In vitro potential antioxidant activity of indigenous yeasts isolated from virgin olive oil.

AIM: The aim of the present study was to investigate the in vitro antioxidant activity of yeast strains isolated from virgin olive oil. METHODS AND RESULTS: The 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free-radical scavenging activity of single cultures of 24 yeast strains belonging to eight species isolated from virgin olive oil was evaluated and compared with that of the reference yeast Saccharomyces boulardii. All the yeasts studied in vitro showed antioxidant activity similar to or superior to that of the reference yeast. The highest antioxidant activity was observed in Nakazawaea wickerhamii, which exceeded the value reached by the reference strain S. boulardii, while the significantly lowest values were observed in the Candida adriatica, Candida diddensiae and Barnettozyma californica strains. Tests performed with virgin olive oil enriched with Wickerhamomyces anomalus and S. boulardii yeast biomasses showed a positive correlation between the microbial biomass used and the percentage of antioxidant activity observed during 60 days of storage. Survival in virgin olive oil was also significantly higher for W. anomalus compared to S. boulardii. CONCLUSIONS: All the oil-borne yeasts studied showed DPPH free-radical scavenging activity in both aqueous and oily media. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the antioxidant activity of the microbiota of virgin olive oil is reported. This activity may indicate the probiotic characteristics of the microbiota.

Use of selected yeast starter cultures in industrial-scale processing of brined Taggiasca black table olives.

The aim of this study was to evaluate the effects of six selected yeast starters on natural Taggiasca black table fermentations, in different brine solutions. The olives were subjected to fermentation in 8% (w/v) NaCl, 12% (w/v) NaCl and 12% (w/v) NaCl brine solutions with 0.3% (w/v) citric acid and inoculated with selected yeast starter strains belonging to the following species: Candida adriatica 1985, Candida diddensiae 2011, Cyteromyces matritensis 2005, Nakazawaea molendini-olei 2004, Saccharomyces cerevisiae 2046 and Wickerhamomyces anomalus 1960. Samples of brines and olives were analysed in the initial phase, then again after 30 and 120 days of fermentation. The yeast starters survived differently during the first 30 days of brine fermentation, depending on the NaCl concentration. After 120 days of fermentation N. molendini-olei 2004 and C. matritensis 2005 failed in the brines with 12% NaCl, while the yeast starter cultures C. diddensiae 2011, C. adriatica 1985 and W. anomalus 1960 showed the best performances in terms of survival and competitiveness towards wild yeasts of the brines. The physicochemical and sensorial analysis suggest a potential positive role of these yeasts during the debittering process of the Taggiasca table olives. Considering the combination between yeast starters and the fermentation conditions, the best indication occurred with the brines containing 12% NaCl acidified with citric acid.

Evaluation of physiological properties of yeast strains isolated from olive oil and their in vitro probiotic trait.

Virgin olive oil contains a biotic fraction represented by rich microbiota, including yeasts. The aim of this study was to investigate some physiological properties and the in vitro probiotic potential of yeast strains previously isolated from Italian virgin olive oil. Eleven yeast strains belonging to the species Candida adriatica, Candida diddensiae, Nakazawaea molendini-olei, Nakazawaea wickerhamii, Wickerhamomyces anomalus, and Yamadazyma terventina were used in this study and compared with the reference yeast Saccharomyces boulardii. Present research has demonstrated that unlike Saccharomyces boulardii which produce only satured and monounsatured fatty acids (MUFAs), the olive oil-borne yeast strains also synthesize polyunsatured fatty acids (PUFAs) in quantities greater than those found in olive oil, which provide health benefits. The survival in gastric and pancreatic juices, which is important for probiotic yeasts because it allows them to cross the human intestinal tract, has reached a maximum of 100% when yeast cells were coated with olive oil. Cholesterol was removed by 50% of the studied yeast strains, and among them, the best results were reached by the strains 2032 and 2033 of W. anomalus which appear the best probiotic candidate in terms of the in vitro probiotic trait evaluated. Further experiments are underway to confirm this findings.

Taggiasca extra virgin olive oil colonization by yeasts during the extraction process.

The opalescent appearance of the newly produced olive oil is due to the presence of solid particles and microdrops of vegetation water in which the microorganisms from the olives' carposphere are trapped. Present research has demonstrated that the microbiota of the fresh extracted olive oil, produced in the mills, is mainly composed of yeasts and to a lesser extent of molds. The close link between the composition of the microbiota of the olives' carposphere undergoing to processing, and that of the microbiota of the newly produced olive oil, concerns only the yeasts and molds, given that the bacterial component is by and large destroyed mainly in the kneaded paste during the malaxation process. Six physiologically homogenous yeast groups were highlighted in the wash water, kneaded paste and newly produced olive oil from the Taggiasca variety which had been collected in mills located in the Liguria region. The more predominant yeasts of each group belonged to a single species called respectively: Kluyveromyces marxianus, Candida oleophila, Candida diddensiae, Candida norvegica, Wickerhamomyces anomalus and Debaryomyces hansenii. Apart from K. marxianus, which was found only in the wash water, all the other species were found in the wash water and in the kneaded paste as well as in the newly produced olive oil, while in the six-month stored olive oil, was found only one physiologically homogeneous group of yeast represented by the W. anomalus specie. These findings in according to our previous studies carried out on other types of mono varietal olive oils, confirms that the habitat of the Taggiascas' extra virgin olive oil, had a strong selective pressure on the yeast biota, allowing only to a few member of yeast species, contaminating the fresh product, to survive and reproduce in it during storage.

Effect of lipolytic activity of Candida adriatica, Candida diddensiae and Yamadazyma terventina on the acidity of extra-virgin olive oil with a different polyphenol and water content.

Previous microbiological research demonstrated the presence of a rich micro-flora composed mainly of yeasts in the suspended fraction of freshly produced olive oil. Some of the yeasts are considered harmful as they can damage the quality of the olive oil through the hydrolysis of the triacylglycerols. Present research has demonstrated that the lipolytic activity of some lipase-producer strains belonging to a yeast species called Candida adriatica, Candida diddensiae and Yamadazyma terventina can be modulated by the water and the polyphenol content of olive oil. Laboratory tests highlighted a substantial increase in free fatty acid in the inoculated olive oil characterized by high water content and low polyphenol concentration. The acidity of the olive oil samples containing 0.06% and 0.31% of water increased significantly by 33% in the lipase-producer yeast strains tested during a period of 2 weeks of incubation at 30 °C. All other yeasts showed strong lipolytic activity in the presence of 1.31% of water - the only exception to this was the C. adriatica 1985 strain. The phenolic compounds typical of olive oil represent another important factor able to condition the viability and the lipolytic activity of the lipase-producer yeasts. From the tests performed on the olive oil characterized by an increasing content of total polyphenols equal to 84, 150 and 510 mg per kg of oil, the percentage of the lipase-producer yeasts able to hydrolyse the triacylglycerols was respectively 100%, 67% and 11%.

Effects of some oil-born yeasts on the sensory characteristics of Italian virgin olive oil during its storage.

The olive oil microbiota, mainly composed of yeasts, is associated with the suspended fraction of freshly produced olive oils. Some olive oil yeasts are considered useful as they are able to hydrolyse the bitter tasting secoiridoid compound of the oil, whereas others are considered harmful as they can damage the quality of the oil. Present research demonstrated the influence of some yeast strains belonging to Candida adriatica, Candida diddensiae and Candida wickerhamii species on the olive oil sensory characteristics during its storage. All the tested yeasts survived in the inoculated extra virgin olive oil and, after four months of storage, the suspended yeast cells recovered from the olive oil varied between 50% and 80% of the initial total yeasts, according to their sedimentation capacity. The mean of five analytical indices (free fatty acids, peroxide value, K232, K270 and ΔK) were quite similar and about 60% of the treated samples analysed after four months of storage, on the basis of these indices, were still classed as extra virgin. Completely different results were obtained from the analyses of volatile and non volatile carbonyl compounds according to the yeast used. In the samples of oil treated with C. adriatica and C. wickerhamii, instead of some strains of C. diddensiae, a lower concentration of C6 volatile carbonyl compounds and polyphenols, responsible for positive oil attributes, were found. The sensory attributes of the treated olive oils varied according to the composition of the volatile and non volatile carbonyl compounds produced with the treatments. "Muddy-sediment", "rancid" or both defects were found in olive oil samples treated with C. adriatica DAPES 1933, C. wickerhamii DAPES 1885 and C. diddensiae DAPES 1912 and 1913 strains, whereas olive oil samples treated with C. diddensiae DAPES 1918 and 1922 after four months of storage were defect-free, and still categorized as extra virgin, according to the requirements of both chemical and sensory quality indices of European Community Regulations.

Distribution of dimorphic yeast species in commercial extra virgin olive oil.

Recent microbiological research has demonstrated the presence of a rich microflora mainly composed of yeasts in the suspended fraction of freshly produced olive oil. Some of the yeasts are considered useful as they improve the organoleptic characteristics of the oil during preservation, whereas others are considered harmful as they can damage the quality of the oil through the hydrolysis of the triglycerides. However, some dimorphic species can also be found among the unwanted yeasts present in the oil, considered to be opportunistic pathogens to man as they have often been isolated from immunocompromised hospital patients. Present research demonstrates the presence of dimorphic yeast forms in 26% of the commercial extra virgin olive oil originating from different geographical areas, where the dimorphic yeasts are represented by 3-99.5% of the total yeasts. The classified isolates belonged to the opportunistic pathogen species Candida parapsilosis and Candida guilliermondii, while among the dimorphic yeasts considered not pathogenic to man, the Candida diddensiae species was highlighted for the first time in olive oil. The majority of the studied yeast strains resulted lipase positive, and can consequently negatively influence the oil quality through the hydrolysis of the triglycerides. Furthermore, all the strains showed a high level of affinity with some organic solvents and a differing production of biofilm in "vitro" corresponded to a greater or lesser hydrophobia of their cells. Laboratory trials indicated that the dimorphic yeasts studied are sensitive towards some components of the oil among which oleic acid, linoleic acid and triolein, whereas a less inhibiting effect was observed with tricaprilin or when the total polyphenols extracted from the oil were used. The observations carried out on a scanning electron microscope (SEM), demonstrated the production of long un-branched pseudohyphae in all the tested dimorphic yeasts when cultivated on nutrient-deficient substrates.

Lipolytic yeasts distribution in commercial extra virgin olive oil.

The olive oil lipase-producing yeasts can lower the quality of the product through the hydrolysis of the triacylglycerols. In this research the olive oil total yeasts were divided into five chromogenic groups, for each group the amount of the lipase-producing yeasts was evaluated and the more active isolates were classed. The trials showed the prevalence of more than three chromogenic yeast groups in the commercial poly-varieties of extra virgin olive oil mixture, whereas in the mono-varieties one, no more than one chromogenic yeast group prevailed according to the olive variety. The ratio of the lipase-producing yeast varied from 33% in the smooth brown chromogenic group to 83% in the wrinkly bluish one. The physiological and genetic analyses of the more active lipase-producing yeast belonging to each chromogenic group allowed us to classify the wrinkly red 1892 strain and the wrinkly bluish 1890 strain as Candida parapsilosis, whereas the smooth white 1885 strain was classed as Candida wickerhamii. The ribosomal (26S) D1/D2 region sequencing results of the wrinkly white 1886 strain and the smooth brown 1887 strain are not identical to that of any known yeast species. They showed 8 substitution and 11 substitution plus 2 indels, respectively, different from Pichia mexicana and Pichia minuta. This is the first report where the human pathogen C. parapsilosis species was found in commercial extra virgin olive oil. The chromogenic group evaluation permitted the rapid preliminary identification of technological and human health important olive oil yeasts.

Lipolytic activity of Williopsis californica and Saccharomyces cerevisiae in extra virgin olive oil.

Inoculation trials performed with three strains of yeasts, isolated from extra virgin olive oil, Williopsis californica 1,639, Saccharomyces cerevisiae 1,525 and Candida boidinii 1,638, demonstrated that some yeast can lower the quality of the oil during storage. Laboratory tests highlighted a substantial increase in the total diglycerides and free fatty acids in the samples of oil inoculated with the lipase-producing strains of yeasts, W. californica 1,639 and S. cerevisiae 1,525, while in the samples of oil inoculated with the lipase-negative strain C. boidinii 1,638 no differences were found in respect to the uninoculated control. The acidity of the extra virgin olive oil, inoculated with the lipase-producing strains W. californica 1,639 and S. cerevisiae 1,525, during 2 weeks of incubation at 30 degrees C increased respectively from 0.62% to 1.50 and 1.62%, exceeding the limit of 0.8% established by current regulations for this commercial category of olive oil, while in the oil inoculated with the lipase-negative strain and in the uninoculated control, the acidity remained constant throughout. Furthermore, the two strains of lipase-producing yeasts also increased the concentration of the 1.3-diglyceride isomer in the oil lowering the values of the total 1.2-diglycerides/total 1.3-diglycerides ratio considered to be an important index of quality for an extra virgin olive oil. The lipolytic activity of lipase-producing strains W. californica 1,639 and S. cerevisiae 1,525 showed an optimum pH of 6 and 7.5 and an optimum temperature of 20 degrees C and 30 degrees C respectively. Nevertheless, the lipolytic activity was negatively influenced by glucose and polyphenols when the concentration was higher than 0.25% and 0.4% (wt/vol) respectively.

Microbiological activity in stored olive oil.

The disappearance of the bitter taste of newly produced olive oil during storage is due to the enzymatic hydrolysis of the bitter-tasting secoiridoid compound known as oleuropein. Current knowledge attributes the enzymatic hydrolysis of the oleuropein to the beta-glucosidase present in the olives. The present study, however, has demonstrated for the first time that oleuropein present in olive oil can be hydrolysed by beta-glucosidase from the yeasts Saccharomyces cerevisiae and Candida wickerhamii. The enzymatic analyses carried out directly on the untreated olive oil and on sterilized olive oil inoculated with the above-mentioned yeasts proved the beta-glucosidase activity through the hydrolysis of both the synthetic substrate p-nitrophenyl-beta-D-glucopyranoside (PNPG) and the oleuropein. The absence of lipases in the isolated S. cerevisiae and C. wickerhamii examined lead us to believe that the yeasts contribute in a positive way towards the improvement of the organological quality of the oil without altering the composition of the triglycerides.

Lipase production by yeasts from extra virgin olive oil.

Newly produced olive oil has an opalescent appearance due to the presence of solid particles and micro-drops of vegetation water from the fruits. Some of our recent microbiological research has shown that a rich micro-flora is present in the suspended fraction of the freshly produced olive oil capable of improving the quality of the oil through the hydrolysis of the oleuropein. Present research however has, for the first time, demonstrated the presence of lipase-positive yeasts in some samples of extra virgin olive oil which can lower the quality of the oil through the hydrolysis of the triglycerides. The tests performed with yeasts of our collection, previously isolated from olive oil, demonstrated that two lipase-producing yeast strains named Saccharomyces cerevisiae 1525 and Williopsis californica 1639 were able to hydrolyse different specific synthetic substrates represented by p-nitrophenyl stearate, 4-nitrophenyl palmitate, tripalmitin and triolein as well as olive oil triglycerides. The lipase activity in S. cerevisiae 1525 was confined to the whole cells, whereas in W. californica 1639 it was also detected in the extracellular fraction. The enzyme activity in both yeasts was influenced by the ratio of the aqueous to the organic phase reaching its maximum value in S. cerevisiae 1525 when the water added to the olive oil was present in a ratio of 0.25% (v/v), whereas in W. californica 1639 the optimal ratio was 1% (v/v). Furthermore, the free fatty acids of olive oil proved to be good inducers of lipase activity in both yeasts. The microbiological analysis carried out on commercial extra virgin olive oil, produced in four different geographic areas, demonstrated that the presence of lipase-producing yeast varied from zero to 56% of the total yeasts detected, according to the source of oil samples. The discovery of lipase-positive yeasts in some extra virgin olive oils leads us to believe that yeasts are able to contribute in a positive or negative way towards the organological quality of the olive oil.

Use of the Most-Probable-Number Technique To Detect Polymyxa betae (Plasmodiophoromycetes) in Soil.

The fungus Polymyxa betae is an obligate parasite of the roots of many plants of the family Chenopodiaceae. In the sugar beet, it acts as a vector of beet necrotic yellow vein virus, the agent of a serious disease known as rhizomania. With indirect methods of analysis, such as bioassay, one can establish only the presence or absence, but not the quantity, of P. betae in soil. A new method based on the technique of the most probable number (MPN) of infective units of P. betae present in the soil was developed on the basis of the biological characteristics of this microorganism. Compared with traditional bioassay methods, the MPN method is suitable for determining the contamination level of P. betae in a soil, and it appears promising for the routine analysis of many soil samples, whether they were affected by rhizomania or presumed noninfested. The instrumentation designed especially for the recovery of viable P. betae from soil with the MPN technique is made from commercially available materials, results in a saving of space during sample incubation, and permits this method to be used for any laboratory analysis.

Hydrolysis of Oleuropein by Lactobacillus plantarum Strains Associated with Olive Fermentation.

Oleuropein (Chemical Abstracts Service registry number 32619-42-4), a bitter-tasting secoiridoid glucoside commonly found in leaves of the olive tree as well as in olives (Olea europaea L.), was found to be hydrolyzed by the beta-glucosidase (EC 3.2.1.2.1) produced by oleuropeinolytic Lactobacillus plantarum-type strains. Three strains, designated B17, B20, and B21, were isolated from the brine of naturally ripe olives not treated with alkali. These strains were rod-shaped forms, grown at a pH 3.5 limit, and tolerated 1% oleuropein and 8% NaCl in the growth medium. The beta-glucosidase produced hydrolyzed 5-bromo-4-chloro-3-indolyl-beta-d-glucopy-ranoside as well as oleuropein. The presence of 2% glucose in the medium inhibited activity by 40 to 50%, depending on the bacterial strain. Chromatographic analysis of the trimethylsilyl derivatives of the products obtained after 7 days of incubation at 30 degrees C of strain B21 showed all the hydrolysis products of oleuropein, i.e., aglycone, iridoid monoterpen, and 3,4-dihydroxyphenylethanol (hydroxytyrosol). Oleuropein and its aglycone after 21 days of incubation decreased to trace levels with the simultaneous increase in concentration of beta-3,4-dihydroxyphenylethanol.