The antileukaemic cell cycle regulatory activities of swainsonine purified from <i>Metarhizium anisopliae</i> fermentation broth

<div><p>Swainsonine is a <i>Metarhizium</i> secondary metabolite known differentially for its specific mannosidase inhibitory, toxic and therapeutic activities. Here, the standard and purified swainsonine from <i>Metarhizium anisopliae</i> fermentation broth were comparatively evaluated for their <i>in situ</i> antileukaemic activities in human promyelocytic cell line, HL-60. Both the standard (IC₅₀ = 6.96 μM) and purified (IC₅₀ = 9.50 μM) compounds inhibited the leukaemic cell proliferation without inflicting cell membrane disruption at 48 h of post-treatment incubation. The DNA cell cycle analysis showed approximately 48.81% and 60.72% of the treated cells arrested in the synthetic phase (S-phase) at 36 and 48 h, respectively, upon treatment with IC₅₀ concentration of the purified swainsonine. However, only 29.62% of cells were arrested in S-phase with standard swainsonine at 48 h, suggesting the comprehensive action of certain other metabolites sharing the similar paradigm of antiproliferative properties in <i>Metarhizium</i> broth extract.</p></div

DataSheet1.DOCX

<p>Notwithstanding its mitosporic nature, an improbable morpho-transformation state i. e., sclerotial development (SD), is vaguely known in Aspergillus oryzae. Nevertheless an intriguing phenomenon governing mold's development and stress response, the effects of exogenous factors engendering SD, especially the volatile organic compounds (VOCs) mediated interactions (VMI) pervasive in microbial niches have largely remained unexplored. Herein, we examined the effects of intra-species VMI on SD in A. oryzae RIB 40, followed by comprehensive analyses of associated growth rates, pH alterations, biochemical phenotypes, and exometabolomes. We cultivated A. oryzae RIB 40 (S1_VMI: KACC 44967) opposite a non-SD partner strain, A. oryzae (S2: KCCM 60345), conditioning VMI in a specially designed “twin plate assembly.” Notably, SD in S1_VMI was delayed relative to its non-conditioned control (S1) cultivated without partner strain (S2) in twin plate. Selectively evaluating A. oryzae RIB 40 (S1_VMI vs. S1) for altered phenotypes concomitant to SD, we observed a marked disparity for corresponding growth rates (S1_VMI < S1)_7days, media pH (S1_VMI > S1)_7days, and biochemical characteristics viz., protease (S1_VMI > S1)_7days, amylase (S1_VMI > nS1)_3–7days, and antioxidants (S1_VMI > S1)_7days levels. The partial least squares—discriminant analysis (PLS-DA) of gas chromatography—time of flight—mass spectrometry (GC-TOF-MS) datasets for primary metabolites exhibited a clustered pattern (PLS1, 22.04%; PLS2, 11.36%), with 7 days incubated S1_VMI extracts showed higher abundance of amino acids, sugars, and sugar alcohols with lower organic acids and fatty acids levels, relative to S1. Intriguingly, the higher amino acid and sugar alcohol levels were positively correlated with antioxidant activity, likely impeding SD in S1_VMI. Further, the PLS-DA (PLS1, 18.11%; PLS2, 15.02%) based on liquid chromatography—mass spectrometry (LC-MS) datasets exhibited a notable disparity for post-SD (9–11 days) sample extracts with higher oxylipins and 13-desoxypaxilline levels in S1_VMI relative to S1, intertwining Aspergillus morphogenesis and secondary metabolism. The analysis of VOCs for the 7 days incubated samples displayed considerably higher accumulation of C-8 compounds in the headspace of twin-plate experimental sets (S1_VMI:S2) compared to those in non-conditioned controls (S1 and S2—without respective partner strains), potentially triggering altered morpho-transformation and concurring biochemical as well as metabolic states in molds.</p

Metabolomics Reveal Optimal Grain Preprocessing (Milling) toward Rice <i>Koji</i> Fermentation

A time-correlated mass spectrometry (MS)-based metabolic profiling was performed for rice <i>koji</i> made using the substrates with varying degrees of milling (DOM). Overall, 67 primary and secondary metabolites were observed as significantly discriminant among different samples. Notably, a higher abundance of carbohydrate (sugars, sugar alcohols, organic acids, and phenolic acids) and lipid (fatty acids and lysophospholipids) derived metabolites with enhanced hydrolytic enzyme activities were observed for <i>koji</i> made with DOM of 5–7 substrates at 36 h. The antioxidant secondary metabolites (flavonoids and phenolic acid) were relatively higher in <i>koji</i> with DOM of 0 substrates, followed by DOM of 5 > DOM of 7 > DOM of 9 and 11 at 96 h. Hence, we conjecture that the rice substrate preprocessing between DOM of 5 and 7 was potentially optimal toward <i>koji</i> fermentation, with the end product being rich in distinctive organoleptic, nutritional, and functional metabolites. The study rationalizes the substrate preprocessing steps vital for commercial <i>koji</i> making

Additional file 2 of Exploring the metabolomic diversity of plant species across spatial (leaf and stem) components and phylogenic groups

Additional file 2. Tentatively identified secondary metabolits in plants based on metabolite profiling with multivariate analysis

Production profiles of variant metabolites from solid-state and submerged fermentation of <i>P</i>. <i>expansum</i> 40815.

<p>The Y-axis of graphs indicates the UPLC-Q-TOF-MS data transformed by log10. (△: Submerged fermentation, ▲: Solid-state fermentation; red: 4 days, orange: 6 days, yellow: 8 days, green: 10 days, blue: 12 days, navy: 14 days, violet: 16 days)</p

Additional file 1 of Exploring the metabolomic diversity of plant species across spatial (leaf and stem) components and phylogenic groups

Additional file 1. Tentatively identified primary metabolits in plants based on metabolite profiling with multivariate analysis

Systematic metabolic profiling and bioactivity assays for bioconversion of Aceraceae family - Fig 1

<p>(A) Partial least-square discriminant analysis score plot based on UHPLC–LTQ-IT-MS/MS datasets, and (B) average antioxidant activity (2,2-diphenyl-1-picrylhydrazyl: DPPH), for the metabolite extracts derived from plant species belonging to the families Aceraceae, Rosaceae, and Asteraceae. The different letters are indicative of statistically significant differences for observed bioactivities according to Duncan’s multiple-range test at <i>p</i> < 0.05.</p

Systematic metabolic profiling and bioactivity assays for bioconversion of Aceraceae family

<div><p>Plants are an important and inexhaustible source of bioactive molecules in food, medicine, agriculture, and industry. In this study, we performed systematic liquid chromatography–mass spectrometry (LC-MS)-based metabolic profiling coupled with antioxidant assays for indigenous plant family extracts. Partial least-squares discriminant analysis of LC-MS datasets for the extracts of 34 plant species belonging to the families Aceraceae, Asteraceae, and Rosaceae showed that these species were clustered according to their respective phylogenies. In particular, seven Aceraceae species were clearly demarcated with higher average antioxidant activities, rationalizing their application for bioconversion studies. On the basis of further evaluation of the interspecies variability of metabolic profiles and antioxidant activities among Aceraceae family plants, we found that <i>Acer tataricum</i> (TA) extracts were clearly distinguished from those of other species, with a higher relative abundance of tannin derivatives. Further, we detected a strong positive correlation between most tannin derivatives and the observed higher antioxidant activities. Following <i>Aspergillus oryzae</i>-mediated fermentative bioconversion of <i>Acer</i> plant extracts, we observed a time-correlated (0–8 days) linear increase in antioxidant phenotypes for all species, with TA having the highest activity. Temporal analysis of the MS data revealed tannin bioconversion mechanisms with a relatively higher abundance of gallic acid (m/z 169) accumulated at the end of 8 days, particularly in TA. Similarly, quercetin precursor (glycoside) metabolites were also transformed to quercetin aglycones (m/z 301) in most <i>Acer</i> plant extracts. The present study underscores the efficacy of fermentative bioconversion strategies aimed at enhancing the quality and availability of bioactive metabolites from plant extracts.</p></div

(A) Orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot and (B) loading S-plots of different cultures in the UPLC-Q-TOF-MS based dataset.

<p>The significantly different metabolites (▲) according to different fermentation condition are highlighted in the S-plot. The numbering of metabolites is identical to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149012#pone.0149012.t001" target="_blank">Table 1</a>. (△: Submerged fermentation, ▲: Solid-state fermentation, red: 4 days, orange: 6 days, yellow: 8 days, green: 10 days, blue: 12 days, violet: 14 days, black: 16 days. 1: N.I.1, 2: N.I.2, 3: N.I.3, 4:: N.I.4, 5: Agonodepside B, 6: Rotiorin, 7: Verrucosidin, 8: N.I.5, 9: Andrastin A, 10: N.I.6, 11: N.I.7, 12: N.I.8, 13: Ochrephilone, 14: Andrastin C, 15: N.I.9)</p

Presentation_1_Comprehensive Secondary Metabolite Profiling Toward Delineating the Solid and Submerged-State Fermentation of Aspergillus oryzae KCCM 12698.pdf

<p>Aspergillus oryzae has been commonly used to make koji, meju, and soy sauce in traditional food fermentation industries. However, the metabolic behaviors of A. oryzae during fermentation in various culture environments are largely uncharacterized. Thus, we performed time resolved (0, 4, 8, 12, 16 day) secondary metabolite profiling for A. oryzae KCCM 12698 cultivated on malt extract agar and broth (MEA and MEB) under solid-state fermentation (SSF) and submerged fermentation (SmF) conditions using the ultrahigh performance liquid chromatography-linear trap quadrupole-ion trap-mass spectrometry (UHPLC-LTQ-IT-MS/MS) followed by multivariate analyses. We observed the relatively higher proportions of coumarins and oxylipins in SSF, whereas the terpenoids were abundant in SmF. Moreover, we investigated the antimicrobial efficacy of metabolites that were extracted from SSF and SmF. The SSF extracts showed higher antimicrobial activities as compared to SmF, with higher production rates of bioactive secondary metabolites viz., ketone-citreoisocoumarin, pentahydroxy-anthraquinone, hexylitaconic acid, oxylipins, and saturated fatty acids. The current study provides the underpinnings of a metabolomic framework regarding the growth and bioactive compound production for A. oryzae under the primarily employed industrial cultivation states. Furthermore, the study holds the potentials for rapid screening and MS-characterization of metabolites helpful in determining the consumer safety implications of fermented foods involving Koji mold.</p