Athanasios Mallouchos

Variation of the Chemical Profile and Antioxidant Behavior of Rosmarinus officinalis L. and Salvia fruticosa Miller Grown in Greece

In this study, the essential oil and the phenolic composition along with the antioxidant activity of R. officinalis L. and S. fruticosa Miller, collected in Zakynthos island (Ionian Sea, Greece), were investigated. The essential oil composition of the plants was characterized by the presence of 1,8-cineole. Mean values of the antioxidant activities of rosemary and sage essential oils indicated slight differences. The antioxidant activity of sage oil was correlated with the oxygenated sesquiterpenes and diterpenes concentrations. Concerning the methanolic extracts, a close relationship between the phenolic content and the development stage during vegetative cycle of these plants was observed. The identified flavonoids, except rutin, seemed to increase with the advancement of developmental stages, while phenolic acids followed an opposite pattern. The antioxidant activity was correlated with the amount of total phenolic content.

Wine fermentations by immobilized and free cells at different temperatures. Effect of immobilization and temperature on volatile by-products

A biocatalyst was prepared by immobilization of Saccharomyces cerevisiae, strain AXAZ-1, on delignified cellulosic material (DCM) and gluten pellets (GP). Repeated batch fermentations were conducted using these biocatalysts and free cells, separately, at different temperatures. The volatile constituents were extracted with dichloromethane and the extracts were subsequently analyzed by HRGC/MS. Wines produced by DCM contained higher amounts of esters, at every temperature studied, whereas those produced by GP biocatalyst contained higher amounts of alcohols. Free cells and DCM biocatalyst gave wines with similar contents of alcohols. Wines produced by DCM biocatalyst gave better ratios of esters to alcohols and had dominating fruity aromas. GC/MS analysis proved that cell immobilization did not create serious changes in qualitative composition of the wine aroma. As regards the quantitative profile, the combined effect of temperature and immobilization resulted in the production of wines with more fruity character because of the higher ratio of esters to alcohols.

Investigation of the antioxidant behavior of air- and freeze-dried aromatic plant materials in relation to their phenolic content and vegetative cycle.

The total phenolic and flavonoid content of the aerial parts of five aromatic plants harvested at different periods was estimated, and their antioxidant capacity was evaluated. Major phenolic compounds present in their extracts were determined by RP-HPLC. The results demonstrated different amounts of total phenolic compounds and various degrees of antioxidant activity depending on the plant species, the time of harvest, and the drying method employed. Extracts from air-dried Mentha viridis L., Origanum majorana L., and Rosmarinus officinalis L. demonstrated the greatest efficacy during the flowering stage, in which the identified flavonoids were found in significantly higher amounts, whereas phenolic acids were found in their lowest concentration. Extracts from air-dried Laurus nobilis L. and Foeniculum vulgare Mill were less efficient in terms of antioxidant activity, with the highest values being observed during the early fruiting stage. This stage was characterized by the lowest flavonoid content and high phenolic acid content, except for L. nobilis L. extracts. Overall, the amount of identified phenolic acids did not vary considerably within the investigated year. The total phenolic concentration in all plant extracts decreased significantly when freeze-dried rather than air-dried samples were used. The HPLC analysis further supported the above for most of the phenolic compounds present in the extracts, except for hydroxybenzoic acids, which were better retained during the freeze-drying process.

The dynamics of the HS/SPME-GC/MS as a tool to assess the spoilage of minced beef stored under different packaging and temperature conditions

The aim of the current study was to assess meat spoilage through the evolution of volatile compounds using chemometrics. Microbiological and sensory assessment, pH measurement and headspace solid phase microextraction gas chromatography/mass spectroscopy (headspace SPME-GC/MS) analysis were carried out in minced beef stored aerobically and under modified atmosphere packaging (MAP) at 0, 5, 10, and 15 °C. It was shown that the HS/SPME-GC/MS analysis provided useful information about a great number of volatile metabolic compounds detected during meat storage. Many of the identified and semi-quantified compounds were correlated with the sensory scores through the use of chemometrics, depicting possible spoilage indicators such as 2-pentanone, 2-nonanone, 2-methyl-1-butanol, 3-methyl-1-butanol, ethyl hexanoate, ethyl propanoate, ethyl lactate, ethyl acetate, ethanol, 2-heptanone, 3-octanone, diacetyl, and acetoin. Finally, the applied GC/MS global models were able to estimate the microbial counts of the different microorganisms and the sensory scores of a meat sample regardless of storage conditions (i.e. packaging and temperature).

Microbiological spoilage and investigation of volatile profile during storage of sea bream fillets under various conditions.

Volatile organic compound (VOC) profile was determined during storage of sea bream (Sparus aurata) fillets under air and Modified Atmosphere Packaging (MAP - CO2/O2/N2: 60/10/30) at 0, 5 and 15°C. Microbiological, TVB-N (Total Volatile Base Nitrogen) and sensory changes were also monitored. Shelf-life of sea bream fillets stored under air was 14, 5 and 2days (d) at 0, 5 and 15°C respectively, while under MAP was 18, 8, and 2d at 0, 5 and 15°C respectively. At the end of shelf life, the total microbial population ranged from 7.5 to 8.5logcfu/g. Pseudomonas spp. were among the dominant spoilage microorganisms in all cases, however growth of Brochothrix thermosphacta and Lactic Acid Bacteria (LAB) were favoured under MAP compared to air. TVB-N production was favoured at higher temperatures and under air compared to lower temperatures and MAP. TVB-N increased substantially from the middle of storage and its value never reached concentrations higher than 30-35mgN/100g, which is the legislation limit, making it a poor chemical spoilage index (CSI). A lot of alcohols, aldehydes, ketones and ethyl esters that were detected in the present study have been reported as bacterial metabolites, others as products of chemical oxidation while others as aroma constituents. VOCs such as 3-methylbutanal, acetic acid, ethanol, ethyl esters of isovaleric and 2-methylbutyric acids, 1-penten-3-ol, 1-octen-3-ol and cis-4-heptenal appeared from the early or middle stages and increased until the end of storage. From those only 3-methylbutanal, acetic acid, ethanol and the ethyl esters have been reported as microbial origin, making them potential CSI candidates of sea bream fillets.

Performance of two potential probiotic Lactobacillus strains from the olive microbiota as starters in the fermentation of heat shocked green olives

The performance of two potential probiotic Lactobacillus strains from olive microbiota, namely L. pentosus B281 and L. plantarum B282 was assessed as starter cultures in Spanish-style fermentation of heat shocked green olives cv. Halkidiki. Two different initial salt levels were studied, 10% (w/v) and 8% (w/v) NaCl, and the brines were inoculated with (a) L. pentosus B281, (b) L. plantarum B282, and (c) a mixture of both strains. A spontaneous fermentation was also taken into account as control treatment. Prior to brining, olives were heat shocked at 80 °C for 10 min to reduce the level of the indigenous microbiota on olive drupes and facilitate the dominance of the inoculated cultures. Microbiological, physicochemical and sensory analyses were conducted throughout fermentation. The composition of LAB population and the evolution of added inocula were assessed by Pulsed Field Gel Electrophoresis (PFGE). The final population of LAB was maintained above 6 log cycles in olive flesh. Both L. pentosus B281 and L. plantarum B282 were able to dominate over indigenous LAB, albeit strain B281 exhibited higher recovery percentages (100 or 94.7% for B281 and 58.8% or 55.0% for B282 in 10% or 8% NaCl, respectively). L. pentosus B281 also dominated over L. plantarum B282, when the two strains were co-inoculated in olive fermentations. The sensory assessment showed higher preference for inoculated fermentations of L. pentosus and L. plantarum separately in 8% NaCl, followed by the L. plantarum in 10% NaCl. The present study showed that probiotic strains L. pentosus B281 and L. plantarum B282, may offer a great potential for use as functional starter cultures in olive fermentation and deliver a promising probiotic food to the consumer.

Conversion of biodiesel‐derived glycerol into biotechnological products of industrial significance by yeast and fungal strains

Oleochemical activities (e.g. biodiesel production, fat saponification) generate annually very high amounts of concentrated glycerol‐containing waters (called crude glycerol) as the principal residues of these processes. Crude glycerol is an industrial residue the valorization of which attracts remarkable and constantly increasing interest. In the current investigation, biodiesel‐derived glycerol was employed as substrate for yeast and fungal strains cultivated under nitrogen‐limited conditions in shake flasks. Glucose was employed as reference substrate. Several yeasts (Candida diddensiae, Candida tropicalis, Pichia ciferrii, Williopsis saturnus, Candida boidinii, and Candida oleophila) rapidly assimilated glucose and converted it into ethanol, despite aerobic conditions imposed, and were Crabtree‐positive. None of these yeasts produced ethanol during growth on glycerol or accumulated significant quantities of lipid during growth on glucose or glycerol. Only Rhodosporidium toruloides produced notable lipid quantities from glucose and to lesser extent from glycerol. Yarrowia lipolytica LFMB 20 produced citrate ≈58 g/L growing on high‐glucose media, while on high‐glycerol media ≈42 g/L citrate and ≈18 g/L mannitol. During growth on glucose/glycerol blends, glycerol was assimilated first and thereafter glucose was consumed. Fungi produced higher lipid quantities compared with yeasts. High lipid quantities were produced by Mortierella ramanniana, Mucor sp., and mainly Mortierella isabellina, with glycerol being more adequate for M. ramanniana and glucose for Mucor sp. and M. isabellina. M. isabellina ATHUM 2935 produced lipids of 8.5 g/L, 83.3% w/w in dry cell weight (DCW) and conversion yield per unit of glucose consumed ≈0.25 g/g. The respective values on glycerol were 5.4 g/L, 66.6% w/w in DCW and ≈0.22 g/g. Lipids of all microorganisms were analyzed with regards to their fatty acid composition, and M. isabellina presented the closest similitude with rapeseed oil. Crude lipids produced by this fungus and extracted with chloroform/methanol blend, were composed mostly of triacylglycerols, thus indicating that these solvents are adequate for triacylglycerol extraction.

Production of added-value metabolites by Yarrowia lipolytica growing in olive mill wastewater-based media under aseptic and non-aseptic conditions

Yarrowia lipolytica ACA‐YC 5033 was grown on glucose‐based media in which high amounts of olive mill wastewaters (OMWs) had been added. Besides shake‐flask aseptic cultures, trials were also performed in previously pasteurized media while batch bioreactor experiments were also done. Significant decolorization (∼58%) and remarkable removal of phenolic compounds (∼51% w/w) occurred, with the latter being amongst the highest ones reported in the international literature, as far as yeasts were concerned during their growth on phenol‐containing media. In nitrogen‐limited flask fermentations the microorganism produced maximum citric acid quantity ≈19.0 g/L [simultaneous yield of citric acid produced per unit of glucose consumed (YCit/Glc)≈0.74 g/g]. Dry cell weight (DCW) values decreased at high phenol‐containing media, but, on the other hand, the addition of OMWs induced reserve lipid accumulation. Maximum citric acid concentration achieved (≈52.0 g/L; YCit/Glc≈0.64 g/g) occurred in OMW‐based high sugar content media (initial glucose added at ≈80.0 g/L). The bioprocess was successfully simulated by a modified logistic growth equation. A satisfactory fitting on the experimental data occurred while the optimized parameter values were found to be similar to those experimentally measured. Finally, a non‐aseptic (previously pasteurized) trial was performed and its comparison with the equivalent aseptic experiment revealed no significant differences. Yarrowia lipolytica hence can be considered as a satisfactory candidate for simultaneous OMWs bioremediation and the production of added‐value compounds useful for the food industry.

Lipid production and characterization by Mortierella (Umbelopsis) isabellina cultivated on lignocellulosic sugars

To study and characterize the lipids produced by Mortierella (Umbelopsis) isabellina, during its growth on mixtures of glucose and xylose.

Effect of osmotic dehydration of olives as pre-fermentation treatment and partial substitution of sodium chloride by monosodium glutamate in the fermentation profile of Kalamata natural black olives

This study examined the effect of osmotic dehydration of Kalamata natural black olives as pre-fermentation treatment in combination with partial substitution of NaCl by monosodium glutamate (MSG) on the fermentation profile of olives. Osmotic dehydration was undertaken by immersing the olives in 70% (w/w) glucose syrup overnight at room temperature. Further on, three different mixtures of NaCl and MSG with/without prior osmotic dehydration of olives were investigated, namely (i) 6.65% NaCl - 0.35% MSG (5% substitution), (ii) 6.30% NaCl - 0.70% MSG (10% substitution), (iii) 5.95% NaCl - 1.05% MSG (15% substitution), and (iv) 7% NaCl without osmotic dehydration (control treatment). Changes in the microbial association (lactic acid bacteria [LAB], yeasts, Enterobacteriaceae), pH, titratable acidity, organic acids, sugars, and volatile compounds in the brine were analyzed for a period of 4 months. The final product was subjected to sensory analysis and the content of MSG in olives was determined. Results demonstrated that osmotic dehydration of olives prior to brining led to vigorous lactic acid processes as indicated by the obtained values of pH (3.7-4.1) and acidity (0.7-0.8%) regardless of the amount of MSG used. However, in non-osmotically dehydrated olives, the highest substitution level of MSG resulted in a final pH (4.5) that was beyond specification for this type of olives. MSG was degraded in the brines being almost completely converted to γ-aminobutyric acid (GABA) at the end of fermentation. Finally, the sensory assessment of fermented olives with/without osmotic dehydration and at all levels of MSG did not show any deviation compared to the control treatment.