Pichia Membranifaciens Research Articles

A novel high-level phenyllactic acid fungal producer, Kodamaea ohmeri w5 screened from fermented broad bean-chili-paste

Phenyllactic acid (PLA) is a broad-spectrum and efficient antimicrobial phenolic acid with potential applications in the food industry. Previous studies have demonstrated that fungi may be ideal producers of PLA. In this study, 15 fungi screened from Doubanjiang with the ability to produce PLA were first reported, including Wickerhamomyces anomalus, Candida etchellsii, Candida parasitosis, Pichia kudriavzevii, Pichia membranifaciens and Kodamaea ohmeri. Among them, K. ohmeri w5 had the highest PLA yield, producing up to 7160 mg/L PLA in shake flask fermentation with phenylalanine as substrate, which was more than ten times higher than the PLA produced by wild-type LAB under the similar conditions. In addition, K. ohmeri w5 was able to grow under extreme hypertonic conditions of 20 % NaCl (w/v) and 50 % glucose (w/v) as well as produce 57.12 ± 0.42 and 1609.22 ± 36.26 mg/L of PLA, respectively. Furthermore, the fermentation supernatant of K. ohmeri w5 demonstrated direct inhibitory effects against foodborne pathogenic microorganisms, Aspergillus flavus and Bacillus cereus. However, the inhibitory effect was weaker than that of the PLA standard at the same concentration. Further, 12,497,932 bp of w5 genome-wide information was obtained by sequencing and assembling. And its gene model was predicted based on transcriptomic evidence, which showed that a total of 7 genes related to PLA synthesis were identified in the w5 genome. Based on qRT-PCR, structure prediction, and molecular docking, a potentially key genetic resource from K. ohmeri w5 for PLA production was uncovered. The results will provide novel producers of PLA and its potential genetic resources.

Kombucha with yam: Comprehensive biochemical, microbiological, and sensory characteristics

Consumer demand for functional foods has increased, helping to popularize and increase the consumption of Kombucha. Other substrates have been used together with tea to improve the functional and sensory properties of the beverage. Thus, this study evaluated the comprehensive biochemical, microbiological, and sensory characteristics of kombuchas fermented with green tea (Camellia sinensis) and different concentrations of yam (0, 10, and 20 % w/v). Based on pre-tests to detect the best concentration of yam in the beverage (10, 20, 30, and 40 %) and fermentation time (5, 7, and 14 days),the concentrations of 10 and 20 % of yam and five days of fermentation were selected through pH, °Brix, and sensory analysis. During the kombucha fermentation, there was a decrease in °Brix and pH. Sucrose, glucose, fructose, citric, and succinic acids were related to the beginning of fermentation, and lactic and acetic acids were more related to the end of fermentation in the treatment containing 20 % yam. The fermentation time did not change the color of the kombucha. Fatty acids, phenols, terpenoids, and alcohols were the volatile groups with the most compounds identified. Only two yeast genera were identified (Brettanomyces bruxellensis and Pichia membranifaciens), and bacteria of the genera Acetobacter, Lactobacillus, Pantoea, Pseudomonas, Azospirillum, and Enterobacter. The beverage control showed less turbidity and more clear. The fruity descriptor was more perceived in treatments with yam. However, the perception of the apple descriptor decreases as the yam concentration increases. The yam’s concentration alters the kombucha’s microbiota and sensory characteristics, mainly appearance and acidity. Kombucha fermentation using yam extract is viable, and the product is sensorially accepted. However, technological improvements, such as yam flour, could be made mainly for appearance and taste attributes.

Effects of ultrasound and microwave treatments on oenological yeast microbiota: comparison with a flash pasteurisation

SummaryThe control of yeast biota in wine could be a challenge, as different genera could coexist with different effects on wine quality; generally, SO2 plays an important role in this context, while other approaches have been proposed. In this article, two Microwave (MW) and two Ultrasound treatments (US) were tested and compared to a flash pasteurisation on seven yeast strains (Saccharomyces cerevisiae, Saccharomyces bayanus, Brettanomyces spp., Schizosaccharomyces pombe, Hanseniaspora osmophila, Zygosaccharomyces bailii, Pichia membranifaciens); viable count (viability) and yeast growth kinetic (vitality) were assessed. The treatments reduced viable count by 27%, with a higher biocidal effect of US, depending on yeast and treatment conditions; they also impaired yeast growth causing a delay, which is a significant increase of the lag phase, while the effect on the growth rate or the maximum OD attained in the steady state was less significant. In conclusion, the US generally exerted a stronger effect than MW and flash pasteurisation and Schizo. pombe and Z. bailii were the less resistant yeasts. A partial uncoupling was found between vitality and viability for Z. bailii, while in Schizo. pombe the delay in the growth was generally related to the decrease in the cell count immediately after the treatment.

DIVERSITY AND PHYTATE-DEGRADING POTENTIAL OF YEAST MICROORGANISMS ISOLATED FROM SOURDOUGH

Phytases, which perform the stepwise hydrolysis of phytic acid to myo-inositol and inorganic phosphate, are used worldwide to reduce phosphorus pollution and improve nutrition in monogastric animals and humans. Yeasts isolated from their natural environments represent rich and still underexplored sources of industrially valuable enzymes, including phytases; therefore, they are widely studied for the production of these enzymes. In this regard, thirteen yeast pure cultures were isolated from the microbial consortium of four types of sourdough obtained during the natural fermentation of different grain-based flours. Ten of the newly isolated yeast strains were selected as potential phytase producers based on their growth in liquid culture media with sodium phytate as the sole source of phosphorus. Using 18S rDNA and D1/D2 26S rDNA analyses, the species affiliation of the selected isolates was established. They referred to seven yeast species from 3 families, with the most significant representation of the family Saccharomycetaceae.Intracellular phytate-degrading activity was found in 8 isolates, the highest being in Nakaseomyces glabratus strain 7-4. The highest level of extracellular phytase was measured in Pichia membranifaciens strain 5-2. Both isolates showed significant antioxidant capacity higher than those of ascorbic acid.

Antibacterial mechanism of thermoultrasound combined with potassium sorbate against Pichia membranifaciens

The effect of the antimicrobial mechanism of ultrasound (US) in combination with heat (T) and potassium sorbate (PS) treatment on Pichia membranifaciens (P. membranifaciens) is investigated in this study. It is found that ultrasound combined with heat (thermoultrasound [TS]) exhibits a synergistic inactivation efficiency, and the combinations of US, T, and TS with PS also improve inactivation efficacy through reductions of 0.32 ± 0.02 log, 0.73 ± 0.04 log, and 4.50 ± 0.04 log in P. membranifaciens achieved using US + PS, T + PS, and TS + PS, respectively. Further, use of the TS + PS treatment leads to cytoplasmic leakage and a decrease in dehydrogenase activity, as well as a significant increase in cell membrane permeability, causing membrane depolarization and the inward flow of extracellular calcium ions. In addition, the cell membrane composition is detected by infrared spectroscopy, demonstrating that TS + PS treatment results in a decrease in lipid and protein content and an increase in phospholipid content in the cell membrane. Scanning and transmission electron microscopy reveals a deformed cell structure and damage to cell membrane integrity after TS + PS treatment. In addition, the expression of heat shock protein (Hsp82 and Hsp70) genes is shown to decrease after TS and TS + PS treatment compared to the T and T + PS treatment groups, indicating cellular homeostasis has been broken and cells are responding to the damage by activating the protective gene expression program.

Mechanism of lactic acid synthesis during strong-flavor baijiu fermentation revealed by transcriptomic analysis

Lactic acid is one of the backbone organic acids in Chinese baijiu. Its content affects both the fermentation process and the flavor and quality of baijiu. However, contribution of different microorganisms to lactic acid synthesis and the metabolism of lactic acid during strong-flavor baijiu fermentation have not yet been clarified. In this study, microbes and pathways involved in lactate synthesis during strong-flavor baijiu fermentation were revealed by RNA-seq. It was found that lactate was synthesized under microaerobic and anaerobic conditions, and both nitrogen and carbon metabolisms were involved. In the process of nitrogen metabolism, Acetobacter malorum, Acetobacter pasteurianus, Komagataeibacter xylinus, Pichia membranifaciens and Saccharomyces cerevisiae synthesized d-lactate. Torulaspora delbrueckii synthesized lactate through homolactic fermentation under microaerobic condition. In the process of strong-flavor baijiu fermentation, lactate is mainly synthesized by Lactobacillus through heterolactic fermentation. Lactobacillus ozensis and Lactobacillus diolivorans were identified as two major lactate producers and they synthesized d-lactate under anaerobic condition. l-lactate could be synthesized by T. delbrueckii, L. ozensis and Lactobacillus acetotolerans. The results demonstrated a comprehensive and clarified mechanism of lactic acid synthesis during strong-flavor baijiu fermentation, and could provide a theoretical basis for regulation of lactic acid synthesis in the process of baijiu fermentation.

Antagonistic yeast and trehalose-enriched gelatin film: A bioactive antifungal packaging film for cherry tomato preservation

To extend the shelf-life of cherry tomatoes infected with Botrytis cinerea, this study analyzed the effect of Pichia membranifaciens (PM) on a composite film of gelatin (Gel) and trehalose (Tre), and explored the effect of different concentrations of trehalose on the yeast in the bioactive film and its control effect on cherry tomatoes. Scanning electron microscopy showed that Tre/Gel-based film can be applied as effective carriers to protect the cellular integrity of PM. Fourier-transform infrared spectroscopy, thermogravimetric analysis, and tests of mechanical properties showed that there was interaction between PM and the Tre/Gel base film, which maintained the thermal stability of the base film. The addition of PM reduced the water vapor permeability, hygroscopicity, and moisture content of the Tre/Gel base film, effectively extending the active film's storage time. This study also explored the growth curve of PM and found that 15% trehalose was effective in maintaining PM activity and could result in a 30-day survival rate of 62% for PM in Tre/Gel films, with 72.41% inhibition of B. cinerea. Finally, the PM/Tre/Gel film significantly reduced the incidence of decay in cherry tomatoes and improved the storage quality and fungal resistance of the fruit. Thus, PM/Tre/Gel films may be applied as a new antifungal packaging material for the preservation of fresh fruit.

Biological Control Mechanisms of Non‐Saccharomyces Yeasts against Filamentous Fungal Pathogens within Rosaceae Family

Pathogenic fungi can cause significant annual losses in the quality and quantity of fruits belonging to Rosaceae family during pre‐ and postharvest. Synthetic fungicides are considered the main method for controlling diseases in food crops; however, they pose a great risk to human health and to the environment. As a result, biological control methods, which are based on living microorganisms to reduce the population or to inhibit the growth of pathogens, have arisen as a safe alternative. In this review, we highlight the principal mechanisms (e.g., nutrition and space competition, mycoparasitism, induced resistance and growth enhancement, production of volatile organic compounds, and antibiosis) utilized by the most frequently reported non‐Saccharomyces biocontrol agents (BCAs) yeasts: Meyerozyma guilliermondii, Pichia membranifaciens, Wickerhamomyces anomalus, Metschnikowia pulcherrima, Hanseniaspora uvarum, and Aureobasidium pullulans, against the common pathogenic fungi found within Rosaceae family: Penicillium expansum, Botrytis cinerea, and Monilinia spp. Understanding BCAs’ importance and mode of action is a necessary step in order to reduce the detrimental effect of spoilage fungi in the agriculture and food industry, by achieving a more sustainable and safer control of them.

THE EFFECT OF COMBINATIONS OF SOURDOUGH LACTIC ACID BACTERIA AND YEASTS ON WHEAT BREAD QUALITY: EVALUATION IN TERMS OF MICROBIOLOGICAL, RHEOLOGICAL, TEXTURAL AND VOLATILE COMPONENTS

In this study, co-cultivation of lactic acid bacteria (Lb. plantarum 45MK-30, Lb. fermentum 29GT-19) and yeasts (Saccharomyces cerevisiae 06B-39, Kazachstania humilis KCO-60 and Pichia membranifaciens 11G-11) on the quality of sourdough bread the effect was studied. Two different fermentation conditions were used for this (I and II fermentation). While cell density and pH value of microorganisms were close to each other in I fermentation, a decrease in these values was observed in II fermentation. Also, all doughs exhibited elastic behavior. It can be said that the textural feature of sourdough breads is softer compared to both controlled acidified (CA) bread and non-sourdough (flat dough, CY) bread. Another parameter examined in breads was the evaluation of volatile components by HS-GC / MS. It was observed that breads produced by co-cultivation had more volatile components than plain dough bread.

Cyberlindnera jadinii and Kluyveromyces lactis, two fungi used in food processes, have potential probiotic effects on gut inflammation.

The food industry has always used many strains of microorganisms including fungi in their production processes. These strains have been widely characterized for their biotechnological value, but we still know very little about their interaction capacities with the host at a time when the intestinal microbiota is at the center of many pathologies. In this study, we characterized five yeast strains from food production which allowed us to identify two new strains with high probiotic potential and beneficial effects in a model of intestinal inflammation.

Antifungal activities of leaf extracts from the Capsicum genus

The emergence of microorganisms resistant to antimicrobials has driven the search for new substances aimed at the control and prevention of pathogens. Plant antimicrobial peptides have biotechnological applications in the development of drugs and agricultural inputs for this purpose. The present work aims to evaluate the antifungal activity of extracts containing peptides obtained from the leaves of Capsicum plants. The accessions UENF 1626, 1632 and 1381 and the cultivar Carioquinha were used. The leaves were macerated, and then extraction was performed using a 60% methanol and dichloromethane solution in a 1:1 ratio. The hydromethanolic phase was separated and used for testing after evaporation of methanol. All extracts have a similar electrophoretic profile on tricine gel stained with Coomassie Blue, showing 2 bands of approximately 6.5 and 3.5 kDa. Inhibition of fungal growth with 24 h of culture was performed in liquid culture medium in 96-well plates with reading at 620 nm using extracts with a protein concentration of 200 mg mL-1. The filamentous fungi Fusarium solani, Fusarium lateritium, and Colletotrichum lindemuthianum and the yeasts Candida albicans, Candida buinensis, Candida tropicalis, Saccharomyces cerevisiae, Pichia membranifaciens and Kluyveromyces marxianus were used. The extract with the greatest antifungal potential was the leaves of Capsicum annuum cv. Carioquinha, inhibiting the growth of F. solani by 29%, C. lindemuthianum by 15%, Candida buinensis by 22% and K. marxianus by 24%. Therefore, the extract has peptides with biotechnological potential.

Multi-omics characterization of the microbial populations and chemical space composition of a water kefir fermentation.

In recent years, the popularity of fermented foods has strongly increased based on their proven health benefits and the adoption of new trends among consumers. One of these health-promoting products is water kefir, which is a fermented sugary beverage based on kefir grains (symbiotic colonies of yeast, lactic acid and acetic acid bacteria). According to previous knowledge and the uniqueness of each water kefir fermentation, the following project aimed to explore the microbial and chemical composition of a water kefir fermentation and its microbial consortium, through the integration of culture-dependent methods, compositional metagenomics, and untargeted metabolomics. These methods were applied in two types of samples: fermentation grains (inoculum) and fermentation samples collected at different time points. A strains culture collection of ∼90 strains was established by means of culture-dependent methods, mainly consisting of individuals of Pichia membranifaciens, Acetobacter orientalis, Lentilactobacillus hilgardii, Lacticaseibacillus paracasei, Acetobacter pomorum, Lentilactobacillus buchneri, Pichia kudriavzevii, Acetobacter pasteurianus, Schleiferilactobacillus harbinensis, and Kazachstania exigua, which can be further studied for their use in synthetic consortia formulation. In addition, metabarcoding of each fermentation time was done by 16S and ITS sequencing for bacteria and yeast, respectively. The results show strong population shifts of the microbial community during the fermentation time course, with an enrichment of microbial groups after 72h of fermentation. Metataxonomics results revealed Lactobacillus and Acetobacter as the dominant genera for lactic acid and acetic acid bacteria, whereas, for yeast, P. membranifaciens was the dominant species. In addition, correlation and systematic analyses of microbial growth patterns and metabolite richness allowed the recognition of metabolic enrichment points between 72 and 96h and correlation between microbial groups and metabolite abundance (e.g., Bile acid conjugates and Acetobacter tropicalis). Metabolomic analysis also evidenced the production of bioactive compounds in this fermented matrix, which have been associated with biological activities, including antimicrobial and antioxidant. Interestingly, the chemical family of Isoschaftosides (C-glycosyl flavonoids) was also found, representing an important finding since this compound, with hepatoprotective and anti-inflammatory activity, had not been previously reported in this matrix. We conclude that the integration of microbial biodiversity, cultured species, and chemical data enables the identification of relevant microbial population patterns and the detection of specific points of enrichment during the fermentation process of a food matrix, which enables the future design of synthetic microbial consortia, which can be used as targeted probiotics for digestive and metabolic health.

Sour Beer as Bioreservoir of Novel Craft Ale Yeast Cultures.

The increasing demand for craft beer is driving the search for novel ale yeast cultures from brewing-related wild environments. The focus of bioprospecting for craft cultures is to identify feral yeasts suitable to imprint unique sensorial attributes onto the final product. Here, we integrated phylogenetic, genotypic, genetic, and metabolomic techniques to demonstrate that sour beer during aging in wooden barrels is a source of suitable craft ale yeast candidates. In contrast to the traditional lambic beer maturation phase, during the aging of sour-matured production-style beer, different biotypes of Saccharomyces cerevisiae dominated the cultivable in-house mycobiota, which were followed by Pichia membranifaciens, Brettanomyces bruxellensis, and Brettanomyces anomalus. In addition, three putative S. cerevisiae × Saccharomyces uvarum hybrids were identified. S. cerevisiae feral strains sporulated, produced viable monosporic progenies, and had the STA1 gene downstream as a full-length promoter. During hopped wort fermentation, four S. cerevisiae strains and the S. cerevisiae × S. uvarum hybrid WY213 exceeded non-Saccharomyces strains in fermentative rate and ethanol production except for P. membranifaciens WY122. This strain consumed maltose after a long lag phase, in contrast to the phenotypic profile described for the species. According to the STA1+ genotype, S. cerevisiae partially consumed dextrin. Among the volatile organic compounds (VOCs) produced by S. cerevisiae and the S. cerevisiae × S. uvarum hybrid, phenylethyl alcohol, which has a fruit-like aroma, was the most prevalent. In conclusion, the strains characterized here have relevant brewing properties and are exploitable as indigenous craft beer starters.

Exploring yeast diversity of dry-salted naturally black olives from Greek retail outlets with culture dependent and independent molecular methods

In the present study, the physicochemical (pH, water activity, moisture content, salt concentration) classical plate counts (total viable counts, yeasts, lactic acid bacteria, Staphylococcus aureus, Pseudomonas spp., Enterobacteriaceae) and amplicon sequencing of naturally black dry-salted olives obtained from different retail outlets of the Greek market were investigated. According to the results, the values of the physicochemical characteristics presented great variability among the samples. Specifically, pH and water activity (aw) values ranged between 4.0 and 5.0, as well as between 0.58 and 0.91, respectively. Moisture content varied between 17.3 and 56.7 % (g Η2Ο/100 g of olive pulp), whereas salt concentration ranged from 5.26 to 9.15 % (g NaCl/100 g of olive pulp). No lactic acid bacteria, S. aureus, Pseudomonas spp. and Enterobacteriaceae were detected. The mycobiota consisted of yeasts that were further characterized and identified by culture-dependent (rep-PCR, ITS-PCR, and RFLP) and amplicon target sequencing (ATS). Pichia membranifaciens, Candida sorbosivorans, Citeromyces nyonsensis, Candida etchelsii, Wickerhamomyces subpelliculosus, Candida apicola, Wickerhamomyces anomalus, Torulaspora delbrueckii and Candida versatilis were the dominant species according to ITS sequencing (culture-dependent), while ATS revealed the dominance of C. etchelsii, Pichia triangularis, P. membranifaciens, and C. versatilis among samples. The results of this study demonstrated considerable variability in quality attributes among the different commercial samples of dry-salted olives, reflecting a lack of standardization in the processing of this commercial style. However, the majority of the samples were characterized by satisfactory microbiological and hygienic quality and complied with the requirements of the trade standard for table olives of the International Olive Council (IOC) for this processing style in terms of salt concentration. In addition, the diversity of yeast species was elucidated for the first time in commercially available products, increasing our knowledge on the microbial ecology of this traditional food. Further investigation into the technological and multifunctional traits of the dominant yeast species may result in better control during dry-salting and enhance the quality and shelf-life of the final product.

Microbial Communities of Flor Velums and the Genetic Stability of Flor Yeasts Used for a Long Time for the Industrial Production of Sherry-like Wines

Flor yeast strains represent a specialized group of Saccharomyces cerevisiae yeasts used for the production of sherry-like wines by biological wine aging. We sequenced the genome of the industrial flor yeast strain I-329 from a collection of microorganisms for winemaking “Magarach” and the metagenomes of two flor velums based on this strain and continuously maintained for several decades. The winery uses two processes for the production of sherry-like wine: batch aging and a continuous process similar to the criaderas–solera system. The 18S rRNA gene profiling and sequencing of metagenomes of flor velums revealed the presence of the yeasts Pichia membranifaciens and Malassezia restricta in minor amounts along with the dominant S. cerevisiae I-329 flor yeast. Bacteria Oenococcus oeni and Lentilactobacillus hilgardii together accounted for approximately 20% of the velum microbiota in the case of a batch process, but less than 1% in the velum used in the continuous process. Collection strain I-329 was triploid for all chromosomes except diploid chromosomes I and III, while the copy numbers of all chromosomes were equal in industrial velums. A comparative analysis of the genome of strain I-329 maintained in the collection and metagenomes of industrial velums revealed only several dozens of single nucleotide polymorphisms, which indicates a long-term genetic stability of this flor yeast strain under the harsh conditions of biological wine aging.

Bacterial and fungal microbiota of mould‐ripened cheese produced in Konya

Bacterial and fungal diversities of 24 mould‐ripened cheeses originating from Konya‐Türkiye were examined by metagenomic analysis. Firmicutes phylum, Enterococcus, Clostridium sensu stricto and Lactobacillus (Levilactobacillus) genera were the dominant bacteria. Ascomycota phylum and Penicillium and Pichia genera and Penicillium roqueforti and Pichia membranifaciens species were dominant fungi. Enterococcus faecium (n = 30) and Enterococcus faecalis (n = 6) were identified, and all strains were susceptible to penicillin, ampicillin, vancomycin, teicoplanin, chloramphenicol and linezolid. The highest resistance (n = 14) was against rifampin. Tetracycline resistance was determined in two strains. Biofilm‐forming ability was found in nine E. faecium and 1 E. faecalis. E. faecium strains revealed 40–88.9%, and E. faecalis showed 59.2–100% homology by pulsed field gel electrophoresis.

Characterization of kefir yeasts with antifungal capacity against Aspergillus species.

Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them-belonging to S. cerevisiae and P. kudriavzevii species-generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.

Non-Saccharomyces Are Also Forming the Veil of Flor in Sherry Wines

Biological ageing is an essential process for obtaining some distinctive Sherry wines, such as Fino and Manzanilla. It occurs after the fermentation of the grape must due to the appearance of a biofilm on the surface of the wine called “veil of flor”. Yeasts belonging to the Saccharomyces cerevisiae species mainly comprise such biofilm. Although other species have also been found, these have been traditionally considered spoilage. Indeed, it has even been hypothesised that they may not be able to form biofilm on their own under such conditions. In the present work, four different non-Saccharomyces yeasts isolated from barrels in the Jerez area under biological ageing have been characterised through their physiological abilities, including extracellular enzymatic and biofilm-forming capabilities. Results showed not only a surprising ethanol tolerance, above 15.5% in all cases, but also a significant degree of extracellular enzyme production, highlighting the urease and proteolytic activities found in Pichia manshurica, as well as lipolytic activity in Pichia kudriavzevii, Pichia membranifaciens and Wicherhamomyces anomalus. As a conclusion, these non-Saccharomyces could be very interesting in the oenological field, beyond improving the organoleptic characteristics as well as technological features in these wines.

Application of Bioactive Coatings with Killer Yeasts to Control Post-Harvest Apple Decay Caused by Botrytis cinerea and Penicillium italicum.

A new method was proposed to produce alginate bio-films containing Pichia membranifaciens and Wickerhamomyces anomalus killer yeast to control the post-harvest fungal decay in organic apples caused by Botrytis cinerea and Penicillium italicum. Coatings with W. anomalus killer yeast effectively controlled the growth of P. italicum during storage at 22 °C. W. anomalus killer yeast incorporated in alginate reduced the P. italicum incidence from 90% (control) to 35% after 14 days of storage at 22 °C. Alginate biofilms with W. anomalus or P. membranifaciens also limited the incidence of the fungal decay of apples inoculated with B. cinerea compared with the control fruits, although the antagonistic capability against B. cinerea was lower than against P. italicum. The survival of W. anomalus cells in alginate coating was higher than P. membranifaciens. The incorporation of killer yeasts into alginate had no significant effect on the mechanical properties (tensile strength, percent elongation at break) of alginate coating, however, they increased the thickness of the biofilm. The bioactive coating reduced the fruit weight loss and had no significant effects on the fruit firmness during storage at 2 °C. As organic apples, produced without any synthetic fungicides, are especially prone to fungal decay during storage, the proposed alginate biofilms containing killer yeast seem to be a very promising solution by offering non-chemical, biological control of post-harvest pathogens.

Exploring microbial communities of Spanish-style green table olives of Conservolea and Halkidiki cultivars during modified atmosphere packaging in multi-layered pouches through culture-dependent techniques and metataxonomic analysis

Τhe aim of this study was to investigate the microbiological and physicochemical changes of pitted green olives (cvs. Conservolea and Halkidiki) elaborated in the Spanish style method and packaged in two types of multi-layered pouches under nitrogen atmosphere (100% N2) for a period of 12 months. Moreover, the evolution of microbial consortia at the beginning (0 days), middle (180 days) and final (360 days) time points of storage was elucidated by plating, genotyped and identified through RAPD-PCR and ITS region amplicon sequencing, respectively, and subsequently metataxonomic analysis (for fungal communities only). Results showed that no enterobacteria could be detected on olive drupes, whereas the dominant microbiota from the onset of storage in both pouches consisted of LAB in populations ranging between ca. 4.2–6.6 log CFU/g. Although yeasts were initially enumerated at ca. 5.5 log CFU/g, they declined rapidly and could not be detected by plate counting after 30 days. The pH values increased from 4.11 to 4.24 and 4.03 to 4.12 at the beginning and end of storage for cvs. Halkidiki and Conservolea green olives, respectively. The total color difference index (ΔΕ*) presented a perceivable change in visual color (ΔΕ* > 2.0) only in cv. Conservolea olives during storage. Finally, molecular fingerprinting RAPD-PCR and 16S amplicon based identification revealed the dominance of five LAB species, namely Pediococcus ethanolidurans, Lactiplantibacillus pentosus, Lentilactobacillus rapi, Lentilactobacillus parafarraginis and Lentilactobacillus buchneri. Regarding the identification of yeasts, the metagenetic amplicon sequencing approach revealed fungal complexity in the olive samples. Pichia manshurica and Pichia membranifaciens prevailed during the first and middle stages of storage, whereas at the final stage higher complexity was noticeable.