Solid-state cultivation of multiple industrial strains of koji mold on different Thai unpolished rice cultivars: biotransformation of phenolic compounds and their effects on antioxidant activity.

Colored rice is abundant in polyphenols, and koji molds have potential for biotransformation. This study aimed to produce Thai-colored rice koji to study its polyphenolic biotransformation. Four industrial koji mold strains: Aspergillus oryzae 6001, A. oryzae 6020, A. sojae 7009, and A. luchuensis 8035, were cultivated on unpolished Thai-colored rice (Riceberry and Sangyod), unpolished Thai white rice (RD43), and polished Japanese white rice (Koshihikari). We discovered that koji molds grew on all the rice varieties. Methanol extracts of all rice kojis exhibited an approximately 2-fold or greater increase in total phenolic content and DPPH antioxidant activity compared to those of steamed rice. Moreover, quercetin, quercetin-3-O-glucoside, isorhamnetin-3-O-glucoside, ferulic acid, caffeic acid, protocatechuic acid, vanillic acid, (+)-catechin, and (-)-epicatechin content increased in Riceberry and Sangyod koji samples. Consequently, Aspergillus solid-state cultivation on unpolished Thai-colored rice exhibited higher functionalization than the cultivation of unpolished Thai white rice and polished Japanese white rice.

Aerobic bioconversion of C-glycoside mangiferin into its aglycone norathyriol by an isolated mouse intestinal bacterium.

Norathyriol is an aglycone of a xanthonoid C-glycoside mangiferin that possesses different bioactive properties useful for humans compared to mangiferin. Mangiferin is more readily available in nature than norathyriol; thus, efficient mangiferin conversion into norathyriol is desirable. There are a few reports regarding mangiferin C-deglycosylation because of the C-C bond resistance toward acid, alkaline, and enzyme hydrolysis. In this study, we isolated a mangiferin-deglycosylating bacterium strain KM7-1 from the mouse intestine. 16S rDNA sequencing indicated that KM7-1 belongs to the Bacillus genus. Compared to the taxonomically similar bacteria, the growth characteristic of facultative anaerobic and thermophilic resembled, yet only Bacillus sp. KM7-1 was able to convert mangiferin into norathyriol. Resting cells of Bacillus sp. KM7-1 obtained from aerobic cultivation at 50 °C showed high norathyriol formation from 1 m m of mangiferin. Norathyriol formation can be conducted either under aerobic or anaerobic conditions, and the reaction depended on time and bacterial amount.

Biotransformation of ß-Mangostin by an Endophytic Fungus of Garcinia mangostana to Furnish Xanthenes with an Unprecedented Heterocyclic Skeleton.

Biotransformation of ß-mangostin (1) by the endophytic fungus Xylaria feejeensis GM06 afforded hexacyclic ring-fused xanthenes with an unprecedented hexacyclic heterocylic skeleton. ß-Mangostin (1) was transformed to two diastereomeric pairs of enantiomers, mangostafeejin A [(-)-2a/(+)-2b)] and mangostafeejin B [(-)-3a/(+)-3b)]. The chemical structures of the transformation products were elucidated by analysis of NMR and MS data, and the structure of mangostafeejin A [(-)-2a/(+)-2b)] was confirmed by single-crystal X-ray diffraction analysis. The absolute configurations of 3a and 3b were established on the basis of calculated and measured ECD data using the ECD spectra of 2a and 2b as models. The fungal biotransformation described herein provides an effective method to convert an abundant achiral plant natural product scaffold into new chiral heterocyclic scaffolds representing expanded chemical diversity for biological activity screening.

Unique antimicrobial spectrum of ophiobolin K produced by Aspergillus ustus.

A co-cultivation study of two fungal strains showed that Aspergillus ustus could inhibit Aspergillus repens growth. The bioactive compound responsible for the observed activity was purified and identified as a sesterterpene, ophiobolin K. Ophiobolin K exhibited marked inhibition against both fungi and bacteria, especially A. repens, A. glaucus and gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus.

Antifungal and Ichthyotoxic Sesquiterpenoids from Santalum album Heartwood.

In our continuing study on a survey of biologically active natural products from heartwood of Santalum album (Southwest Indian origin), we newly found potent fish toxic activity of an n-hexane soluble extract upon primary screening using killifish (medaka) and characterized α-santalol and ß-santalol as the active components. The toxicity (median tolerance limit (TLm) after 24 h at 1.9 ppm) of α-santalol was comparable with that of a positive control, inulavosin (TLm after 24 h at 1.3 ppm). These fish toxic compounds including inulavosin were also found to show a significant antifungal effect against a dermatophytic fungus, Trichophyton rubrum. Based on a similarity of the morphological change of the immobilized Trichophyton hyphae in scanning electron micrographs between treatments with α-santalol and griseofulvin (used as the positive control), inhibitory effect of α-santalol on mitosis (the antifungal mechanism proposed for griseofulvin) was assessed using sea urchin embryos. As a result, α-santalol was revealed to be a potent antimitotic agent induced by interference with microtubule assembly. These data suggested that α-santalol or sandalwood oil would be promising to further practically investigate as therapeutic agent for cancers as well as fungal skin infections.

Disease Burden and Progression in Patients with New-Onset Mild Cognitive Impairment and Alzheimer's Disease Identified from Japanese Claims Data: Evidence from the LIFE Study.

BACKGROUND: Accurate epidemiological data on mild cognitive impairment (MCI) and Alzheimer's disease (AD) can inform the development of prevention and control measures, but there is a lack of such data in Japan. OBJECTIVE: To investigate the disease burden and progression in patients with new-onset MCI or AD in Japan. METHODS: Using claims data, this multi-region cohort study was conducted on new-onset MCI and AD patients in 17 municipalities from 2014 to 2021. To characterize the patients, we investigated their age, comorbidities, and long-term care (LTC) needs levels at disease onset according to region type (urban, suburban, or rural). Disease burden was examined using health care expenditures and LTC expenditures, which were estimated for 1, 2, and 3 years after disease onset. Kaplan-Meier curves were plotted for AD progression in new-onset MCI patients and death in new-onset AD patients. RESULTS: We analyzed 3,391 MCI patients and 58,922 AD patients. In MCI and AD patients, health care expenditures were high in the first year ($13,035 and $15,858, respectively), but had declined by the third year ($8,278 and $10,414, respectively). In contrast, LTC expenditures (daily living support) steadily increased over the 3-year period (MCI patients: $1,767 to $3,712, AD patients: $6,932 to $9,484). In the third year after disease onset, 30.9% of MCI patients developed AD and 23.3% of AD patients had died. CONCLUSIONS: This provides an important first look at the disease burden and progression of MCI and AD in Japan, which are high-priority diseases for a rapidly aging population.

MS/MS fragmentation-guided search of TMG-chitooligomycins and their structure-activity relationship in specific ß-N-acetylglucosaminidase inhibition.

The reducing tetrasaccharide TMG-chitotriomycin (1) is an inhibitor of ß-N-acetylglucosaminidase (GlcNAcase), produced by the actinomycete Streptomyces anulatus NBRC13369. The inhibitor shows a unique inhibitory spectrum, that is, selectivity toward enzymes from chitin-containing organisms such as insects and fungi. Nevertheless, its structure-selectivity relationship remains to be clarified. In this study, we conducted a structure-guided search of analogues of 1 in order to obtain diverse N,N,N-trimethylglucosaminium (TMG)-containing chitooligosaccharides. In this approach, the specific fragmentation profile of 1 on ESI-MS/MS analysis was used for the selective detection of desired compounds. As a result, two new analogues, named TMG-chitomonomycin (3) and TMG-chitobiomycin (2), were obtained from a culture filtrate of 1-producing Streptomyces. Their enzyme-inhibiting activity revealed that the potency and selectivity depended on the degree of polymerization of the reducing end GlcNAc units. Furthermore, a computational modeling study inspired the inhibitory mechanism of TMG-related compounds as a mimic of the substrate in the Michaelis complex of the GH20 enzyme. This study is an example of the successful application of a MS/MS experiment for structure-guided isolation of natural products.

New analogs of pochonicine, a potent ß-N-acetylglucosaminidase inhibitor from fungus Pochonia suchlasporia var. suchlasporia TAMA 87.

Three novel analogs of pochonicine (1) were isolated from a solid fermentation culture of the fungal strain Pochonia suchlasporia var. suchlasporia TAMA 87, and their structures were elucidated as 7-deoxypochonicine (2), 6-deoxypochonicine (3), and 6,7-dideoxypochonicine (4). These analogs were found to possess the same stereochemistry as pochonicine. Comparison of ß-N-acetylglucosaminidase (GlcNAcase) inhibitory activity between these analogs and pochonicine suggested that the C-6 hydroxy group of pochonicine was essential to its potent GlcNAcase inhibitory activity and that the C-7 hydroxy group also contributed to the activity, but to a lesser extent than the C-6 hydroxy group.

A new asteltoxin analog with insecticidal activity from Pochonia suchlasporia TAMA 87.

A new asteltoxin analog, named asteltoxin H (1), was isolated by the solid-state fermentation of the fungus Pochonia suchlasporia var. suchlasporia TAMA 87. The chemical structure of 1 was deduced by spectroscopic methods, including 1D and 2D NMR, HRESIMS, and UV-Vis analyses. Compound 1 showed insecticidal activity against prepupae of the blowfly, Lucilia sericata, with an LD50 value of 0.94 µg/mg prepupal body weight.

Efficient production of recombinant tannase in Aspergillus oryzae using an improved glucoamylase gene promoter.

A tannase-encoding gene, AotanB, from Aspergillus oryzae RIB40 was overexpressed in A. oryzae AOK11 niaD-deficient mutant derived from an industrial strain under the control of an improved glucoamylase gene promoter PglaA142. The recombinant tannase, designated as rAoTanBO, was produced efficiently as an active extracellular enzyme. Purified rAoTanBO showed a smeared band with a molecular mass of approximately 80-100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rAoTanBO had a molecular mass of 65 kDa, after treatment with endo-ß-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30-35°C and pH 6.0. The tannase activity of purified rAoTanBO towards natural and artificial substrates was 2-8 folds higher than that of the recombinant enzyme produced by Pichia pastoris, designated as rAoTanBP. N-terminus of the mature rAoTanBP had six more amino acids than the N-terminus of the mature rAoTanBO. Kinetic analyses showed that rAoTanBO had higher catalytic efficiency (kcat/Km) than rAoTanBP. rAoTanBO was stable up to 60°C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP.

Pochonicine, a polyhydroxylated pyrrolizidine alkaloid from fungus Pochonia suchlasporia var. suchlasporia TAMA 87 as a potent beta-N-acetylglucosaminidase inhibitor.

A new polyhydroxylated pyrrolizidine alkaloid designated as pochonicine (1) was isolated from a solid fermentation culture of the fungal strain Pochonia suchlasporia var. suchlasporia TAMA 87. The structure of 1 was determined using NMR and MS techniques as (1R*, 3S*, 5S*, 6S*, 7R*, 7a S*)-5-acetamidomethyl-3-hydroxymethyl-1,6,7-trihydroxypyrrolizidine. Pochonicine (1) showed potent inhibition against beta-N-acetylglucosaminidases (GlcNAcases) of various organisms including insects, fungi, mammals, and a plant but no inhibition against beta-glucosidase of almond, alpha-glucosidase of yeast, or chitinase of Bacillus sp. The GlcNAcase inhibitory activity of pochonicine (1) was comparable to nagstatin, a potent GlcNAcase inhibitor of natural origin.

TMG-chitotriomycin, an enzyme inhibitor specific for insect and fungal beta-N-acetylglucosaminidases, produced by actinomycete Streptomyces anulatus NBRC 13369.

A novel beta-N-acetylglucosaminidase (GlcNAcase) inhibitor named TMG-chitotriomycin (1) was isolated from the culture filtrate of Streptomyces anulatus NBRC13369. The strain produced 1 only when colloidal chitin was used as the sole carbon source in the production medium. The structure of 1 was determined by spectral and constitutive sugar analyses of the corresponding alditol derivatives to be an equilibrated mixture of alpha-d-N,N,N-triMeGlcNH2-(1,4)-beta-d-GlcNAc-(1,4)-beta-d-GlcNAc-(1,4)-d-GlcNAc and its C-2 epimer of the reducing end residue. TMG-chitotriomycin (1) showed potent and selective inhibition of insect and fungal GlcNAcases with no inhibition of mammalian and plant GlcNAcases. In contrast, the known GlcNAcase inhibitor nagstatin potently inhibited all GlcNAcases. It should be emphasized that synthesized d-N,N,N-triMeGlcNH2, which is the component sugar of 1, showed no inhibition of the insect Spodoptera litura GlcNAcase. These results suggest that the (GlcNAc)3 unit positioned at the reducing end of 1 is essential for its enzyme inhibitory activity. The unique inhibitory spectrum of 1 will be useful to study chitinolytic systems and to develop selective fungicides or pesticides.

Use of wood vinegar to enhance 5-aminolevulinic acid production by selected Rhodopseudomonas palustris in rubber sheet wastewater for agricultural use.

This study aimed to produce inexpensive 5-aminolevulinic acid (ALA) in a non-sterile latex rubber sheet wastewater (RSW) by Rhodopseudomonas palustris TN114 and PP803 for the possibility to use in agricultural purposes by investigating the optimum conditions, and applying of wood vinegar (WV) as an economical source of levulinic acid to enhance ALA content. The Box-Behnken Design experiment was conducted under microaerobic-light conditions for 96 h with TN114, PP803 and their mixed culture (1:1) by varying initial pH, inoculum size (% v/v) and initial chemical oxygen demand (COD, mg/L). Results showed that the optimal condition (pH, % inoculum size, COD) of each set to produce extracellular ALA was found at 7.50, 6.00, 2000 for TN114; 7.50, 7.00, 3000 for PP803; and 7.50, 6.00, 4000 for a mixed culture; and each set achieved COD reduction as high as 63%, 71% and 75%, respectively. Addition of the optimal concentration of WV at mid log phase at 0.63% for TN114, and 1.25% for PP803 and the mixed culture significantly increased the ALA content by 3.7-4.2 times (128, 90 and 131 µM, respectively) compared to their controls. ALA production cost could be reduced approximately 31 times with WV on the basis of the amount of levulinic acid used. Effluent containing ALA for using in agriculture could be achieved by treating the RSW with the selected ALA producer R. palustris strains under the optimized condition with a little WV additive.

Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste.

The aim of this work was to study chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. The culture produced two biosurfactants, a and b, which showed strong activity and were identified as L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate or Rha-Rha-C10-C10 and L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydodecanoate or Rha-Rha-C10-C12, respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as SDS and Tween 80. Rhamnolipid a showed significant antiproliferative activity against human breast cancer cell line (MCF-7) at minimum inhibitory concentration (MIC) at 6.25 microg/mL while rhamnolipid b showed MIC against insect cell line C6/36 at 50 microg/mL.

Enzymatic conversion-based method for screening cyclic dipeptide-producing microbes.

We developed a method for screening cyclic dipeptide-producing microbes by enzymatic conversion. In this method, cyclic dipeptides are detected by the combination of: (i) conversion of cyclic dipeptides to dehydro cyclic dipeptides by cyclo(Leu-Phe) oxidase and (ii) detection of the dehydro derivative by UV spectrophotometry using TLC or HPLC analysis based on the absorbance change caused by the conversion. Using this method, the actinomycete strain A8 was isolated as a cyclic dipeptide-producing strain. The cyclic dipeptides were purified from the microbial extract by enzymatic detection-guided fractionation, and their structures were determined to be cyclo(L-Phe-L-Pro) and cyclo(L-Pro-L-Tyr) by spectroscopic methods.

Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste.

The aim of this work was to study chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. The culture produced two biosurfactants, a and b, which showed strong activity and were identified as L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate or Rha-Rha C10-C10 and L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydodecanoate or Rha-Rha C(10)-C(12), respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as SDS and Tween 80. Rhamnolipid a showed significant antiproliferative activity against human breast cancer cell line (MCF-7) at minimum inhibitory concentration (MIC) at 6.25 microg/mL while rhamnolipid b showed MIC against insect cell line C6/36 at 50 microg/mL.

Characterization of exopolymeric substances from selected Rhodopseudomonas palustris strains and their ability to adsorb sodium ions.

Removal of Na(+) by binding with exopolymeric substances (EPS) from Rhodopseudomonas palustris TN114 and PP803 was investigated. The moderate negative correlation pairs (rp) between remaining Alcian blue and amount of Na(+) adsorbed on EPS from strains TN114 and PP803 were -0.652 and -0.609. Both strains showed positive relationships between the amounts of EPS produced and bacterial growth. EPS from strain PP803 had a higher efficiency in removing Na(+) than the EPS from strain TN114 based on their EC50 values (1.79 and 1.49 mg/mL for TN114 and PP803, respectively). The principal component from EPS of strain PP803 which was responsible for salt removal was purified and it was identified as a polysaccharide (≈18 kDa) mainly composed of galacturonic acid. Overall results suggested that EPS is a key factor that our strains used to bind Na(+) allowing their survival in high NaCl concentrations.

A novel potent cell cycle inhibitor dehydrophenylahistin--enzymatic synthesis and inhibitory activity toward sea urchin embryo.

A novel dehydrogenated cyclic dipeptide named as dehydrophenylahistin (deltaPLH) was effectively prepared from a fungal metabolite (-)-phenylahistin by the enzymatic conversion catalyzed by the cell-free extract of Streptomyces albulus KO-23, an albonoursin-producing actinomycete. deltaPLH exhibited more than 1,000 times as high potent inhibitory activity toward the first cleavage of sea urchin embryos as (-)-phenylahisitn which has been reported to be a cell cycle inhibitor and more than 10,000 as high as albonoursin, indicating that deltaPLH is a promising leading compound for anticancer drugs.

A potent insect chitinase inhibitor of fungal origin.

A water-soluble polysaccharide was isolated from the culture filtrate of a fungal strain, Sphaeropsis sp. TNPT116-Cz, as a novel insect chitinase inhibitor. It was purified to chromatographic homogeneity by ethanol precipitation, anion-exchange and gel filtration chromatography. Its molecular weight was estimated to be 16 kDa by gel filtration HPLC. Monosaccharide analysis showed that it contained glucose, galactose, N-acetylglucosamine and a deoxysugar. This polysaccharide showed potent and specific inhibitory activity against Spodoptera litura chitinase with an IC50 value of 28 nM.