Yeasts and Lactic Acid Bacteria for Panettone Production: An Assessment of Candidate Strains

The recovery of yeasts and lactic acid bacteria (LAB) involved in sourdough fermentation is the first step in the selection of starters with suitable technological aptitude and capable of producing desired aromas and/or aromatic precursors. In this work, two sourdoughs samples (MA and MB) and the derived doughs (samples A and B) were collected from a bakery during artisanal Panettone manufacture. Yeasts and bacteria were isolated at different fermentation steps on selective agar media. A total of 77 isolates were obtained and characterized. Representative strains of yeasts and LAB were identified by sequencing the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, respectively. Moreover, the volatile organic compounds (VOCs) produced in the collected samples were detected and correlated to the species found in the same samples. The results highlighted the occurrence of Kazachstania humilis in both samples A and B, while Saccharomyces cerevisiae strains were detected only in samples B. Among LAB, Fructilactobacillus sanfranciscensis was the main species detected in both sourdoughs. Furthermore, strains belonging to the species Lactiplantibacillus plantarum, Furfurilactobacillus rossiae, Lactobacillus parabuchneri, Leuconostoc citreum, and Leuconostoc mesenteroides were assessed in the dough samples.

Adhesion Properties, Biofilm Forming Potential, and Susceptibility to Disinfectants of Contaminant Wine Yeasts

In this study, yeasts isolated from filter membranes used for the quality control of bottled wines were identified and tested for their resistance to some cleaning agents and potassium metabisulphite, adhesion to polystyrene and stainless-steel surfaces, and formation of a thin round biofilm, referred to as a MAT. A total of 40 strains were identified by rRNA internal transcribed spacer (ITS) restriction analysis and sequence analysis of D1/D2 domain of 26S rRNA gene. Strains belong to Pichia manshurica (12), Pichia kudriavzevii (9), Pichia membranifaciens (1), Candida sojae (6), Candida parapsilosis (3), Candida sonorensis (1), Lodderomyces elongisporus (2), Sporopachydermia lactativora (3), and Clavispora lusitaniae (3) species. Regarding the adhesion properties, differences were observed among species. Yeasts preferred planktonic state when tested on polystyrene plates. On stainless-steel supports, adhered cells reached values of about 6 log CFU/mL. MAT structures were formed only by yeasts belonging to the Pichia genus. Yeast species showed different resistance to sanitizers, with peracetic acid being the most effective and active at low concentrations, with minimum inhibitory concentration (MIC) values ranging from 0.08% (v/v) to 1% (v/v). C. parapsilosis was the most sensible species. Data could be exploited to develop sustainable strategies to reduce wine contamination and establish tailored sanitizing procedures.

Biodiversity of non-Saccharomyces yeasts associated with spontaneous fermentation of Cabernet Sauvignon wines from Shangri-La wine region, China

Shangri-La is a wine region that has the highest altitude vineyards in China. This is the first study investigated the biodiversity of non-Saccharomyces yeasts associated with spontaneous fermentation of Cabernet Sauvignon wines produced from two sub-regions (Lancang River and Jinsha River) of Shangri-La. The culturable yeasts were preliminarily classified based on their colonial morphology on the Wallerstein Laboratory nutrient agar plates. Yeast species were identified by the sequencing of the 26S rRNA D1/D2 region and the 5.8S rRNA ITS region. Twenty-five non-Saccharomyces yeast species belonging to sixteen genera were isolated and identified in Shangri-La wine region. Candida, Hanseniaspora, Pichia, and Starmerella were found in both sub-regions, but the Lancang River showed more diverse yeast species than the Jinsha River. Shangri-La not only exhibited high diversity of non-Saccharomyces yeasts, and furthermore, seven species of non-Saccharomyces yeasts were exclusively found in this region, including B. bruxellensis, D. hansenii, M. guilliermondii, S. vini, S. diversa, T. delbrueckii and W. anomalus, which might play an important role in distinctive regional wine characteristics. This study provide a relatively comprehensive analysis of indigenous non-Saccharomyces yeasts associated with Cabernet Sauvignon from Shangri-La, and has significance for exploring ‘microbial terroir’ of wine regions in China.

Production and Optimization of Xylanase and α-Amylase from Non-Saccharomyces Yeasts (Pichia membranifaciens)

The xylanolytic and amylolytic yeasts were qualitatively determined by Cong red xylan agar and soluble starch agar plates, respectively. The most xylanase and α-amylase inducible strain (AUN-02) was selected and identified using PCR amplification of 26S rRNA gene and sequence analysis. The comparison of the alignment results and phylogenetic analysis of the sequences of the isolated yeast to published rRNA gene sequences in GenBank, confirmed the identification of the isolate as Pichia membranifaciens. Xylanase and α-amylase production by isolated P. membranifaciens were investigated at different pH values (4-8), temperature degrees (20-45°C), incubation time (1-7 days) and various substrates.A higher production of xylanase (38.8 U/mL) and a-amylase (28.7 U/mL) was obtained after 4 days of fermentation of P. membranifaciens. Higher activity of xylanase (36.83 U/mL) and a-amylase (27.7 U/mL) was obtained in the fermentation of P. membranifaciens in a culture medium adjusted to pH 7.0. The optimum temperature showed maximum xylanase and a-amylase activity (42.6 and 32.5 units/mL, respectively) was estimated at 35 °C. The xylanase and a-amylase activities of P. membranifaciens were estimated and compared for the different substrates tested. The strain revealed 100% relative activity of xylanase and a-amylase on beechwood and potato starch, respectively. The affinity of enzymes towards substrate was estimated using Km values. The Km values of xylanase and α-amylase increased in the order of pH’s 7.0, 6.0 and 4.5 (0.85, 1.6 and 3.4 mg xylan/mL and 0.22, 0.43 and 2.8 mg starch/mL, respectively). the yeast P. membranifaciensis is suitable for produce neutral xylanase and α-amylase enzymes. So, it could be used as a promising strain for production of these enzymes in industrial field.

Yeasts in native fruits from Brazilian neotropical savannah: occurrence, diversity and enzymatic potential

Cerrado is the second largest phytogeographic domain in Brazil, with a huge ethnobotany variety, including fruit species that stand out for their economic, industrial, biotechnological and medicinal potential. The objective of this study was to characterize the diversity of culturable yeasts and their potential for the production of hydrolytic enzymes in fruits of 13 species of native plants of the Cerrado in Brazil. Sequencing the 26S rRNA gene identified the isolates. The enzymatic potential was evaluated using specific substrates for the enzymes amylases, cellulases, proteases, and pectinases. Nine of the 13 fruit species analyzed showed yeast growth, totaling 82 isolates, identified in 26 species. The phylum Ascomycota predominated over Basidiomycota. The fruits of Butia capitata presented the highest species richness. Candida and Meyerozyma were the most frequent genera. About 57% of the isolates were able to produce at least one of the enzymes analyzed. The species Papiliotrema flavescens, Hanseniaspora meyeri, Meyerozyma guilliermondii, and Rhodotorula mucilaginosa produced all the enzymes tested. The results were found to expand the knowledge about the yeast communities present in fruits of the Cerrado native plants, evidencing the presence of species shared among the plants, and their potential for biotechnological use in the future.

Exploring the Microbial Community of Traditional Sourdoughs to Select Yeasts and Lactic Acid Bacteria

Sourdoughs represent an awesome example of ecosystem in which yeasts and lactic acid bacteria (LAB) interact with each other, defining the characteristics of the final product in terms of composition, texture, taste and flavor. Therefore, the identification of dominant yeasts and LAB involved in the fermentation process can lead to the selection of starters with suitable fermentation aptitude and capable of producing desired aromas and/or aromatic precursors. In this work, two sourdoughs samples (A and B) for Panettone production were collected from an artisan bakery. Yeasts and bacteria were isolated at different fermentation steps on selective agar media. A total of 120 isolates were obtained and firstly characterized by conventional microbiological methods. Afterward, genomic DNA was extracted from the cultures, and (GTG)5-PCR fingerprinting analysis was carried out to reduce the redundance among the isolates. Representative yeasts and LAB strains, having a unique profile, were identified by sequencing the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, respectively. The results highlighted the occurrence of Kazachstania humilis and Fructilactobacillus sanfranciscensis in both sourdoughs. Among LAB, also some other strains belonging to Lactobacillus genus were found. Moreover, Saccharomyces cerevisiae and Staphylococcus spp. strains were detected in sample B. In this study, a pool of yeasts and LAB strains for producing starter cultures with specific technological traits for sourdoughs production was obtained.

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis in Caenorhabditis elegans

Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m5C at position C2381 on the 26S rRNA in Caenorhabditis elegans. Here, we show that NSUN-1 is writing the second known 26S rRNA m5C at position C2982. Depletion of nsun-1 or nsun-5 improved thermotolerance and slightly increased locomotion at midlife, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodeled leading to reduced production of collagens, loss of structural integrity of the cuticle, and impaired barrier function. We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal development and physiology.

Yeasts Associated with Various Amazonian Native Fruits

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis in Caenorhabditis elegans

Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m5C at position C2381 on the 26S rRNA in Caenorhabditis elegans.Here, we show that NSUN-1 is writing the second known 26S rRNA m5C at position C2982. Depletion of nsun-1 or nsun-5 improved locomotion at midlife and resistance against heat stress, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodelled leading to reduced production of collagens, loss of structural integrity of the cuticle and impaired barrier function.We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal physiology.

Isolation and Screening of Microbial Isolates from Kombucha Culture for Bacterial Cellulose Production in Sugarcane Molasses Medium

Kombucha tea is a traditional fermented beverage of Manchurian origins which is made of sugar and tea. The fermentation involves the application of a symbiotic consortium of bacteria and yeast (SCOBY) in which their metabolites provide health benefits for the consumer and subsequently allow the product to protect itself from contamination. Additionally, kombucha tea fermentation also produces a byproduct in the form of a pellicle composed of cellulose (Bacterial Cellulose, BC). Compared to plant cellulose, BC properties are more superior, which makes it industrially important. However, BC production at industrial scale has been faced with many challenges, including low yield and high fermentation medium cost. Many researchers have focused their studies on the use of alternative low cost media, such as molasses, which is a by-product of sugar refining process. To maximize the BC production in molasses medium, it is important to select the microbial strains that can grow and produce BC at high yield in molasses. This study aimed to isolate and characterize BC-producing bacteria and a dominant yeast from kombucha culture which had been previously adapted in molasses medium. The isolation of bacteria was performed using Nutrient Agar (NA) and Hestrin and Schramm (HS) supplemented with cycloheximide, while yeast was isolated using Potato Dextrose Agar (PDA) supplemented with chloramphenicol. The most dominant colonies were isolated and then subjected to microscopic observation for morphological analysis. The pure bacteria and yeast isolates were then identified by sequencing the 16S rRNA gene and D1/D2 region of the 26S rRNA, respectively. The bacteria isolates obtained were all from closely related genera: Komagataeibacter sp. DS1MA.62A, Komagataeibacter xylinus, Komagataeibacter saccharivorans, Komagataeibacter xylinus and Gluconacetobacter saccharivorans. The single isolated yeast was identified as Brettanomyces bruxellensis. This study helps to elucidate the BC-producing species which thrive in molasses medium for potential use in the BC production using molasses as alternative cheap carbon source. Also, the study revealed that the co-culture of Komagataeibacter sp. DS1MA.62A and B. bruxellensis could produce BC from molasses supplemented with caffeine and acetate buffer at an average yield of 27.7±1.83 g/L. Keywords: Kombucha, SCOBY, bacterial cellulose, Acetobacter, Komagataeibacter, Brettanomyces, alternative medium, molasses, caffeine, acetate buffer.