Morphogenesis modulation of Rhizopus oryzae by peptone enhances lipase production during submerged fermentation

To establish a specific and sensitive TaqMan multiplex real-time quantitative PCR (RT-qPCR) assay to rapidly detect Mucorales. Customized oligonucleotide primers and corresponding detection probes were developed using the 18S ribosomal RNA gene sequences of Rhizomucor spp., Lichtheimia spp., and Mucor spp./Rhizopus spp. A multiplex RT-qPCR assay was established using TaqMan fluorescent probes, and its specificity, repeatability, and sensitivity were evaluated. The method was also used for the detection of simulated Mucorales-positive clinical samples and on samples from clinical patients possibly having fungal infections. The established TaqMan fluorescent probe-based multiplex RT-qPCR assay accurately detected Mucorales with high sensitivity, showing a minimum detection limit of 1fg/μL. The assay also showed high specificity and accurately detected organisms representing the four main genera in Mucorales (i.e., Rhizomucor miehei, Lichtheimia corymbifera, Mucor circinelloides, and Rhizopus oryzae). Additionally, no cross-reactivity was observed with 21 non-targeted strains (including viruses, bacteria, fungi, and Cunninghamella bertholletiae). The reproducibility of the assay was confirmed, with a relative standard deviation within groups of less than 1%. The assay was able to detect Mucorales pathogens (i.e., Rhizomucor spp., Lichtheimia spp., Mucor spp., and Rhizopus spp.) in simulated positive clinical specimens of blood, urine, lavage fluid, and sputum. Among 56 specimens from clinical patients with suspected fungal infections that were tested using the assay, one positive specimen was detected, consistent with the findings from sequencing and fungal culture identification. A highly specific and sensitive TaqMan fluorescent probe-based multiplex RT-qPCR detection method for Mucorales was established that has promising applications in the early and rapid diagnosis of Mucorales pathogens in clinical specimens.

β-Glucan from Tibetan highland barley (THB) is an excellent edible gel polysaccharide due to its unique hypoglycemic and antioxidant activities. However, direct extraction of β-glucan from THB exhibits low yields with higher costs. Given that highland barley spent grain (BSG) is a byproduct of the brewing process and is frequently considered waste, the efficient extraction of its β-glucan could promote high-value repurposing of BSG. In this study, 2.74% β-glucan (BSG-B) was extracted from Rhizopus oryzae (R. oryzae)-fermented BSG, which is lower than those from THB (THB-B: 4.62%) yet enabled value-added utilization of BSG. The molecular weight of BSG-B was 5.24 × 106 Da, which significantly increased by 124.89% compared to that of THB-B. Fourier-transform infrared (FT-IR) spectroscopy showed similar absorption peaks in BSG-B and THB-B, except for structural modifications in the β-glucan pyranose ring induced by the fermentation of R. oryzae. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) indicated that BSG-B possesses a more compact structure and lower aggregation heights compared to THB-B. Moreover, BSG-B demonstrated superior antioxidant capacities to THB-B in NO/DPPH/ABTS/reducing power assays, and lower apparent viscosity and oil adsorption capacity, likely attributed to the fermentation of R. oryzae. This study establishes a foundation for extracting higher-molecular-weight antioxidant β-glucan from BSG.

Mucormycosis is a life-threatening infection caused by fungi of the Mucorales order, typically associated with immunocompromised hosts, but increasingly reported in immunocompetent individuals. This study investigated fungal burden, Th1/Th17 inflammatory profiles, and organ-specific dynamics in immunocompetent BALB/c mice intravenously infected with Rhizopus oryzae, Mucor circinelloides, or Rhizomucor pusillus. Colony-forming units were quantified in spleen, liver, and kidney at multiple time points, while serum cytokines and oxidative stress markers were analyzed. The results showed fungal persistence primarily in the spleen, accompanied by species-specific Th1/Th17 responses: R. oryzae induced the highest inflammatory response among all groups, with maximal cytokine production observed on day 7, particularly for IL-17A (352.58 pg/mL). In contrast, M. circinelloides exhibited its peak cytokine levels earlier, reaching the highest TNF-α concentration on day 3 (425.43 pg/mL). Meanwhile, R. pusillus triggered an early but moderate inflammatory response, with a maximum TNF-α value of 372.62 pg/mL detected on day 1, followed by clearance. Correlation analysis highlighted distinct immunological patterns, with IL-10 acting as a negative regulator of inflammation, while TNF-α and IL-17A reflected infection intensity depending on species and timing. The spleen emerged as a key organ coordinating immune responses during systemic infection. These findings reveal that mucormycosis in immunocompetent hosts triggers complex, species-dependent immune dynamics beyond classical immunosuppression, emphasizing the need to consider host–pathogen interactions when developing targeted antifungal strategies.

Corn is one of the most widely cultivated cereal crops and is rich in antioxidant compounds, especially phenolics. However, many of these are bound to cell wall components, requiring pre-treatment for release. Solid-state fermentation (SSF) with Rhizopus oryzae has been used to enhance antioxidant capacity in grains and legumes, though its application in pigmented corn (PC) has not been reported. This study evaluated R. oryzae growth on PC via SSF and its effect on phenolic compound release and antioxidant capacity (AC). Variables such as temperature, pH, inoculum, and medium salts were tested for their influence on phenolic release and AC. Nutrient changes in PC due to SSF were also examined. HPLC-MS was used to analyze the phenolic compounds’ profile. R. oryzae grew effectively on PC, increasing total phenolic content (TPC) and AC by 131 and 50%, respectively. The pH was found to negatively impact phenolic release. The SSF also raised protein content by 10% and reduced carbohydrates and fiber by 3 and 8%. Thirteen phenolic compounds were identified, including Feruloyl tartaric acid ester and p-Coumaroyl tartaric acid glycosidic ester, with known anti-inflammatory properties. This process offers a sustainable method for enhancing the functional properties of pigmented corn.

The growing demand for aquaculture has driven the search for sustainable practices and utilization of agro-industrial residues. Brown rice bran, an abundant and low-cost by-product, has emerged as a promising raw material. This dissertation aimed to evaluate solid-state fermentation (SSF) of rice bran using the fungus Rhizopus oryzae and the yeast Saccharomyces cerevisiae with the goal of improving its nutritional value for use in diets formulated for zebrafish (Danio rerio). Proximate composition analyses revealed the strong biotransformation potential of Rhizopus oryzae. Fermentation with this fungus resulted in a significant 36.45% increase in protein content, a 51.62% increase in total polyphenols, and a 13.7% reduction in lipid content. In an in vivo experiment, zebrafish fed a diet containing rice bran fermented by R. oryzae showed the best zootechnical performance, with higher weight gain, specific growth rate, and improved feed conversion. Gene expression analysis showed a significant difference only for glut6, which is related to glucose transport. In summary, the fermentation of brown rice bran with Rhizopus oryzae represents an effective strategy to enhance its nutritional profile, establishing it as a viable alternative for the formulation of more sustainable and efficient diets in aquaculture.

Commercial fungal lipases from Rhizopus oryzae, Rhizopus niveus, Aspergillus niger, Rhizomucor miehei, and Candida rugosa were immobilized via physical adsorption onto Accurel MP 1000, a hydrophobic polypropylene support. The effects of enzyme concentration, pH, temperature, and glutaraldehyde post-treatment were systematically evaluated. Immobilization generally enhanced enzyme stability, which was further improved in several cases by glutaraldehyde crosslinking. The immobilized preparations retained over 50% of their initial activity for 3-6 cycles, and 7-10 cycles following glutaraldehyde treatment. While soluble enzymes lost nearly all activity within three months at 5 °C and 25 °C and retained only 5-20% at -20 °C, the immobilized forms preserved 50-100% of their activity under all storage conditions tested. Immobilized lipases also exhibited improved thermal stability at 60 °C by general increments between 1.3 and 1.8 times compared to soluble lipases. Increased tolerance to pH fluctuations was observed in most immobilized enzymes, particularly from R. oryzae, R. niveus, R. miehei, and C. rugosa. Organic solvent tolerance of the immobilized enzymes showed highest stability in hexane (66-100% residual activity after 4 h incubation). Glutaraldehyde treatment affected solvent stability of immobilized lipases in enzyme and solvent dependent manner. These findings demonstrate the improved stability and applicability of the produced biocatalysts in varying reaction environments.

The research presented in this manuscript aimed to conduct complex studies on epoxy composites filled with modified biowaste. This work discusses the entire process: first, the preparation and analysis of oak waste flour used as an additive to epoxy materials based on Epidian 6; then, obtaining and characterizing epoxy composites containing 5 wt.% of biowaste; and finally, the determination of the influence of wood filler and modification performed using selected ionic liquids (tetradecyltrihexylphosphonium bis(trifluoromethylsulfonyl)amide (IL-1), tetradecyltrihexylphosphonium bis(2,4,4-trimethylpentyl)phosphinate (IL-2), and 1-ethyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)imide) (IL-3)) on the susceptibility of epoxy composite to the enzymatic degradation. The enzymatic degradation was performed for four weeks using the lipase enzymes (Porcine Pancreas and Rhizopus Oryzae). The epoxy composition EP6-WF_IL-2, containing 5 wt.% of wood flour modified with IL-2, was characterized by the best mechanical parameters in terms of bending strength and flexural modulus (65.64 MPa and 1855.3 MPa, respectively). During enzymatic biodegradation, the lowest susceptibility to enzymatic degradation, regardless of the incubation conditions, was observed in samples of EP6-WF_IL-3 epoxy composition containing wood flour modified with methyltrioctylammonium bis(trifluoromethylsulfonyl)imide.

Arabica coffee is recognized as a high-value plantation commodity, favored for its distinctive flavor profile. However, its relatively high caffeine content can cause certain side effects in some individuals, such as sleep disturbances or increased blood pressure. One approach to reducing caffeine content without compromising flavor quality is fermentation using microorganisms. Rhizopus oryzae is known to produce enzymes capable of altering the chemical composition of coffee, making it a potential method for lowering caffeine levels while enhancing sensory quality. This study aimed to examine changes in physical properties and caffeine content of Arabica coffee powder subjected to fermentation with Rhizopus oryzae at concentrations of 1%, 2%, 3%, and 4% w/w, compared to a non-fermented control sample. The fermentation process was carried out for 24 hours at room temperature. Analyses included organoleptic evaluation, moisture content measurement, and caffeine determination using UV-Vis spectrophotometry. Results indicated that fermentation with Rhizopus oryzae at a 4% concentration yielded the most optimal outcome, with caffeine reduced to 0.95%, moisture content at 0.94%, and a final pH of 4.8. The brewed coffee from this treatment also exhibited a distinctive aroma, light brown color, and a balanced bitterness. Overall, fermentation with Rhizopus oryzae proved effective in reducing caffeine levels while maintaining and improving the sensory quality of Arabica coffee.

Abstract This study investigated effects of mold fermentation on the texture and flavor of soy-nut cheese analogs (SNCA) using Mucor wutungkiao (SNCA-M), Rhizopus oryzae (SNCA-R), and their combination (SNCA-M+R) over 20-day fermentation period. The results found that all cheese analogs showed decreasing trends in protein content, fat content, pH, springiness, and cohesiveness throughout fermentation. Notably, the moduli, hardness, and chewiness peaked on day 3 before declining. The rate of these changes was more pronounced in SNCA-M+R than in SNCA-M or SNCA-R. All samples achieved their higest sensory scores on day 5 of fermentation. At this stage, the fermented analogs showed significantly higher free amino acid (FFA) content and volatile flavor compound concentrations, along with reduced levels of beany flavor substances compared to unfermented controls. Among the variants, SNAC-M+R displayed the highest sensory score and FFAs content, as well as the lowest concentration of beany flavor compounds.

The insoluble dietary fiber (IDF) of pomegranate peel residue was treated by mixed solid-state fermentation (SSF) of Aspergillus niger and Rhizopus oryzae. The content of bound polyphenols (BP) reached a maximum of 26.50 mg GAE/g DW after 5 days of fermentation, which was 3.22-fold higher than that of the unfermented group. The BP release during fermentation was strongly correlated with the activities of CMCase, xylanase and β-glucosidase. A total of 35 polyphenolic compounds and catabolic metabolites were identified, and potential biotransformation pathways were proposed. Structural characterization of IDF before and after fermentation via SEM, XRD, and TGA revealed that the degradation of cellulose and disruption of hemicellulose and hemicellulose-lignin complexes played a key role in BP release. Moreover, the postfermentation BP in vitro showed enhanced antioxidant capacity and pronounced inhibition of α-amylase/α-glucosidase. Collectively, the mixed SSF significantly promote the BP release, facilitating pomegranate peel valorization.

Abstract Brewer’s spent grain (BSG) is a protein- and fiber-rich biomass with minimal food functionality that limits its usability in foods. Solid-state fermentation (SSF) of BSG using the filamentous fungus Rhizopus oryzae increased the protein content from 16.8 to 23.3% of which 95% is mycoprotein. Alkaline extraction combined with ultrasonication was used to produce protein extracts from BSG and fermented BSG (F-BSG) having 44.9% and 46.2% protein content, respectively. In addition, the protein extracts had a lower total fiber content while composition of fibers changed to have a higher proportion of soluble fibers than insoluble fibers. LC–MS/MS analysis provided a detailed biophysical characterization of the proteins to shed light on the differences inferred by fermentation and protein extraction. This revealed an enrichment in the F-BSG protein extract of the mycoprotein “hydrophobic surface binding protein” that contributes hydrophobic properties to the sample. BSG and F-BSG exhibited poor protein solubility (below 15%) in the pH range 3–8, while the F-BSG protein extract displayed very high protein solubility (85%). The F-BSG extract stood out from the non-fermented and non-extracted samples in terms of smaller particle size, higher surface hydrophobicity, and improved oil holding capacity. Furthermore, the F-BSG protein extract displayed improved functionality by producing 534% foam overrun with desired bubble morphology and by stabilizing a coarse emulsion. In summary, by combining detailed characterization and functional analyses, we have shown the potential of SSF using a food-grade fungi combined with protein extraction as a bioprocessing technology to upcycle side streams into climate-neutral protein-rich ingredients.

Potatoes (Solanum tuberosum L) are nourishing tubers, highly perishable and susceptible to microbial spoiling and waste due to a lack of adequate storage strategies. This study isolated and identified the fungus species responsible for Solanum tuberosum rotting and determined the pathogenicity of each fugal isolate on healthy Irish potato tubers. Total of thirty (30) spoiled Irish potato tubers were collected from three (3) separate sellers: Badariya, Bello Way, and Gwadangaji areas of Birnin Kebbi. Serial dilution of the samples were performed and inoculated on sterile PDA medium using pour plate method of inoculation. The plates were incubated at room temperature (270C) for six (6) days. The isolates were identified based on their macroscopic and microscopic characteristics. The total fungal count for Bello Way, Badariya, and Gwadangaji was 5.7 ± 4.05, 5.7 ± 3.52, and 5.0 ± 2.65 TFC/g, respectively. Mold isolates discovered were Aspergillus niger 9 (39.13%), Fusarium solani 6 (26.09%), Penicillium digitatum 4 (17.39%), Rhizopus oryzae 1 (4.34%), and Mucor spp 2 (8.70%). Among the other isolates, Aspergillus niger had the highest frequency and percentage of 9 (39.13%). The five fungal isolates had varying average degrees of pathogenicity, with Aspergillus niger causing the most extensive and rapid spoiling (6.8 cm), followed by Fusarium solani (6.2 cm), Penicillium digitatum (6.0 cm), Mucor spp. (4.6 cm), and Rhizopus oryzae (4.5 cm). In conclusion, mold is responsible for the spoilage of Irish potatoes marketed within Birnin Kebbi; therefore, proper handling of potato tubers is required to mitigate fungal infection.

Ions and low molecular weight components in sulfite pulp mills strongly reduce or boost lipases activity on lipophilic wood extractives.

Molecular arrangements that accompany binding of rice xylanase inhibitor protein OsXIP and the Rhizopus oryzae GH11 xylanase RXyn2.

The objective of this study was the evaluation of fungal solid-state fermentation (SSF) for the production of alginate lyase and extraction of uronic acids from Sargassum sp. For this purpose, the fungi Trichoderma asperellum, Aspergillus oryzae, and Rhizopus oryzae were applied (alone or combined) to Sargassum sp. biomass through SSF (107 spores gbiomass−1, 30 °C, and 7 days of treatment). In general, individual SSF with all three fungi degraded the biomass, achieving a marked synergy in the production of cellulase, laminarinase, and alginate lyase activities (especially for the last one). Trichoderma was the most efficient species in producing laminarinase, whereas Rhizophus was the best option for producing alginate lyase. However, when dual combinations were tested, the maximal values of alginate lyase activities were reached (13.4 ± 0.2 IU gbiomass−1 for Aspergillus oryzae and Rhizopus oryzae). Remarkably, uronic acids were the main monomeric units from algal biomass solubilization, achieving a maximum yield of 14.4 mguronic gbiomass−1, with the A + R condition being a feasible, eco-friendly alternative to chemical extraction of this monomer. Additionally, the application of all the fungal pretreatments drastically decreased the total phenolic content (TPC) in the biomass from 369 mg L−1 to values around 44–84 mg L−1, minimizing the inhibition for possible subsequent biological processes in which the residual solid can be used.

This study aims to develop tempeh (Rhizopus oryzae) into tempeh flour to be utilized as the main ingredient in the production of a traditional Indonesian cookie known as kastengel. Tempeh is a rich source of plant-based protein and dietary fiber, yet its application in the processed food industry, particularly in dry baked goods such as cookies, remains underexplored. This innovation is expected to serve as a healthy food alternative while supporting the diversification of local food products. The research employed a Research and Development (R&D) approach, encompassing several key stages: processing tempeh into flour through slicing, drying, grinding, and sieving; formulating kastengel dough using tempeh flour as the base ingredient; and evaluating the final product through both organoleptic (sensory) testing and nutritional analysis. Organoleptic testing involved 10 panelists, including lecturers and students, who assessed the product based on color, taste, aroma, and texture. Nutritional content was analyzed at the Manado Industrial Research and Standardization Agency. The results showed that kastengel made from tempeh flour had a light brown color, a savory-sweet flavor, a distinctive yet pleasant aroma, and a crunchy texture. Laboratory tests revealed the nutritional composition of the product to include 46.12% carbohydrates, 29.82% fat, and 3.92% protein, indicating its potential as a nutritious food option. Most panelists reported a favorable response toward the product. In conclusion, tempeh flour can serve as an effective substitute for wheat flour in the production of kastengel cookies. The final product, branded as “Kastengel Eka,” demonstrates that tempeh-based food innovations can be well-accepted in terms of sensory qualities and possess promising potential as a functional, nutritious, and marketable food product.

Tea production and processing enterprises in several industrialized and developing nations are generating a huge byproduct and solid waste, thereby posing a distress and pervasive issues. Consequently, the global research community has started focusing on the exploration of sustainable ways that would repurpose tea waste (TW) in numerous utilizations. This research focuses on the beneficial usage of TW biomass in bioethanol production along with the sustainable reutilization of tea industry waste in a sustainable manner. Bioethanol is a clean and renewable energy resource that has gained escalated concentration as a prospective substitute for non-renewable and conventional petroleum-based fuels. In this research field waste, industrial waste and spent tea waste of Camelia sinensis were explored for their potential to produce bioethanol by Kluveromyces marxianus NCIM 3465 and Rhizopus oryzae NCIM 1387. It was found that both Kluveromyces marxianus NCIM 3465 and Rhizopus oryzae NCIM 1387 produced maximum ethanol (437.69 ± 1.22 mg ml–1 and 500.87 ± 2.42 mg ml–1 respectively) from 10 per cent (wt/v) HNO3 pretreated field waste. Kluveromyces marxianus NCIM 3465 produced maximum amount of ethanol 341.45 ± 2.02 mg ml–1 whereas Rhizopus oryzae NCIM 1387 (F) produced maximum amount of ethanol 388.14 ± 2.82 mg ml–1, respectively in 6 and 10 per cent (wt/v) HNO3 pretreated industrial waste. Maximum amount of ethanol was generated from spent tea waste by Kluveromyces marxianus NCIM 3465 and Rhizopus oryzae NCIM 1387 when this waste was pretreated with 6 per cent (wt/v) HNO3 viz. 515.82 ± 3.80 and 499.86 ± 2.72 mg ml–1, respectively

Objectives. The work set out to describe conditions for the purification of a model fermentation broth for cultivating the lactic acid-containing micromycete Rhizopus oryzae from impurities of inorganic salts using ion-exchange resins under dynamic conditions. Methods. The solutions collected for analysis were examined using precipitation titration to determine the concentration of chlorides along with a qualitative reaction with Nessler’s reagent to ascertain the presence of ammonium ions. The concentration of lactic acid was evaluated spectrophotometrically using iron(III) chloride. The total nitrogen content was determined by high-temperature catalytic combustion on a Formacs HT TOC/TN Analyzer (Netherlands). The content of trace elements and macroelements in the samples was determined using an iCAP 6300 Duo inductively coupled plasma emission spectrometer (United Kingdom). Results. Purification of the model broth under the described conditions was carried out by successive filtration through the cation exchanger KU-2-8 in the H-form and subsequently through a mixture of weakly basic A847 and strongly basic AV-17-8 anion exchangers in the OH-form taken in a one-to-one ratio. The breakthrough of impurity ions into the solution was shown to occur after passing 30-fold and 10-fold volumes of the model broth relative to the volume of the cation-exchange and anion-exchange resins, respectively. The dynamic exchange capacity prior to breakthrough was determined as follows: 0.35 mmol-eq/cm3 for the anion-exchange column and 1.61 mmol-eq/cm3 for the cation-exchange column. The following parameters were defined as column regeneration modes: 3-fold excess of 2 M H2SO4, 10-fold excess of distilled H2O for cation exchange; for anion exchange, 3-fold excess of 2 M NaOH and 20-fold excess of H2O. Conclusions. The conducted studies showed that purification of the model fermentation broth of Rhizopus oryzae can be successfully implemented using ion-exchange resins. The model fermentation broth passing successively through cation-exchange and anion-exchange columns was shown to be purified from impurities of mineral salts while maintaining the concentration of lactic acid.

Structure-activity relationship studies of tanzawaic acid A, an antifungal agent against Rhizopus oryzae.