Identification of Brucella spp. isolates and discrimination from the vaccine strain Rev.1 by MALDI-TOF mass spectrometry.

Brucellosis' surveillance and control programs require robust laboratory techniques that can reliably identify and biotype Brucella strains and discriminate between vaccine and field infection. In the recent years, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has revolutionized the routine identification of several microorganisms in clinical microbiology laboratories. Nevertheless, its application on Brucella spp. identification is limited since there are no reference spectra in the commercial databases, due to the microorganism's potential bioterrorist use. In this study, a custom MALDI-TOF MS reference library was constructed and its performance on identification at species level was evaluated using 75 Brucella spp. isolates. Furthermore, distinct peak biomarkers were detected for biovar assignment and discrimination from vaccine strain Rev.1. Analysis of mass peak profiles allowed Brucella accurate identification at genus and species level (100%) with no misidentifications. Despite the high intrageneric similarity, MALDI-TOF MS database succeeded in classifying at biovar level, 47 out of 62 B. melitensis bv. 3 isolates (75.81%), whereas all B. melitensis strains, except for one, were correctly discriminated from vaccine strain Rev.1. MALDI-TOF MS appeared to be a rapid, cost-effective and reliable method for the routine identification of brucellae which reduces time consumption in pathogen identification and could replace in the near future the current conventional and molecular techniques. Its ability to differentiate vaccine from field infection could facilitate brucellosis' monitoring systems contributing in the effective control of the disease.

Application of triplex-PCR with an innovative combination of 3 pairs of primers for the detection of milk's animal origin in cheese and yoghurt.

In this research communication we describe an innovative protocol that combines three pairs of primers, two from the literature and one designed in our laboratory, for application in triplex-PCR on somatic cell DNA to enable identification of the species origin (cow, sheep, goat) of cheeses and yogurts with a detection limit of 0.1%. Mislabeling was detected in 15 out of 40 cheeses and in 18 out of 40 yogurts tested. The suggested procedure is a quick and reliable tool for identifying the animal origin of cheeses and yogurts and it can be used to certify product reliability on the domestic and international market. Additionally, in combination with a serological test it can offer a reliable tool for detecting the presence of cow's whey.

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.

Physicochemical Characteristics, Fatty Acids Profile and Lipid Oxidation during Ripening of Graviera Cheese Produced with Raw and Pasteurized Milk.

The production of cheese can be made from either pasteurized or non-pasteurized milk, depending on the country or dietary habits. In this work, the effect of pasteurization of milk on the progress of the physicochemical properties, fatty acids profile and lipid oxidation of cheese throughout a maturation period of 90 days is presented. This research was carried out on two types of Graviera cheese produced in Greece, one made from raw milk and the other from pasteurized milk. The proximal composition of each sample was evaluated, the fatty acids profile was analyzed by Gas Chromatography, whereas lipid oxidation was determined on the basis of the formation of malondialdehyde (MDA). Significant differences (p < 0.05) in the values of pH, fat and density between raw and pasteurized milk were observed. The physicochemical parameters during the ripening of the cheeses showed significant differences according to the type and the stage of maturation. Specifically, the two types of cheese differed significantly (p < 0.05) in terms of pH, protein, fat in dry matter (FDM), and water-soluble nitrogen/total nitrogen (WSN/TN). Although the fatty acids profile was similar for the two types of cheese, differences were observed during the ripening stages as well as between the milk and the final product. The lipid oxidation levels increased during maturation, whereas they seemed to be lower in the pasteurized cheeses. Therefore, it can be concluded that the use of raw or pasteurized milk affects the physicochemical characteristics, fatty acids profile and lipid oxidation of Graviera cheese during ripening.

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.

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.

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.

Detection of ß-Lactams and Chloramphenicol Residues in Raw Milk-Development and Application of an HPLC-DAD Method in Comparison with Microbial Inhibition Assays.

The present study was carried out to assess the detection sensitivity of four microbial inhibition assays (MIAs) in comparison with the results obtained by the High Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) method for antibiotics of the ß-lactam group and chloramphenicol in fortified raw milk samples. MIAs presented fairly good results when detecting ß-lactams, whereas none were able to detect chloramphenicol at or above the permissible limits. HPLC analysis revealed high recoveries of examined compounds, whereas all detection limits observed were lower than their respective maximum residue limits (MRL) values. The extraction and clean-up procedure of antibiotics was performed by a modified matrix solid phase dispersion procedure using a mixture of Plexa by Agilent and QuEChERS as a sorbent. The HPLC method developed was validated, determining the accuracy, precision, linearity, decision limit, and detection capability. Both methods were used to monitor raw milk samples of several cows and sheep, obtained from producers in different regions of Greece, for the presence of examined antibiotic residues. Results obtained showed that MIAs could be used effectively and routinely to detect antibiotic residues in several milk types. However, in some cases, spoilage of milk samples revealed that the kits' sensitivity could be strongly affected, whereas this fact does not affect the effectiveness of HPLC-DAD analysis.

Effect of water deficit on leaf phenolic composition, gas exchange, oxidative damage and antioxidant activity of four Greek olive (Olea europaea L.) cultivars.

The olive tree (Olea europaea L.) is often exposed to severe water stress during the summer season. In this study, we determined the changes in total phenol content, oleuropein and hydroxytyrosol in the leaves of four olive cultivars ('Gaidourelia', 'Kalamon', 'Koroneiki' and 'Megaritiki') grown under water deficit conditions for two months. Furthermore, we investigated the photosynthetic performance in terms of gas exchange and chlorophyll a fluorescence, as well as malondialdehyde content and antioxidant activity. One-year-old self-rooted plants were subjected to three irrigation treatments that received a water amount equivalent to 100% (Control, C), 66% (Field Capacity 66%, FC(66)) and 33% (Field Capacity 33%, FC(33)) of field capacity. Measurements were conducted 30 and 60 days after the initiation of the experiment. Net CO(2) assimilation rate, stomatal conductance and F(v)/F(m) ratio decreased only in FC(33) plants. Photosynthetic rate was reduced mainly due to stomatal closure, but damage to PSII also contributed to this decrease. Water stress induced the accumulation of phenolic compounds, especially oleuropein, suggesting their role as antioxidants. Total phenol content increased in FC(33) treatment and oleuropein presented a slight increase in FC(66) and a sharper one in FC(33) treatment. Hydroxytyrosol showed a gradual decrease as water stress progressed. Malondialdehyde (MDA) content increased due to water stress, mostly after 60 days, while antioxidant activity increased for all cultivars in the FC(33) treatment. 'Gaidourelia' could be considered as the most tolerant among the tested cultivars, showing higher phenolic concentration and antioxidant activity and lower lipid peroxidation and photochemical damage after two months of water stress. The results indicated that water stress affected olive tree physiological and biochemical parameters and magnitude of this effect depended on genotype, the degree of water limitation and duration of treatment. However, the severity as well as the duration of water stress might exceed antioxidant capacity, since MDA levels and subsequent oxidative damage increased after two months of water deficit.