Proteomic characterization of peanut flour fermented by Rhizopus oryzae.

Fermentation alters the protein content and composition of foods. To characterize fungal catabolism of peanut proteins, defatted peanut flour was fermented by Rhizopus oryzae (R. oryzae) for up to 48 h and evaluated by SDS-PAGE, mass spectrometry, and antibody binding. A clear change in peanut protein migration was observed by SDS-PAGE after 16 h of fermentation. Mass spectrometric analysis indicated changes in allergen peptides and R. oryzae proteins. Several low molecular weight allergen fragments produced during fermentation were identified by mass spectrometry. Immunoassays using anti-peanut allergen antibodies demonstrated reduced allergen content as early as 16 h of fermentation. However, ELISA with peanut allergic IgE indicated only slightly reduced allergen binding even after 48 h. These results indicate that while R. oryzae fermentation efficiently metabolizes peanut allergens, significant IgE binding remains in lower molecular mass peptides, and therefore R. oryzae fermented peanut products would not be safe for peanut allergic individuals.

Transcriptome analysis of Rhizopus oryzae seed pellet formation using triethanolamine.

Rhizopus oryzae (R. oryzae) can effectively produce organic acids, and its pellet formation in seed cultures has been shown to significantly enhance subsequent fermentation processes. Despite advances in strain development, simple and effective methods for inducing pellet morphology and a basic understanding of the mechanisms controlling this process could facilitate substantial increases in efficiency and product output. Here, we report that 1.5% triethanolamine (TEOA) in seed culture medium can activate the growth of R. oryzae spores in compact and uniform pellets which is optimal for fermentation conditions. Analysis of fermentation kinetics showed that the production of fumaric and L-malic acid increases 293% and 177%, respectively. Transcriptomic analysis revealed that exposure of R. oryzae to 1.5% TEOA during the seed culture activated the phosphatidylinositol and mitogen-activated protein kinase signaling pathways. Theses pathways subsequently stimulated the downstream carbohydrate-active synthases and hydrolases that required for cell wall component synthesis and reconstruction. Our results thus provide insight into the regulatory pathways controlling pellet morphology germane to the viability of seed cultures, and provide valuable reference data for subsequent optimization of organic acid fermentation by R. oryzae.

Comparative host transcriptome in response to pathogenic fungi identifies common and species-specific transcriptional antifungal host response pathways.

Candidiasis, aspergillosis, and mucormycosis cause the majority of nosocomial fungal infections in immunocompromised patients. Using an unbiased transcriptional profiling in PBMCs exposed to the fungal species causing these infections, we found a core host response in healthy individuals that may govern effective fungal clearance: it consists of 156 transcripts, involving canonical and non-canonical immune pathways. Systematic investigation of key steps in antifungal host defense revealed fungal-specific signatures. As previously demonstrated, Candida albicans induced type I and Type II interferon-related pathways. In contrast, central pattern recognition receptor, reactive oxygen species production, and host glycolytic pathways were down-regulated in response to Rhizopus oryzae, which was associated with an ER-stress response. TLR5 was identified to be uniquely regulated by Aspergillus fumigatus and to control cytokine release in response to this fungus. In conclusion, our data reveals the transcriptional profiles induced by C. albicans, A. fumigatus, and R. oryzae, and describes both the common and specific antifungal host responses that could be exploited for novel therapeutic strategies.

Microwave-assisted extraction of chitosan from Rhizopus oryzae NRRL 1526 biomass.

Microwave-assisted extraction (MAE) of chitosan from dried fungal biomass of Rhizopus oryzae NRRL1526, obtained by culturing on potato dextrose broth (PDB), was performed and the optimal conditions required were identified using statistical analysis for the first time in this study. This microwave-assisted extraction (MAE) was compared against the conventional autoclave assisted method of chitosan extraction. The full factorial experimental design was used to investigate the impact of operating parameters of MAE, microwave power (100 W-500 W), and duration (10 min-30 min), on alkaline insoluble material (AIM) yield, chitosan yield, and degree of deacetylation (DDA). The effect of operating conditions was then evaluated using full factorial data analysis and optimum condition for MAE of chitosan was identified using response surface methodology to be 300 W and 22 min. This optimum condition identified was then further evaluated and the chitosan obtained characterized. Higher chitosan yield of 13.43 ± 0.3% (w/w) of fungal biomass was obtained when compared to that obtained, 6.67% ± 0.3% (w/w) of dry biomass, for the conventional extraction process. MAE yielded chitosan of higher degree of deacetylation, 94.6 ± 0.9% against 90.6 ± 0.5% (conventional heating), but the molecular weight was observed to be similar to that obtained by using conventional autoclave heating. MAE of chitosan was observed to yield a higher quantity of chitosan when compared to conventional extraction process and obtained chitosan exhibited a higher degree of deacetylation as well as molecular weight. The lower energy consumption of 0.11 kW h for MAE (5 kW h for conventional process) and the concomitant reduction in the energy bill to 1.1 cents from 50 cents, in addition to the above results, show that microwave irradiation is a more efficient and environment-friendly means to obtain chitosan from fungal biomass.

Efficient regioselective acylation of quercetin using Rhizopus oryzae lipase and its potential as antioxidant.

The present investigation describes the regioselective enzymatic acylation of quercetin with ferulic acid using Rhizopus oryzae lipase. Optimization of reaction parameters resulted in 93.2% yield of the ester synthesized using 750IU of lipase in cyclo-octane at a temperature of 45°C. The reaction was successfully carried out upto 25g scale. The ester synthesized was analyzed by (1)H Nuclear magnetic resonance spectroscopy. The ester synthesized (quercetin ferulate) showed higher antiradical activity as compared to ascorbic acid using the 2,2-diphenyl-1-picrylhydrazyl radical method. These results on enzyme-catalyzed acylation of quercetin might be used to prepare and scale-up other flavonoids derivatives.

Omics-based approaches reveal phospholipids remodeling of Rhizopus oryzae responding to furfural stress for fumaric acid-production from xylose.

In order to relieve the toxicity of furfural on Rhizopus oryzae fermentation, the molecular mechanism of R. oryzae responding to furfural stress for fumaric acid-production was investigated by omics-based approaches. In metabolomics analysis, 29 metabolites including amino acid, sugars, polyols and fatty acids showed significant changes for maintaining the basic cell metabolism at the cost of lowering fumaric acid production. To further uncover the survival mechanism, lipidomics was carried out, revealing that phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and polyunsaturated acyl chains might be closely correlated with R. oryzae's adapting to furfural stress. Based on the above omics analysis, lecithin, inositol and soybean oil were exogenously supplemented separately with an optimized concentration in the presence of furfural, which increased fumaric acid titer from 5.78g/L to 10.03g/L, 10.05g/L and 12.13g/L (increased by 73.5%, 73.8% and 110%, respectively). These findings provide a methodological guidance for hemicellulose-fumaric acid development.