A. Govaris

The antimicrobial effect of oregano essential oil, nisin and their combination against Salmonella Enteritidis in minced sheep meat during refrigerated storage

The antimicrobial effect of oregano essential oil (EO) at 0.6 or 0.9%, nisin at 500 or 1000 IU/g, and their combination against Salmonella Enteritidis was studied in minced sheep meat during storage at 4 degrees or 10 degrees C for 12 days. Sensory evaluation showed that the addition of oregano EO at 0.6 or 0.9% in minced sheep meat was organoleptically acceptable, and attribute scores were higher for the EO at 0.6 than 0.9%. According to compositional analysis of the oregano EO, the phenols carvacrol (80.15%) and thymol (4.82%) were the predominant components. Treatment of minced sheep meat with nisin at 500 or 1000 IU/g, proved insufficient to act against S. Enteritidis. The combination of the oregano EO at 0.6% with nisin at 500 IU/g showed stronger antimicrobial activity against S. Enteritidis than the oregano EO at 0.6% but lower than the combination with nisin at 1000 IU/g, which in turn was lower than that of the oregano EO at 0.9%. In its turn, oregano EO at 0.9% showed lower antimicrobial activity than its combinations with nisin at 500 or 1000 IU/g, which showed a bactericidal effect against the pathogen. The inhibition percentages of all treatments against S. Enteritidis at 10 degrees C were higher than those at 4 degrees C.

The antimicrobial effect of thyme essential oil, nisin and their combination against Escherichia coli O157:H7 in minced beef during refrigerated storage

The antimicrobial effect of thyme essential oil (EO) at supplementation levels of 0.3%, 0.6% or 0.9%, nisin at 500 or 1000IU/g, and their combination, on Escherichia coli O157:H7 was examined in both tryptic soy broth (TSB) and minced beef meat. EO at 0.3% possessed a weak antibacterial activity against the pathogen in TSB, whereas at 0.9% showed unacceptable organoleptic properties in minced meat. Thus, only the level of 0.6% of EO was further examined against the pathogens in minced meat. Treatment of minced beef meat with EO at 0.6% showed an inhibitory activity against E. coli O157:H7 during storage at 10°C, but not at 4°C. Treatment of minced beef meat or TSB with nisin at 500 or 1000IU/g did not show any antibacterial activity against E. coli O157:H7. The combination of EO at 0.6% and nisin at 500 or 1000IU/g showed an additive effect against the pathogen, which was higher during storage at 10°C than at 4°C.

The effects of dietary oregano essential oil and α-tocopheryl acetate on lipid oxidation in raw and cooked turkey during refrigerated storage.

The effects of dietary oregano essential oil and α-tocopheryl acetate supplementation on the susceptibility of raw and cooked turkey breast and thigh meat to lipid oxidation during refrigerated storage for 9 days were examined. Thirty 12-week-old turkeys were divided into five groups and fed a basal diet containing 30 mg α-tocopheryl acetate kg(-1) feed as control, or basal diet plus 200 mg α-tocopheryl acetate kg(-1), or basal diet plus 100 mg oregano oil kg(-1), or basal diet plus 200 mg oregano oil kg(-1), or basal diet plus 100 mg oregano oil and 100 mg α-tocopheryl acetate kg(-1), for 4 weeks prior to slaughter. Lipid oxidation was assessed by monitoring malondialdehyde formation in raw and cooked meat at 0, 3, 6 and 9 days of refrigerated storage, through use of a third-order derivative spectrophotometric method. Results showed that all dietary treatments significantly (P<0.05) increased the stability of both raw and cooked turkey meat to lipid oxidation compared with the control. Oregano oil at 200 mg kg(-1) was significantly (P<0.05) more effective in delaying lipid oxidation compared to the level of 100 mg kg(-1), equivalent to α-tocopheryl acetate at 200 mg kg(-1), but inferior (P<0.05) to oregano oil plus α-tocopheryl acetate at 100 mg kg(-1) each, which in turn was superior (P<0.05) to all dietary treatments, indicating a synergistic effect. Thigh muscle was more susceptible to oxidation compared with breast muscle in all treatments, although it contained α-tocopherol at significantly (P<0.05) higher levels.

Dietary versus post-mortem use of oregano oil and/or α-tocopherol in turkeys to inhibit development of lipid oxidation in meat during refrigerated storage

The dietary and post-mortem uses of oregano oil in turkeys to inhibit development of lipid oxidation in breast and thigh meat during refrigerated storage were investigated. Using minced meat, patties were prepared from turkey meat post-mortem added with either 200 mg oregano oil or α-tocopherol/kg, meat from turkeys dietary supplemented with either 200 mg oregano oil or α-tocopheryl acetate/kg feed, and control meat. All patties were cooked, placed in a refrigerated cabinet at 4°C, and lipid oxidation was assessed by monitoring malondialdehyde formation after 3, 6 and 9 days of storage. Treatments significantly (P<0.05) retarded lipid oxidation in both breast and thigh meat patties at all storage times compared with controls. The dietary supplementation of either oregano oil or α-tocopheryl acetate exhibited the highest antioxidative activity compared with the other treatments. Post-mortem addition of either oregano oil or α-tocopherol to the minced meat also retarded lipid oxidation in the prepared patties compared with controls; however, this effect was inferior to that of the dietary supplementation even though the post-mortem α-tocopherol supplemented meat contained 90-fold more α-tocopherol than patties from the dietary supplemented meat. Thigh meat was more susceptible to oxidation than breast meat, although the former contained α-tocopherol at markedly higher levels. Supplementing the diet with 200 mg oregano oil/kg, α-tocopherol levels in the breast and thigh meat significantly (P<0.05) increased compared with control. This increase could not be attributed to the α-tocopherol already present in the oregano oil since post-mortem addition of oregano oil to control breast and thigh meat at the same dose could not actually increase the α-tocopherol concentrations.

Occurrence of aflatoxin M1 in raw and market milk commercialized in Greece

From December 1999 to May 2000, 114 samples of pasteurized, ultrahigh temperature-treated (UHT) and concentrated milk were collected in supermarkets, whereas 52 raw milk samples from cow, sheep and goat were obtained from different milk producers all over Greece. Sample collection was repeated from December 2000 to May 2001 and concerned 54 samples of pasteurized milk, 23 samples of bulk-tank raw milk and 55 raw milk samples from cow, sheep and goat. The total number of samples analysed for aflatoxin M1 (AFM1) contamination by immunoaffinity column extraction and liquid chromatography was 297. In the first sampling, the incidence rates of AFM1 contamination in pasteurized, UHT, concentrated and cow, sheep and goat raw milk were 85.4, 82.3, 93.3, 73.3, 66.7 and 40%, respectively, with only one cow raw milk and two concentrated milk samples exceeding the EU limit of 50 ng l-1. In the second sampling, the incidence rates of AFM 1 contamination in pasteurized, bulk-tank and cow, sheep and goat raw milk were 79.6, 78.3, 64.3, 73.3 and 66.7%, respectively, with only one cow and one sheep raw milk samples exceeding the limit of 50 ng l-1. The results suggest that the current regulatory status in Greece is effective.

Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece

The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx(2)-positive, five of which were also stx(1)-positive. The remaining isolate was positive only for the stx(1) gene. All stx-positive isolates (whether positive for stx(1), stx(2) or stx(1) and stx(2)) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.

Distribution and stability of aflatoxin M1 during processing, ripening and storage of Telemes cheese.

Telemes cheeses were produced using milk that was artificially-contaminated with aflatoxin M1 at the levels of 0.050 and 0.100 microg/l. The cheeses produced in the two cheese-making trials were allowed to ripen for 2 months and stored for an additional 4 months to simulate commercial production of Telemes cheese. Concentrations of aflatoxin M1 in whey, curd, brine, and the produced cheeses were determined at intervals by liquid chromatography and fluorometric detection coupled with immunoaffinity column extraction. Concentrations of aflatoxin M1 in the produced curds were found to be 3.9 and 4.4 times higher than those in milk, whereas concentrations in whey were lower than those in curd and milk. Aflatoxin M1 was present in cheese at higher concentrations at the beginning than at the end of the ripening/storage period, and it declined to concentrations 2.7 and 3.4 times higher than those initially present in milk by the end of the sixth month of storage. Concentrations of aflatoxin M1 in brine started low and increased by the end of the ripening/storage period but only a portion of the amounts of aflatoxin M1 lost from cheese was found in the brine. Results showed that Telemes cheeses produced from milk containing aflatoxin M1 at a concentration close to either the maximum acceptable level of 0.05 microg/l set by the European union (EU) or at double this value, will contain the toxin at a level that is much lower or slightly higher, respectively, than the maximum acceptable level of 0.250 microg of aflatoxin M1/kg cheese set by some countries.

Distribution and stability of aflatoxin M1 during production and storage of yoghurt

Yoghurt from cows milk artificially contaminated with aflatoxin M1 (AFM1) at levels of 0.050 and 0.100 g l-1 was fermented to reach pHs 4.0 and 4.6. Yoghurt fermented to pH 4.6 was also used for preparing strained yoghurt. Yoghurts were stored at 4°C for up to 4 weeks. Analysis of AFM1 in milk, yoghurt, strained yoghurt and yoghurt whey was carried out using immunoaffinity column extraction and liquid chromatography coupled with fluorometric detection. AFM1 levels in yoghurt samples showed a significant decrease (p < 0.01) compared with those initially added to milk. Growth of culture lactic acid bacteria was not affected in the AFM1 contaminated yoghurts, with the exception of Streptococcus thermophilus that showed a significantly (p < 0.01) lower increase in the yoghurt containing the toxin at high concentration. Following fermentation, AFM1 was significantly lower (p < 0.01) in yoghurts with pH 4.0 than in yoghurts with pH 4.6 at both contamination levels. During refrigerated storage, AFM1 was rather more stable in yoghurts with pH 4.6 than with pH 4.0. The percentage loss of the initial amount of AFM1 in milk was estimated at about 13 and 22% by the end of the fermentation, and 16 and 34% by the end of storage for yoghurts with pHs 4.6 and 4.0, respectively. The percentage distribution ratio of AFM1 in strained yoghurt/yoghurt whey of the initial toxin present in the yoghurt was about 90/10 and 87/13 for the lower and the higher contamination levels, respectively.

The incorporation of dehydrated rosemary leaves in the rations of turkeys and their impact on the oxidative stability of the produced raw and cooked meat.

Thirty-six 12-week-old turkeys were distributed into six groups and were raised for 4 weeks on rations containing 0%, 0.5% or 1.0% dehydrated rosemary leaves as antioxidant in the presence of α-tocopheryl acetate from 10 to 300 mg/kg. Following slaughtering, breast and thigh meat samples, raw or cooked, from all six groups were collected to be refrigerated at 4°C for 9 days. All stored samples were submitted to analysis for their concentration in malondialdehyde (MDA), a lipid oxidation marker, and α-tocopherol. The results showed that the rations containing 300 mg/kg α-tocopheryl acetate increased the mean α-tocopherol content of the breast and thigh significantly (P <0.05) compared with the respective control values. No significant (P>0.05) changes could be observed in the α-tocopherol content of breast and thigh of turkeys consuming rations containing up to 1% dehydrated rosemary leaves. The refrigeration of the meats led to spontaneous increase in the MDA content of the breast and thigh meat samples. Samples from turkeys fed rations containing 300 mg/kg α-tocopheryl acetate showed the lowest mean levels of MDA after the 9-day refrigerated period. The incorporation of rosemary in the rations led to a modest decrease in the formation of MDA in the meats compared with the respective mean control values. The combination of α-tocopheryl acetate and rosemary was not associated with an additional decrease in MDA formation.

Behavior of Escherichia coli O157:H7 during the Manufacture and Ripening of Feta and Telemes Cheeses

Pasteurized whole ewes and cows milk was used in the manufacture of Feta end Telemes cheeses, respectively, according to standard procedures. In both cases, the milk had been inoculated with Escherichia coli O157:H7 at a concentration of ca. 5.1 log CFU/ml and with thermophilic or mesophilic starter cultures at a concentration of ca. 5.3 to 5.6 log CFU/ml. In the first 10 h of cheesemaking, the pathogen increased by 1.18 and 0.82 log CFU/g in Feta cheese and by 1.56 and 1.35 log CFU/ g in Telemes cheese for the trials with thermophilic and mesophilic starters, respectively. After 24 h of fermentation, a decrease in E. coli O157:H7 was observed for all trials. At that time, the pH was reduced to 4.81 to 5.10 for all trials. Fresh cheeses were salted and held at 16 degrees C for ripening until the pH was reduced to 4.60. Cheeses were then moved into storage at 4 degrees C to complete ripening. During ripening, the E. coli O157:H7 population decreased significantly (P < or = 0.001) and finally was not detectable in Feta cheese after 44 and 36 days and in Telemes cheese after 40 and 30 days for the trials with thermophilic and mesophilic starters, respectively. The estimated times required for one decimal reduction of the population of E. coli O157:H7 after the first day of processing were 9.71 and 9.26 days for Feta cheese and 9.09 and 7.69 days for Telemes cheese for the trials with thermophilic and mesophilic starters, respectively.