, blood, penis and antler products.The multiplex PCR described in this study, proved to be very sensitive and reliable in species identification, could be considered as a further improvement of traditional methods based assay for the identification of deer products.     

Inactivation of a non-pathogenic strain of E. coli by ionising radiation

Ionising radiation is an effective method to reduce pathogenic E. coli O157:H7 in meat and poultry products. Radiation sensitivity of bacteria, however, depends on several factors. After applying an irradiation dose of 1 kGy to cultures of the non-pathogenic strain of E. coli, DSM 498, grown and irradiated in nutrient broth, reductions of 3–4 decimal units were achieved (D₁₀ = 0.27 kGy). If grown on minced turkey meat, however, reduction rates were lower (D₁₀ = 0.47 kGy). Even lower reduction rates were obtained during irradiation of frozen meat (D₁₀ = 0.72 kGy) compared to treatments at cooling temperatures (D₁₀ = 0.48 kGy). For data evaluation, both, first order reduction kinetics and the Weibull model were compared. The results emphasise the necessity to determine inactivation kinetics in food matrices of target extrinsic factors (e.g. temperature).     

Prevalence,genetic diversity and antimicrobial susceptibility of Listeria monocytogenes isolated from open-air food markets in Greece

A total of 210 food samples originating from milk products, ready-to-eat salads, raw meat and raw meat products purchased in ten open-air market places in Thessaloniki, Greece, were analyzed for the presence of Listeria monocytogenes. Thirty (14.3%) contained L. monocytogenes with the highest prevalence in raw meat (27.5%), raw meat products (18%) and cheese (8%). The strains were susceptible to 16 antimicrobials as determined by microbroth dilution, except one strain which displayed resistance to tetracycline (MIC > 32 μg/ml). This strain carried the tetracycline resistance gene tet(M). Pulsed-field gel electrophoresis (PFGE) revealed a low genetic diversity among the isolates, irrespective of their origin. This suggests that dominant L. monocytogenes clones are widespread in different food product types in open-air food markets in Greece. The high prevalence of L. monocytogenes in these products indicates that appropriate hygienic measures and periodic bacteriological controls are also necessary in open-air food markets to reduce contamination with food-borne pathogens. Greek specialties made with raw meat and raw milk may contain L. monocytogenes and should not be consumed by persons at risk.     

Meat species identification based on the loop mediated isothermal amplification and electrochemical DNA sensor

An easy, rapid and sensitive method of detection of the presence of meat species in raw or processed foods is important from cultural, religious, health and commercial perspectives. In this study we have tried to distinguish species-specificity in control and processed pork, chicken and bovine meats using loop mediated isothermal amplicons (LAMP) and disposable electrochemical printed (DEP) chips. LAMP is a nucleic acid amplification method that amplifies target DNA with high specificity, efficiency and rapidity under isothermal condition (63 °C). Electrochemical genosensor with the DEP chips detects the amplicons by Linear Sweep Voltammetry (LSV) observation of DNA–Hoechst33258 interaction on the chip surface. Hoechst33258 interacts with DNA in solution without immobilization of DNA onto the electrode surface eliminating the time consuming probe immobilization step. Our method is more specific and free of unwanted amplifications compared to Multiplexed PCR (M-PCR) method and gave limits of detection of ∼20.33 ng/μl (3 × 10⁴ copies/reaction), ∼78.68 pg/μL (3 × 10² copies/reaction) and ∼23.63 pg/μL (30 copies/reaction) for pork, chicken and bovine species, respectively. Our method of detection is quick, taking only an hour, and it may be useful for food administration laboratories to carry out meat species identification in raw and processed foods.     

Analysis of total aerobic viable counts in samples of raw meat using fluorescence-based probe and oxygen consumption assay

A simple test for determining total aerobic viable counts in raw meat is presented. Homogenates of meat samples are prepared in full PBW medium, dispensed in the wells of 96-well plate together with the oxygen-sensing probe, Redlight, covered with oil and monitored on a fluorescent reader at 30 °C. The probe produces characteristic sigmoidal profiles of fluorescence reflecting depletion of sample dissolved oxygen, with onset time indicating the initial microbial load. The test provides rapid and accurate results (1 and 12 h for contamination levels of 10⁸ and 10³ cfu/g, respectively) and correlates well with the ISO:4833:2003 method (r = 0.86), which make it useful as alternative to conventional culture methods for the quick, high throughput determination of TVC (30 °C) in meat samples.     

A potential methodology for differentiation of ciguatgoxin-carrying species of moray eel

A food poisoning incident due to ingestion of an unknown moray eel occurred in Taiwan. To identify the species of causative moray eel implicated in food poisoning, a 376-bp fragment sequence of cytochrome b gene was successfully obtained from four species of moray eel by using a pair of primers (L newest-1/H 15149). This fragment could be amplified using fish meat treated with different heating processes (100 °C for 30 min, 100 °C for 60 min, 121 °C for 15 min, and 121 °C for 30 min). After sequencing, it was found that no variation in sequence was detected among individuals within each species. The species of causative moray eel implicated in food poisoning was judged as Gymnothorax javanicus based on sequence analysis. In addition, using restriction enzyme analysis, including Taq I and Sau96 I, could distinguish these four species of moray eel and identify the fish species of poisoning sample as G. javanicus. The toxicity of viscera in 24 specimens of four moray eel species was not detected, but the food poisoning sample was found to be toxic. Overall, this study proved that DNA sequence and restriction enzyme analyses are useful in identifying that the causative moray eel species was G. javanicus.     

Biogenic amines content,histamine-forming bacteria,and adulteration of pork and poultry in tuna dumpling products

Nine tuna dumpling products were purchased at nine retail markets in southern Taiwan. Occurrence of biogenic amines, histamine-forming bacteria, and adulteration of pork and poultry were determined. This study showed the high contents of aerobic plate count, total coliform and Escherichia coli in tested tuna dumpling products. Average content of various biogenic amines in all tested samples was less than 2.0 mg/100 g. Fifteen histamine-producing bacterial strains isolated from tested samples produced 8.7–466 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH). Assay of multiplex polymerase chain reaction (PCR) revealed adulteration rates were 88.9% (8/9) and 33.3% (3/9) for pork and poultry, respectively, in tuna dumpling. In addition, six samples of tuna dumpling meat were identified as Thunnus obesus, while other three samples were identified as Thunnus thynnus.     

Survival of Listeria monocytogenes,and enterotoxin-producing Staphylococcus aureus and Staphylococcus pasteuri,during two types of biltong-manufacturing processes

Biltong is a ready-to-eat (RTE) meat product, produced from raw muscle meat by marination, curing and drying. The aim of this study was to determine the survival of three representative bacterial pathogens, (Listeria monocytogenes, Staphylococcus aureus and Staphylococcus pasteuri) throughout the manufacturing process of biltong. Beef portions were surface inoculated with selected isolates and prepared using either a traditional, home-style or modern, manufacturing method prior to drying at 25 °C for 96 h. These two methods differed in their marination process. Samples were taken every 12 h and bacteria enumerated by plate counts. Biltong produced using the modern method was associated with lower counts compared to product produced using the traditional technique. Less than 1 Log CFU/g L. monocytogenes cells were detected in final product and cell counts decreased rapidly throughout both processes. By contrast, 2–4 Log CFU/g cells of Staphylococcus strains survived in the final product. The latter finding may hold future foodborne illness implications, as these strains of Staphylococcus were enterotoxin-producers.     

A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species

A novel common primer multiplex PCR (CP-M-PCR) was applied to detect four kinds of meats (chicken, cattle, pig and horse) as raw materials. A common adapter was designed in the 5′-end of species-specific reverse primers which matched with the species-specific DNA sequences for each species and also used as the common primer (CP). CP-M-PCR primers were designed to uncover different length fragments of 239, 292, 412, and 451 bp from chicken, cattle, pig and horse meats, respectively. The bands of specific DNA fragments amplified by CP-M-PCR method still appeared until the concentration of species-specific primers diluted to 0.015 pmol and primer sensitivity was increased by 100 times compared with conventional multiplex PCR without CP. CP-M-PCR detection limit of the DNA samples was 0.1 ng (36.4 copies) for single kind of meat as well as four kinds of meats. CP-M-PCR method simplified the PCR reaction system and conquered the disparate amplified efficiency from different primers. The CP-M-PCR method could be widely applied in practical detection for simultaneous identification of other meat species and their products.     

Biogenic amines content,histamine-forming bacteria and adulteration of bonito in tuna candy products

Thirty-four tuna candy products sold in retail markets and supermarkets in Taiwan were purchased and tested to determine the biogenic amine, histamine-forming bacteria, and adulteration of bonito meat. The levels of pH value, water activity (Aw), water content, total volatile basic nitrogen (TVBN), aerobic plate count (APC) and total coliform (TC) in all samples ranged from 5.3 to 6.1, 0.47 to 0.65, 7.37% to 17.32%, 12.1 to 54.6 mg/100 g, <1 to 6.2 log CFU/g and <3 to 1700 MPN/g, respectively. None of these sample contained Escherichia coli. The average content of various biogenic amines in all tested samples was less than 1.0 mg/100 g. Four histamine-producing bacterial strains isolated from tested samples produced 1.02–1.74 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH) after incubation at 35 °C for 24 h. Assay of multiplex polymerase chain reaction (PCR) revealed that adulteration rate of bonito meat was 26.5% (9/34) in tuna candy samples. Tuna species in tuna candy samples was identified as Thunnus albacares for 29 samples (85.3%), Thunnus alalunga for four samples (11.8%) and Thunnus obesus for one sample (2.9%) by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP).     

Rapid and sensitive identification of buffalo’s,cattle’s and sheep’s milk using species-specific PCR and PCR–RFLP techniques

For the rapid, specific and sensitive identification of buffalo’s, cattle’s and sheep’s milk, species-specific PCR and PCR–RFLP techniques were developed. DNA from small amount of fresh milk (100 μL) was extracted to amplify the gene encoding species-specific repeat (SSR) region and the mitochondrial DNA segment (cytochrome-b gene). PCR amplification size of the gene encoding SSR region was 603 bp in both buffalo’s and cattle’s milk, while in sheep’s milk was 374 bp. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique was used to discriminate between buffalo’s and cattle’s milk. Restriction analysis of PCR–RFLP of the mitochondrial cytochrome-b segment (359 bp) analysis showed difference between buffalo’s and cattle’s milk. Where, the fragment length (bp) generated by TaqI PCR–RFLP were 191 and 168, whereas no fragments were obtained in cattle’s milk for cytochrome-b gene (359 bp). The proposed PCR and PCR–RFLP assays rep resent a rapid and sensitive method applicable to the detection and authentication of milk species-specific.     

Characteristics of Batzos cheese made from raw,pasteurized and/or pasteurized standardized goat milk and a native culture

Experimental cheeses were made from raw (R), raw with starter (RS), pasteurized with starter (PS) and standardized, pasteurized with starter (PSS) goat milk to study the influence of the starter and pasteurization on the quality of Batzos cheese. Lactococcus lactis subsp lactis strains from raw milk cheese used as starter proliferated significantly (P < 0.05) only in PSS cheese. Heat treatment lowered (P < 0.05) the levels of most microbial groups. However, TC and LAB counts were higher (P < 0.05) in PSS cheese and this was accompanied by lower (P < 0.05) pH; thus, a higher rate of decrease of undesirable microorganisms in PSS cheese was recorded. Degradation of αs-casein was in R > RS and in PS > PSS, while a small reduction of β-casein during ripening and storage was recorded. The proportions of aminoacids and lipolysis products increased throughout ripening and storage.     

A Turkey survey of hygiene indicator bacteria and Yersinia enterocolitica in raw milk and cheese samples

In this research, a total of 200 dairy (raw milk, cheese) samples obtained from Ankara, were examined for the presence of Yersinia spp., total coliform and Escherichia coli. As expected, raw milk 55% (55/100) were significantly contaminated with Yersinia spp., than cheese samples 14% (14/100). Y. enterocolitica was the most commonly isolated species, and was recovered from 47.3% in raw milk 35.7% in cheese samples. The other Yersinia spp. were identified as Y. frederiksenii (31.0%, 21.4%), Y. kristensenii (12.7%), Y. intermedia (7.2%, 7.1%) and atypical Yersinia spp. (1.8%, 35.7%) in raw milk and cheese samples, respectively. All the samples of cheese examined were negative for Y. kristensenii. All Y. enterocolitica strains tested gave negative results in the autoagglutination tests and crystal violet binding test.Furthermore total coliform bacteria and E. coli were investigated in these samples. According to the analysis results, most of the samples containing Yersinia spp.were found high number of viable count cell total coliform than E. coli. Total coliform bacteria and E. coli, were detected (3.0 × 10⁴ cfu/ml and 1.5 × 10⁴ cfu/ml) in four milk samples containing Y. enterocolitica while, they were detected (2.5 × 10⁴ cfu/ml and 1.0 × 10⁴ cfu/ml) in eight milk samples containing Y. enterocolitica. In the present study, total coliform bacteria and E. coli were detected >1.1 × 10⁵ cfu/g in six cheese samples containing Y. enterocolitica and atypical Yersinia spp. The results indicate that Yersinia spp. is more likely to be isolated from foods with a high level of coliforms than from foods with low coliform counts.     

A comparison of microbial contamination on sheep/goat carcasses in a modern Indian abattoir and traditional meat shops

The microbial load on sheep/goat carcasses was investigated in Deonar abattoir and traditional meat shops in Mumbai. A total of 96 swab samples from carcass sites were collected and analysed from the abattoir, while 144 swab samples from carcass sites were analysed from three meat shops. These samples were processed for total viable count (TVC) and differential counts. The average TVC after flaying, evisceration and washing in the abattoir was 5.51 ± 0.36, 6.06 ± 0.53 and 5.13 ± 0.58 CFU/cm², respectively. Pooled average TVC in the shops after flaying, evisceration and washing was 5.83 ± 0.42, 6.48 ± 0.27 and 6.17 ± 0.41 log CFU/cm², respectively. Statistical analysis revealed a highly significant difference (P < 0.01) among TVC counts after washing between abattoir and the shops. The highest prevalence of Micrococcus spp. and S. epidermidis was noticed during various operations in both the abattoir and the shops. Although Salmonella spp. could not be isolated from any of the carcass sites in the abattoir, in the shops it showed 16.4% prevalence at all the sites irrespective of operations. Overall study revealed that level of contamination in the traditional meat shops was significantly higher compared to the abattoir. However, the microbial contamination in the abattoir is also high if we compare these results to the reports from developed countries and do not conform to EU specifications. The findings of this study reflect the hygiene status of meat production in the developing world.     

Some hazards associated with the production of a popular roasted meat (tsire) in Zaria,Nigeria

Some hazards associated with the entire production process of tsire (a local kebab) were identified in three production centres. Microbiological analyses showed tsire to have highest aerobic plate count of log₁₀ 6.27; B. cereus count was highest at log₁₀ 3.30 cfu/g; Clostridium perfringens count was highest at log₁₀ 2.92 cfu/g; Staphylococcal count was highest at log₁₀ 3.96 cfu/g; Coliform count was highest at log₁₀ 4.08 cfu/g; yeast and mould count was highest at log₁₀ 2.49 cfu/g. The proximate analysis showed tsire to averagely have 11.87% moisture, 31.77% protein, 23.16% fat and 2.43% salt. The critical appraisal of the production process indicated potential hazards in the raw meat, environmental contamination as well as post-process handling contamination from humans and the environment. The nature of microorganisms associated with tsire production as shown by this study calls for worry from the public health standpoint. In the light of this, efforts should be made by public health services with regard to improving its production in order to reduce the associated hazards.     

Effects of marinating on the formation of polycyclic aromatic hydrocarbons (benzo[a]pyrene,benzo[b]fluoranthene and fluoranthene) in grilled beef meat

The study was conducted to investigate the effect of marinating on the generation of polycyclic aromatic hydrocarbons (benzo[a]pyrene, benzo[b]fluoranthene and fluoranthene) in grilled beef meat. Seven marinade treatments containing 1) basic marinade, which include sugar, water, onion, turmeric, lemon grass, salt, garlic, coriander and cinnamon, 2) basic–oil, 3) Commercial marinade. 4) basic–oil–lemon juice, 5) basic–lemon juice, 6) basic–oil–tamarind and 7) commercial–tamarind at four time intervals (0, 4, 8 and 12 h) were applied on meat samples before charcoal grilling. Tandem solid-phase extraction (SPE) was used to clean up the samples. A high performance liquid chromatography (HPLC) with fluorescence detector was used for PAHs analysis. The study showed significant (p < 0.05) reduction (70%) of PAH in beef samples treated with the acidic marinade (containing 1.2% lemon juice). The basic–lemon > basic > basic–oil–lemon > basic–oil was the best order of marinade treatment. The duration of marinating was not a significant (p > 0.05) factor in PAH reduction.     

Microbial profile of buffalo sausage during processing and storage

A study was made on the microbial levels of buffalo sausage during preparation and storage at 4 ± 1 °C. Microbial counts in raw minced meat were, total plate count (TPC) (log cfu/g) 5.41 ± 0.25; coliforms (MPN/g) 23.2; Staphylococcus aureus (log cfu/g) 1.57 ± 0.11; yeasts and molds (log cfu/g) 2.29 ± 0.07 and lactic acid bacteria (LAB) (log cfu/g) 0.60 ± 0.20. Sausage emulsion showed similar trend in microbial counts with minimal microbial contamination during the preparation of emulsion. Cooked buffalo sausage gave the following microbial counts: TPC (log cfu/g) 3.75 ± 0.31; coliforms (MPN/g) 0.2; LAB (log cfu/g) 0.07 ± 0.01; yeast and molds (log cfu/g) 0.72 ± 0.07. S. aureus, Clostridium perfringens and Bacillus cereus were not detected in cooked sausages. These results indicate that steam cooking for 45 min followed in the study was effective in reducing the microbial counts substantially. The investigation revealed that shelf life of cooked buffalo sausage was 31 days in either vacuum or CO₂ at 4 ± 1 °C. The results indicated that spoilage of vacuum packed cooked buffalo sausage was likely due to LAB while microflora other than LAB may be responsible for spoilage of CO₂ packed cooked buffalo sausage. The study suggests that measures such as low initial microbial counts, hygienic precautions during preparation of sausage, steam cooking for 45 min, vacuum or CO₂ packing and storage at 4 ± 1 °C would control the microbial growth and provide wholesomeness and safety to the buffalo sausage.     

Authentication of meat and commercial meat products from common pigeon (Columba livia) woodpigeon (Columba palumbus) and stock pigeon (Columba oenas) using a TaqMan real-time PCR assay

A species-specific real-time polymerase chain reaction (PCR) assay using TaqMan^® probes has been developed for the identification of meat and meat products from common pigeon (Columba livia), woodpigeon (Columba palumbus) and stock pigeon (Columba oenas). The method combines the use of species-specific primers and TaqMan^® probes that amplify small fragments (amplicons < 200 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141 bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species, demonstrated the suitability of the assay for the detection of the target DNAs. The PCR assay reported in this work could be useful in inspection programs to verify the correct labelling of raw and heat-treated pigeon meat products.     

Pentocin 31-1, a novel meat-borne bacteriocin and its application as biopreservative in chill-stored tray-packaged pork meat

Pentocin 31-1 was produced by Lactobacillus pentosus 31-1, isolated from the traditional China fermented Xuan-Wei Ham. In this work, study on its application as biopreservative in chill-stored nonvacuum-tray-packaged pork meat was carried out. Pentocin 31-1 was prepared by pH-adsorption in 5 l stainless steel fermentor. Each 200 ml semi-purified bacteriocin was obtained from one fermentation, and the specific activity was 1280 AU/ml. The effects of pentocin 31-1 on microbiological counts, physicochemical change and sensory quality of chilled pork in the period of preservation at 4 °C was investigated. Results showed that pentocin 31-1 could substantially inhibit the accumulation of VBN and generally suppress the growth of microflora, especially Listeria and Pseudomonas, during chilled pork storage. Microbiological counts, physicochemical parameters and sensory characteristics of the treatments (40 AU/ml pentocin 31-1 or 75 AU/ml nisin) had exceeded the limitation of Chinese hygienic standard for fresh meat of livestock by day 15. 80 AU/ml pentocin could extend the shelf life to 15 days and the meat showed good sensory characteristics. These results suggest the potential of pentocin 31-1 as a biopreservative in tray-packaged chilled pork storage.     

Aqueous extracts of Hibiscus sabdariffa calyces as an antimicrobial rinse on hot dogs against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus

Contamination of ready-to-eat meat products by foodborne pathogens is a major concern in the food industry. Novel methods to control foodborne pathogens are made necessary by continuing outbreaks as well as the development of antibiotic-resistant pathogens. Hibiscus sabdariffa extracts could be useful as a natural source of antimicrobial rinse on ready-to-eat products to control pathogens. In this study, lyophilized Hibiscus flower extracts were examined for their antimicrobial activity as a rinse on all-beef hot dogs against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus (MRSA). Beef hot dogs were dip inoculated in overnight cultures of 1:1 mixtures of L. monocytogenes strains Scott A and 101 or MRSA strains ATCC 33591 and ATCC 33593 and were placed at 4 °C overnight to allow for bacterial attachment. Hot dogs were rinsed with extracts (120, 240 mg/mL) or water (control) for 5, 15, 30, or 60 min and then plated immediately (0 h; no storage) or stored at 4 °C overnight and plated at 24 h. Serial dilutions were plated in duplicate on both TSA and selection media, Modified Oxford (Listeria) or Baird Parker (MRSA), and the entire experiment was replicated 3 times. Higher extract concentrations, longer rinse times, and longer storage times were the most effective at inhibiting and/or killing L. monocytogenes and MRSA on hot dogs. L. monocytogenes was reduced to ca. 1.5 log CFU/g while MRSA was reduced to undetectable levels following rinsing of hot dogs with extracts at 240 mg/mL for 60 min and stored for 24 h. Both L. monocytogenes and MRSA were reduced ca. 2 log CFU/g following rinsing of hot dogs with extracts at 120 mg/mL for 60 min and stored for 24 h. This research demonstrates the effectiveness of Hibiscus extracts against L. monocytogenes and MRSA as an antimicrobial rinse on ready-to-eat meat products.