Development of a genetic method for the identification of salmon,trout, and bream in seafood products by means of PCR–RFLP and FINS methodologies

In the present study, two alternative methods for identifying 13 salmon, trout and bream species were developed. Both of them are based on polymerase chain reaction (PCR) amplification of a cytochrome b gene fragment. Subsequently, different techniques were assayed to assign the PCR amplicons previously obtained to particular species. The first one is based on the restriction fragment length polymorphism (RFLP) and includes three endonucleases for generating species-specific restriction profiles, while the second one is based on the phylogenetic analysis of DNA sequences. The main novelty of this work lies in the applicability of the developed methods to all kinds of processed products, including those undergoing intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 25 commercial samples including some that had been subjected to intensive thermal treatment, allowing the detection of those incorrectly labeled (16%). Therefore, these methods are useful to check the fulfillment of labeling regulations for seafood products, verify the correct traceability in commercial trade, and for fisheries control.     

Identification of gadoid species (Pisces, Gadidae) by sequencing and PCR–RFLP analysis of mitochondrial 12S and 16S rRNA gene fragments

We applied PCR–RFLP (Polymerase Chain Reaction – Restriction Fragment Length Polymorphism) analysis to identify seven gadoid species of different biogeographical origin and commercial relevance, namely Gadus morhua (Atlantic ocean); Trisopterus minutus capelanus, Trisopterus minutus minutus, Molva elongata, Phycis blennoides, Micromesistius poutassou (Atlantic ocean and Mediterranean sea); Theragra chalcogramma (Pacific ocean). Two DNA fragments belonging to mitochondrial 12S and 16S rRNA genes of about 430 and 630 bp, respectively, were isolated by PCR amplification. Their direct sequencing showed a significant genotypic diversity among gadoid species, useful for species identification. Digestion of 16S rRNA gene PCR fragment with MvaI or Bsh1285I restriction enzymes, followed by agarose gel electrophoresis of the cleaved products, yielded specific restriction profiles that enabled direct, visual identification of the species analyzed. This PCR–RFLP method allowed a clear and rapid discrimination of the gadoid species studied.     

Molecular identification of cephalopod species by FINS and PCR-RFLP of a cytochrome b gene fragment

Identification of commercial species is a relevant issue to assure the correct labeling of seafood products. In this work two different molecular techniques, FINS (Forensically Informative Nucleotide Sequencing) and PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) were developed to identify 8 cephalopod species (families Loliginidae and Ommastrephidae) employing a fragment of the cytochrome b gene. DNA amplification for all of the species was carried out with a new set of specific primers designed in this study for cephalopods. FINS is a technique based on DNA sequencing, while PCR-RFLP allows direct species identification by comparing specific DNA restriction patterns. Both techniques are useful for cephalopod species identification. 17 food products (mainly "squid rings") were analyzed and the species employed for their manufacture identified by FINS.     

A rapid and high-throughput real-time PCR assay for species identification: application to stockfish sold in Italy

In some parts of Italy, there is a tradition of eating special, highly prized species of cod, fished and dried in Norway. In order to safeguard the value of this food product, in 2005, the Italian government legislated that the commercial term “stockfish” can only be attributed to Gadus morhua (Gm). In this study, we present an improved real-time PCR assay for efficient identification of Gm with respect to other gadiforms of commercial interest. The method is applied to 437 stockfish samples, collected in various Italian regions, in order to verify whether labelling regulations had been respected and to report instances of fraud in the Italian stockfish market. The PCR method employed here allowed rapid and economical identification of the species, with a very high percentage of correct identifications.     

Meat species identification and Halal authentication analysis using mitochondrial DNA

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.     

Development of a method for the identification of scombroid and common substitute species in seafood products by FINS

In the present work, a method for the authentication of scombroid products was developed, by means of FINS (Forensically Informative Nucleotide Sequencing) technique (Polymerase Chain Reaction (PCR) followed by phylogenetic analysis). The methodology developed allows the identification of most important scombroid species using the mitochondrial cytochrome b as molecular marker. Due to the different commercial value of the species belonging to this family, substitutions between species in seafood products can take place.     

Development of a polymerase chain reaction assay for species identification of goose and mule duck in foie gras products

Polymerase chain reaction amplification of a conserved region of the -actin gene has been used for the specific identification of goose (Anser anser) and mule duck (Anas platyrhynchos×Cairina moschata) foie gras. Universal primers were used for the amplification of a DNA fragment containing three introns and four exons of the -actin gene in goose and mule duck. Sequence analysis of the amplified fragments was necessary for the design of forward species-specific primers in the goose and mule duck -actin genes. The use of species-specific forward primers, together with a reverse universal primer, produced amplicons of different length, allowing clear identification of goose and mule duck foie gras samples. Analysis of experimental mixtures demonstrated that 1% of duck can be easily detected in goose foie gras using the PCR method developed here. This genetic marker can be very useful for the accurate identification of these two species in foie gras products.     

Development of a rapid mitochondrial DNA extraction method for species identification in milk and milk products

Isolation of mitochondrial DNA (mtDNA) from milk offers an effective way to monitor aspects of quality control and traceability to ensure food safety. A few methods of DNA isolation from milk have been reported, but many of them are time consuming and expensive. Here, we report a rapid, simple, and efficient method of mtDNA extraction from raw and processed milk (pasteurized, retorted, and UHT milk) to generate substrate for analysis using any PCR analysis platform. Various techniques used for the separation of mitochondria were explored and combined with a sodium dodecyl sulfate method for mtDNA extraction from raw and processed milk. The optimized protocol supports the efficient amplification of mtDNA independent of sample origin and is sufficiently straightforward to allow its widespread adoption by industry.     

抑制副溶血性弧菌的乳酸菌筛选鉴定及其生物学特性研究

目的:丰富功能性乳酸菌资源库,寻找具有副溶血性弧菌拮抗能力的优良乳酸菌出发菌株。方法:采用牛津杯抑菌试验筛选具有广谱抑菌潜力的乳酸菌菌株,通过生长代谢性能、胃肠液耐受性能、耐盐性、抗生素敏感性、抑菌谱等指标探讨其生物学特性。结果:以副溶血性弧菌为指示菌,筛选得到6株乳酸菌,经形态学、生理生化、分子生物学鉴定,分别归类于类干酪乳酪杆菌、发酵黏液乳杆菌和植物乳植物杆菌。其中,类干酪乳酪杆菌A1抑菌活性最佳,24 h内菌落总数超过1×10⁹ CFU/mL,发酵液pH值稳定在4.1左右,经人工模拟胃液处理2 h后,存活率为54.61%,再经人工模拟肠液处理8 h后,存活率仍可达45.46%,经10%NaCl胁迫处理24 h后,活菌总数>1×10⁵ CFU/mL。同时,类干酪乳酪杆菌A1细菌素粗提物对13种致病菌呈良好抑菌活性,具有广谱抑菌潜力,且对8种常见抗生素未见耐药性。结论:筛选得到了能够抑制副溶血性弧菌且生物学特性优良的类干酪乳酪杆菌A1。     

基于可视化和实时荧光环介导等温扩增技术快速鉴定日本红菇

目的 利用可视化和实时荧光环介导等温扩增（LAMP）技术,建立日本红菇的快速鉴别方法。方法 基于日本红菇内转录间隔区的基因序列设计了特异性的LAMP引物,在23种蘑菇物种间进行特异性验证,通过检测10 ng/μL~1 fg/μL系列浓度的基因组DNA,评估方法的灵敏度。结果 设计的LAMP引物可特异性鉴别日本红菇,不与其他蘑菇种类发生交叉反应。建立的LAMP方法检测灵敏度为1%混合蘑菇样本,或终浓度为2 pg/μL的日本红菇DNA。结论 本方法可用于新鲜/干制蘑菇及烹饪后蘑菇的检测,检测时间<1 h,其中可视化方法无需专业仪器设备,适用于日本红菇的现场快速鉴别。     

Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon

NADPH, a major reducing power in microorganisms, is mostly generated from the pentose phosphate (PP) pathway by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) expressed by the zwf and gnd genes, respectively. The characteristics of these two genes in Escherichia coli were compared after their re-introduction into the parent strain for over-expression. zwf encoding G6PDH increased the level of NADPH 3 folds compared to gnd encoding 6PGDH. An excess of NADPH depressed cell growth mainly due to the inhibition of citrate synthase in the TCA cycle. Recombinant plasmids containing zwf or gnd co-integrated with the phbCAB operon from Ralstonia eutropha were constructed, and introduced into E. coli for the oddball biosynthesis of PHB. The amount of PHB increased after enforcing the genes; especially the zwf gene an increase of around 41%, due to the rise in NADPH and the depressed TCA cycle, leading to the metabolic flux of intermediates to the pathway for the biosynthesis of PHB.     

沙门菌、金黄色葡萄球菌、副溶血性弧菌的选择性共增菌培养基的研制

目的 研制一种可同时对沙门菌、金黄色葡萄球菌及副溶血性弧菌进行富集的共增菌培养基SSV,达到同时检测3种食品中常见的食源性致病菌的目的。方法 根据3株菌的营养需求,选择不同的培养基组分进行单因素实验,确定选择性共增菌培养基的配方,通过OD600 nm、受损菌复苏、多重聚合酶链式反应(PCR)、培养基选择性4个方面对SSV培养基进行验证。结果 确定增菌培养基的成分为：蛋白胨10.0 g、KH₂PO₄ 1.5 g、NaCl 15.0 g、LiCl 1.0 g,Na₂S₂O₃ 5.0 g、去氧胆酸钠0.05 g、甘露醇1.0 g、丙酮酸钠5.0 g,蒸馏水1 000 mL。SSV培养基能同时富集以上3株菌,37℃培养16 h后,3株菌的菌体浓度均达到107 CFU/mL及以上;SSV培养基对于受损的目标菌有良好的复苏效果,复苏后OD₆₀₀ nm较于复苏前增长了3倍;同时多重PCR、培养基选择性也得到很好的验证。结论共增菌培养基SSV可用于同时培养沙门菌、金黄色葡...     

Co-amplification and sequencing of a cytochrome b fragment affecting the identification of cattle in PCR-RFLP food authentication studies

Food authentication studies based on PCR-RFLP analysis are frequently targeted to well-conserved mitochondrial sequences, such as certain regions of the cytochrome b (cytb) gene. The use of mitochondrial L14841/H15149 universal cytb-targeted primers in PCR-RFLP assays revealed the existence of a complex restriction pattern in three genetically-unrelated Iberian (Northern Spain) cows, this being due to the simultaneous co-amplification of two 359 bp cytb fragments. Microsatellite analysis of 11 bovine-specific loci confirmed no familiar linkage among the animals investigated. Both co-amplification products were successfully separated by specific cleavage with endonucleases RsaI and MvaI, which allowed the recovery of each amplification product, respectively. The new co-amplified cytb fragment described in this study was successfully sequenced, and exhibited a significantly high homology (95.1–99.3% range) with respect to mitochondrial sequences previously described for a Bos indicus specimen and for another two Asian Bos taurus, this underlining that its presence in cattle may be more extended than initially thought. In contrast, the homology with the cytb sequence widely accepted for B. taurus was only 89.6%. The results presented in this work imply that food authentication studies by PCR-RFLP analysis may be complicated in the case of cattle by the co-amplification of two different cytb fragments.     

Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use

A set of simple in vitro tests (identification by species-specific PCR, genetic diversity, phage sensitivity, growth and viability in milk, resistance to salts and flavor compounds, bacterial interactions, tolerance to simulated gastric juice and bile, bile salts deconjugation, hydrophobicity and β-galactosidase and antibacterial activities), that can be carried out in almost every laboratory of microbiology, mainly in developing countries where there is often limited access to sophisticated techniques, allowed us to identify, among 19 intestinal human isolates, a potential candidate for new probiotic dairy foods for the local market. Lactobacillus gasseri LgF_37/1 performed well in the culture media used for the enumeration of probiotic bacteria in argentinian dairy products. This strain showed also high tolerance to the technological challenges assessed, bile salts resistance, the capacity to produce bacteriocin-like metabolites, to inhibit pathogenic bacteria, to deconjugate bile salts and high hydrophobicity. Further in vivo research should be carried out with this strain before claiming probiotic properties for it. However, the use of a set of simple in vitro techniques proved to be important to determine which strains should undergo future and more complex studies.     

Bacteriocin-producing Lactobacillus strains isolated from poto poto, a Congolese fermented maize product, and genetic fingerprinting of their plantaricin operons

Thirty one bacteriocin-producing Lactobacillus isolates were identified among 135 lactobacilli isolated from the Congolese fermented maize product poto poto, during the preparation and from the finished product. Using species-specific PCR and 16S rRNA gene sequencing, 28 and 3 isolates were identified as L. plantarum and L. fermentum, respectively. Cluster analysis of RAPD-PCR fingerprints revealed two main groups (G1 and G2) plus the L. fermentum isolate C4-13. Group G1 contained 23 isolates with a similarity coefficient >74.5%, and could be divided in two subgroups (G1-1, G1-2) each with several branches, plus the L. plantarum isolate C11. Group G2 contained 8 isolates with a similarity coefficient >86%, with two main branches. Using PCR amplification with specific primers, several genes of the plantaricin cluster found in L. plantarum C11 were identified in the isolates. The number of genes that were detected varied between the strains. The L. fermentum isolate EC11 also contained the plnDEFG genes. PCR amplification of DNA from isolates with primers directed to the upstream and downstream region of the plantaricin cluster generated an amplicon identical to that obtained with DNA from the control strain L. plantarum WCFS1. Amplification products from the positive strains were used for restriction analysis with HindIII, EcoRI and KpnI in separate reactions. Cluster analysis of restriction profiles revealed high similarities for EcoRI and HindII digest profiles, and an identical profile for all KpnI digests. The L. fermentum EC11 isolate clustered with L. plantarum strains in a group with a high correlation coefficient. The results suggest a low degree of diversity in the plantarincin gene cluster. However, other strains that tested positive for individual plantaricin genes may present great heterogeneity in the plantaricin operons. Because of their broad spectra of inhibition (including Escherichia coli, Salmonella enterica, Enterobacter aerogenes, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis), isolates from the present study could be used to improve the safety and storage stability of poto poto.     

Development of a rapid, simple paddle-style dipstick dye immunoassay specific for Listeria monocytogenes

We have developed two types of new paddle-style dipstick dye immunoassays. The first is genus Listeria specific and the second is specific to Listeria monocytogenes. They are based respectively, on antisera raised against heat-killed L. monocytogenes cells and against internalin B crude extract, a virulence protein found only in the pathogenic L. monocytogenes. The minimum detectable level for L. monocytogenes is 2×10⁷ CFU ml⁻¹ for strain number 88/049 in pure culture. Detection is unaffected by the presence of high numbers (approximately log 8.0 CFU/ml) of the other microorganisms tested. When the dipsticks were applied to milk samples inoculated with L. monocytogenes reference material (ALM92), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of milk and ice cream food samples.     

Development of a SCAR marker for the identification of Olea europaea L.: A newly detected adulterant in commercial Mediterranean oregano

A recent pharmacognostic survey on the European food market highlighted a previously unreported adulteration of Mediterranean oregano. Dried crushed leaves, silvery grey in colour but devoid of glandular hairs and with unequivocal xerophytic adaptations were copiously spotted (20–30% w/w) in a number of commercial samples. Microscopical investigations narrowed the range of suspect candidates to Olea europaea L. and a method based on Sequence-Characterised Amplified Regions markers (SCARs) was developed from Random Amplified Polymorphic DNA markers (RAPDs) specific for O. europaea, in order to authenticate the contaminant and set up a fast, sensitive, reliable and low-cost screening of dried commercial Mediterranean oregano samples. The method enabled the unequivocal detection of low amounts (up to 1%) of olive leaves in both artificial and commercial batches, allowing the preemptive rejection of suspect samples and reducing the number of samples to be subjected to more careful pharmacognostic analyses. The relatively short dimension of the amplicons is suitable for the analysis of potentially degraded DNA obtained from dried and processed commercial plant material and given their specificity the method may be enforceable also in case of forensic disputes even in case of finely ground material.     

Application of molecular identification tools for Lactobacillus, with a focus on discrimination between closely related species: A review

Lactobacillus is among the most important GRAS food lactic acid bacteria, with nearly 140 species at present, mostly of industrial importance. Being part of the natural flora of a range of food products like raw milk, fermented dairy products, fruits, vegetables, meat products they also serve as starters for a number of fermented food products either to enhance the quality or to add health benefits. These groups of economically important species are often alike in phenotypic and physiological characteristics, probably due to their co-evolution in the same ecological niches; hence they are difficult to be differentiated. This demands advanced methods for their proper identification and characterization. With the advancement of molecular biology, a range of DNA-based molecular techniques has replaced the largely cumbersome phenotypic methods. This review summarizes the various molecular techniques available for detection and identification within the genus Lactobacillus, with special emphasis on the four groups of closely resembling species: L. casei group, L. acidophilus group, L. delbrueckii subspecies, and L. plantarum group. This review also provides insights into current trends for alternative molecular markers other than 16S rRNA to resolve the ambiguity within phylogenetically close species in the genus Lactobacillus.     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovars Typhimurium in iceberg lettuce and the antimicrobial effect of rice vinegar against E. coli O157:H7

The microbiological safety of fresh produce is a significant concern of consumers and industry. After applying at an inoculated level (about 10(6) CFUg(-1)) of E. coli O157:H7 and Salmonella enterica serovars Typhimurium on shredded iceberg lettuce and water samples individually, they were stored at 4 degrees C for 14 days and 22 degrees C for 7 days to monitor the growth and survival of pathogens. The results showed that at the end of 4 degrees C storage, populations of two pathogens in lettuce and water decreased approximately 1 log CFUg(-1). However, microbial levels on shredded lettuce increased 3 logs within 3 days at 22 degrees C. Vinegar (acetic acid) had been used to reduce populations of foodborne pathogens in foods; hence, the antimicrobial effect of rice vinegar on the survival of E. coli O157:H7 in inoculated lettuce (10(4) and 10(7) CFUg(-1)) is examined in this study. Results were observed that the treatment of inoculated lettuce (10(7) CFUg(-1)) with commercial vinegar containing 5% acetic acid (pH 3.0) for 5 min would reduce 3 logs population at 25 degrees C. Less than a 1-log decrease in bacterial numbers was recovered during 5 min exposure to 0.5% (pH 3.26) acetic acid.     

Supercritical fluid and solid–liquid extraction of phenolic antioxidants from grape pomace: a comparative study

Distilled white grape pomace (Vitis vinifera var. Garnacha) was subjected to extraction by using two different methods: (1) solid–liquid extraction (SLE) employing 96% ethanol and water and (2) supercritical extraction (SFE) by running carbon dioxide coupled with ethanol as a modifier. Higher phenolic concentrations of extracts were attained by SFE (∼400 ppm), doubling those obtained by SLE. In the latter, increasing values of both temperature (from 25 to 50 °C) and contact time (from 30 to 90 min) and lower solvent-to-solid ratios (from 5:1 to 1:1) resulted in an enhancement of extraction efficiency. In SFE, the addition of the modifier (8%) was also found to favour the release of phenolic compounds. Antiradical activity values—evaluated by the ability to scavenge DPPH radicals—were, as general, also higher for SFE extracts, although maximum values reached at were similar (73% inhibition versus 68%). Chromatographic profiles confirmed the diverse nature of phenolic species occurring in extracts obtained from both extraction methods. Extracts from SLE contained more proanthocyanidins, whereas SFE ones contained basically gallic acid, catechin and epicatechin. Protection against oil oxidation assayed with two samples confirmed these results.