Ioannis N. Savvaidis

Metal-microbe interactions: contemporary approaches.

Publisher Summary This chapter discusses the methods to study interactions between metals and microorganisms, and demonstrate the necessity of adopting a multidisciplinary approach to tackle this increasingly important and diverse area of microbiology. The multidisciplinary approach surveyed in the chapter involves microbiology, particularly microbial physiology, genetics and molecular biology, bioinorganic chemistry, analytical chemistry, and the application of instrumental techniques. Commonly used methods for the determination of metal concentrations in cells (and in cell compartments or fractions)—that is, those of atomic spectroscopy are discussed. Radiochemical techniques are of particular value in studying the uptake and localization of metals and sometimes offer the only way of studying variations in concentrations of metals in cellular compartments. The use of spectroscopic methods provides information about the ligands, geometries, andthe electronic status of metals coordinated at binding sites, sometimes in living cells. The use of non-invasive techniques is of significant mechanistic potential as they may allow the direct study of ionic and metabolic processes in essentially unperturbed cells. Genetics and molecular biology hold great promise for advancing studies of metal-microbe interactions, particularly those aspects that are currently perceived as especially significant. These include gene regulation by metals (metalcontaining transcription factors and zinc fingers), control of specificity at metalbinding sites and genetic modification of microorganisms for application in biohydrometallurgy. Studies on extracellular precipitates produced by microbial activity or on the complexation of metal ions in supernatant solutions or growth media by release of extracellular ligands are also discussed.

Combined effect of MAP and thyme essential oil on the microbiological, chemical and sensory attributes of organically aquacultured sea bass (Dicentrarchus labrax) fillets.

The present study evaluated the combined effect of Modified Atmosphere Packaging (MAP) using two different gas mixtures (40% CO2/50% N2/10% O2; treatment M1, 60% CO2/30% N2/10% O2, treatment M2), and thyme oil (0.2% v/w, T) used as a natural preservative, on the quality and shelf life extension of fresh filleted sea bass, product of organic aquaculture, during refrigerated storage (4 +/- 0.5 degrees C), for a period of 21 days. Aerobically packaged sea bass fillets (A) were used as control samples. The dominant bacteria in the microflora of sea bass fillets, irrespective of treatment, were the pseudomonads and the H2S-producing bacteria while lactic acid bacteria were also part of the dominant microflora. Total viable counts for fresh sea bass fillets stored aerobically exceeded 7 log CFU/g after 7 days, while treatments A+T, M1, M2 and M2+T reached the same value on days 9, 10, 12 and 19, respectively. Among the chemical indices determined, TBA values were within the good quality limits (2-4 mg MDA/kg), during the sensory shelf lives of sea bass samples, irrespective of treatment. TVB-N proved to be a suitable index for the spoilage of sea bass fillets stored at 4 degrees C. Samples A and A+T, M1, M2, M2+T exceeded the proposed upper TVB-N acceptability limit (10 mg N/100 g) on days 6, 8, 9, 13 and 17 of storage respectively. TMA-N values of the samples A, A+T and M1, M2, M2+T exceeded the proposed limit (4 mg N/100 g) on days 6, 9, 9-10, 13 and 19 of storage, respectively, and correlated well with the microbiological data, indicating that along with TVB-N, TMA-N may serve as a useful index for sea bass fillets spoilage. As regards sensory evaluation, the presence of thyme oil proved to improve the sensory quality of sea bass fillets when used in combination with MAP2, providing a shelf life of 17 days as compared to 6 days of the control samples.

Combined effect of vacuum-packaging and oregano essential oil on the shelf-life of Mediterranean octopus (Octopus vulgaris) from the Aegean Sea stored at 4 °C

The present study evaluated the use of vacuum packaging (alone) or with addition of oregano essential oil (EO), as an antimicrobial treatment for shelf-life extension of fresh Mediterranean octopus stored under refrigeration for a period of 23 days. Four different treatments were tested: A, control sample; under aerobic storage in the absence of oregano essential oil; VP, under vacuum packaging in the absence of oregano essential oil; and VO1, VO2, treated samples with oregano essential oil 0.2 and 0.4% (v/w), respectively, under VP. Of all the microorganisms enumerated, Pseudomonas spp., H2S-producing bacteria and lactic acid bacteria (LAB) were the groups that prevailed in octopus samples, irrespective of antimicrobial treatment. With regard to the chemical freshness indices determined, thiobarbituric acid (TBA) values were low in all octopus samples, as could have been expected from the low fat content of the product. Both trimethylamine nitrogen (TMA-N) and total volatile basic nitrogen (TVB-N) values of oregano treated under VP octopus samples were significantly lower compared to control samples during the entire refrigerated storage period. Based primarily on sensory evaluation (odor), the use of VP, VO1 and VO2 extended the shelf-life of fresh Mediterranean octopus by ca. 3, 11 and 20 days, respectively.

Determination of biogenic amines as their benzoyl derivatives after cloud point extraction with micellar liquid chromatographic separation.

The advantages of micellar cloud point extraction combined with a surfactant-assisted separation in a HPLC system are presented as a method for the effective separation and determination of nine biogenic amines in fish substrates. Benzoyl derivatives of the amines are extracted inside the micelles of a non-ionic surfactant, Triton X-114, and separated with gradient elution micellar liquid chromatography. Quantification was performed by measuring the UV absorbance of the benzene ring at 254 nm. Detection limits of the nine biogenic amines were in the vicinity of 0.01 mg l(-1) which are approximately 10 times lower than those of the conventional method (HPLC-UV) and 100 times lower than those of micellar electrokinetic capillary chromatography. The correlation coefficients of determinations were 0.9911-0.9996. The method was applied for the determination of putrescine, cadaverine, agmatine, tyramine, tryptamine, phenylethylamine, spermine, spermidine and histamine in trout samples. Recovery of the proposed method ranged from 95 to 103.5%.

Combined effect of freeze chilling and MAP on quality parameters of raw chicken fillets

The effect of short-term frozen storage prior to thawing on the quality of freeze-chilled chicken fillets was investigated, as was the effect of modified atmosphere packaging (MAP). Four process treatments were used: (1) fresh chicken chilled at 4 degrees C without previous freezing, (2) freeze-chilled for 7 days and thawed at 4 degrees C, (3) chilled at 4 degrees C packaged under MAP (70% N(2)-30%CO(2)), and (4) packaged under MAP, freeze-chilled for 7 days and thawed at 4 degrees C. Microbiological, chemical and sensory analyses were conducted on samples for a period up to 15 days. Freeze-chilled fillets gave a lower total viable count (TVC) at a given sampling day than chilled fillets. MAP, as expected, delayed microbial growth. The Pseudomonads were the dominant microbial species in fillets under aerobic conditions. MAP reduced the populations of Pseudomonads by 2-4 log cfu/g. Lactic acid bacteria (LAB) and Enterobacteriaceae increased progressively for all treatments throughout storage. Yeasts and molds were inhibited by MAP and by freeze chilling. Total volatile basic nitrogen (TVB-N) values increased rapidly for the chilled fillets but remained significantly lower for the freeze-chilled and the MA-packaged samples. MAP and especially freeze chilling enhanced drip loss. MAP did not affect redness or yellowness of product while freeze chilling decreased product redness. Lightness was not affected by either MAP or freeze chilling. Based on taste, which proved to be the most sensitive sensory attribute, shelf life of product ranged from 6 to 7 days for all treatments leading to the conclusion that freeze chilling is a suitable technology for fresh chicken fillets enabling their distribution as a frozen product and upon subsequent thawing at their final destination, their retail display as chilled products. MAP in combination with freeze chilling had a negligible effect on product quality.

Combined effects of salting, oregano oil and vacuum-packaging on the shelf-life of refrigerated trout fillets.

The present study evaluated the effect of salt, oregano essential oil (EO) and packaging on fresh rainbow trout fillets during storage at 4 degrees C. Treatments included the following: A1 (control samples, unsalted: air packaged), A2 (salted: air packaged), VP1 (salted, vacuum packaged), VP2 (salted, vacuum packaged with added oregano EO 0.2% v/wt), and VP3 salted, vacuum packaged with added oregano EO 0.4% v/wt). Lactic acid bacteria (LAB) (to a greater extent), followed by H(2)S-producing bacteria (including Shewanella putrefaciens), Pseudomonas spp. and Enterobacteriaceae reached higher populations in A1, A2 (as compared to VP1, VP2 and VP3) trout samples. Treatments VP1, VP2 and VP3 produced significantly lower (P < 0.05) total volatile basic nitrogen (TVBN) and trimethylamine nitrogen (TMAN) values as compared to the A1 and A2 samples after day 6 and until end of storage period. Changes in thiobarbituric acid values (TBA) values for A1, A2, VP1, VP2 and VP3 samples were variable, indicative of no specific trend in trout samples, irrespective of packaging in the absence and/or presence of salt and oregano EO. As determined by sensory analysis (overall acceptability attribute) the observed shelf-life of trout fillets was longest for VP2 (16-17 days) followed by VP1 (14 days), A2 (8 days) and control (A1) samples (5 days). The presence of salt and oregano oil (0.2%) in cooked VP1 trout samples produced a distinct but sensorially acceptable pleasant odor, well received by the panellists, in contrast to the combined effect of salt and oregano oil at the higher concentrations (0.4% v/wt) used. Addition of salt (treatment VP1) extended the products shelf-life by 9 days, whereas the combination of salt, oregano EO (0.2% v/wt) under VP conditions (treatment VP2) resulted in a significant shelf-life extension of trout fillets (11-12 days) according to sensory data, as compared to the control sample, kept under aerobic conditions.

Microbiological, biochemical and sensory assessment of mussels (Mytilus galloprovincialis) stored under modified atmosphere packaging

Aims:  To determine the microbiological, biochemical and sensory changes of mussels during storage under aerobic, vacuum packaging (VP) and modified atmosphere packaging (MAP) conditions at 4°C, and to determine shelf‐life of mussels under the same packaging conditions using the above assessment parameters.

Shelf-life extension of refrigerated Mediterranean mullet (Mullus surmuletus) using modified atmosphere packaging.

The present work evaluated the quality and freshness characteristics and the effect of modified atmosphere packaging (MAP) on the shelf-life extension of refrigerated Mediterranean mullet using microbiological, biochemical, and sensory analyses. Fresh open sea red mullet (Mullus surmuletus) were packaged in four different atmospheres: M1, 10%/20%/70% (O2/ CO2/N2); M2, 10%/40%/50% (O2/CO2/N2); M3, 10%/60%/30% (O2/CO2/N2); identical fish samples were packaged in air. All fish were kept under refrigeration (4 +/- 0.5 degrees C) for 14 days. Of the three gas atmospheres, the 10%/40%/50% (M2) and 10%/ 60%/30% (M3) gas mixtures were the most effective for inhibiting growth of aerobic microflora in mullet samples until day 10 of refrigerated storage. H2S-producing bacteria and pseudomonads were part of the mullet microflora and their growth was partly inhibited under MAP conditions. Between these two bacterial groups, H2S-producing bacteria (including Shewanella putrefaciens) were dominant toward the end of the storage period, regardless of the packaging conditions. Brochothrix thermosphacta and lactic acid bacteria were found to be members of the final microbial flora of MAP and air-packaged mullet, whereas the Enterobacteriaceae population was lower than other bacterial groups. Of the chemical freshness indices determined, thiobarbituric acid values were variable in mullet samples irrespective of packaging conditions indicative of no specific oxidative rancidity trend. Based on sensorial data and aerobic plate count, trimethylamine nitrogen and total volatile basic nitrogen limit values in the range of ca. 15 to 23 and 52 to 60 mg N/100 g of fish muscle were obtained, respectively, for mullet packaged under modified atmosphere and air. Sensory analyses (odor and taste attributes) showed that the limit of sensorial acceptability was reached after ca. 6 days for the samples packaged in air, 8 days for the M1 and M3 samples, and after 10 days for the M2 samples. Respective shelf-life extension for fresh whole mullet was ca. 2 days (M1 and M3 gas mixtures), and 4 days (M2 gas mixture).

Formation of biogenic amines and relation to microbial flora and sensory changes in smoked turkey breast fillets stored under various packaging conditions at 4 °C

The present study evaluated: (1) the formation of biogenic amines (BAs) in smoked turkey fillets during storage under aerobic and modified atmosphere packaging (MAP) conditions at 4 degrees C, (2) the relation of BAs to microbial and sensory changes in turkey meat and (3) the possible role of BAs as indicators of poultry meat spoilage. Smoked sliced turkey fillets were stored in air and under vacuum, skin and two modified atmospheres (MAP), M1 (30% CO(2)/70% N(2)) and M2 (50% CO(2)/50% N(2)), at 4+/-0.5 degrees C, for a period of 30 days. The BAs determined were: tryptamine, tyramine, histamine, putrescine, cadaverine, spermidine and spermine. Low levels of BAs were observed throughout the entire storage period, with the exception of histamine, tyramine and tryptamine, for which higher concentrations were recorded. Values for these three BAs were the highest for air-packaged samples (32.9, 25.0 and 4.1mg/kg, respectively) and the lowest for skin-packaged samples (11.9, 4.3 and 2.8 mg/kg, respectively) after 30 days of storage. All microorganism populations increased throughout the storage period, except for Pseudomonas spp. and Enterobacteriaceae, in skin-packaged fillets and modified atmosphere M2, which remained under the method detection limit (<1logCFU/g) until day 30 of storage. Pseudomonas spp. and Enterobacteriaceae for the rest of the packaging treatments remained below 5logCFU/g throughout storage. On the other hand, lactic acid bacteria were dominant throughout the storage period, regardless of the packaging conditions reaching 8.9logCFU/g on day 30 of storage. Mesophiles reached 7logCFU/g after ca. 19-20 days for the air and skin packed samples, 22-23 days for the M2 and vacuum packed samples and 25-26 days for the M1 packed samples. BA values for tryptamine, histamine and tyramine correlated well with both microbiological and sensory analyses data. Tryptamine, histamine and tyramine may be used as chemical indicators of turkey meat spoilage.

Relation of Biogenic Amines with Microbial and Sensory Changes of Whole and Filleted Freshwater Rainbow Trout (Onchorynchus mykiss) Stored on Ice

The biogenic amine (BA) content of whole and filleted rainbow trout was monitored during ice storage for a period of 18 days and related to respective microbial and sensorial changes occurring during the same period. Eight amines, namely, putrescine, cadaverine, tyramine, spermidine, tryptamine, beta-phenylethylamine, spermine, and histamine, were determined. Agmatine was not detected in any of the fish samples. In all cases, concentration of BAs was higher (P < 0.05) in filleted compared with whole trout samples. Pseudomonads, H2S-producing bacteria, and, to a lesser extent, Enterobacteriaceae were the dominant microorganisms in both whole and filleted trout. Higher populations (P < 0.05) of these microorganisms were present in filleted trout compared with whole fish samples. Of the BAs determined, concentration of putrescine, cadaverine, spermidine, tryptamine, and beta-phenylethylamine increased steeply in both whole and filleted trout between days 15 and 18 of storage when pseudomonads and H2S-producing bacteria reached approximately 10(6) to 10(7) CFU/g. For the rest of the BAs, including tyramine, histamine, and spermine, a stepwise increase was recorded throughout the entire storage period. Interestingly, Enterobacteriaceae counts remained below 10(6) throughout the entire storage period, accounting for the lower production of histamine. A putrescine value of 13 to 14 mg/kg and a spermidine value of approximately 7 mg/kg for both the whole and filleted trout obtained after 12 and 9 days, respectively, may be proposed as the upper limit for spoilage initiation (freshness indicator) of fresh rainbow trout based on sensorial and microbiological (total viable count of 10(6) to 10(7)) data. With respect to other amines determined, both tyramine and spermine may also be proposed as freshness indicators preferably for whole trout, whereas tryptamine, beta-phenylethylamine, histamine, and cadaverine produced only during later stages of storage are not suitable as freshness indicators of either whole or filleted trout.