Lipolytic activity of the yeast species associated with the fermentation/storage phase of ripe olive processing

Ripe olives account for ca. 30% of the world's table olive production. Fruits intended for this type of product are preserved in an aqueous solution (acidic water or brine) for several months, where they may undergo a spontaneous fermentation. Enterobacteriaceae and lactic acid bacteria were not detected in the present survey during storage. Thus, the work focused on studying the yeast microflora associated with the ripe olive storage of Manzanilla and Hojiblanca cultivars in acidified brines. A total of 90 yeast isolates were identified by means of molecular methods using RFLP analysis of the 5.8S-ITS rDNA region and sequencing of the D1/D2 domains of the 26S rDNA gene. The two most important species identified in both cultivars were Saccharomyces cerevisiae and Pichia galeiformis, which were present throughout the storage period, while Candida boidinii was detected during the later stages of the process. The species Pichia membranifaciens was detected only in the early stages of the Hojiblanca cultivar. The lipase assays performed with both extracellular and whole cell fractions of the yeast isolates showed that neither of the S. cerevisiae and P. galeiformis species had lipase activity, while the P. membranifaciens isolates showed a weak activity. On the contrary, all C. boidinii isolates gave a strong lipase activity. Change in olive fat acidity was markedly higher in the presence of the yeast population than in sterile storage, indicating that lipases produced by these microorganisms modify the characteristics of the fat in the fruit.     

Exploring the yeast biodiversity of green table olive industrial fermentations for technological applications

In recent years, there has been an increasing interest in identifying and characterizing the yeast populations associated with diverse types of table olive elaborations because of the many desirable technological properties of these microorganisms. In this work, a total of 199 yeast isolates were directly obtained from industrial green table olive fermentations and genetically identified by means of a RFLP analysis of the 5.8S-ITS region and sequencing of the D1/D2 domains of the 26S rDNA gene. Candida diddensiae, Saccharomyces cerevisiae and Pichia membranifaciens were the most abundant yeast species isolated from directly brined Aloreña olives, while for Gordal and Manzanilla cultivars they were Candida tropicalis, Pichia galeiformis and Wickerhamomyces anomalus. In the case of Gordal and Manzanilla green olives processed according to the Spanish style, the predominant yeasts were Debaryomyces etchellsii, C. tropicalis, P. galeiformis and Kluyveromyces lactis. Biochemical activities of technological interest were then qualitatively determined for isolates belonging to all yeast species. This preliminary screening identified two isolates of W. anomalus with interesting properties, such as a strong β-glucosidase and esterase activity, and a moderate catalase and lipolytic activity, which were also confirmed by quantitative assays. The results obtained in this survey show the potential use that some yeast species could have as starters, alone or in combination with lactic acid bacteria, during olive processing.     

Detection and identification of wild yeasts in Champús, a fermented Colombian maize beverage

The aim of this study was to identify and characterise the predominant yeasts in Champús, a traditional Colombian cereal-based beverage with a low alcoholic content.Samples of Champús from 20 production sites in the Cauca Valley region were analysed. A total of 235 yeast isolates were identified by conventional microbiological analyses and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of ITS1-5.8S rDNA-ITS2. The dominant species were: Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia fermentans, Pichia kluyveri var. kluyveri, Zygosaccharomyces fermentati, Torulospora delbruekii, Galactomyces geotrichum and Hanseniaspora spp. Model Champús systems were inoculated with single strains of some isolated sporogenus species and the aromatic profiles were analysed by SPME. Analysis of data showed that Champús strains produced high amounts of esters. The aromatic compounds produced by Saccharomyces and non-Saccharomyces yeasts from Champús can exert a relevant influence on the sensory characteristics of the fermented beverage. The Champús strains could thus represent an important source for new yeast biotypes with potential industrial applications.     

Effects of olive pomace and olive pomace oil on growth performance,fatty acid composition and cardio protective properties of gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax)

Gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) were fed with two experimental diets: olive pomace diet and olive pomace oil diet, in order to examine whether fish oil substitution in fish feeds by olive oil production wastes, such as olive pomace and olive pomace oil, has an influence on growth performance, fatty acid composition and cardio protective properties of fish. Gilthead sea bream fed with both experimental diets exhibited satisfactory growth performance factors while sea bass did not. The total lipids of gilthead sea bream fed with olive pomace diet contained statistically decreased levels of fatty acids, while exhibited the most potent biological activity against platelet aggregation induced by Platelet Activating Factor. These data indicate that olive pomace can be used as a partial substitute of fish oil in fish feed improving its cardio protective properties.     

Lactobacillus pentosus dominates spontaneous fermentation of Italian table olives

Culture-dependent and -independent approaches were applied to identify the bacterial species involved in Italian table olive fermentation. Bacterial identification showed that Lactobacillus pentosus was the dominant species although the presence of Lactobacillus plantarum, Lactobacillus casei, Enterococcus durans, Lactobacillus fermentum and Lactobacillus helveticus was observed. Rep-PCR allowed to obtain strain-specific profiles and to establish a correlation with table olive environment. PCR-DGGE (Denaturing Gradient Gel Electrophoresis) confirmed the heterogeneity of bacterial community structure in fermented table olives as well as the prevalence of L. pentosus. The strains were characterized on the basis of technological properties (NaCl tolerance, β-glucosidase activity and the ability to grow in synthetic brine and in presence of 1 g/100 mL oleuropein). L. pentosus showed a high capacity of adaptation to the different conditions characterizing the olive ecosystem. This species showed the highest percentage of strains able to grow in presence of 10 g/100 mL NaCl, oleuropein and in the synthetic brine. Moreover, all the strains belonging to L. pentosus and L. plantarum species showed a β-glucosidase activity. This study allowed both to identify the main species and strains associated to Italian table olives and to obtain a lactic acid bacteria collection to apply as starter culture in the process of olive fermentation.     

Gas chromatography screening of bioactive phytosterols from mono-cultivar olive oils

Phytosterols (PS) from nine samples of olive oil from Olea europaea L., the Carolea, Cassanese and Coratina mono-cultivars, have been analyzed by gas chromatography. Coratina virgin olive oil (VOO) from the month of November showed highest contents of β-sitosterol (5491 mg kg⁻¹) and Δ⁵ avenasterol (1767 mg kg⁻¹). Olive pomace oil had the lowest total content of all PS, when compared to VOO. These results suggests that, PS can be an important regulatory factor for the functional quality of olive oil along the agro-industry chain from the orchard to nutraceutical.     

Yeast species involved in artisanal cachaça fermentation in three stills with different technological levels in Pernambuco,Brazil

Both the taste and aroma of cachaça, an alcoholic beverage produced by yeast fermentation of sugar cane, are influenced by yeast metabolites and volatiles. The knowledge of yeast population dynamics during the fermentation process will help to establish the basis for quality control of alcoholic beverage. In the present work, the population dynamics of three fermentation processes, with differing levels of technological sophistication, were studied. Saccharomyces cerevisiae was found to be the dominant species, but Candida milleri (Candida humilis), Pichia caribbica, Pichia guilliermondii and Zygosaccharomyces fermentati (Lachancea fermentati) were also significantly involved. In addition, four new yeast species that are not represented in NCBI/EMBL nucleotide database were found. These yeasts were classified as Candida sp., Candida drosophilae-like, Candida ubatubensis-like and Zygosaccharomyces sp. In fermentation trails at laboratory scale, all species were found to contribute to the production of volatiles. Thus it is probable that product quality is strongly dependent on population dynamics during the fermentation process.     

Partitioning of predominant lipophilic bioactives (squalene, α-tocopherol and β-sitosterol) during olive oil processing

The partitioning of predominant lipophilic olive bioactives (squalene, β-sitosterol and α-tocopherol) in olive oil processing was studied for the first time using common integrated olive milling plant. 7% of the oil and 5% of the squalene present in the olives was lost in the last pomace. β-sitosterol and α-tocopherol, which are mainly concentrated in the seed of the olive fruit, was recovered in virgin olive oil to a lesser extent (66%, 67%) while 10% were lost in last pomace. Thus, the loss of β-sitosterol and α-tocopherol was higher than that of squalene and oil, due to nonrecovered bound forms. 19% of the α-tocopherol and 21% of the β-sitosterol loss was unaccounted for, which can be attributed to degradation of α-tocopherol and incomplete recovery of sterols from the olive and pomace matrices. This study provides a basis for process development studies.     

Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias

This paper analyses yeast diversity and dynamics during the production of Asturian cider. Yeasts were isolated from apple juice and at different stages of fermentation in a cellar in Villaviciosa during two Asturian cider-apple harvests. The species identified by ITS-RFLP corresponded to Hanseniaspora valbyensis, Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia guilliermondii, Candida parapsilosis, Saccharomyces cerevisiae and Saccharomyces bayanus/Saccharomyces pastorianus/Saccharomyces kudriavzevii/Saccharomyces mikatae. The species C. parapsilosis is reported here for the first time in cider. The analysis of Saccharomyces mtDNA patterns showed great diversity, sequential substitution and the presence of a small number of yeast patterns (up to 8), present in both harvests. Killer (patterns nos. 22′ and 47), sensitive (patterns nos. 12, 15, 33 and 61) and neutral phenotypes were found among the S. cerevisiae isolates. The detection of β-glucosidase activity, with arbutin as the sole carbon source, allowed two S. cerevisiae strains (patterns nos. 3′ and 19′) to be differentiated by means of this enzymatic activity. Yeast strains producing the killer toxin or with β-glucosidase activity are reported for the first time in autochthonous cider yeasts.     

Stored product pests as models for trapping entomopathogenic fungi from olive tree orchards in Western Greece

We used the following new range of insect species as baits for trapping entomopathogenic fungi: Plodia interpunctella, Rhyzopertha dominica, Tribolium confusum, Trogoderma granarium, Ephestia kuehniella and Tenebrio molitor, which constitute important stored product pests. Soil samples were collected from random points in olive tree orchards in Western Greece using Geographical Information Systems (GIS) and sample point coordinates were located with the Global Positioning System (GPS). Entomopathogenic fungi were identified by sequencing the ITS4 and ITS5 regions of the genomic DNA. Among the isolated fungal species, Beauveria bassiana and Metarhizium anisopliae showed the highest frequency, at 33.8% and 25.4% respectively. They also caused the most infestations of R. dominica and T. confusum whose mortality reached 33–97% and 87–100% respectively. We have also isolated various other fungal taxa such as Aspergillus alliaceus, Aspergillus insuetus, Aspergillus sp., Apophysomyces ossiformis, Chaetomium acropullum, Chaetomium sp., Chaetomium globosum, Chaetomium truncatulum, Trichoderma gamsii and Purpureocillium lilacinum. Our study confirms that T. confusum and R. dominica may be further utilized as baits for entomopathogenic fungi.     

Fungal enzymes as a powerful tool to release antioxidants from olive mill wastewater

Enzymatic hydrolysis of olive mill wastewater compounds (OMW) using culture broth of Aspergillus niger, Trichoderma atroviride and Trametes trogii was tested in order to release free simple phenolic compounds having high antioxidant activity. HPLC analysis of treated and untreated OMW indicated the release of large amounts of free hydroxytyrosol (HT). Enzymatic pre-treatment of OMW by A. niger and T. atroviride broth cultures which are rich in β-glucosidase increased the amount of HT respectively from 0.05 up to 1.1 and 0.50 g/L. However, the use of T. trogii broth culture showed an oxidation of phenolic compounds due to its high laccase activity. The ethyl acetate extract of OMW treated by A. niger broth culture with IC₅₀ of 45 μg/mL showed the strongest antioxidant potency compared to untreated OMW with IC₅₀ of 501 μg/mL.     

Expert system based on computer vision to estimate the content of impurities in olive oil samples

The determination of the content of impurities is a very frequent analysis performed on virgin olive oil samples, but the official method is quite work-intensive, and it would be convenient to have an alternative approximate method to evaluate the performance of the impurity removal process. In this work we develop a system based on computer vision and pattern recognition to classify the content of impurities of the olive oil samples in three sets, indicative of the goodness of the separation process of olive oil after its extraction from the paste. Starting from the histograms of the channels of the Red–Green–Blue (RGB), CIELAB and Hue-Saturation-Value (HSV) color spaces, we construct an initial input parameter vector and perform a feature extraction previous to the classification. Several linear and non-linear feature extraction techniques were evaluated, and the classifiers used were Support Vector Machines (SVMs) and Artificial Neural Networks (ANNs). The best classification rate achieved was 87.66%, obtained using Kernel Principal Components Analysis (KPCA) and a grade-3-polynomial kernel SVM. The best result using ANNs was 82.38%, yielded by the use of Principal Component Analysis (PCA) with the Perceptron.     

Molecular analysis of yeasts from Indonesian cassava and glutinous rice tapé

Tapé is a popular delicacy in Indonesia that is prepared by fermenting starch-rich material. Yeast plays an important role in the making of tapé especially in the aroma and taste of the product. In this study, yeasts were isolated from tapé and subjected to diversity analysis using the restriction fragment length polymorphism (RFLP) technique on 5.8S rRNA encoding gene and the internal transcribed spacer (ITS). A total of 21 yeast isolates were obtained from 3 different types of Indonesian tapé (cassava tapé, white-, and black-glutinous rice tapé). The identified yeast species from cassava tapé were Pichia jadinii, Candida glabrata, and Clavispora lusitaniae. P. fabianii, Rhodotorula mucilaginosa, and Ogataea polymorpha were obtained from white glutinous rice tapé, while Saccharomycopsis fibuligera and Wickerhamomyces anomalus were obtained from black glutinous rice tapé.     

Conventional and enzyme-assisted autolysis during ageing over lees in red wines: Influence on the release of polysaccharides from yeast cell walls and on wine monomeric anthocyanin content

HPLC with refractive index detection (HPLC/RI) was used to study the autolytic release of polysaccharides from the cell walls of six strains of Saccharomyces cerevisiae and the commercial yeast species Sacch. uvarum in a model medium over a nine month period of ageing over lees. The effect of adding β-glucanase was also studied. In the presence of this enzyme autolysis was complete within 2–3 weeks; conventional autolysis needed to proceed for at least five months before large quantities of released polysaccharides could be detected. The final polysaccharide profile of the model medium obtained by enzyme-assisted autolysis was, however, different to that obtained by conventional autolysis, with more fragments of smaller molecular weight and a greater grouping of molecular sizes in the HPLC/RI peaks. The influence on the monomeric anthocyanin content of commercial red wines was examined by HPLC with photodiode array detection (HPLC/PDAD). Ageing over lees without enzymes appeared to have a protective effect on the total monomeric anthocyanin content, while the use of β-glucanase led to a large reduction in their concentration, probably via the undesirable activity of β-glucosidase impurities.     

Estimation of olive oil acidity using FT-IR and partial least squares regression

Olive oil characteristics are directly related to olive quality. Information about olive quality is of paramount importance to olive and olive oil producers, in order to establish its price. Real-time characterization of the olives avoids mixtures of high quality with low quality fruits, and allows improvement of olive oil quality. This work describes an indirect determination of olive acidity and that allows a rapid evaluation of olive oil quality. The applied method combines chemical analysis (30 min Soxhlet olive pomace extraction) in tandem with a spectroscopic technique (FT-IR) and multivariate regression (PLS1). The most suitable calibration model found used SNV pre-processing and was built with 4 Latent Variables giving a RMSECV of 8.7% and a Q² of 0.97. This accurate calibration model allows the estimation of olive acidity using a FT-IR spectrum of the corresponding Soxhlet oil dry extract and therefore is a suitable method for indirect determination of FFA in olives.     

Transfer of phenolic compounds during olive oil extraction in relation to ripening stage of the fruit

The transfer of phenolic compounds of Olea europaea L. cv. Arbequina variety during olive oil extraction in relation to ripening stage was investigated. The parameters of oil extraction by the Abencor system are shown together with mass balances of the products and by products from the olive oil extraction in relation to olive paste. The phenolic compounds in olive paste, pomace, oil and wastewater were identified and measured by HPLC. Throughout the study, the concentrations of simple phenols, secoiridoids and flavonoids were higher in the olive paste and pomace phases than in oil and wastewater phases. High concentrations of 4‐(acetoxyethyl)‐1,2‐dihydroxybenzene (3,4‐DHPEA‐AC) and secoiridoid derivatives such as the dialdehydic form of elenolic acid linked to 3,4‐DHPEA (hydroxytyrosol) or p‐HPEA (tyrosol) (3,4‐DHPEA–EDA, p‐HPEA–EDA, where EDA is elenolic acid dialdehyde) and an isomer of oleuropein aglycone (3,4‐DHPEA–EA, where EA is elenolic acid aldehyde) were found in olive oil, together with lignan compounds. It was observed that 3,4‐DHPEA–EDA was the most abundant polyphenol present in the wastewater phase. This indicates that biotransformation occurred during olive extraction, especially in the crushing and malaxation operations, and reflects the possible chemical changes that lead to the formation of new compounds. Moreover, the distribution of compounds showed their affinities toward different phases. Copyright © 2005 Society of Chemical Industry     

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.     

Using a polynomial model for fungi from table olives

Table olives are one of the most important fermented vegetables in the food industry because of their worldwide economic importance. Enterobacteriaceae, lactic acid bacteria and yeasts are among the most relevant microorganisms involved in olive fermentation determining safety, quality and flavour of the final product. Fungi can play a double role during table olive processing acting as spoilage or desirable microorganisms. This article addresses the effects of some factors (pH, NaCl, temperature, phenols) on the growth/survival of yeasts and moulds, both some wild isolates from olive brine and some yeasts from a public collection (Pichia guilliermondii, P. holstii, Wickerhamomyces anomalus). The research was divided into two steps: a 1st step was aimed at assessing fungal growth by evaluating separately each factor; in a 2nd step, NaCl, pH and temperature were combined through a simple DoE (design of experiment). For this last step, W. anomalus and P. guilliermondii were used as test fungi. Generally, fungi were not inhibited either by temperature, salt or acidic/alkaline pHs. Moreover, the approach of DoE pinpointed that the effect of some factors could experience a shift and change over the time.     

Yeast diversity during tapping and fermentation of palm wine from Cameroon

In the present study, we have investigated the occurrence of yeast flora during tapping and fermentation of palm wine from Cameroon. The yeast diversity was investigated using both traditional culture-dependent and culture-independent methods. Moreover, to characterize the isolates of the predominant yeast species (Saccharomyces cerevisiae) at the strain level, primers specific for δ sequences and minisatellites of genes encoding the cell wall were used. The results confirm the broad quantitative presence of yeast, lactic acid bacteria and acetic acid bacteria during the palm wine tapping process, and highlight a reduced diversity of yeast species using both dependent and independent methods. Together with the predominant species S. cerevisiae, during the tapping of the palm wine the other species found were Saccharomycodes ludwigii and Zygosaccharomyces bailii. In addition, denaturing gradient gel electrophoresis (DGGE) analysis detected Hanseniaspora uvarum, Candida parapsilopsis, Candida fermentati and Pichia fermentans. In contrast to the progressive simplification of yeast diversity at the species level, the molecular characterization of the S. cerevisiae isolates at the strain level showed a wide intraspecies biodiversity during the different steps of the tapping process. Indeed, 15 different biotypes were detected using a combination of three primer pairs, which were well distributed in all of the samples collected during the tapping process, indicating that a multistarter fermentation takes place in this particular natural, semi-continuous fermentation process.