Grape Microbiome: Potential and Opportunities as a Source of Starter Cultures

Grape microbiome is the source of a vastly diverse pool of filamentous fungi, yeast and bacteria that will play a coordinated role for the quality of the produced wines. In recent times, the significance of this pool of microorganisms with a long list of studies of the microbial ecology of grape berries of different geographical origin, cultural practices, grape varieties and climatic conditions has been acknowledged. Similarly, the ongoing microbial evolution of must fermentations has been fully uncovered. All these ecology studies, along with detailed metabolic studies and sensorial characterisations of the produced wines, led to the suggestion of the microbial terroir. These new concepts are today leading worldwide research efforts to the production of unique wines, preserving their historical identity and verifying their quality and geographical origin. This chapter is a quick but thorough and up-to-date review of how autochthonous microbiota highlight the terroir in wines, a comparison of commercial and wild yeast strains and how this biodiversity has been explored. Moreover, technological, physiological and oenological selection criteria will be under consideration. At the end, the positive and negative aspects of wild vinifications, the technological problems of wild strains and some suggestions for the future in starter cultures will be presented

Yeast Ecology of Fermented Table Olives: A Tool for Biotechnological Applications

The table olive is considered to be a traditional fermented vegetable in the Mediterranean countries and its production and consumption is recently spreading all around the world. The presence of yeasts is very important during olive fermentation due to their double role. On one hand, yeasts maintain desirable biochemical activities (lipase, esterase, β-glucosidase, catalase, production of killer factors, etc.) with essential technological applications in this fermented vegetable. On the other hand, spoilage activity may be shown. However, recent studies have reported that yeasts coming from table olives would be a new source of potential probiotics. Indeed, many yeast species found in table olive processing, have been reported to demonstrate such properties. Thus, starter cultures technology will play significant role, not only in olive fermentation by controlling the safety and the quality of the final product, but also in consumer’s health

The Multiple and Versatile Roles of Aureobasidium pullulans in the Vitivinicultural Sector

The saprophytic yeast-like fungus Aureobasidium pullulans has been well documented for over 60 years in the microbiological literature. It is ubiquitous in distribution, being found in a variety of environments (plant surfaces, soil, water, rock surfaces and manmade surfaces), and with a worldwide distribution from cold to warm climates and wet/humid regions to arid ones. Isolates and strains of A. pullulans produce a wide range of natural products well documented in the international literature and which have been regarded as safe for biotechnological and environmental applications. Showing antagonistic activity against plant pathogens (especially post-harvest pathogens) is one of the major applications currently in agriculture of the fungus, with nutrient and space competition, production of volatile organic compounds, and production of hydrolytic enzymes and antimicrobial compounds (antibacterial and antifungal). The fungus also shows a positive role on mycotoxin biocontrol through various modes, with the most striking being that of binding and/or absorption. A. pullulans strains have been reported to produce very useful industrial enzymes, such as β-glucosidase, amylases, cellulases, lipases, proteases, xylanases and mannanases. Pullulan (poly-α-1,6-maltotriose biopolymer) is an A. pullulans trademark product with significant properties and biotechnological applications in the food, cosmetic and pharmaceutical industries. Poly (β-l-malic acid), or PMA, which is a natural biopolyester, and liamocins, a group of produced heavy oils and siderophores, are among other valuable compounds detected that are of possible biotechnological use. The fungus also shows a potential single-cell protein source capacity with high levels of nucleic acid components and essential amino acids, but this remains to be further explored. Last but not least, the fungus has shown very good biocontrol against aerial plant pathogens. All these properties are of major interest in the vitivinicultural sector and are thoroughly reviewed under this prism, concluding on the importance that A. pullulans may have if used at both vineyard and winery levels. This extensive array of properties provides excellent tools for the viticulturist/farmer as well as for the oenologist to combat problems in the field and create a high-quality wine

Microbial composition and physicochemical attributes of traditional feta cheese made in different mountainous regions

The microbiological and compositional characteristics, as well as the volatile organic compounds (VOCs) of traditional Feta cheese produced in three mountainous areas of Greece from milk obtained by animals of local herds fed exclusively with fresh pasture, were studied. The dominant microbial group was Lactic Acid Bacteria (LAB). Their identification with the SDS-PAGE showed that Lc. lactis subsp. lactis prevailed in milk and fresh cheese samples, while Lb. plantarum in mature cheeses. A great variability in the technological properties among the different strains of all areas, was recorded. The results suggested that cheeses from the three areas differ in species composition of NSLAB and their technological properties. Thus, cheeses may mature differently and develop different flavor characteristics. The genetic diversity of NSLAB isolates was assessed by RAPD-PCR and PFGE. The data indicated intraspecies genetic heterogeneity and specificity depending on the production area. In addition, traditional Feta cheese may be the source of different NSLAB genotypes in order to make cheese with specific sensorial attributes, characteristic for each area. The dynamics and the diversity of dominant microbiota in fresh and mature Feta cheese was assessed applying the PCR-DGGE method. DGGE profiles suggested variability in the microbial composition of cheeses within the production area and distinctive differences in band profile of NSLAB in cheeses between the three areas, revealing species that were not isolated during the culturing study. The compositional characteristics, amino acids and short chain organic acids, commonly found in traditional Feta cheese, showed that the area of production influenced each parameter studied. The results suggested that the cheeses may be differentiated, according to their production area, by their compositional characteristics. The VOCs profile and content of the plants, bulk forage, raw milk samples, fresh and mature cheeses, were evaluated. The PCA applied to the 23 collected plant species, could differentiate the two areas with respect to VOCs composition. The milk obtained from the three areas, was poorer in VOCs than the plant species. Common terpenes for plants and milk may be used as bio-markers for linking traditional Feta cheese to the respective grazing pasture, as PCA suggested. Aldehydes were found in higher (P<0.05) amounts for area 1 fresh cheeses, ketones for cheeses from area 2 (P<0.05) and alcohols for cheeses from area 3 (P<0.05). Terpenes were the most dominant group of VOCs, followed by hydrocarbons. In mature cheeses, acids were the first most abundant group of VOC, followed by esters and alcohols. There were substances common for all areas and others appear as discriminants for each individual area of cheese production. There were also common terpenes in mature cheeses from all areas and others characteristic for each area of production. Overall, the VOCs composition of traditional Feta cheese made in three mountainous areas of Greece could differ depending on the production area.Μελετήθηκαν τα μικροβιολογικά και φυσικοχημικά χαρακτηριστικά καθώς επίσης τα πτητικά οργανικά συστατικά (VOCs) της παραδοσιακής Φέτας που παράγεται σε τρεις ορεινές περιοχές της Ελλάδας από γάλα ζώων ντόπιων φυλών που διατρέφονται αποκλειστικά με χλωρά βοσκή. Οι κυρίαρχες ομάδες μικροοργανισμών ήταν τα γαλακτικά βακτήρια. Η ταυτοποίησή τους με SDS-PAGE έδειξε πως στο γάλα και στα φρέσκα τυριά επικρατούσε το Lc. lactis subsp. lactis, ενώ στα ώριμα το Lb. plantarum. Η μελέτη των τεχνολογικών τους ιδιοτήτων έδειξε πως τα τυριά των τριών περιοχών διαφέρουν ως προς τη σύνθεση των NSLAB και τις τεχνολογικές ιδιότητες που διαθέτουν και συνεπώς είναι πιθανό να ωριμάζουν διαφορετικά και να αναπτύσσουν διαφορετικά οργανοληπτικά χαρακτηριστικά. Η γενετική ετερογένεια των απομονώσεων εκτιμήθηκε με την εφαρμογή των RAPD-PCR και PFGE. Τα προφίλ έδειξαν υψηλή γενοτυπική ετερογένεια μεταξύ των στελεχών που ανήκαν στο ίδιο είδος, αλλά και εξειδίκευση ανάλογα με τα τυριά της περιοχής από τα οποία είχαν απομονωθεί. Υποδεικνύεται πως η παραδοσιακή Φέτα μπορεί να αποτελέσει αξιόλογη πηγή διαφορετικών γενοτύπων NSLAB που θα μπορούσαν να αξιοποιηθούν για την παραγωγή τυριών ιδιαίτερων σε κάθε περιοχή. Η δυναμική και η ποικιλομορφία των βακτηριακών κοινοτήτων φρέσκων και ώριμων τυριών, μελετήθηκε με την εφαρμογή της PCR-DGGE. Τα βακτηριακά προφίλ έδειξαν μια μεταβλητότητα στη μικροβιακή σύνθεση των τυριών εντός της περιοχής παραγωγής, καθώς και διακριτές διαφορές στα προφίλ των NSLAB στα τυριά διαφορετικών περιοχών, αναδεικνύοντας είδη που δεν απομονώθηκαν κατά την καλλιεργητική μελέτη. Η μελέτη των φυσικοχημικών χαρακτηριστικών των τυριών, ο καθορισμός των ελεύθερων αμινοξέων και οργανικών οξέων, έδειξε πως η περιοχή παραγωγής των τυριών ασκεί επίδραση στις παραμέτρους που μελετήθηκαν. Το προφίλ και η περιεκτικότητα των φυτών, του νωπού γάλακτος, των φρέσκων και ώριμων τυριών σε VOCs έδειξε διαφορές μεταξύ των τριών περιοχών. Στα 23 φυτικά είδη που συλλέχθηκαν, έγινε ανάλυση με PCA η οποία διαφοροποίησε τις περιοχές με βάση τη σύνθεση των VOCs. Το γάλα των τριών περιοχών ήταν φτωχότερο σε VOCs φυτικής προέλευσης, ενώ κοινά τερπένια σε φυτά και γάλα θα μπορούσαν να χρησιμοποιηθούν ως δείκτες για τη σύνδεση της παραδοσιακής Φέτας με την περιοχή παραγωγής της. Στα φρέσκα τυριά, οι αλδεΰδες ήταν σημαντικά (Ρ<0,05) υψηλότερες για τα τυριά της περιοχής 1, οι κετόνες για την περιοχή 2 (Ρ<0,05), οι αλκοόλες για την περιοχή 3 (Ρ<0,05), ενώ τα τερπένια αποτελούσαν την επικρατέστερη ομάδα VOCs, ιδιαίτερα για τα τυριά των περιοχών 1 και 3. Στα ώριμα τυριά, τα οξέα ήταν η επικρατέστερη ομάδα VOCs και ακολουθούνταν από τους εστέρες και τις αλκοόλες, οι οποίες βρέθηκαν σε σημαντικά υψηλότερη (Ρ<0,05) ποσότητα στα τυριά της περιοχής 2. Βρέθηκαν ενώσεις κοινές για τα τυριά όλων των περιοχών και άλλες που χαρακτήριζαν τυριά μιας μόνο περιοχής. Παρόμοια, υπήρχαν τερπένια κοινά στα ώριμα τυριά και των τριών περιοχών και άλλα που φαίνεται να αποτελούν χαρακτηριστικές ενώσεις για τα τυριά της κάθε περιοχής. Συνεπώς, η σύνθεση των VOCs της παραδοσιακής Φέτας που παράγεται σε τρεις ορεινές περιοχές της Ελλάδας διαφέρει, ανάλογα με την περιοχή παραγωγής της, ενώ διαφορές εντοπίστηκαν και μεταξύ των τυριών της ίδιας περιοχής

The Multiple and Versatile Roles of Aureobasidium pullulans in the Vitivinicultural Sector

The saprophytic yeast-like fungus Aureobasidium pullulans has been well documented for over 60 years in the microbiological literature. It is ubiquitous in distribution, being found in a variety of environments (plant surfaces, soil, water, rock surfaces and manmade surfaces), and with a worldwide distribution from cold to warm climates and wet/humid regions to arid ones. Isolates and strains of A. pullulans produce a wide range of natural products well documented in the international literature and which have been regarded as safe for biotechnological and environmental applications. Showing antagonistic activity against plant pathogens (especially post-harvest pathogens) is one of the major applications currently in agriculture of the fungus, with nutrient and space competition, production of volatile organic compounds, and production of hydrolytic enzymes and antimicrobial compounds (antibacterial and antifungal). The fungus also shows a positive role on mycotoxin biocontrol through various modes, with the most striking being that of binding and/or absorption. A. pullulans strains have been reported to produce very useful industrial enzymes, such as &beta;-glucosidase, amylases, cellulases, lipases, proteases, xylanases and mannanases. Pullulan (poly-&alpha;-1,6-maltotriose biopolymer) is an A. pullulans trademark product with significant properties and biotechnological applications in the food, cosmetic and pharmaceutical industries. Poly (&beta;-l-malic acid), or PMA, which is a natural biopolyester, and liamocins, a group of produced heavy oils and siderophores, are among other valuable compounds detected that are of possible biotechnological use. The fungus also shows a potential single-cell protein source capacity with high levels of nucleic acid components and essential amino acids, but this remains to be further explored. Last but not least, the fungus has shown very good biocontrol against aerial plant pathogens. All these properties are of major interest in the vitivinicultural sector and are thoroughly reviewed under this prism, concluding on the importance that A. pullulans may have if used at both vineyard and winery levels. This extensive array of properties provides excellent tools for the viticulturist/farmer as well as for the oenologist to combat problems in the field and create a high-quality wine

Enterococci Isolated from Cypriot Green Table Olives as a New Source of Technological and Probiotic Properties

&Tau;able olive is one of the main fermented vegetable worldwide and can be processed as treated or natural product. Lactic Acid Bacteria (LAB) are responsible for the fermentation of treated olives. The aim of this work was to study the technological characteristics and the potential probiotic properties of LAB isolated from Cypriot green table olives. This is the first comprehensive report on the isolation and characterization of LAB isolates retrieved from Cypriot green table olives. From a collection of 92 isolates from spontaneously fermenting green olives, 64 g positive isolates were firstly identified to genus level using biochemical tests, and secondly to species level using multiplex species specific polymerase chain reaction (PCR) amplifications of the sodA gene. Moreover, each of our isolates were tested for their technological and probiotics properties, as well as for their safety characteristics, using biochemical and molecular methods, in order to be used as starter cultures. Finally, to discriminate the most promising isolates on the base of their technological and probiotics properties, Principal component analysis was used. All the isolates were identified as Enteroccocus faecium, having interesting technological properties, while pathogenicity determinants were absent. Principal component analysis showed that some isolates had a combination of the tested parameters. These findings demonstrate that enteroccoci from Cypriot table olives should be considered as a new source of potential starter cultures for fermented products, having possibly promising technological and probiotic attributes

Assessment of microbial diversity of the dominant microbiota in fresh and mature PDO Feta cheese made at three mountainous areas of Greece

The aim of the present study was to assess the dynamics and the diversity of dominant microbiota in fresh and mature traditional Feta cheese produced in three mountainous areas and for that purpose a combined approach of culture-independent examination with conventional genotypic typing of the predominant NSLAB microbiota were applied. The microbial communities were monitored by PCR-DGGE on seven fresh and nine mature Feta cheese samples made by three artisanal producers. DGGE profiles suggested variability in the microbial composition of cheeses within the production area and distinctive differences in band profile of NSLAB in cheeses between the areas. PCR-DGGE analysis showed that Lactococcus spp. were the most widespread bacteria, Streptococcus macedonicus was detected often and, among lactobacilli, Lactobacillus plantarum prevailed. The diversity of NSLAB isolates from fresh and mature cheeses was assessed by RAPD-PCR and PFGE. Typing data indicated intraspecies genetic heterogeneity and specificity to the production area. Our results point to the conclusion that the manufacturing environment influence the dynamic and the diversity of microbial groups developed in the cheese and possibly the cheese flavour. In addition, the traditional Feta cheese may be a source of different NSLAB genotypes to make cheese with sensorial peculiarities appropriate for each area

Raw donkey milk as a source of Enterococcus diversity: Assessment of their technological properties and safety characteristics

The diversity, the technological and the safety aspects, and the probiotic potential of enterococci isolated from raw donkey milk were studied, in order to determine their potential to be used as starters/adjuncts cultures in dairy products. Gram-positive, catalase negative bacteria (257) were isolated using selective microbiological media from eleven raw milk samples, collected over seven months from a donkey farm in Cyprus. All isolates were identified by phenotypic and molecular techniques and further tested for their technological and probiotic properties. Furthermore, in order to assess their suitability as starter/adjunct cultures, their susceptibility to antibiotics, the absence of virulence factors, the lack of haemolytic activity, and production of biogenic amines were also investigated. The safety profile of the isolates revealed that their great majority were susceptible to clinically important antibiotics (i.e. vancomycin) and production of biogenic amines (i.e. tyramine) while the presence of some virulence genes occurred in a few isolates. The results obtained in this study showed that Enterococci in raw donkey milk should not pose a risk to human health, while they possess interesting technological characteristics which could be exploited as potential starter/adjunct cultures for food fermentations

Microbiological Characteristics of Trachanas, a Traditional Fermented Dairy Product from Cyprus

The purpose of this study was to characterize the autochthonous microbiota of Cypriot Trachanas, a traditional fermented ewes’ milk product. For this reason, 12 samples of raw and fermented milk as well as natural starter culture were collected in order to count, isolate, and identify the main species present during Trachanas fermentation. In total, 198 colonies were retrieved and 163 were identified by sequencing analysis at species level. Lactic acid bacteria (LAB) were the predominant group, followed by yeasts. Lactococcus, Lactobacillus, and Enterococcus were frequently isolated from raw milk, and Lactobacillus casei/paracasei predominated in the starter culture. Lactococcus lactis was isolated in high frequency (27.9% of the isolates) at the beginning, while Lactobacillus spp. (20%) and Saccharomyces unisporus (17.9%) were isolated at the end of fermentation. After assessing their technological potential, selected strains could be used as starters to ferment milk for artisanal Trachanas production

Microbiological Characteristics of Trachanas, a Traditional Fermented Dairy Product from Cyprus

The purpose of this study was to characterize the autochthonous microbiota of Cypriot Trachanas, a traditional fermented ewes’ milk product. For this reason, 12 samples of raw and fermented milk as well as natural starter culture were collected in order to count, isolate, and identify the main species present during Trachanas fermentation. In total, 198 colonies were retrieved and 163 were identified by sequencing analysis at species level. Lactic acid bacteria (LAB) were the predominant group, followed by yeasts. Lactococcus, Lactobacillus, and Enterococcus were frequently isolated from raw milk, and Lactobacillus casei/paracasei predominated in the starter culture. Lactococcus lactis was isolated in high frequency (27.9% of the isolates) at the beginning, while Lactobacillus spp. (20%) and Saccharomyces unisporus (17.9%) were isolated at the end of fermentation. After assessing their technological potential, selected strains could be used as starters to ferment milk for artisanal Trachanas production