Jyotiranjan Bal

Mycoflora dynamics analysis of Korean traditional wheat-based nuruk.

The growing popularity of traditional Korean alcoholic beverages has led to a demand for quality enhancement of the traditional starter culture nuruk, which consists primarily of wheat. Therefore, this study focused on mycoflora characterization and the temporal variations in traditional wheat-based nuruks fermented at two representative traditional temperature conditions for 30 days. Nuruk A was fermented at a constant temperature of 36°C for 30 days and nuruk B was fermented at a high initial temperature of 45°C for 10 days followed by 35°C for 20 days. The average mycoflora load in the two different nuruk conditions did not vary significantly between the 0 and 30 day cultures, and a maximum load of 8.39 log CFU/g was observed for nuruk A on culture day 3 and 7.87 log CFU/g for nuruk B on culture day 30. Within two samples, pH was negatively correlated with temporal changes in mycoflora load. The pH of nuruk A was significantly lower than that of nuruk B at all of the time points evaluated. Culture-dependent characterization led to the identification of 55 fungal isolates belonging to 9 genera and 15 species, with the most prominent genera comprising Lichtheimia, Penicillium, Trametes, Aspergillus, Rhizomucor, and Mucor. A total of 25 yeast isolates were characterized belonging to 6 genera and 7 species, the most prominent among which were Rhodotorula, Pichia, Debaryomyces, Saccharomycopsis, and Torulospora. Mycofloral community dynamics analysis revealed that both samples A and B varied considerably with respect to the fungal communities over a span of 30 days.

Pyrosequencing reveals bacterial diversity in Korean traditional wheat-based nuruk

The emerging global importance of Korea’s alcoholic beverages emphasizes the need for quality enhancement of nuruk, a traditional Korean cereal starter that is used extensively in traditional brewing. Apart from fungi and yeasts, bacteria known to be ubiquitously present are also a part of the nuruk ecosystem and are known to influence fermentation activity by influencing fermentation favorable factors. In the current study, bacterial diversity and temporal variations in the traditional wheat-based nuruk, fermented at two representative temperature conditions for 30 days, along with two commercial wheat-based nuruk samples for comparison analysis were evaluated using libraries of PCR amplicons and 454 pyrosequencing targeting of the hypervariable regions V1 to V3 of the 16S rRNA gene. A total of 90,836 16S reads were analyzed and assigned to a total of 314, 321, and 141 Operational Taxonomic Units (OTUs) for nuruk A, B, and C, respectively. Diversity parameters clearly indicated nuruk B to be more diverse in terms of bacterial composition than nuruk A. Taxonomic assignments indicated that nuruk A was dominated by phylum Cyanobacteria, whereas nuruk B was dominated by phylum Actinobacteria. For both nuruk A and B, members of the phylum Firmicutes mostly converged into the family Bacillaceae; these microorganisms might be present in negligible numbers at the beginning but became significant as the fermentation progressed. The commercial samples were predominated by phylum Firmicutes, which is composed of Lactobacillaceae and Leoconostocaceae. The findings of this study provide new insights into understanding the changes in bacterial community structure during traditional nuruk starter production.

Taxonomic Characterization, Evaluation of Toxigenicity, and Saccharification Capability of Aspergillus Section Flavi Isolates from Korean Traditional Wheat-Based Fermentation Starter Nuruk

Abstract The most economically important species used in a wide range of fermentation industries throughout Asia belong to Aspergillus section Flavi, which are morphologically and phylogenetically indistinguishable, with a few being toxigenic and therefore a major concern. They are frequently isolated from Korean fermentation starters, such as nuruk and meju. The growing popularity of traditional Korean alcoholic beverages has led to a demand for their quality enhancement, therefore requiring selection of efficient non-toxigenic strains to assist effective fermentation. This study was performed to classify the most efficient strains of Aspergillus section Flavi isolated from various types of traditional wheat nuruk, based on a polyphasic approach involving molecular and biochemical evaluation. A total of 69 strains were isolated based on colony morphology and identified as Aspergillus oryzae/flavus based on internal transcribed spacer and calmodulin gene sequencing. Interestingly, none were toxigenic based on PCR amplification of intergenic regions of the aflatoxin cluster genes norB-cypA and the absence of aflatoxin in the culture supernatants by thin-layer chromatography analysis. Saccharification capability of the isolates, assessed through α-amylase and glucoamylase activities, revealed that two isolates, TNA24 and TNA15, showed the highest levels of activity. Although the degrees of variation in α-amylase and glucoamylase activities among the isolates were higher, there were only slight differences in acid protease activity among the isolates with two, TNA28 and TNA36, showing the highest activities. Furthermore, statistical analyses showed that α-amylase activity was positively correlated with glucoamylase activity (p < 0.001), and therefore screening for either was sufficient to predict the saccharifying capacity of the Aspergillus strain.

Global DNA Methylation in the Chestnut Blight Fungus Cryphonectria parasitica and Genome-Wide Changes in DNA Methylation Accompanied with Sectorization

Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK) of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase), demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

Effects of initial moisture content of Korean traditional wheat-based fermentation starter nuruk on microbial abundance and diversity

The brewing of makgeolli, one of Korea’s most popular alcoholic beverages that is gaining popularity globally, is facilitated by nuruk, a traditional Korean cereal starter. The nuruk microbiome greatly influences the fermentation process as well as the nutritional, hygienic, and aromatic qualities of the product. This study is a continuation of our efforts to examine nuruk biodiversity at a depth previously unattainable. In this study, microfloral dynamics in wheat-based nuruk C, composed of traditional ingredients such as barley, green gram, and wheat and fermented under various internal moisture contents of 20% (C20), 26% (C26), and 30% (C30), was evaluated using 454 pyrosequencing during the 30-day fermentation process. Rarefaction analysis and alpha diversity parameters indicated adequate sampling. C20 showed the greatest fungal richness and diversity, C20 and C26 exhibited similar bacterial richness and diversity, while C30 had low fungal and bacterial richness. Fungal taxonomic assignments revealed that the initial moisture content caused selective enrichment of Aspergillus candidus with a decreasing trend during fermentation, whereas Saccharomycetales sp. exhibited increasing relative abundance with increasing moisture content from day 6 of the fermentation process. Depending on initial moisture level, changes in bacterial communities were also observed in the genera Streptomyces, Bacillus, and Staphylococcus, with decreasing trends whereas Saccharopolyspora exhibited a sigmoidal trend with the highest abundance in C26. These findings demonstrate the possible impact of initial moisture content of nuruk on microfloral richness, diversity, and dynamics; this study is thus a step toward our ultimate goal of enhancing the quality of nuruk.

Evaluation of cell-surface displayed synthetic consensus dengue EDIII cells as a potent oral vaccine candidate

BackgroundDengue is a rapidly spreading mosquito borne tropical viral disease affecting hundreds of millions of people across the globe annually. The dengue virus (DENV) includes four genetically distinct serotypes that cause serious life-threatening infections, including dengue hemorrhagic fever/dengue shock syndrome. Dengue vaccine development is complicated by the possibility of vaccine-enhanced severe dengue disease due to antibody-dependent enhancement by pre-existing cross-reactivity, as well as homotypic antibodies. Thus, the development of an efficacious dengue vaccine conferring simultaneous and durable immunity to each of the four DENV serotypes has not yet been developed despite years of research. For mass immunization in deeply affected resource-limited countries, oral vaccination is considered more beneficial than conventional approaches. Therefore, in a continuing effort towards designing economical and potent vaccine candidates, the current study applied yeast surface display technology to develop an oral dengue vaccine candidate using whole recombinant yeast cells displaying the recombinant fusion protein of M cell targeting ligand Co1 fused to the synthetic consensus dengue envelope domain III (scEDIII). Female Balb/c mice were orally fed with recombinant yeast cells and immunogenicity in terms of systemic and mucosal immune responses was monitored.ResultsImmunofluorescence microscopy with dengue specific antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG antibody clearly showed that recombinant protein Co1-scEDIII-AGA was localized on the cell surface of the respective clones in comparison with scEDIII-Co1 and Mock cells with no fluorescence. Oral dosage applications of surface displayed Co1-scEDIII-AGA stimulated a systemic humoral immune response in the form of dengue-specific serum IgG, as well as a mucosal immune response in the form of secretory immunoglobulin A (sIgA). Antigen-specific B cell responses in isolated lymphoid cells from the spleen and Peyer’s patches further supported an elevated mucosal immune response. In addition, surface displayed Co1-scEDIII-AGA feeding elicited strong immune responses in comparison with scEDIII-Co1 and Mock following intraperitoneal booster with purified scEDIII antigen.ConclusionsSurface displayed preparations of Co1-scEDIII-AGA induced strong immunogenicity compared with non-displayed scEDIII-Co1. Prior studies have supported the neutralization potential of scEDIII constructs against all four serotypes. Thus, the oral administration of genetically engineered yeast whole cells displaying biologically active Co1-scEDIII fusion protein without any further processing shows prospective as a potent oral vaccine candidate against dengue viral infection.