Effect of oil-born yeasts on the quality of extra-virgin olive oils of Basilicata region.

The olive oil microbiota mainly consists of yeasts, which may positively or negatively affect the physicochemical and sensory features of product. In this study, 17 yeast strains belonging to Candida boidinii, Lachancea fermentati, Nakazawaea molendinolei, N. wickerhamii and Schwanniomyces polymorphus species were collected during olive oil production, identified and tested for the ability to ferment sugars, to grow at low temperatures, for the occurrence of different enzymatic activities, for the tolerance and degradation of phenolic compounds, radical scavenging activities, biofilm formation, survival to simulated gastro-intestinal (GIT) tract. Yeasts were also inoculated in extra virgin olive oils (EVOO; from Leccino and Coratina cultivar) to evaluate their survival and their effect on EVOO quality (changes in analytical indices) during 6-months of storage. Most of strains were able to grow at 15°C, while the ability to ferment different sugars was strain-specific. All strains had ß-glucosidase activity, while none exhibited lipolytic activity; peroxidase was widespread among the strains, while protease activity was strain-dependent. Esterase and the ability to hydrolyse oleuropein and form hydroxytyrosol was present only in N. wickerhamii strains. All strains were able to survive in olive mill wastewater, used as a model of phenolic compounds-rich matrix. A potential biofilm formation was observed only in N. wickerhamii, while the ability to scavenge radical and to cope with GIT-associated stresses were strain-dependent. High levels of survival were observed for almost strains (except S. polymorphus), in both Leccino and Coratina samples. Yeasts limited the acidity rise in olive oils, but overtime they contributed to increase the parameters related to oxidative phenomena (i.e. peroxides, K232, K270), resulting in a declassification of EVOOs. The total phenolic content (TPC) was correlated to the presence of yeasts and, at the end of storage period (6 months) inoculated samples had significantly lower concentrations compared to the control oils. This study confirms that yeasts are able to survive in olive oils and, therefore, the control of their occurrence during extraction process and storage conditions is needed to obtain high-quality products and to maintain the standards of EVOO classification.

Adherence to the traditional Mediterranean diet in a population of South of Italy: factors involved and proposal of an educational field-based survey tool.

We developed and validated a tool assessing the adherence to the Mediterranean diet based on current nutritional recommendations for health promotion (MEDI-Quest). Four-hundred-and-eleven clinically healthy subjects completed both the MEDI-Quest-score and the validated MedDietScore (MDS). A good accordance between MEDI-Quest and MDS was observed (R = 0.638), as well as for the percentage of individuals having similar scores with both methods for each food group. Regarding socio-cultural-factors, higher adherence was observed in people with older-age (OR = 1.030, p < .0001), more qualifier-employment (OR = 1.136, p < .005), higher-income (OR = 1.925, p < .0001), interest in reading-food-labels (OR = 2.057, p < .0001), and having frequent physical activity (OR = 1.608, p < .05). In conclusion, our findings show that (1) the MEDI-Quest score well correlate with MDS, and (2) the adherence to the Mediterranean diet is not particularly high due to less cultural roots. It could be necessary to improve people's knowledge about food and nutrition, taking into consideration believes embedded in their family, tradition and religion.

Aerobic and respirative growth of heterofermentative lactic acid bacteria: A screening study.

Heterofermentative lactic acid bacteria (76 strains) belonging to Lactobacillus, Leuconostoc and Weissella species which are important in fermentation, spoilage or as probiotics were screened in a factorial experiment for their ability to grow, produce catalase and consume oxygen in aerobiosis or in anaerobiosis, with or without supplementation with hemin and/or menaquinone in a medium containing glucose as a carbohydrate source. Aerobiosis improved growth with a few exceptions. The effect of supplementation with heme and/or menaquinone was strain specific and clear evidence of heme-boosted respiration was found in some cases. Heme-catalase was produced by strains of L. brevis, W. minor and Leuc. mesenteroides; some strains of the latter species produced non-heme catalase. Shaken flasks experiments showed that aerobic growth resulted in increased maximum growth rate and in a limited increase in biomass. Heme supplementation during aerobic growth resulted in a further increase in growth rate and final biomass only for a few strains; this was often related to catalase, which was also responsible for increased tolerance of H2O2. In both experiments we found evidence of heme toxicity, especially in anaerobiosis and in absence of menaquinone. Dose response curves for aerobic growth in the presence of combinations of hemin and menaquinone were non-monotonic, with growth stimulation at low doses of heme (<2.5 mg/l) and toxicity at higher doses. Menaquinone at 0.25-8 mg/l increased growth stimulation and partially reduced toxicity.

Factors affecting gene expression and activity of heme- and manganese-dependent catalases in Lactobacillus casei strains.

Catalases reduce oxidative stress by degrading hydrogen peroxide to molecular oxygen and water. The presence of heme-dependent or manganese-dependent catalases was observed for a long time in lactic acid bacteria (LAB) but, to date, knowledge on the factors affecting gene expression and enzymatic functionality are limited to a very few strains. In this study, the effect of atmosphere of incubation (not aerated static growth vs aerated shaken growth) and supplementation with Fe2+, hemin, Mn2+ or their combinations on the catalase production of respiration-competent strain Lactobacillus casei N87 was evaluated using a 24 factorial design. Kinetics of growth, enzymatic activity, tolerance of oxidative stress and expression of heme- and Mn-catalase genes were assessed. A phylogenetic analysis of heme- and Mn-catalase sequences retrieved for all published LAB genomes was performed. The presence of cofactors, especially when combined, improved biomass production in L. casei N87 in both aerated and not aerated conditions. The genome of L. casei N87 harboured sequences for both catalases and hemin and Mn supplementation was crucial for gene expression and enzyme functionality. Iron and oxygen had an additive stimulatory effect. Tolerance of oxidative stress was higher in aerated cultures supplemented with hemin and/or Mn, because of high catalase activities. The presence of both enzymes was confirmed in other respirative strains of L. casei. Clustering of catalase sequences reflected in most of cases the phylogenetic distance between LAB genomes, but in other cases significant differences were found within the same genus, indicating a different evolutionary history. The occurrence of both genes is rare in LAB genomes. The exploitation of LAB with both heme- and Mn-catalases may ensure protection from oxidative stress in different conditions and may be relevant for several food (reduction of oxidative processes on food components) and health (prevention of human diseases) related applications.

Investigation of Factors Affecting Aerobic and Respiratory Growth in the Oxygen-Tolerant Strain Lactobacillus casei N87.

Aerobic and respiratory cultivations provide benefits for some lactic acid bacteria (LAB). Growth, metabolites, enzymatic activities (lactate dehydrogenase; pyruvate and NADH oxidases, NADH peroxidase; catalase), antioxidant capability and stress tolerance of Lactobacillus casei N87 were evaluated in anaerobic, aerobic and respiratory (aerobiosis with heme and menaquinone supplementation) batch cultivations with different dissolved oxygen (DO) concentrations. The expression of pox (pyruvate oxidase) and cydABCD operon (cytochrome bd oxidase complex) was quantified by quantitative Real Time polymerase chain reaction. Respiration increased biomass production compared to anaerobiosis and unsupplemented aerobiosis, and altered the central metabolism rerouting pyruvate away from lactate accumulation. All enzymatic activities, except lactate dehydrogenase, were higher in respiratory cultures, while unsupplemented aerobiosis with 60% of DO promoted H2O2 and free radical accumulation. Respiration improved the survival to oxidative and freeze-drying stresses, while significant numbers of dead, damaged and viable but not cultivable cells were found in unsupplemented aerobic cultures (60% DO). Analysis of gene expression suggested that the activation of aerobic and respiratory pathways occurred during the exponential growth phase, and that O2 and hemin induced, respectively, the transcription of pox and cydABCD genes. Respiratory cultivation might be a natural strategy to improve functional and technological properties of L. casei.

Assessment of aerobic and respiratory growth in the Lactobacillus casei group.

One hundred eighty four strains belonging to the species Lactobacillus casei, L. paracasei and L. rhamnosus were screened for their ability to grow under aerobic conditions, in media containing heme and menaquinone and/or compounds generating reactive oxygen species (ROS), in order to identify respiratory and oxygen-tolerant phenotypes. Most strains were able to cope with aerobic conditions and for many strains aerobic growth and heme or heme/menaquinone supplementation increased biomass production compared to anaerobic cultivation. Only four L. casei strains showed a catalase-like activity under anaerobic, aerobic and respiratory conditions and were able to survive in presence of H2O2 (1 mM). Almost all L. casei and L. paracasei strains tolerated menadione (0.2 mM) and most tolerated pyrogallol (50 mM), while L. rhamnosus was usually resistant only to the latter compound. This is the first study in which an extensive screening of oxygen and oxidative stress tolerance of members of the L. casei group has been carried out. Results allowed the selection of strains showing the typical traits of aerobic and respiratory metabolism (increased pH and biomass under aerobic or respiratory conditions) and unique oxidative stress response properties. Aerobic growth and respiration may confer technological and physiological advantages in the L. casei group and oxygen-tolerant phenotypes could be exploited in several food industry applications.