Indigenous Lactic Acid Bacteria Isolated from Raw Graviera Cheese and Evaluation of Their Most Important Technological Properties.

The aim of the present study was to characterize LAB isolates from raw-milk cheeses, to evaluate some of their technological properties and to select a few 'wild' LAB strains that could potentially be used as starter cultures. LAB strains were isolated and identified from raw milk, curd, and cheese at 30, 60, and 90 days of ripening. A total of 100 strains were isolated, 20 from each phase of ripening. All isolates were tested for acidification ability, curd formation, and aroma production at 32 °C and 42 °C after 24 and 48 h. Following the acidification test, 42 strains were selected for identification and characterization of their technological properties. A high proportion of lactic acid bacteria and Gram + cocci were found throughout the cheese-making process. Enterococci reached their maximum proportion on the 7th day of ripening while Lactobacilli increased significantly during the first month of ripening. Forty-two strains were identified by phenotypic, biochemical, and molecular techniques. Lactococci were predominant in raw milk and curd while Lactobacilli in the ripening of the cheese. Four LAB strains including one Leuconostoc pseudomenteroides, two Lacticaseibacillus paracasei subsp. paracasei and one Enterococcus hirae, were proposed for their potential use as starters or secondary cultures.

Hygiene and Safety of Hard Cheese Made from Raw Cows' Milk.

This study was conducted to evaluate the microbiological status of cheese made from unpasteurized cows' milk, to examine the safety of the cheese and to observe the changes that occurred in its microbial community during ripening and storage. Furthermore, the pH, the moisture and salt concentration were also monitored throughout processing, ripening and storage. Seven cheesemaking trials took place along with the microbiological and physicochemical analysis of the milk, curd and cheese produced. The milk used for the cheesemaking, two curd samples before the heating and two after the heating, two cheese samples at days 3, 7, 15, 30, 60 and 90 were subjected to microbiological analysis for total mesophilic bacterial count (for milk only), Enterobacteriaceae, E. coli, Staphylococcus, Salmonella, Listeria, and Clostridium. The microbiological quality of raw milk was found to be good. It was initially slightly above the EU limit but improvements associated with farm biosecurity and milking equipment hygiene led to a significantly improved milk quality. A small increase in the prevalence of indicator microorganisms in curd and cheese samples was observed for the first few days, followed by a relatively stable condition as manufacturing proceeded and throughout the ripening of the final product. In two cheesemaking trials, Clostridium perfringens and Salmonella spp. were detected, the first originating from the milk and the second from the environment. The use of good-quality raw milk under sanitary conditions, the application of good manufacturing practices and a maturation period in a controlled environment were found to be the necessary prerequisites for the production of safe raw cheese products.

Comparative Evaluation of Three Commercial Elisa Kits Used for the Detection of Aflatoxins B1, B2, G1, and G2 in Feedstuffs and Comparison with an HPLC Method.

Various analytical techniques for detecting mycotoxins have been developed in order to control their concentration in food and feed. Conventional analytical approaches for mycotoxin identification include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC). Rapid methods for mycotoxin analysis are also becoming increasingly relevant. One of the most common rapid methods for determining these compounds is the enzyme-linked immunosorbent assay (ELISA). The current study aimed to compare three available ELISA kits for the detection and quantification of aflatoxins B1, B2, G1, and G2 in spiked feed samples at known quantities. All three ELISA kits were validated and showed good performance with high recovery rates and LOD and LOQ values lower than the MRL. The developed HPLC-FL method was validated for all the compounds determining the accuracy, precision, linearity, decision limit, and detection capability with fairly good results. Unknown feed samples (corn, silage, pellet, barley, wheat, soya, and sunflower) were also tested using the best ELISA kit and HPLC, and the results were compared. Both ELISA and HPLC were proven to be suitable methods for mycotoxin analysis. The analytical technique should be determined primarily by the availability and number of samples.

Determination of Aflatoxin M1 in Raw Milk Using an HPLC-FL Method in Comparison with Commercial ELISA Kits-Application in Raw Milk Samples from Various Regions of Greece.

The highly toxic Aflatoxin M1 (AFM1) is most often detected in milk using an Enzyme-Linked-Immunosorbent Assay (ELISA) for screening purposes, while High-Performance Liquid Chromatography with Fluorescence Detector (HPLC-FL) is the reference method used for confirmation. The aim of the present study was the comparison between three commercially available ELISA kits and a newly developed HPLC-FL method for the determination of the AFM1 in milk samples. The developed HPLC-FL method was validated for the AFM1 and Aflatoxin M2 (AFM2), determining the accuracy, precision, linearity, decision limit, and detection capability with fairly good results. All three ELISA kits were also validated and showed equally good performance with high recovery rates. Moreover, the Limit Of Detection (LOD) and Limit Of Quantification (LOQ) values were found to be significantly lower than the Maximum Residue Limit (MRL) (50 ng kg-1). After the evaluation of all three commercial kits, the ELISA kit with the optimum performance along with the HPLC method was used for the determination of AFM1 in raw cow's, goat's, and sheep's milk samples (396) obtained from producers in different regions of Greece. The evaluation of both methods showed that this ELISA kit could be considered as a faster and equally reliable alternative method to HPLC in routine analysis for the determination of AFM1 in milk.

Investigating the Association Between the Caecal Microbiomes of Broilers and Campylobacter Burden.

One of the major transmission routes for the foodborne bacterial pathogen Campylobacter is undercooked poultry meat, contaminated from intestinal contents during processing. In broilers, Campylobacter can grow to very high densities in the caeca, and is often considered to be a commensal or an opportunistic pathogen in poultry. Reduction of caecal loads of Campylobacter may assist in lowering incidence rates of Campylobacter food poisoning. To achieve this, there needs to be a better understanding of the dynamics of Campylobacter colonization in its natural niche, and the effect of the local microbiome on colonization. Previous studies have shown that the microbiome differed between Campylobacter colonized and non-colonized chicken intestinal samples. To characterize the microbiome of Campylobacter-colonized broilers, caecal samples of 100 randomly selected birds from four farms were analyzed using amplified 16S rRNA gene sequences. Bacterial taxonomic analysis indicated that inter-farm variation was greater than intra-farm variation. The two most common bacterial groups were Bacteroidetes and Firmicutes which were present in all samples and constituted 29.7-63.5 and 30.2-59.8% of the bacteria present, respectively. Campylobacter was cultured from all samples, ranging from 2 to 9 log10 CFU g-1. There was no clear link between Campylobacter counts and Firmicutes, Bacteroidetes, or Tenericutes levels in the 16S rRNA operational taxonomic unit (OTU)-based analysis of the caecal microbiome, but samples with high Campylobacter counts (>9 log CFU g-1) contained increased levels of Enterobacteriaceae. A decrease in Lactobacillus abundance in chicken caeca was also associated with high Campylobacter loads. The reported associations with Lactobacillus and Enterobacteriaceae match changes in the intestinal microbiome of chickens and mice previously reported for Campylobacter infection, and raises the question about temporality and causation; as to whether increases in Campylobacter loads create conditions adverse to Lactobacilli and/or beneficial to Enterobacteriaceae, or that changes in Lactobacilli and Enterobacteriaceae levels created conditions beneficial for Campylobacter colonization. If these changes can be controlled, this may open opportunities for modulation of chicken microbiota to reduce Campylobacter levels for improved food safety.

Genotyping of Listeria monocytogenes isolates from poultry carcasses using high resolution melting (HRM) analysis.

An outbreak situation of human listeriosis requires a fast and accurate protocol for typing Listeria monocytogenes. Existing techniques are either characterized by low discriminatory power or are laborious and require several days to give a final result. Polymerase chain reaction (PCR) coupled with high resolution melting (HRM) analysis was investigated in this study as an alternative tool for a rapid and precise genotyping of L. monocytogenes isolates. Fifty-five isolates of L. monocytogenes isolated from poultry carcasses and the environment of four slaughterhouses were typed by HRM analysis using two specific markers, internalin B and ssrA genes. The analysis of genotype confidence percentage of L. monocytogenes isolates produced by HRM analysis generated dendrograms with two major groups and several subgroups. Furthermore, the analysis of the HRM curves revealed that all L. monocytogenes isolates could easily be distinguished. In conclusion, HRM was proven to be a fast and powerful tool for genotyping isolates of L. monocytogenes.

A novel closed-tube method based on high resolution melting (HRM) analysis for authenticity testing and quantitative detection in Greek PDO Feta cheese.

Animal species identification of milk and dairy products has received increasing attention concerning food composition, traceability, allergic pathologies and accurate consumer information. Here we sought to develop an easy to use and robust method for species identification in cheese with emphasis on an authenticity control of PDO Feta cheese products. We used specific mitochondrial DNA regions coupled with high resolution melting (HRM) a closed-tube method allowing us to detect bovine, ovine and caprine species and authenticate Greek PDO Feta cheese. The primers successfully amplified DNA isolated from milk and cheese and showed a high degree of specificity. HRM was proven capable of accurately identifying the presence of bovine milk (not allowed in Feta) down to 0.1% and also of quantifying the ratio of sheep to goat milk mixture in different Feta cheese commercial products. In conclusion, HRM analysis can be a faster, with higher resolution and a more cost effective alternative method to authenticate milk and dairy products including PDO Feta cheese and to quantitatively detect its sheep milk adulterations.

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis.

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220bp and 77bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.