Nuclear DNA barcodes for cod identification in mildly-treated and processed food products

Gadoids are a group of fish with historical importance in the fishing industry. The high demand for cod is one of the reasons why cod products are often mislabelled, and numerous observations have been made on the replacement of Atlantic cod (Gadus morhua) by cheaper species or its illegal capture in contravention of fish quotas. Fish species identification is traditionally based on morphological features, but this may be difficult in case of heat-treated or processed products, or where the species look similar, as in the Gadoid group. DNA-based approaches (using either nuclear or mitochondrial DNA) are most commonly used in this case, due to their high specificity and to the high resilience of the target molecules to food processing techniques. In this article, we identified, using an automated screening approach, novel barcode regions and their associated primers in the nuclear genome, to be used for the efficient identification of Gadoids. The barcode regions were tested on official and commercial samples, raw or mildly treated products, like frozen, or salted, as well as pre-cooked complex mixtures and processed samples, using next-generation sequencing (NGS) technique. The method proposed could complement existing fish identification strategies in establishing an efficient framework to detect and prevent frauds along the food chain.     

Technological and flavour potential of cultures isolated from traditional Greek cheeses – A pool of novel species and starters

This study explored the microbiota of Formaella, Kopanisti, Feta and Mana cheeses. A total of 133 wild lactic acid bacteria were isolated and classified phenotypically. Mesophilic lactobacilli were the most abundant group. Thermophilic lactobacilli and thermophilic cocci were the best milk acidifiers, whereas thermophilic lactobacilli were the most proteolytic isolates. Higher peptidolytic and esterolytic activities were obtained with thermophilic cocci. Only five isolates were lipolytic, whereas none was able to catabolize citrate. Fast gas chromatography−mass spectrometry analysis of the metabolites produced and subsequent principal components analysis revealed segregated groups of isolates in accordance with the phenotypic ones. Electronic nose analysis revealed similar results. Lactobacillus rennini and Lactobacillus acidipiscis were found to be the sole microbial species in Kopanisti cheese and Mana. These isolates produced alcohols and aldehydes as major volatile compounds, as a result of secondary amino acid catabolism.     

Enterococcus and Lactobacillus contamination of raw milk in a farm dairy environment

Enterococci and lactobacilli are ubiquitously found in the intestinal microflora of humans and animals. The aim of the present study was to determine the importance of bovine faeces as a source of these organisms in raw milk. One hundred and fifty six putative enterococci and 362 lactobacilli were isolated from bovine faeces (n=26), cows' teats, raw milk, the milking machine and the milking environment on one farm. The clonal relationships of each group were investigated using Pulsed-Field Gel Electrophoresis and representatives of the different clusters were identified by repetitive DNA element (rep)-PCR fingerprinting, protein profiling, phenylalanyl-tRNA synthase (pheS) sequence analysis or 16S rDNA gene sequencing. Lactobacilli were present at approximately 3 orders of magnitude greater than enterococci in the bovine faeces. The majority of the bovine faecal enterococcal isolates were identified as Aerococcus viridans. Seven teat isolates belonged to a potential novel Aerococcus sp. and one bovine faecal isolate to a potential second novel Aerococcus sp. The lactobacilli present in the bovine faeces were predominantly Lactobacillus mucosae and Lactobacillus brevis, with small numbers of Lactobacillus plantarum. Only one Enterococcus (a strain of E. casseliflavus) out of 76 and one Lactobacillus (a strain of L. parabuchneri/kefir) out of 247 of the bovine faecal isolates was found in the milk. The major source of these bacteria in the milk was the milking equipment.     

Development of an ELISA Reverse-Based Assay to Assess the Presence of Mycotoxins in Cereal Flour

The first successful application of the ELISA reverse method and device (ER m&d), in the context of food safety and traceability, was developed in our laboratories to detect and quantify CP4EPSPS and Cry1AB genetically modified related proteins in soy and maize samples, respectively. To prove the versatility and the transferability of the technology, the ER was here applied to assess the presence of mycotoxins in cereals. Appropriate protocols were developed to assess the presence of deoxynivalenol and ochratoxin A and the ER was tested on contaminated wheat samples. In particular, deoxynivalenol (DON) and ochratoxin A (OTA) were detected in a range of values from 430 to 5,000 μg kg⁻¹ and from 1.7 to 10 μg kg⁻¹, respectively. The assays were optimized to reach a limit of quantification equal to 550 and 1.8 μg kg⁻¹ for DON and OTA, respectively.     

Combinatory SYBR® Green Real-Time PCR Screening Approach for Tracing Materials Derived from Genetically Modified Rice

Two real-time PCR approaches for the detection of genetically modified (GM) rice were tested: (1) a combination of SYBR® Green real-time PCR methods detecting the 35S promoter (P-35S) of Cauliflower Mosaic Virus, the nopaline synthase terminator (T-nos) of Agrobacterium tumefaciens and the Bacillus thuringiensis (Bt) CryIAb/Ac toxins and (2) a P-35S/T-nos duplex TaqMan® real-time PCR system. Both systems correctly recognized their respective targets in control samples of Bt63, Kefeng6 and KMD1 insect-resistant and LLRice62 and LLRice601 herbicide-resistant rice. Due to its lesser specificity but broader genetically modified organism (GMO) coverage capacity, the SYBR® Green real-time PCR approach was further tested in more detail. Melting curve, capillary and agarose gel electrophoresis analyses indicated that the P-35S, T-nos and CryIAb/Ac targets in the GM rice events are similar to the corresponding targets present in many known GMOs. High-resolution melting analysis showed that the CryIAb/Ac targets of the GM rice events Bt63 and Kefeng6 matched best the corresponding Bt11 CryIAb sequence. Digital PCR analysis on genomic DNA from the GM rice Bt63 and Kefeng6 events indicated that both GMO contained multiple inserts. Sensitivity tests showed that all SYBR® Green real-time PCR methods could detect their targets at less than an estimated five copies per reaction. Finally, it was shown that these SYBR® Green real-time PCR methods could detect low levels of their targets in rice consignments originating from China. Together, these results demonstrated that a ‘P-35S and T-nos and CryIAb/Ac’ combinatory SYBR® Green real-time PCR screening is highly suited to trace the respective targets including the possible presence of Bt63, Kefeng6 and KMD1 GM rice materials in food products.     

Identification and characterization of Enterococcus spp. in Greek spontaneous sausage fermentation

A total of 108 enterococcal strains previously isolated from spontaneously fermented sausages were identified using phenotypic traits, pulsed-field gel electrophoresis (PFGE), and sequencing of the 16S rRNA gene. The proteolytic and lipolytic activities of these isolates and their ability to decarboxylate lysine, tyrosine, ornithine, and histidine and to produce antimicrobial compounds also were assessed. All strains were identified as Enterococcus faecium, and a lack of correlation between data derived from phenotypic and those derived from genotypic techniques was evident. Wide strain diversity was revealed by both phenotypic properties and PFGE strain typing results. Few strains were present in all batches, suggesting a possible persistence in the respective production plants. Neither proteolytic nor lipolytic activities were detected, and none of the strains decarboxylated lysine, tyrosine, ornithine, or histidine. A total of 42 E. faecium strains inhibited in vitro growth of Listeria monocytogenes, which suggests possible contribution of these strains to the safety of the end product and possible utilization of these strains as protective cultures.     

Lactic acid bacteria population dynamics during minced beef storage under aerobic or modified atmosphere packaging conditions

A total of 266 lactic acid bacteria (LAB) have been isolated from minced beef stored at 0, 5, 10 and 15 °C aerobically and under modified atmosphere packaging consisting of 40% CO₂–30% O₂–30% N₂ in the presence MAP (+) and absence MAP (−) of oregano essential oil. Sequencing of their 16S rRNA gene along with presence of the katA gene demonstrated dominance of the LAB microbiota by Leuconostoc spp. during aerobic storage at 5, 10 and 15 °C, as well as during MAP (−) and MAP (+) storage at 10 and 15 °C; Lactobacillus sakei prevailed during aerobic storage at 0 °C, as well as at MAP (−) and MAP (+) storage at 0 and 5 °C. The sporadic presence of other species such as Leuconostoc mesenteroides, Weisella viridescens, Lactobacillus casei and Lactobacillus curvatus has also been determined. Pulsed-Field Gel Electrophoresis of high molecular weight genomic DNA revealed the dynamics of the isolated LAB strains. Prevalence of Leuconostoc spp. was attributed to one strain only. On the other hand, packaging conditions affected Lb. sakei strain spoilage dynamics.