Enhanced Production of Two Bioactive Isoflavone Aglycones in

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Enhanced Production of Two Bioactive Isoflavone Aglycones in Astragalus membranaceus Hairy Root Cultures by Combining Deglycosylation and Elicitation of Immobilized Edible Aspergillus niger Jiao Jiao, Qing-Yan Gai, Li-Li Niu, Xi-Qing Wang, Na Guo, Yu-Ping Zang, and Yu-Jie Fu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03148 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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Journal of Agricultural and Food Chemistry

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Enhanced Production of Two Bioactive Isoflavone Aglycones in Astragalus

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membranaceus Hairy Root Cultures by Combining Deglycosylation and

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Elicitation of Immobilized Edible Aspergillus niger

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Jiao Jiao, †,‡ Qing-Yan Gai, †,‡ Li-Li Niu, ‡ Xi-Qing Wang, ‡ Na Guo, ‡ Yu-Ping Zang, ‡

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and Yu-Jie Fu, *,§

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‡

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University, Harbin 150040, People’s Republic of China

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§

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Forestry University, Beijing 100083, People’s Republic of China

Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry

Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing

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*Corresponding authors: Y.-J. Fu

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Tel./Fax: +86-451-82190535

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E-mail: [email protected], [email protected].

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†

These authors contributed equally to this work

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ACS Paragon Plus Environment


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ABSTRACT A co-cultivation system of Astragalus membranaceus hairy root cultures

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(AMHRCs) and immobilized food-grade fungi was established for the enhanced

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production of calycosin (CA) and formononetin (FO). The highest accumulations of

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CA (730.88 ± 63.72 µg/g DW) and FO (1119.42 ± 95.85 µg/g DW) were achieved in

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34 day-old AMHRCs co-cultured with immobilized A. niger (IAN) for 54 h, which

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were 7.72- and 18.78-fold higher than CA and FO in non-treated control, respectively.

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IAN deglycosylation could promote the formation of CA and FO by conversion of

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their glycoside precursors. IAN elicitation could intensify the generation of

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endogenous signal molecules involved in plant defense response, which contributed to

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the significantly up-regulated expression of genes in CA and FO biosynthetic pathway.

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Overall, the coupled culture of IAN and AMHRCs offered a promising and effective in

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vitro approach to enhance the production of two health-promoting isoflavone

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aglycones for possible nutraceutical and pharmaceutical uses.

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KEY WORDS: edible fungi, elicitation, deglycosylation, hairy root cultures,

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isoflavone aglycones, antioxidant activity

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INTRODUCTION

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Astragalus membranaceus is an economically important leguminous crop widely

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cultivated in China. Its roots have been acknowledged as traditional folk medicines in

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East Asian areas, and as functional foods/nutraceuticals in the United States and

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European countries 1–3. Like most of leguminous plants, A. membranaceus contains

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diverse isoflavones, among which calycosin (CA), formononetin (FO),

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calycosin-7-O-β-D-glucoside (CAG) and formononetin-7-O-β-D-glucoside (FOG)

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possess various health-enhancing benefits including antioxidant, anti-inflammatory,

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antiviral, anti-fatigue, hematopoietic, neuroprotective and estrogenic activities 4, 5.

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However, it is well documented that isoflavones in their aglycone forms are more

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metabolically active in intestines resulting in better bioavailability than their

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glycosides 6–9, which suggested that the intake of isoflavone aglycone-rich foodstuffs

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might be more beneficial for human health. In this respect, it is not surprising that the

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two isoflavone aglycones, CA and FO, have frequently been used as indices for the

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quality evaluation of A. membranaceus roots 10.

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In recent years, the production of bioactive secondary metabolites by plant

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cell/organ cultures is an attractive alternative to the classical extraction of whole plant

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materials, in which the quality of phytochemicals is often fluctuating due to

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environmental, ecological and climatic variations 11, 12. Moreover, Food and

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Agriculture Organization of the United Nations (FAO) endorses plant cell/organ

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culture techniques as feasible tools to produce high-value natural compounds for food

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purposes 13, 14. In this context, we have established a reliable in vitro culture platform,

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i.e. A. membranaceus hairy root cultures (AMHRCs), that could supersede field

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cultivated plants for the efficient production of CA and FO 15. Generally, isoflavones

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can function as phytoalexins or phytoanticipins that are strongly inducible and

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sensitive to environmental stresses 16. Thus, application of external elicitors can further

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trigger the biosynthesis of CA and FO in AMHRCs by inducing plant defense

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responses. In view of the bio-safety of products, it is recommendable to use elicitors of

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biological origin for the enhancement of CA and FO accumulation in AMHRCs. Aspergillus niger and Aspergillus oryzae are widely used in the food fermentation

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industry for the production of wine, soy sauce, vinegar, soybean paste, etc., and they

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are approved as GRAS (Generally Recognized as Safe) fungi by the United States

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Food and Drug Administration 17, 18. The both food-grade fungi have proven to be

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promising and effective elicitors for the enhanced production of health-promoting

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compounds (resveratrol, ginsenosides, rosmarinic acid, glycyrrhizic acid, etc.) in plant

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seedlings or plant cell/organ cultures 19–22. However, in these reports, extracts of fungal

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mycelia and culture filtrates are always prepared as elicitors rather than the direct

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utilization of live fungi. Factually, A. niger and A. oryzae are capable of secreting

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extracellular glucosidases, and this characteristic can make them act as effective

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biocatalysts for the deglycosylation of glycosides to their aglycones 23. It is worth

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mentioning that CA and FO can be obtained by the hydrolysis of glucosyl residues in

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their precursors (CAG and FOG) 24. Recently, immobilization of fungal spores has

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opened a new avenue for the bio-production of various bioactive compounds, which is

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associated with advantages of operational stability, reusability and scale-up feasibility

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25, 26

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immobilized Aspergillus (elicitation and deglycosylation) for promoting CA and FO

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production in AMHRCs.

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. Based on the foregoing, an interesting possibility is to exploit the dual abilities of

In the present study, the coupled culture of AMHRCs and immobilized food-grade

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Aspergillus was established in an attempt to enhance the production of CA and FO by

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fungal elicitation and deglycosylation. Initially, the feasibility of immobilized A. niger

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(IAN) and immobilized A. oryzae (IAO) for promoting FO and CA accumulation in

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AMHRCs was investigated. Subsequently, IAN and IAO were compared by

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monitoring the levels of CA and FO as well as their precursors (CAG and FOG) in

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AMHRCs along a time course from 0 to 72 h. Moreover, the generation of endogenous

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defense signal molecules in AMHRCs upon the selected IAN treatment was

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investigated. Meanwhile, the expression of associated genes involved in CA and FO

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biosynthetic pathway was determined. Additionally, antioxidant activities of enzymes

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and extracts from AMHRCs before and after IAN treatment were also evaluated. To

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the best of our knowledge, there is no report on the combination of elicitation and

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deglycosylation by immobilized food-grade fungi to enhance isoflavone aglycone

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production in plant in vitro cultures.

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MATERIALS AND METHODS

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Hairy Root Line and Aspergillus Strains

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All experiments were conducted using an A. membranaceus hairy root line II

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(AMHRL II, Figure 1A) with the high productivity of isoflavones established

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previously by our laboratory 15. AMHRCs (Figure 1B) were initiated by culturing

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AMHRL II under the optimal conditions as described previously 15. Two food-grade

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Aspergillus strains (A. niger 3.3883 and A. oryzae 3.951) were purchased from the

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Institute of Microbiology, Heilongjiang province, China. Pure plate cultures of both

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fungi were grown on potato dextrose agar (PDA) medium, and incubated at 30 ± 1 °C

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till sporulation (Figure 1C and Figure 1D). Fungal spores were collected and counted

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for the following immobilization experiment.

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Co-cultivation of AMHRCs and Immobilized Edible Aspergillus

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IAN or IAO were prepared by immobilization of their spores in Ca-alginate gel (CG) beads. The detailed operation procedures were performed according to our

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previous report 25, 26. For co-cultivation, IAN or IAO beads were transferred into a

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series of 250 mL Erlenmeyer’s flasks containing AMHRCs (34 day-old) with 100 mL

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of fresh culture medium (Figure 1E), and these flask cultures were then incubated on

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an orbital shaker at 120 rpm and maintained under continuous darkness. On the basis

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of preliminary studies (data not shown), the incubation temperature (30 °C), spore

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amount of IAN or IAO load (ca.104 spores/ flask), and initial pH value of media (7.0)

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were determined as the appropriate co-cultivation parameters. For control, non-treated

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AMHRCs and CG-treated AMHRCs (addition of CG beads without fungal spores)

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underwent the same culture conditions. To evaluate the feasibility of the co-cultivation

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system, 34 day-old AMHRCs were initially treated with IAN and IAO for 2 days

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under the pre-determined parameters. Additionally, a 72 h time course of non-, IAN-

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and IAO-treated AMHRCs was conducted at a series of time points (0, 6, 12, 18, 24,

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30, 36, 42, 48, 54, 60 and 72, h). After co-cultivation (Figure 1F), the harvested hairy

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roots were rinsed by distilled water, and divided into three parts for the respective

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extraction of isoflavones, endogenous signal molecules and total RNA. Meanwhile,

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media were also collected for the liquid-liquid extraction of isoflavones. Moreover, the

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immobilized fungus beads could be simply recovered by filtration, washed with sterile

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water, and used for the next cycle to evaluate their reusability.

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LC-MS/MS Analysis

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Isoflavones extraction from AMHRCs and sample preparation for LC-MS/MS

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analysis were performed as previously described 27. The simultaneous determination of

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four target isoflavones (CAG, FOG, CA and FO) was conducted by a LC-MS/MS

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method with selected reaction monitoring (SRM) mode as established before 15. The

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precursor ion–product ion combinations of m/z 445.2 → 283.0, m/z 428.8 → 266.9,

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m/z 283.0 → 268.0 and m/z 267.0 → 252.0 were adopted for the identification and

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quantification of CAG, FOG, CA and FO, respectively. The content of each analyte was

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calculated by the corresponding calibration curve, and expressed as microgram per

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gram based on the dry weight (DW) of root samples.

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Quantification of Endogenous Signal Molecules

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Fresh hairy root samples were homogenized thoroughly by an Ultra turrax system

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(IKA Co., Germany). The extraction and determination of endogenous nitric oxide

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(NO) from the resulting homogenates was conducted by an established method

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described by Zhou et al. 28. Quantification of NO was expressed as micromole per

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gram based on the FW of root samples. Additionally, fresh hairy root samples were

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ground under liquid nitrogen using a mortar and pestle until fine powders were

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obtained. The extraction and determination of endogenous salicylic acid (SA) and

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jasmonic acid (JA) from the resulting powders was performed according to an

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established method reported by Segarra et al. 29. Quantification of SA and JA was

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expressed as nanogram per gram based on the fresh weight (FW) of root samples.

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Quantitative Real-time PCR (qRT-PCR) Analysis

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Total RNA was extracted from frozen hairy root samples using a MiniBEST Plant

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RNA Extraction Kit (TaKaRa, Dalian, China), and RNA was reverse-transcribed to

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cDNA using a PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China). Specific

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primers of genes involved in CA and FO biosynthetic pathway were designed

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according to our previous report 27. The reaction solution for qRT-PCR assay was

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prepared with a SYBR Premix Ex Taq™ II Kit (TaKaRa, Dalian, China) following the

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manufacturer’s guidelines. The qRT-PCR amplification procedure was performed as

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previously described 27. 18S was used as the internal reference gene, and the relative

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expression level of each target gene was quantified by the ∆∆CT method 30.

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Determination of Antioxidant Activity

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Activities of two antioxidant enzymes including superoxide dismutases (SOD)

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and catalase (CAT) in fresh hairy root samples were measured following the methods

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described by Arbona et al. 31, and activities of SOD and CAT were expressed as units

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per mg of protein that was detected in enzyme extracts. Additionally, non-enzymatic

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antioxidant properties of extracts (NEAPE) from AMHRCs was determined by the

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β-carotene/linoleic acid oxidation method reported by Simic et al. 32, and NEAPE

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activity was calculated as the β-carotene protection ratio of the tested sample relative

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to the control.

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Statistical Analysis

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All experiments were conducted in triplicate, and results were given as averages ±

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standard deviations. All statistical analyses were carried out using the SPSS statistical

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software 17.0 (SPSS Inc, Chicago, USA). One-way analysis of variance with Tukey’s

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test was used to determine significant differences between multiple groups of data at P

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values < 0.05.

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RESULTS AND DISCUSSION

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Feasibility of IAN/IAO Treatment for Enhancing FO and CA Yield in AMHRCs

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In our previous report, AMHRCs initiated by culturing a high-productive A.

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membranaceus hairy root line (AMHRL II) at day 34 exhibited the maximum

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productivity of isoflavone derivatives (CAG, FOG, CA and FO), independent of the

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elicitor used 15. Accordingly, 34 day-old AMHRCs was adopted as the ideal system

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processed by immobilized Aspergillus to further promote the production of two target

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isoflavone aglycones (CA and FO) in this work. Moreover, in most of the studies done

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to date to boost phytochemicals production in plant cell/organ cultures treated by fungi,

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extracts of fungal mycelia and culture filtrates have always been utilized 33. However,

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there are very few studies available where live fungus cells are used as elicitor. Thus,

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34 day-old AMHRCs were initially co-cultured with IAN and IAO for 2 days under

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the pre-determined parameters to evaluate whether IAN and IAO treatment can

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enhance CA and FO accumulation in this work.

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As shown in Figure 2, the levels of two target isoflavone aglycones in AMHRCs

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challenged by IAN (658.69 ± 54.51 µg/g DW of CA and 998.45 ± 86.92 µg/g DW of

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FO) and IAO (466.77 ± 33.02 µg/g DW of CA and 613.78 ± 51.14 µg/g DW of FO)

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were much higher as compared to those in non-treated AMHRCs (96.44 ± 1.83 µg/g

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DW of CA and 61.29 ± 0.91 µg/g DW of FO) and CG-treated AMHRCs (95.71 ± 2.60

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µg/g DW of CA and 62.55 ± 3.28 µg/g DW of FO). Moreover, there were no

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significant differences in CA and FO yield between non-treated AMHRCs and CG

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-treated AMHRCs, which clearly eliminated the interference of the immobilization

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matrix (CG) on phytochemicals production in AMHRCs. As expected, the application

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of IAN and IAO treatment was feasible for the augmented production of CA and FO in

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AMHRCs, which also opened a gate to perform the following studies.

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Time-course of Isoflavone Yield in AMHRCs Co-cultured With IAN/IAO

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For the accurate comparison of IAN and IAO treatment on isoflavone profiles in

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AMHRCs, yields of CA and FO as well as their glycosides (CAG and FOG) were

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monitored along a time course from 0 to 72 h. Moreover, the dynamic study was also

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favorable to investigate and understand the dual effects (elicitation and deglycosylation)

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of immobilized Aspergillus in AMHRCs.

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In comparison with non-treated control, the accumulation pattern of all analytes in

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AMHRCs challenged by IAN and IAO was similar at 6−24 h post fungal treatment,

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exhibiting a gradual increase tendency (Figure 3). Due to the natural pathogenicity of

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microbes to host plants, fungi are capable of producing various pathogenesis-related

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substances (proteins and carbohydrates as well as their derivatives) that can induce

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hypersensitive responses in plant cells, thus quickly prompting the phytoalexin

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production during the initial stages of fungal infection 33. In this regard, the present

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results were consistent with the aforementioned description. Interestingly, the yields of

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two isoflavone glycosides (CAG and FOG) were observed to decrease synchronously

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from 24 to 36 h in IAN-treated AMHRCs and from 24 to 42 h in IAO-treated

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AMHRCs, and kept at low levels afterwards (Figure 3). Under the constant attacks of

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potential pathogens, plants cells are able to transfer the secondary metabolites

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excessively accumulated in cytoplasm to the extracellular region, which can diminish

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the toxicity expected when the intracellular secondary metabolites are present at high

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levels, and also is conducive to defense the pathogen invasion 34. It is worth

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mentioning that A. niger and A. oryzae are capable of secreting extracellular

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β-glucosidase that can hydrolyze glucoside moieties, whereby isoflavones glycosides

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can be effectively conversed into their aglycones 23. As inferred, the β-glucosidase

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secreted from IAN and IAO could exercise their deglycosylation function for the

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hydrolysis of glucoside residues in CAG and FOG to produce CA and FO in the

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culture media of AMHRCs, which might be the reason for the continuous decrease of

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CAG and FOG over the periods of 24 to 42 h in this study. In contrast to isoflavones

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glycosides, the yields of two target isoflavone aglycones (CA and FO) still maintained

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an increasing trend from 24 to 54 h in IAN-treated AMHRCs and from 24 to 48 h in

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IAO-treated AMHRCs, and kept high levels toward the end of the time course (Figure

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3). Indeed, the increased yields of CA and FO could be ascribed to the deglycosylation

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effect of IAN and IAO. However, the actual yields of CA and FO at each time point in

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the conversion process (24 to 42 h) were much higher than their forecasted values that

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were calculated from the deglycosylation of CAG and FOG (data not shown), which

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suggested that the elicitation effect of IAN and IAO mainly contributed to the

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significant enhancement in yields of CA and FO.

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Additionally, it is clearly observed from Figure 3 that IAN was superior as against

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IAO for promoting CA and FO production in AMHRCs during the overall time course.

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More specifically, the highest yields of CA (730.88 ± 63.72 µg/g DW) and FO

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(1119.42 ± 95.85 µg/g DW) in IAN-treated AMHRCs was achieved at the time point of

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54 h, which were significantly higher than the maximum levels of CA (481.03 ± 41.22

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µg/g DW) and FO (625.60 ± 38.77 µg/g DW) in IAO-treated cultures obtained at 48 h.

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This can be explained by the fact that different species of microbes have significantly

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different abilities to induce phytoalexins synthesis in plants. Moreover, the optimal

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accumulations of CA and FO in IAN-treated AMHRCs increased 7.72- and 18.78-fold

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as against CA (94.71 ± 3.66 µg/g DW) and FO (59.62 ± 2.64 µg/g) in non-treated

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control (54 h), respectively. The remarkable enhancement of CA and FO could be

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appreciated by comparing LC-MS/MS chromatograms of extracts from non- and

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IAN-treated AMHRCs (Figure 4).

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Signal Molecule Generation in AMHRCs in Response to IAN Treatment

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Generally, the invasion from pathogens (fungi, bacteria, viruses, etc.) can be

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recognized by specific receptors localized to plasma membranes of plant cells, which

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will activate signaling cascades and prompt the release of endogenous signal molecules

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(terming as danger-associated molecular patterns) in cytosol that ultimately trigger

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defensive secondary metabolism and thereby enhance phytoalexin biosynthesis 35. NO,

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SA and JA are vital endogenous signal molecules involved in plant defense regulatory

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systems against pathogen attack 36. To verify whether the generation of signal

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molecules would be enhanced in AMHRCs in response to IAN treatment, the contents

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of NO, SA and JA in fresh root samples harvested at 0 h, 6 h, 12 h, 18 h, 24 h, 36 h,

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and 42 h post-treatment were determined in this work.

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As shown in Figure 5A, NO generated immediately in IAN-treated AMHRCs,

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reached its peak value (229.41 ± 35.66 µmol/g FW) at 6 h, and declined rapidly to the

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control level afterwards. As exhibited in Figure 5B, the SA content in IAN-treated

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AMHRCs was observed to be highest (481.22 ± 57.93 ng/g FW) at 12 h, and reverted

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to the control level after 36 h. As displayed in Figure 5C, the JA generation in

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IAN-treated AMHRCs began to increase at 18 h, achieved the maximum level (28.38 ±

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3.04 ng/g FW) at 36 h, and decreased gradually afterwards. Obviously, these signal

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molecules accumulated transiently after IAN elicitation, and the enhanced generation

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of NO, SA and JA occurred sequentially.

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In most case, NO burst is considered to be an early response of plant cells to

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fungal pathogens, which can also mobilize the generation of other signal molecules

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such as SA, JA, ethylene, etc. 36. Additionally, SA can accumulate rapidly at the

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infection site following fungal invasion, due to its function as an inducer of systematic

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acquired resistance in plant-pathogen interactions 35. Moreover, SA is reported to

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antagonize JA biosynthesis in plant responses to pathogen/pest attacks, and SA

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reaching a certain level often suppresses the endogenous production of JA, thus

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leading to JA accumulation being later than SA 37. Overall, the results presented here

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concluded that the sequentially transient accumulation of endogenous NO, SA and JA

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constituted an important line in the defense responses of IAN treatment, which might

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contribute to the up-regulated expression of associated genes in CA and FO

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biosynthetic pathway.

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Biosynthetic Gene Expression in AMHRCs Underlying IAN Treatment

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To further investigate the molecular events following the aforementioned signal

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transduction underlying IAN treatment, the transcriptional profiles of eight genes

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encoding enzymes that are involved in CA and FO biosynthetic pathway (Figure 6), i.e.

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phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate

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coenzyme A ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR),

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chalcone isomerase (CHI), isoflavone synthase (IFS) and isoflavone 3’-hydroxylase

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(I3’H), were determined by qRT-PCR. Hairy root samples collected from IAN-treated

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AMHRCs at different time intervals (18 h, 36 h, 54 h and 72 h) were applied for

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qRT-PCR analysis in this work.

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As shown in Figure 6, all investigated genes were significantly up-regulated in

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IAN-treated AMHRCs during the period from 18 h to 54 h, which indicated that the

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elevated production of CA and FO might be achieved through the enhanced

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transcription of these biosynthetic genes. Interestingly, the highest expression levels of

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upstream genes in the biosynthetic pathway, i.e. PAL, C4H, 4CL, CHS, CHR and CHI,

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were found at the time points before 54 h that was necessary for the optimal

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accumulation of CA and FO. This was ascribed to a typical metabolic phenomenon

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that a time lag exists between the upstream gene expression and the downstream

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product synthesis 38. However, the transcriptional profiles of two downstream genes

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(IFS and I3’H) were consistent with the contents of CA and FO over the time from 18

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h to 54 h. Particularly, the expression level of IFS gene in IAN-treated AMHRCs at 54

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h was found to be highest among all tested genes, i.e. 32.35-fold higher relative to

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control.

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Factually, IFS is the critical checkpoint that can direct metabolic entry to

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isoflavonoid biosynthesis 39. The significant induction of IFS transcription here

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suggested that it might play a vital role in the up-regulation of CA and FO production

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in AMHRCs following IAN treatment. This was consistent with our previous report

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that IFS was a crucial regulatory gene controlling isoflavonoid biosynthesis in

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AMHRCs elicited by methyl jasmonate 40. Additionally, the expression levels of all

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biosynthetic genes were observed to be considerably repressed at 72 h in comparison

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with control, which can be explained that the prolonged exposure of plant cells/organs

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to pathogens would lead to the excessive hypersensitive responses that were

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characterized by the rapid metabolic damage or cell death in extreme cases 36.

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Antioxidant Activity in AMHRCs Following IAN Treatment

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A significant characteristic of reactions in plant cells attacked by fungal

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pathogens is the sudden overproduction of reactive oxygen species (ROS), which can

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be toxic to invading pathogens but also cause damages to nucleic acids, proteins and

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lipids in host cells viz. oxidative stress 34. In this work, the hairy root tissues of

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IAN-treated AMHRCs exhibited a dark yellow color (Figure 7A) in comparison with

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the white root tissues observed in non-treated control cultures (Figure 7B), which

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indicated a conclusive evidence of oxidative stress following the fungal attack. Plant

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cells are equipped with an efficient antioxidant defense system comprised of

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antioxidant enzymes and non-enzymatic antioxidant metabolites that can work in

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concert to detoxify the detrimental effects of ROS mediated oxidative stress 41.

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Accordingly, activities of two representative antioxidant enzymes (SOD and CAT) as

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well as non-enzymatic antioxidant properties of extracts (NEAPE) from hairy root

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samples were evaluated in this work at 0 h, 18 h, 36 h, 54 h and 72 h post-treatment to

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clarify the antioxidant response of AMHRCs following IAN treatment.

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As shown in Figure 7C, the SOD activity in IAN-treated AMHRCs was noticed to

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increase strongly during the first 18 h, while it decreased rapidly afterwards. As

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exhibited in Figure 7D, the CAT activity increased gradually in IAN-treated AMHRCs,

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achieved its maximum value at 36 h, and decreased gradually afterwards. As displayed

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in Figure 7E, the NEAPE level in IAN-treated AMHRCs was not significantly changed

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during the early elicitation period, began to increase at 36 h, and entered a platform

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phase after 54 h. Comparison of antioxidant enzyme activities showed the higher

352

activity of SOD as against CAT in IAN-treated AMHRCs during the early phase of

353

post-elicitation (0-18 h). Factually, SOD can directly dismutate O2•– into H2O2, which

354

is considered to be the first line of defense against ROS overproduction in plants

355

suffering from environmental stresses, while CAT is an indispensable enzyme

356

responsible for the subsequent dismutation of H2O2 into H2O and O2 41. Additionally,

357

the results in this work indicated an early up-regulation of SOD and CAT activity (0-36

358

h), while NEAPE level were enhanced at the end of post-elicitation (54-72 h).

359

Generally, plants utilize antioxidant enzymes as the premier contributors for ROS

360

detoxification during stressed conditions. However, the depletion of antioxidant

361

enzyme activity needs to be compensated by the action of non-enzymatic antioxidant

362

metabolites 41.

363

It is noteworthy that IAN-treated AMHRCs showed the highest NEAPE level

364

(3.31-fold higher relative to control) at 54 h post-treatment where the accumulation of

365

CA and FO was maximum. Accordingly, it was inferred that the increase in the content

366

of CA and FO might contribute to the enhancement in antioxidant activity of

367

IAN-treated samples. Factually, CA and FO as two representative isoflavone algycones

368

in A. membranaceu exhibit much better antioxidant activity than their glycosides

369

(CAG and FOG), which is ascribed to the presence of more phenolic hydroxyls that

370

can act as hydrogen/electron donors to neutralize ROS or peroxyl free radicals 42.

371

Moreover, it is reported that leguminous plants challenged by fungal pathogens can

372

drastically induce isoflavone aglycone biosynthesis, thus leading to the significant

373

increase in antioxidant activity 43. Furthermore, stress-induced ROS overproduction

374

can be counteracted by other antioxidant metabolites as diverse as ascorbic acid,

375

glutathione, praline, tocopherol, carotenoid, etc. 41. Therefore, the higher antioxidant

15



376

property of extracts from IAN-treated AMHRCs was likely attributed to the synergistic

377

effects and redox interactions among the aforementioned antioxidants.

378

Overall, the elevation of SOD and CAT activity and NEAPE level within

379

IAN-treated AMHRCs not only suggested a positive-feedback response to fight the

380

oxidative stress, but also indicated an attempt to maintain the cellular redox status to

381

minimize the destructive consequences of oxidative stress. Also, this study

382

demonstrated the potential role of AMHRC extracts rich in CA and FO as natural

383

antioxidant additives for possible nutraceutical and pharmaceutical uses.

384

Reusability of IAN Beads in AMHRCs

385

Investigation of the reusability of immobilized microorganisms is necessary for

386

the evaluation of their practical application potential. In this work, the reusability of

387

IAN beads in AMHRCs was checked by the determination of CA and FO yield during

388

10 successive batches. After 5 cycles, 74.06% and 81.87% of the initial CA and FO

389

yield was still achieved in IAN-treated AMHRCs (Figure 8A), respectively, which

390

indicated the acceptable reusability performance of IAN beads. Comparison of the

391

photographs of IAN beads before (Figure 8B) and after 5 cycles (Figure 8C) revealed

392

that the shape of the recovered beads was nearly unchanged. However, the beads after

393

multiple uses exhibited a light yellow color, which was likely to be ascribed to the

394

inherent adsorption properties of the matrix of IAN beads, i.e. CG that might adsorb

395

the colored metabolites form AMHRCs. For further reducing costs of the overall

396

process, the improvement in the reusability of IAN beads through other immobilization

397

methods will be the next challenge.

398

In conclusion, this work provided a safe and efficient method for the high-level

399

production of two bioactive isoflavone aglycones (CA and FO) using a well-controlled

400

co-cultivation system of food-grade IAN and AMHRCs. During the co-cultivation

16


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401

process, the enhanced yield of CA and FO might be partly due to the IAN

402

deglycosylation, but mainly owing to the IAN elicitation. In detail, IAN elicitation

403

could trigger the sequentially transient accumulation of endogenous signal molecules

404

NO, SA and JA, thus leading to the transcriptional activation of genes involved in CA

405

and FO biosynthetic pathway, which ultimately boosted the accumulation of two target

406

isoflavone aglycones in AMHRCs. Moreover, the up-regulation of SOD and CAT

407

activity followed by the enhancement of NEAPE level suggested a positive-feedback

408

response to detoxify the harmful ROS within AMHRCs challenged by IAN elicitation.

409

Furthermore, the satisfactory reusability of IAN beads indicated that the proposed

410

approach could offer an economic way for industrial applications. Thus, it is highly

411

expected to utilize this promising co-cultivation system for the scale-up production of

412

CA and FO by the aid of bioreactor technology in the future.

413 414 415 416 417 418 419 420 421 422 423 424 425

17



426 427

ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial supports by National Key R&D

428

Program of China (2017YFD0600205), Fundamental Research Funds for the Central

429

Universities (2572017DA04), Heilongjiang Province Science Foundation for Youths

430

(QC2017012), Scientific Research Start-up Funds for Talents Introduction of Northeast

431

Forestry University (1020160010), and Double First-rate Special Funds (41112432).

432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450

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592 593 594 595 596 597 598 599 600

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FIGURE CAPTIONS

602 603

Figure 1. (A) Cultivation of the high-productive AMHRL II on MS solid medium; (B)

604

AMHRCs obtained by culturing AMHRL II for 34 days in MS liquid medium; (C) A.

605

niger 3.3883 colony on PDA medium; (D) A. oryzae 3.951 colony on PDA medium; (E)

606

co-cultivation of IAN beads with 34 day-old AMHRCs at the initial stage; (F)

607

co-cultivation of IAN beads with 34 day-old AMHRCs for 54 h.

608 609

Figure 2. Effects of non-, CG, IAN- and IAO-treatments on yields of CA and FO in

610

34-day-old AMHRCs (spores loaded per flask ca.104, incubation temperature 30 °C,

611

initial pH value 7.0 and time 48 h). Non, non-treated AMHRCs; CG, CG-treated

612

AMHRCs; IAN, IAN-treated AMHRCs; IAO, IAO-treated AMHRCs. Mean ± SD

613

values not sharing the same lowercase letters are significantly different (P < 0.05).

614 615

Figure 3. A 72 h time course of isoflavone profile in 34-day-old AMHRCs under non-,

616

IAN- and IAO-treatments (spores loaded per flask ca.104, incubation temperature

617

30 °C and initial pH value 7.0). Mean ± SD values not sharing the same lowercase

618

letters are significantly different (P < 0.05).

619 620

Figure 4. Representative LC–MS/MS with SRM total ion chromatograms of extracts

621

from non- and IAN-treated AMHRCs.

622 623

Figure 5. Accumulation of endogenous signal molecules including NO (A), SA (B)

624

and JA (C) in non- and IAN-treated AMHRCs at different time-points (0, 6, 12, 18, 24,

625

36 and 42, h). Mean ± SD values not sharing the same lowercase letters are

25



626

significantly different (P < 0.05).

627 628

Figure 6. Transcriptional profiles of eight enzymatic genes involved in CA and FO

629

biosynthetic pathway in IAN-treated AMHRCs at different time-points (18, 36, 54 and

630

72, h). PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL,

631

4-coumarate coenzyme A ligase; CHS, chalcone synthase; CHR, chalcone reductase;

632

CHI, chalcone isomerase; IFS, isoflavone synthase; I3’H, isoflavone 3’-hydroxylase.

633

Mean ± SD values not sharing the same lowercase letters are significantly different (P

634

< 0.05).

635 636

Figure 7. Phenotypes of AMHRCs before (A) and after (B) IAN-treatment; activities

637

of SOD (C), CAT (D) and NEAPE (E) in non- and IAN-treated AMHRCs at different

638

time-points (0, 18, 36, 54 and 72, h). Mean ± SD values not sharing the same

639

lowercase letters are significantly different (P < 0.05).

640 641

Figure 8. (A) Reusability of the recovered IAN beads during 10 successive batches;

642

photographs of IAN beads (B) before and (C) after 5 cycles. Mean ± SD values not

643

sharing the same lowercase letters are significantly different (P < 0.05).

644 645 646 647 648 649 650 651

26


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Figure 1

27



Figure 2

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Figure 3

29



Figure 4

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Figure 5

31



Figure 6

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Figure 7

33



Figure 8

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Graphic for table of contents

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Enhanced Production of Two Bioactive Isoflavone Aglycones in

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