Fabrication and characterization of Rhizochitosan and its incorporation with platelet concentrates to promote wound healing.

Composite dressing composed of Rhizochitosan and Regenplex™ to promote wound healing were assessed. Rhizochitosan was fabricated by deacetylation of Rhizochitin, which obtained by simply depigmenting sporangium-free mycelial mattress produced from Rhizopus stolonifer F6. Physicochemical characterizations of Rhizochitosan demonstrated that it contained 13.5% chitosan with a water-absorption ability of 35-fold dry weight and exhibiting hydrogel nature after hydration. In a wound-healing study on SD rats with full-thickness injury, the composite dressing had a better healing effect than those for each individual components and control group and wound even healed as functional tissue instead of scar tissue. The underlying mechanism of the composite beneficial to wound remodeling is likely attributable to a more reduction level of matrix metalloproteinase (MMP)-9 expression in early stage and a higher MMP-2 expression level in a later stage of healing process. Conclusively, the composite dressing demonstrated to be highly beneficial to the healing of full-thickness injury wound.

A phenylalanine dynamic switch controls the interfacial activation of Rhizopus chinensis lipase.

The catalytic mechanism of most lipases involves a step called "interfacial activation" which significantly increases lipases activity beyond the critical micellar concentration (CMC) of substrate. In the present study, Rhizopus chinensis lipase (RCL) was used as a research model to explore the mechanism of lipase interfacial activation beyond the CMC. Molecular dynamic (MD) simulations indicated the open- and closed-lid transitions and revealed that Phe113 was the critical site for RCL activation by its dynamic flipping. Such putative switch affecting interfacial activation has not been reported in lipase so far. The function of Phe113 was subsequently verified by mutation experiments. The F113W mutant increases the lipase catalytic efficiency (1.9 s-1·µM-1) to 280% at the optimum temperature (40 °C) and pH 8.5 with the addition of 0.12 µg protein in the 200 µL reaction system. MD simulations indicated that the fast flipping rate from the closed to the open state, the high open state proportion, and the exposure of the catalytic triad are the main reasons for the lipase activation. The mutual corroboration of simulations and site-directed mutagenesis results revealed the vital role of Phe113 in the lipase activation.

Mucoricin is a ricin-like toxin that is critical for the pathogenesis of mucormycosis.

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Fungal mediated biotransformation of melengestrol acetate, and T-cell proliferation inhibitory activity of biotransformed compounds.

Glomerella fusaroide, and Rhizopus stolonifer were effectively able to transform the steroidal hormone melengestrol acetate (MGA) (1) into four (4) new metabolites, 17α-acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (2), 17α-acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-1,4,6-triene-3,20-dione (3), 17α-acetoxy-6,7α-epoxy-6ß-methyl-16-methylenepregna-4,6-diene-3,20-dione (4), and 17α-acetoxy-11ß,15ß-dihydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (5). All these compounds were structurally characterized by different spectroscopic techniques. The objective of the current study was to assess the anti-inflammatory potential of melengestrol acetate (1), and its metabolites 2-5. The metabolites and the substrate were assessed for their inhibitory effects on proliferation of T-cells in vitro. The substrate (IC50 = 2.77 ± 0.08 µM) and its metabolites 2 (IC50 = 2.78 ± 0.07 µM), 4 (IC50 = 2.74 ± 0.1 µM), and 5 (IC50 = < 2 µM) exhibited potent T- cell proliferation inhibitory activities, while compound 3 (IC50 = 29.9 ± 0.09 µM) showed a moderate activity in comparison to the standard prednisolone (IC50 = 9.73 ± 0.08 µM). All the metabolites were found to be non-toxic against 3T3 normal cell line. This study thus identifies some potent compounds active against T-cell proliferation. Their anti-inflammatory potential, therefore, deserves to be further investigated.

Rhizopus oligosporus and Lactobacillus plantarum Co-Fermentation as a Tool for Increasing the Antioxidant Potential of Grass Pea and Flaxseed Oil-Cake Tempe.

Tempe-type fermentation originating from Indonesia can enhance the antioxidant activity of plant material. However, this biological potential depends on substrates and applied microorganisms. This study aimed to determine whether co-fermentation with Rhizopus oligosporus and Lactobacillus plantarum improved antioxidant activity of tempe obtained from grass pea seeds with flaxseed oil-cake addition (up to 30%). For this purpose, substances reacting with Folin-Ciocalteu reagent and free radicals scavenging potential were measured in water-soluble fractions and dialysates from simulated in vitro digestion. Additionally, the water-soluble phenolic profile was estimated. The higher level of water-extractable compounds with antioxidant activity was determined in co-fermentation products than in fungal fermentation products. Moreover, the fermentation process with the use of L. plantarum contributed to a greater accumulation of some phenolic acids (gallic acid, protocatechuic acid) in tempe without having a negative effect on the levels of other phenolic compounds determined in fungal fermented tempe. During in vitro digestion simulating the human digestive tract, more antioxidant compounds were released from products obtained after co-fermentation than fungal fermentation. An addition of 20% flaxseed oil-cake and the application of bacterial-fungal co-fermentation, can be considered as an alternative tool to enhance the antioxidant parameters of grass pea tempe.

Signaling pathways associated with macrophage-activating polysaccharide isolated from the fermentation liquor of Rhizopus nigricans.

Our previous reports showed that the structural features and immunologic enhancement of polysaccharide (EPS1-1) from Rhizopus nigricans. However, the molecular mechanism in cellular immunomodulatory of EPS1-1 remains unclear. Here the experiments for the molecular mechanisms of EPS1-1 on the peritoneal macrophages were performed. The results demonstrated that the expression of TLR4 was significantly improved by EPS1-1. Subsequently, the phosphorylation of p38MAPK, ERK1/2, JNK and IKKα/ß were promoted. Moreover, EPS1-1 enhanced the expressions of IL-2, TNF-α and iNOS in EPS1-1-induced macrophages which were pretreated with MAPK signaling pathway inhibitors, and reduced the blocking effects of the inhibitors to the expressions of p-p38MAPK, p-ERK1/2 and p-IKKα/ß. Therefore, these results illustrated that EPS1-1 could improve the immune functions of peritoneal macrophages by promoting the gene expressions of IL-2, TNF-α and iNOS via the MAPK and NF-κB signaling pathways.

Selective inhibition of Rhizopus eumelanin biosynthesis by novel natural product scaffold-based designs caused significant inhibition of fungal pathogenesis.

Melanin is a dark color pigment biosynthesized naturally in most living organisms. Fungal melanin is a major putative virulence factor of Mucorales fungi that allows intracellular persistence by inducing phagosome maturation arrest. Recently, it has been shown that the black pigments of Rhizopus delemar is of eumelanin type, that requires the involvement of tyrosinase (a copper-dependent enzyme) in its biosynthesis. Herein, we have developed a series of compounds (UOSC-1-14) to selectively target Rhizopus melanin and explored this mechanism therapeutically. The compounds were designed based on the scaffold of the natural product, cuminaldehyde, identified from plant sources and has been shown to develop non-selective inhibition of melanin production. While all synthesized compounds showed significant inhibition of Rhizopus melanin production and limited toxicity to mammalian cells, only four compounds (UOSC-1, 2, 13, and 14) were selected as promising candidates based on their selective inhibition to fungal melanin. The activity of compound UOSC-2 was comparable to the positive control kojic acid. The selected candidates showed significant inhibition of Rhizopus melanin but not human melanin by targeting the fungal tyrosinase, and with an IC50 that are 9 times lower than the reference standard, kojic acid. Furthermore, the produced white spores were phagocytized easily and cleared faster from the lungs of infected immunocompetent mice and from the human macrophages when compared with wild-type spores. Collectively, the results suggested that the newly designed derivatives, particularly UOSC-2 can serve as promising candidate to overcome persistence mechanisms of fungal melanin production and hence make them accessible to host defenses.

Exopolysaccharides isolated from Rhizopus nigricans induced colon cancer cell apoptosis in vitro and in vivo via activating the AMPK pathway.

Colorectal cancer (CRC) is a leading cause of cancer-related human deaths. The exopolysaccharide (EPS1-1), isolated from Rhizopus nigricans, has been described as exhibiting anti-tumor and pro-apoptotic activity against CRC, although the underlying mechanism is poorly understood. Herein, we investigate how EPS1-1 induces apoptosis of CRC cells in vitro and in vivo. Our results show that, in vitro, EPS1-1 suppressed cell growth and facilitated apoptosis in a dose- and time-dependent manner by activating the AMP-activated protein kinase (AMPK) pathway in mouse colon cancer CT26 cells. However, treatment with small interfering RNAs (siRNAs) targeting AMPKα or with compound C, an AMPK inhibitor, interfered with the pro-apoptosis effects of EPS1-1. We also show that EPS1-1 initiated the release of reactive oxygen species (ROS) and liver kinase B1 (LKB1), both of which are necessary signals for AMPK activation. Furthermore, EPS1-1-mediated apoptosis is regulated by inactivation of mammalian target of rapamycin complex 1 (mTORC1) and activation of the jun-NH2 kinase (JNK)-p53 signaling axis dependent on AMPK activation. In vivo, azoxymethane/dextran sulfate sodium (AOM/DSS)-treated CRC mice, when administered EPS1-1, exhibited activation of the AMPK pathway, inhibition of mTORC1, and accumulation of p53 in tumor tissues. Collectively, these findings suggest that EPS1-1-induced apoptosis relies on the activation of the AMPK pathway. The present study provides evidence suggesting that EPS1-1 may be an effective target for development of novel CRC therapeutic agents.

Genome Mining and Heterologous Expression Reveal Two Distinct Families of Lasso Peptides Highly Conserved in Endofungal Bacteria.

Genome mining identified the fungal-bacterial endosymbiosis Rhizopus microsporus-Mycetohabitans (previously Burkholderia) rhizoxinica as a rich source of novel natural products. However, most of the predicted compounds have remained cryptic. In this study, we employed heterologous expression to isolate and characterize three ribosomally synthesized and post-translationally modified peptides with lariat topology (lasso peptides) from the endosymbiont M. rhizoxinica: burhizin-23, mycetohabin-16, and mycetohabin-15. Through coexpression experiments, it was shown that an orphan gene product results in mature mycetohabin-15, albeit encoded remotely from the core biosynthetic gene cluster. Comparative genomics revealed that mycetohabins are highly conserved among M. rhizoxinica and related endosymbiotic bacteria. Gene knockout and reinfection experiments indicated that the lasso peptides are not crucial for establishing symbiosis; instead, the peptides are exported into the environment during endosymbiosis. This is the first report on lasso peptides from endosymbiotic bacteria.

[Preparation and antitumor activity of sulfated exopolysaccharide from Rhizopus nigricans].

OBJECTIVE: To explore the effect of the composition ratio on substitution of sulfate group in sulfated exopolysaccharide (EPS) from Rhizopus nigricans and how sulfate modification affects the anti-tumor activity of EPS. METHODS: We used a chlorosulfonic acid-pyridine method to modify EPS and analyzed the effect of esterification ratio on the degree of sulfate substitution using barium chloride turbidimetry. The sulfate groups binding with EPS were analyzed with infrared spectrum analysis. CCK-8 assay was used to evaluate the inhibitory effect of EPS sulfate (SEPS) on the proliferation of human colon cancer HCT 116 cells, and annexin V-FITC/PI double staining was used to assess the pro-apoptotic effect of SEPS in the cells. RESULTS: The esterifying agent and EPS at the composition ratios of 1:1 and 2:1 resulted in sulfate substitution of 0.98% (SEPS-1) and 1.18% (SEPS-2), respectively, and the substitution was improved by increasing the ratio of the esterifying agent (P < 0.05). Infrared spectrum analysis showed that the S=O stretching vibration absorption peak of -OSO3- appeared near 1249 cm-1, indicating that the sulfate group combined with EPS to form sulfate. CCK-8 assay showed that SEPS-1 produced stronger inhibitory effects on the proliferation of HCT 116 cells than EPS within the concentration range of 0.02-0.10 mg/L (P < 0.05). At the concentrations of 0.04-0.08 mg/L, SEPS-2 showed a lower anti-tumor activity than SEPS-1 (P < 0.05). SEPS-1 also showed stronger pro-apoptotic effect than EPS, and as its concentration increased, SEPS-1 dose-dependently increased the ratio of early apoptotic cells and necrotic cells; the cells treated with 0.06, 0.08 and 0.10 mg/mL SEPS-1 showed early apoptotic rates of 6.38%, 11.8% and 12.5%, and late apoptotic and necrotic rates of 5.26%, 8.04% and 6.80%, respectively. CONCLUSIONS: The composition ratio of the esterifying agent has a direct impact on the degree of substitution of EPS, which can be improved by increasing the ratio of the esterifying agent. Sulfate modification of EPS can enhance its antitumor activity, which, however, is not directly related with the degree of substitution.

In Situ Ring Contraction and Transformation of the Rhizoxin Macrocycle through an Abiotic Pathway.

A Rhizopus sp. culture containing an endosymbiont partner ( Burkholderia sp.) was obtained through a citizen-science-based soil-collection program. An extract prepared from the pair of organisms exhibited strong inhibition of Ewing sarcoma cells and was selected for bioassay-guided fractionation. This led to the purification of rhizoxin (1), a potent antimitotic agent that inhibited microtubule polymerization, along with several new (2-5) and known (6) analogues of 1. The structures of 2-6 were established using a combination of NMR data analysis, while the configurations of the new stereocenters were determined using ROESY spectroscopy and comparison of GIAO-derived and experimental data for NMR chemical shift and 3 JHH coupling values. Whereas compound 1 showed modest selectivity for Ewing sarcoma cell lines carrying the EWSR1/ FLI1 fusion gene, the other compounds were determined to be inactive. Chemically, compound 2 stands out from other rhizoxin analogues because it is the first member of this class that is reported to contain a one-carbon-smaller 15-membered macrolactone system. Through a combination of experimental and computational tests, we determined that 2 is likely formed via an acid-catalyzed Meinwald rearrangement from 1 because of the mild acidic culture environment created by the Rhizopus sp. isolate and its symbiont.

Modelling the growth of pear postharvest fungal isolates at different temperatures.

The effect of temperature on the mycelium growth kinetics of four postharvest fungal isolates (i.e., Penicillium expansum, Alternaria alternata, Botrytis cinerea and Rhizopus stolonifer) was assessed. A cardinal model with inflection (CMI) was used to describe the effect of the temperature on the growth rate (µ) and the lag time (λ) of each isolate. Cardinal temperature values such as Tmin, Tmax and Topt were estimated and isolates were sorted according to their growth rate and lag time duration. Additionally, model validation was performed on a medium prepared from mashed pear pulp and on artificially wound-inoculated pear fruits. P. expansum was shown to be the most psychotrophic fungus with the lowest estimated Tmin = -8.78. Model validation on pear pulp agar showed growth rate over-prediction in the case of R. stolonifer and B. cinerea but a good correlation in the case of P. expansum and A. alternata. In vivo experiments on pear fruits showed discrepancies from the synthetic and the simulated counterparts for all the fungi with the only exception of P. expansum.

Monohexosylceramides from Rhizopus Species Isolated from Brazilian Caatinga: Chemical Characterization and Evaluation of Their Anti-Biofilm and Antibacterial Activities.

Monohexosylceramides (CMHs) are highly conserved fungal glycosphingolipids playing a role in several cellular processes such as growth, differentiation and morphological transition. In this study, we report the isolation, purification and chemical characterization of CMHs from Rhizopus stolonifer and R. microspores. Using positive ion mode ESI-MS, two major ion species were observed at m/z 750 and m/z 766, respectively. Both ion species consisted of a glucose/galactose residue attached to a ceramide moiety containing 9-methyl-4,8-sphingadienine with an amidic linkage to a hydroxylated C16:0 fatty acid. The antimicrobial activity of CMH was evaluated against Gram positive and Gram negative bacteria using the agar diffusion assay. CMH from both Rhizopus species inhibited the growth of Bacillus terrae, Micrococcus luteus (M. luteus) and Pseudomonas stutzeri (P. stutzeri) with a MIC50 of 6.25, 6.25 and 3.13 mg/mL, respectively. The bactericidal effect was detected only for M. luteus and P. stutzeri, with MBC values of 25 and 6.25 mg/mL, respectively. Furthermore, the action of CMH on the biofilm produced by methicillin-resistant Staphylococcus aureus (MRSA) was analyzed using 12.5 and 25 mg/mL of CMH from R. microsporus. Total biofilm biomass, biofilm matrix and viability of the cells that form the biofilm structure were evaluated. CMH from R. microsporus was able to inhibit the MRSA biofilm formation in all parameters tested.

Discrimination and growth tracking of fungi contamination in peaches using electronic nose.

A non-destructive method for detection of fungal contamination in peaches using an electronic nose (E-nose) is presented. Peaches were inoculated with three common spoilage fungi, Botrytis cinerea, Monilinia fructicola and Rhizopus stolonifer and then stored for various periods. E-nose was then used to analyze volatile compounds generated in the fungi-inoculated peaches, which was then compared with the growth data (colony counts) of the fungi. The results showed that changes in volatile compounds in fungi-inoculated peaches were correlated with total amounts and species of fungi. Terpenes and aromatic compounds were the main contributors to E-nose responses. While principle component analysis (PC1) scores were highly correlated with fungal colony counts, Partial Least Squares Regression (PLSR) could effectively be used to predict fungal colony counts in peach samples. The results also showed that the E-nose had high discrimination accuracy, demonstrating the potential use of E-nose to discriminate among fungal contamination in peaches.

Beneficial effects of extracellular polysaccharide from Rhizopus nigricans on the intestinal immunity of colorectal cancer mice.

An extracellular polysaccharide (EPS1-1) extracted from the fermentation broth of Rhizopus nigricans has been proved to enhance the immunity of immunosuppressed mice. The purpose of this study was to investigate the beneficial effects of EPS1-1 on the intestinal immunity of mice with colorectal cancer induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). The results showed that EPS1-1 could resist hydrolysis in an artificial stomach. Oral EPS1-1 modulated gut microbiota and increased the concentration of total short-chain fatty acids (SCFAs) in the feces of colorectal cancer mice compared with the AOM/DSS only-treated mice. Furthermore, EPS1-1 increased the villus length, ratio of villus length and crypt depth in colonic tissues, and improved the number of acid mucus-secreting goblet cells in mice with colorectal cancer. These findings suggest that EPS1-1 might play an important role in the improvement of intestinal function in mice with colorectal cancer, which indicate its strong potential as efficient bio-secure immunotherapy for clinical applications or adjuvant drug.

N-Glycosylation Engineering to Improve the Constitutive Expression of Rhizopus oryzae Lipase in Komagataella phaffii.

Our previous studies demonstrated that the N-glycans in Rhizopus chinensis lipase (RCL) was important for its secretion. In order to improve the secretion of Rhizopus oryzae lipase (ROL) under the control of the GAP promoter in Komagataella phaffii, two extra N-glycosylation sites were introduced in ROL according to the position of the N-glycosylation sites of RCL by sequence alignment. The results indicated that the secretion level of ROL was strongly improved by N-glycosylation engineering, and the highest value of extracellular enzyme activity was increased from 0.4 ± 0.2 U/mL to 207 ± 6 U/mL in a shake flask. In the 7-L fermenter, the extracellular enzyme activity of the mutant (2600 ± 43 U/mL) and the total protein concentration (2.5 ± 0.2 g/L) were 218- and 6.25-fold higher than these of the parent, respectively. This study presents a strategy for constitutive recombinant expression of ROL using the GAP promoter combined with N-glycosylation engineering, providing a potential enzyme for application in the food industry.

The impact of Rhizopus oryzae cultivation on rice bran: Gamma-oryzanol recovery and its antioxidant properties.

This study evaluated the effect of the solid state cultivation (SSC) time of rice bran by Rhizopus oryzae on γ-oryzanol recovery and its antioxidant properties. Gamma-oryzanol was extracted with organic solvents and its extracts were characterized by GC-FID and HPLC-UV. The antioxidant capacity was assessed by DPPH and ABTS+ assays, ß-carotene/linoleic acid system, and reduction of oxidation in lipid system. The biomass showed the γ-oryzanol recovery increased by 51.5% (20.52mg/g), and 5.7% in polyunsaturated fatty acids. The γ-oryzanol major components changing in their profile. The γ-oryzanol extract from biomass (72h) showed the greatest DPPH inhibition (59.0%), while 90.5% inhibition of oxidation of ß-carotene/linoleic acid system, and 30% reduction of the indicators of oxidation in olive oil was observed in the one cultivated at 96h, these behaviors were confirmed by PCA analyses. SSC provides an increase in the γ-oryzanol recovery followed by improving of the functional properties of rice bran.

Bidirectional immunomodulating activity of fermented polysaccharides from Yupingfeng.

Accumulating evidence has proven the immunomodulating activity of Yupingfeng. This study compared the immunomodulatory activity in vitro of the unfermented Yupingfeng dreg polysaccharides (UYDP) with that of the fermented Yupingfeng dreg polysaccharides (FYDP) obtained using Rhizopus oligosporus SH. Results consistently elucidated the duality of the immunomodulatory roles of UYDP and FYDP in regulating proliferation, and cytokines expressions in murine lymphocytes and macrophages. Compared with UYDP, FYDP effectively enhanced the proliferation of lymphocytes and promoted mRNA expression of inducible nitric oxide synthase (iNOS), IL-6, TNF-α, INF-γ, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and TLR-4 in lymphocytes and macrophages. Moreover, compared with UYDP, FYDP effectively normalized cell proliferation and downregulated mRNA expression levels of pro-inflammatory cytokines, NF-κB, TLR-4, and iNOs in lipopolysaccharide-induced chronic inflammation cells. The results revealed that the bidirectional immunomodulatory effects in vitro of UYDP and FYDP, and the bi-directional immunomodulatory activity of FYDP is superior over that of UYDP. Moreover, more studies in vivo that needs to be studied further.

Immunomodulatory Properties of Filamentous Fungi Cultivated through Solid-State Fermentation on Rapeseed Meal.

Water extracts from solid-state fermentation (SSF) on rapeseed meal using filamentous fungi exhibit interesting immunomodulatory activities in vitro. Immunomodulation was determined by the capacity of the compounds to activate blood neutrophils and to influence cytokine production in human peripheral blood mononuclear cells (PBMC) and mouse bone marrow-derived macrophages (BMDM). Among the strains tested, Aspergillus sojae mycelium and SSF extracts were the most promising in terms of enhancing the immune response. The filamentous fungus was also successfully cultivated in a pre-pilot bioreactor with forced aeration. The results indicated that the extracts not only activated blood neutrophils but also significantly modulated IL-1ß cytokine levels with lipopolysaccharide (LPS)-stimulated PBMC and BMDM without any cytotoxicity in immune cells. IL-1ß was down-regulated in a dose-dependent manner in the presence of A. sojae crude mycelium and SSF extract with PBMC, which indicated that there was an anti-inflammatory activity, whereas IL-1ß secretion was up-regulated in the presence of stimulated BMDM with the highest concentration that was tested (100 µg/mL). The non-fermented rapeseed had no effect at the same concentration. SSF culture, as a natural product, may be a good source for the development of functional feed with an immunostimulating effect or could potentially be used in medicinal applications.

Metabolism of Quercetin and Naringenin by Food-Grade Fungal Inoculum, Rhizopus azygosporus Yuan et Jong (ATCC 48108).

Rhizopus azygosporus Yuan et Jong (ATCC 48108), a starter culture for fermented soybean tempeh, produces ß-glucosidases that cleave flavonoid glycosides into aglycones during fermentation. However, recent data suggest that fermentation of a flavonoid glycoside-rich extract with this strain did not result in the production of aglycones. Thus, in this paper, flavonoid metabolism of this strain was investigated. Incubation of flavonoid aglycones, naringenin and quercetin, with R. azygosporus resulted in the production of flavonoid glucosyl-, hydroxyl-, and sulfo-conjugated derivatives. Naringenin was completely metabolized within 96 h into eriodictyol sulfate and eriodictyol glucoside, whereas quercetin was partially metabolized into quercetin glucoside, diglucoside, sulfate, and glucosyl-sulfate. Most of these metabolites were found to be excreted by the fungi into the culture medium. Toxicity analysis revealed that incubation with both quercetin and naringenin did not exert inhibitory effects on fungal growth. This study presents an interesting mechanism of fungal detoxification of flavonoids in foods.