Appl Microbiol Biotechnol (2015) 99:4629–4643 DOI 10.1007/s00253-015-6491-7

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Production of pinoresinol diglucoside, pinoresinol monoglucoside, and pinoresinol by Phomopsis sp. XP-8 using mung bean and its major components Yan Zhang & Junling Shi & Zhenhong Gao & Ruiming Yangwu & Huanshi Jiang & Jinxin Che & Yanlin Liu

Received: 25 June 2014 / Revised: 7 February 2015 / Accepted: 17 February 2015 / Published online: 25 March 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Phomopsis sp. XP-8 is an endophytic fungus that has the ability to produce pinoresinol diglucoside (PDG) in vitro and thus has potential application for the biosynthesis of PDG independent of plants. When cultivated in mung bean medium, PDG production was significantly improved and pinoresinol monoglucoside (PMG) and pinoresinol (Pin) were also found in the culture medium. In this experiment, starch, protein, and polysaccharides were isolated from mung beans and separately used as the sole substrate in order to explore the mechanism of fermentation and identify the major substrates that attributed to the biotransformation of PDG, PMG, and Pin. The production of PDG, PMG, and Pin was monitored using high-performance liquid chromatography (HPLC) and confirmed using HPLC-MS. Activities of related enzymes, including phenylalanine ammonia-lyase (PAL), transcinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) were analyzed and tracked during the cultivation. The reaction system contained the compounds isolated from mung bean in the designed amount. Accumulation of phenylalanine, cinnamic acid, p-coumaric acid, PDG, PMG, and Pin and the activities of PAL, C4H, and 4CL were measured during the bioconversion. PMG was found only when mung bean Y. Zhang : Z. Gao : R. Yangwu : H. Jiang : J. Che College of Food Science and Engineering, Northwest A&F University, 28 Xinong Road, Yangling, Shaanxi Province 712100, China J. Shi (*) Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an, Shaanxi Province 710072, China e-mail: [email protected] Y. Liu College of Enology, Northwest A&F University, 28 Xinong Road, Yangling, Shaanxi Province 712100, China

polysaccharide was analyzed, while production of PDG and Pin were found when both polysaccharide and starch were analyzed. After examining the monosaccharide composition of the mung bean polysaccharide and the effect of the different monosaccharides had on the production of PMG, PDG, and Pin, galactose in mung bean polysaccharide proved to be the major factor that stimulates the production of PMG. Keywords Phomopsis sp. XP-8 . Bioconversion . Pinoresinol diglucoside . Pinoresinol monoglucoside . Pinoresinol

Introduction (+)-Pinoresinol (Pin), one of the simplest lignans, is the precursor of other dietary lignans (Elder 2014). It is widely found in flaxseed and other seeds, as well as fruits, vegetables, and beverages (Landete 2012). Pinoresinol diglucoside [(+)-1pinoresinol 4,4′-di-O-β- D -glucopyranoside] (PDG) and pinoresinol monoglucoside [(+)-pinoresinol-4-O-β- D glucopyranoside] (PMG) are glycosides of Pin that are more soluble and stable in water solutions. PDG is the major antihypertensive element found in water extracted from the bark of Tu-Chung (Eucommia ulmoides Oliv.), a popular Chinese herb medicine used for controlling abnormal blood pressure (Charles et al. 1976; Luo et al. 2010). Also, in B16 melanoma cells that have been stimulated by αmelanocyte-stimulating hormone (α-MSH), both PDG and PMG are capable of inhibiting melanogenesis. PMG and Pin have inflammatory responses in LPS-activated microglia (Jung et al. 2010), high inhibitory activity against cyclic adenosine monophosphate phosphodiesterase in vitro (Tsukamoto et al.

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1984), and antifungal effects (Hwang et al. 2010). Three of them are also valuable as potential skin-whitening agents (Akihisa et al. 2013). More importantly, PDG, PMG, and Pin are precursors of enterolactone (Xie et al. 2003), which is a compound that has significant preventative effects against various cancers, including breast (Xie et al. 2013; Pietinen et al. 2001), prostate, cervical, and colon cancers (Adlercreutz 2002). Pin, PMG, and PDG are primarily found and reported in plants, since they are plant lignans or derivatives thereof. They are normally produced via the phenylpropanoid pathway in plants (Vermerris and Nicholson 2006). Occurrence of them in the metabolism of microorganisms was rarely reported until it was found that Phomopsis sp. XP-8, an endophytic fungus isolated from bark of Tu-Chung, produced PDG when cultivated in vitro (Shi et al. 2012). Mung bean is a rich source of phenylalanine (Randhir and Shetty 2007) and phenolic antioxidants that are biosynthesized through phenylpropanoid pathway (Randhir et al. 2004). In our preliminary experiments, when Phomopsis sp. XP-8 was cultivated in mung bean medium, PDG production was significantly improved and accumulation of Pin and PMG was detected. Occurrence of Pin and its glycosides has not been found in mung beans although Pin has been widely found as the major type of lignan in fruits and vegetables (Landete 2012). In addition, mung bean is widely grown and easily obtained around the world (Randhir et al. 2004). Bioconversion of Pin, PDG, and PMG from mung bean or its compounds should have advantage in low cost. However, the composition, normally, the content of each compound in mung bean, varies with mung bean variety and the place of production. This would cause fluctuation among different batches of microbial fermentation. Therefore, it is essential to understand the major mung bean compounds that influence the production of Pin, PMG, and PDG by Phomopsis sp. XP-8 and thus guide the regulation of medium composition or develop a modified medium to stabilize the fermentation processing. Starch, water-soluble polysaccharides, and protein are reported as the major compositions in mung bean (Mubarak 2005). Theoretically, they may also influence the production of Pin, PMG, and PDG by providing precursors of these products. Therefore, in this study, we separately isolated water-soluble polysaccharides, starch, and protein from mung beans and then used them as the sole substrate during bioconversion to identify the major compounds that attributed to the biosynthesis of Pin, PMG, and PDG by Phomopsis sp. XP-8.

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Mung bean (Vigna radiata (L.) R. Wilczak) used in the study was bought from the local market in Yangling City, Shaanxi Province, China. Chromatographical purity of phenylalanine (Phe) (Sigma, St Louis, MO, USA), cinnamic acid and p-coumaric acid (98 %; Aladdin, Shanghai, China), PDG, PMG, and Pin (≥99 %; National Institutes for Food and Drug Control, Beijing, China) were used as the standards (dissolved by methyl alcohol) in the measurements. CoA-SH (Sigma) and glucose-6-phosphate sodium salt (G-6-PNa2) and ATP (MP Biomedicals, Santa Ana, CA, USA) were used in the enzyme reactions to detect enzyme activity. Monosaccharides used in the experiment include rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), galactose (Gal), and glucose (Glu) (Sigma, St Louis, MO, USA). Isolation and purification of water-soluble polysaccharide, starch, and protein from mung bean The main chemical compositions (starch, water-soluble polysaccharides, and protein) of mung bean grain medium after sterilized at 121 °C for 30 min were analyzed according to the method reported by Mubarak (2005). Water-soluble polysaccharides were extracted from mung beans according to method reported by Yang et al. (2008). Starch was extracted from mung beans according to previously reported method (Maisa et al. 2013). Protein isolate from mung beans was prepared using the method described by ElAdawy (2000). Preparation of Phomopsis sp. XP-8 cells Phomopsis sp. XP-8 was grown at 28 °C on potato dextrose agar (PDA) plates for 5 days, and then, three pieces of mycelia (5 mm in diameter, obtained from 5-day-old PDA culture ) was inoculated into 100 mL liquid potato dextrose broth (PDB) in a 250-mL flask and cultivated at 28 °C in a rotary shaker (180 rpm). After 4 days, the cells were collected by centrifugation at 1136×g for 10 min at 4 °C using a refrigerated centrifuge (HC-3018R, Anhui USTC Zonkia Scientific Instruments Co., Ltd., Anhui, China). The cells were washed two times with sterile water and used to bioconverse PDG, PMG, and Pin throughout the study. Conditions for bioconversion

Materials and methods Microorganism, mung bean, and chemicals Phomopsis sp. XP-8, obtained from the China Center for Type Culture Collection (Wuhan, China) (code: Phomopsis sp. CCTCC M 209291), was used in the study.

The medium for bioconversion contained 0.15 g/L glucose (pH 7). For bioconversion, the prepared Phomopsis sp. XP-8 cells were added at 10 g cells (wet weight) per 100-mL medium (in a 250-mL flask). The bioconversion was carried out for 48 h at 28 °C and 180 rpm in a ZHWY-2102 constant temperature shaker (Shanghai WitCity Analyzing Instrument Manufactory Co., Ltd., Shanghai, China).

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After bioconversion, cells and medium were separately collected from the culture by filtration with intermediate speed qualitative filter paper (GB/T1914-2007, 102, Hangzhou Whatman-Xinhua Filter Paper Co., Ltd., Hangzhou, China) and used for further research on enzyme activities and the accumulation of Phe, cinnamic acid, p-coumaric acid, PDG, PMG, and Pin. Bioconversion of PDG, PMG, and Pin from different components isolated from mung bean In the bioconversion medium containing 100 g/L Phomopsis sp. XP-8 cells, the isolated starch, polysaccharide, protein and their combinations, and whole mung bean powder were separately added and sterilized at 121 °C for 30 min before bioconversion. After bioconversion for 56 h, production of PDG, PMG, and Pin was analyzed and identified. The medium without mung bean partials, but containing Phomopsis sp. XP-8 cells, was used as the control. Each treatment was conducted in duplicate, and the mean values are presented with standard deviations. Intermediates accumulated during bioconversion of PDG, PMG, or/and Pin from mung bean polysaccharide and starch In the bioconversion medium, mung bean polysaccharide (35 g/L) or starch (30 g/L) was added and sterilized at 121 °C for 30 min before bioconversion. Then, 100 g/L Phomopsis sp. XP-8 cells were added. During bioconversion, accumulation of Phe, cinnamic acid, and p-coumaric acid inside and outside cells were monitored at 8, 16, 24, 32, 40, 48, 56, 64, 72, and 80 h. The effects of starch concentration (5, 10, 15, 20, 25, 30, 35, and 40 g/L) and polysaccharide concentration (5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 g/L) on the results were also tested at 48 and 56 h, respectively. Production of PDG, PMG, and Pin outside cells was also monitored throughout the bioconversion with all treatments. Each treatment was conducted in duplicate. The mean values are presented with standard deviation. Distribution of the enzymes in phenylpropanoid pathway in culture of Phomopsis sp. XP-8 Distribution of the enzymes involved in biosynthesis of PDG, PMG, and Pin in Phomopsis sp. XP-8 was estimated according to the capability of the crude enzyme extracts from the culture of outside and inside cells after cultivation for 56 h in the bioconversion medium with mung bean starch or polysaccharide as substrates. Namely, the bioconversion culture (100 mL) with mung bean starch or mung bean polysaccharide as the substrate was collected after 56 h and separated into cells and liquid phase by filtration through a quantitative filter paper. Into the liquid phase, ammonium sulfate was added up to 75 % saturation to precipitate the crude enzymes. The precipitation was

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carried out overnight at 4 °C, and then, the precipitated protein was collected by centrifugation for 10 min at 13,363×g. After washing twice with a Tris–HCl (Three hydroxymethyl aminomethane hydrochloride) buffer (pH 8.6), the protein sediment, which represents the extracellular enzyme extracts, was dissolved in 8 mL Tris–HCl buffer (pH 8.6). The obtained cells were washed twice with Tris–HCl buffer (pH 8.6) and homogenized with 6 mL Tris–HCl buffer (pH 8.6) in an ice bath using a mortar. After centrifugation at 3340×g for 10 min, the supernatant, which represents the intracellular enzyme extract, was collected and made up to 8 mL with Tris–HCl buffer. he overall activity of the obtained enzyme extracts to convert mung bean starch or polysaccharide to PDG, PMG, and Pin was analyzed by adding 5-mL enzyme extracts into 100-mL medium containing 30 g/L mung bean starch or 35 g/L polysaccharide. After 5 h, the bioconversion was immediately stopped by cooling the culture from 28 to 4 °C. A bioconversion medium containing 5 mL distilled water, instead of enzyme extracts, was used as the blank control. Activities of phenylalanine ammonia lyase (PAL, E.C.4.3.1.5), cinnamate 4-hydroxylase (C4H, EC1.14.13.11), and 4-coumarate-CoA ligase (4CL, EC 6.2.1.12) in the enzyme extracts were also tested to identify the existence of these enzymes in Phomopsis sp. XP-8. Each treatment was conducted in duplicate. The mean values and standard deviations are presented. Activity of phenylpropanoid pathway enzymes during the bioconversion with mung bean polysaccharide and starch as substrates At 8, 16, 24, 32, 40, 48, 56, 64, 72, and 80 h during the bioconversion with 35 g/L mung bean polysaccharide or 30 g/L mung bean starch as a substrate, enzymes were extracted from Phomopsis sp. XP-8 cells and the activities of PAL, C4H, and 4CL were measured. The effects of starch concentration (5, 10, 15, 20, 25, 30, 35, and 40 g/L) and mung bean polysaccharide concentration (5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 g/L) on activities of PAL, C4H, and 4CL were also investigated at 48 and 56 h, respectively, during the bioconversion. Each treatment was conducted in duplicate. Measurement of enzyme activities Enzyme activities were measured using the prepared enzyme extracts in Tris–HCl buffer, pH 8.6. Protein concentration of the enzyme extracts was determined using the visible spectrophotometer, UVmini-1240 (Shimadzu, Kyoto, Japan), according to the Bradford method with bovine serum albumin (BSA) as the standard. All enzyme assays were conducted twice using freshly prepared extracts. At the end of the assays, the reaction system was centrifuged at 13,363×g for 10 min and the absorbance (OD value) of the supernatant was determined

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using the visible spectrophotometer, UVmini-1240. One unit (U) of the enzyme was defined as an increase of 0.01 of OD value per hour, and the enzyme activity was expressed as the units of enzyme per milligram protein (U/mg). The presented results are averages of duplicates. PAL, C4H, and 4CL activity was measured according to Zhang et al. (2013). Bioconversion of PDG, PMG, and Pin using monosaccharides as substrate Monosaccharide components of mung bean polysaccharide were obtained from the results reported by Lai et al. (2010). Mung bean polysaccharide was reported to mainly consist of mannose (43.74 %) and galactose (23.52 %); it also contains rhamnose (11.61 %), arabinose (11.21 %), xylose (3.60 %), and glucose (5.11 %) (Lai et al. 2010). Therefore, each of these six monosaccharide (10 g/L) was either individually added or in conjunction with mung bean starch (30 g/L) to the bioconversion medium and sterilized at 121 °C for 30 min before 100 g/L Phomopsis sp. XP-8 cells were added to initiate the bioconversion. The effect of the combination of glucose, arabinose, galactose, xylose, and mannose on the production of PDG, PMG, and/or Pin was also tested by adding these monosaccharides together to the bioconversion medium without mung starch because they promoted the bioconversion process. The addition of rhamnose was not considered in the combination effect tests due to its inhibitory effect on the bioconversion process. Two different kinds of combinations were designed to make comparison with the results obtained from single monosaccharide effect: combination 1, each monosaccharide was added at an equal amount of 2 g/L to make a total amount of 10 g/L, same to that used in the monosaccharide effect tests; combination 2, five monosaccharides were added at different amounts—10 g/L glucose, 5 g/L arabinose, 6 g/L galactose, 8 g/L xylose, and 2 g/L mannose, corresponding to the optimal conditions for overall production of PDG, PMG, and Pin in the optimization experiments. All the bioconversions were carried out at 28 °C and 180 rpm. Production of PDG, PMG, and Pin were identified and analyzed after 56 h. A medium containing only Phomopsis sp. XP-8 cells without any substrates was used as the blank control. Each treatment was conducted in duplicate, and mean values and standard deviations were calculated. Measurement and identification of intermediates and production during bioconversion In all treatments, measurement of PDG was only performed for the cell-free liquid phase according to previous result that accumulation of PDG was only found outside Phomopsis sp. XP-8 cells in liquid culture (Shi et al. 2012). Accumulated

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amount of PMG, Pin, Phe, cinnamic acid, and p-coumaric acid were measured both inside and outside the cells in the bioconversion system. Before high performance liquid chromatography (HPLC) measurements, Phomopsis sp. XP-8 cells were crushed with quartz sand for 10 min in an ice bath and then mixed with the cell-free liquid culture for extraction of PMG, Pin, Phe, cinnamic acid, and p-coumaric acid. For preparation of samples for measurement of PDG, PMG, Pin, cinnamic acid, and p-coumaric acid, twofold volume of 95 % ethanol was added in the mixture of crushed cells and cell-free liquid culture. After kept overnight at 4 °C, supernatant of the mixture was obtained after centrifugation at 5000×g for 10 min and then vacuum-evaporated at 0.09 MPa, 50 °C to remove ethanol and water. The dried residue was dissolved in 4 mL methanol (chromatographic grade; Sigma), filtered through a Millex®-HV filter membrane (0.45 μM, 13-mm diameter; Millipore, Billerica, MA, USA), and then directly subjected to HPLC measurement. Samples for measurements of phenylalanine were directly filtered through a Millex®-HV filter membrane (0.45 μmol/L, 13mm diameter; Millipore, Billerica, MA, USA), and then subjected to HPLC measurement. The concentrations of PDG, PMG, Pin, Phe, cinnamic acid, and p-coumaric acid in the reaction system were calculated by dividing the reading values obtained according to the standard curves in HPLC by 50, because the sample was concentrated by 50 times before the HPLC measurements. Concentrations of PDG, Pin, PMG, Phe, cinnamic acid, and p-coumaric acid were simultaneously determined using a Shimadzu Essentia LC-15C analytical HPLC system (Shimadzu) equipped with an LC-15C pump, a SIL10AF automated sample injector, an SPD-15C dual-wavelength detector, a Shimadzu Wondasil C18-column (250× 4.6 mm), and LC Solution software (Shimadzu). The column was operated at 35 °C. The mobile phase consisted of acetonitrile (chromatographic grade; Sigma) (solvent A) and ddH2O (solvent B). A multistep gradient was used for all analyses as follows: 0–10 min, 90 % (v/v) B; 10– 20 min, 80 % B; 20–30 min, 30 % B; 30–50 min, 90 % B. The flow rate was 1 mL/min, and the sample injection volume was 10 μL. The detection wavelength was 227, 226, 229, 280, 290, and 330 nm for PDG, Pin, PMG, Phe, cinnamic acid, and p-coumaric acid, respectively. Standard PDG, Pin, PMG, cinnamic acid, and p-coumaric acid (chromatographic grade; Sigma) were prepared in methanol solution; standard Phe (chromatographic grade; Sigma) was prepared in water solution. Identification of PDG, PMG, and Pin was firstly performed using thin-layer chromatography (TLC), using glass plates (20×20 cm) coated with GF254 silica gel (0.25-mm thickness). The developing solvent contained ethyl acetate, methyl alcohol, chloroform, and water at ratio of 6:5:2:0.1. Electrospray

8.76±0.25 1.25 ±0.22 0 10.74±0.15 10.75±0.15 10.76±0.25

0

Data are average and standard deviation of duplicates. The dates were obtained after bioconversion for 56 h

The abbreviations in the table indicate dry cell weight (DCW), Pinoresinol (Pin), Pinoresinol diglucoside (PDG), and Pinoresinol monoglucoside (PMG)

0 0 0.50 ±0.13 0 1.56±0.13 1.55±0.13 1.57±0.25 0 4.45±0.21 0.45 ±0.15 0 5.47±0.15 5.49±0.15 5.53±0.25 1.09±0.15 3.82±0.15 1.53±0.16 1.28±0.15 4.10±0.15 4.12±0.15 4.35±0.16 0 54.66±0.05 8.75±0.05 26.62±0.05 63.35±0.05 90.03±0.05 100 Blank control Mung bean starch Mung bean polysaccharide Mung bean protein Combination of starch and polysaccharide Combination of three components Mung bean crude powder

0 16.40±0.05 2.62±0.05 7.99±0.05 19.05±0.05 27.00±0.05 30±0.05

PMG (mg/L) PDG (mg/L) DCW (g/L) Addition amount (g/L) Percentage in mung bean (%)

The main chemical components and their respective percentages found in mung bean grain medium after sterilization at 121 °C for 30 min were identified as follows: starch 54.63 %, watersoluble polysaccharide 8.74 %, protein 26.6 %, fiber 4.61 %, fat 1.82 %, and ash 3.54 %. Therefore, these components were added into the bioconversion system in the ratio close to the above results. As shown in Table 1, when 30 g/L mung bean powder was analyzed in the bioconversion system, accumulations of PDG (5.53 mg/L), PMG (1.57 mg/L), and Pin (10.76 mg/L) were identified. When the isolated components of mung bean starch, polysaccharide, and protein were separately added in the bioconversion medium, Phomopsis sp. XP-8 converted starch to PDG (4.45 mg/L) and Pin (8.76 mg/L) and converted polysaccharide to PDG (0.45 mg/L), PMG (0.5 mg/L), and Pin (1.25 mg/L). No production of PDG, PMG, or Pin was found when protein was used as the sole substrate. Therefore, carbon sources should be the essential component for bioconversion of PDG, PMG, and Pin. When mung bean starch and polysaccharide were used together, production of PDG (5.47 mg/L), PMG (1.56 mg/ L), and Pin (10.74 mg/L) were simultaneously obtained. When isolated starch, polysaccharide, and protein were used together in the ratio as found in mung bean powder, production of PDG (5.49 mg/L), PMG (1.55 mg/L), and Pin (10.75 mg/L) produced values almost identical to those when only starch and polysaccharide were used together. When compared to the percentages produced from the bioconversion of whole mung bean powder, 80 % of PDG and 81 % of Pin were produced and no PMG was produced when starch was analyzed as the only substrate. When polysaccharide was analyzed as the only substrate, 8 % of PDG, 32 % of PMG, and 12 % of Pin were produced. Notably, when starch and polysaccharide were analyzed in conjunction, 98.9 % of PDG, 99.4 % of

Substrate

Bioconversion of PDG, PMG, and Pin from different components isolated from mung beans

Production of pinoresinol diglucoside, pinoresinol monoglucoside and pinoresinol by Phomopsis sp. XP-8 cells using different components of mung beans

Results

Table 1

ionization mass spectrometry (ESI−) method according to Shi et al. (2012) was also used to identify PDG, PMG, and Pin. Productions of cinnamic acid and p-coumaric acid and Phe were firstly identified using TLC. The TLC analysis was performed according to the method described by Kim et al. (2011) with modification using glass plates (20×20 cm) coated with GF254 silica gel (0.25-mm thickness). The developing solvent contained methylbenzene, ethyl acetate, and acetic acid at ratio of 5:3:2. Identification of cinnamic acid and pcoumaric acid was also identified using ESI− method in a Thermo LTQ XL ion trap HPLC-MS (Thermo Fisher Scientific Inc., USA) according to Zhang et al. (2013).

4633 Pin (mg/L)

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PMG, and 99.8 % of Pin were produced. This indicates that mung bean polysaccharide is the essential factor in PMG production; starch could increase this effect. It also indicates that starch is the major component that caused production of PDG and Pin; the addition of polysaccharide promoted this effect. Protein could not be directly converted to PDG, PMG, or Pin and also contributed little to their production when it was collectively analyzed with starch and polysaccharide. Therefore, it seems that the biosynthesis of PDG, PMG, and Pin by Phomopsis sp. XP-8 is barely influenced by protein metabolism. In order to explore the intrinsic effects of different mung bean components during the biosynthesis of PDG, PMG, and Pin, the intermediates accumulated during conversion were analyzed and the results interpreted as follows.

Fig. 1 The cell weight and accumulation of phenylalanine during reaction in the resting cell system with mung bean starch as the substrate. The used condition is 30 g/L mung bean starch in a and reaction time of 56 h in b, respectively. The signals in a and b indicate Phe (triangle) and dry cell weight (square)

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Accumulation of intermediates and products and enzyme activities during bioconversion with mung bean starch as substrate According to the reported biosynthesis pathways, starch can be degraded to glucose and then converted to phenylalanine by the shikimic acid pathway. Phe is then converted into lignans through many steps with cinnamic acid, p-coumaric acid, pcoumaroyl-CoA, caffeate, ferulate, feruloyl-CoA, coniferyl aldehyde, and coniferyl alcohol as intermediates (Donnez et al. 2009; Suzuki and Umezawa 2007). Phenylalanine/tyrosine ammonialyase (PAL/TAL), trans-C4H, and 4CL are the key enzymes involved in the biosynthesis pathway. Specifically, PAL converts phenylalanine to cinnamic acid, C4H converts cinnamic acid to

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p-coumaric acid and cinnamoyl-CoA to p-coumaroyl-CoA, and 4CL converts cinnamic acid to cinnamoyl-CoA and p-coumaric acid to p-coumaroyl-CoA with the aid of ATP and coenzyme A. p-Coumaric acid or p-coumaroyl-CoA is converted to coniferyl alcohol, then to lignans (KEGG PASSWAY MAP http://www. kegg.jp/kegg-bin/highlight_pathway?scale=1.0&map= map01061&keyword=pinoresinol). Finally, coniferyl alcohol is converted to Pin by a dirigent protein (Suzuki and Umezawa 2007). The pathway from Pin to PDG and PMG has not been clearly illustrated. As expected, during the bioconversion evaluating mung bean starch as the only substrate, accumulation of phenylalanine was found (Fig. 1), as well as cinnamic acid and pcoumaric acid, production of both Pin and PDG also occurred Fig. 2 The accumulation of cinnamic acid, p-coumaric acid, Pin, and PDG during reaction in the resting cell system with mung bean starch as the substrate. The used condition is 30 g/L mung bean starch in a and reaction time of 56 h in b, respectively. The signals in a and b indicate cinnamic acid (inverted triangle), p-coumaric acid (diamond), Pin (triangle), and PDG (square)

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(Fig. 2), and activities of PAL, C4H, and 4CL (Fig. 3) were also detected in water solution. Combining the curves shown in Figs. 1, 2, and 3 together, it can be observed that all detected intermediates, products, and enzyme activities showed a “saddle pattern” effect with the extension of bioconversion time and the increase in starch concentration. The amount of Phe, cinnamic acid, and p-coumaric acid reached the highest value at 32 h, earlier than the peak time of Pin and PDG (48 to 56 h), indicating that Phe, cinnamic acid, and pcoumaric acid are primary metabolites and Pin and PDG are secondary metabolites in the related biosynthesis pathway. The peak time of PAL and C4H activities appeared at 32 h, coinciding with the highest accumulation of their substrates and products: Phe, cinnamic acid, and p-cinnamic acid. The

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peak time of 4CL activity (40 h) was later than that of C4H and PAL activities, but earlier than that of Pin and PDG. This is consistent with the placement of 4CL in the phenylalanine pathway: positioned after PAL and C4H, but before the formation of Pin and PDG. Additionally, according to the results of the effect of mung bean starch concentration (measured at 56 h), all detected factors and enzyme activities reached the highest values at 30 g/L of mung bean starch (Figs. 2 and 3). It should be mentioned that a significant increase of cell weight was found during the bioconversion with starch as a substrate and reached the highest value at 48 h and 30 g/L starch (Fig. 1). The increase of cell growth might be another factor that caused the highest value of Pin detection at 48 h. Also to be noted, during the bioconversion, the amount of Phe (875 mg/L, 32 h) was always significantly higher than that of cinnamic acid (15.00 mg/L, 32 h) and p-coumaric acid (0.60 mg/ L, 32 h), and the production of Pin (11.83 mg/L, 48 h) was always higher than that of PDG (8.29 mg/L, 56 h). The activity of PAL was always higher than that of 4CL and C4H. C4H activity always had the lowest activity among the three enzymes. This was consistent with the higher accumulation of cinnamic acid than that of p-cinnamic acid, because both high PAL and low C4H would cause the accumulation of cinnamic acid in the biosynthesis pathway. Similarly, higher 4CL activity than C4H activity might be the intrinsic reason that caused the lowest accumulation of p-coumaric acid during the bioconversion.

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highest values (7.7, 8.5, and 5.5 mg/L, respectively) at 56 h, which is much faster than the value yielded in other chemicalbased media (11.65 mg/kg PDG after 216 h) (Shi et al. 2012). The highest activity of C4H was found at 32 h; the highest activity of PAL occurred at 32–40 h; and the highest activity of 4CL occurred at 40 h. All detected intermediates, products, and enzyme activities reached their highest value at 35 g/L mung bean polysaccharide when the bioconversion was stopped at 56 h (the time when products Pin, PDG, and PMG reached their highest values). The peak time of cinnamic acid was also consistent with that of PAL (32–40 h) and C4H activity (32 h), and the peak time of Phe and p-coumaric acid was consistent with that of PAL and 4CL (40 h), indicating the increase effect of substrate concentration on its corresponding enzyme activity then the activated enzyme promote the accumulation of related production: high accumulation of Phe in the medium may stimulate the PAL activity, then high PAL activity caused the high

Accumulation of intermediates and products and enzyme activities with mung bean polysaccharide as substrate Similar to that found in the mung bean starch experiments, during the bioconversion evaluating mung bean polysaccharide as the only substrate in water solution, accumulations of phenylalanine (Fig. 4), cinnamic acid, and p-coumaric acid were detected (Fig. 5). Production of Pin and PDG (Fig. 5) and activities of PAL, C4H, and 4CL (Fig. 6) were detected as well. Likewise, with the extension of bioconversion time and the increase of polysaccharide concentration, all detected intermediates, products, and enzyme activities showed a saddle pattern effect. However, different from those found in the starch experiments, the increase of cell weight in the polysaccharide experiments was much lower and even decreased after 40 h. In addition, accumulation of PMG was found in the polysaccharide experiments, but not found in the starch experiments. Differences were also found in the peak times and highest values of intermediates, products, and enzyme activities. As shown in Fig. 4, the highest value of Phe (775 mg/L) appeared at 40 h and cell weight reached the highest value (2.75 g/L) at 40 h. Taking the data shown in Figs. 5 and 6 together, it was discovered that cinnamic acid reached the highest value (12.50 mg/L) at 32 h, whereas p-coumaric acid peaked (0.60 mg/L) at 40 h. Pin, PDG, and PMG reached their

Fig. 3 Change of enzyme activities during reaction in the resting cell system with mung bean starch as the substrate. The data were obtained at 30 g/L mung bean starch in a and after 56 h in b. The signals in the figures indicate enzymes 4CL (triangle), C4H (diamond), and PAL (square)

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Fig. 4 The cell weight and accumulation of phenylalanin during reaction in the resting cell system with the substrate. The condition is 35 g/L mung bean polysaccharide in a and reaction time of 56 h in b, respectively. The signals in a and b indicate Phe (triangle) and dry cell weight (square)

accumulation of cinnamic acid; similarly, high accumulation of cinnamic acid may promote the C4H activity then convert to be p-coumaric acid, then the concentration of p-coumaric acid reached the highest point at 40 h which may be related with the highest 4CL activity at 40 h. During the whole bioconversion period, the accumulation of Phe was still much higher than that of cinnamic acid and pcoumaric acid, and the activity of PAL was much higher than that of 4CL and C4H. C4H still showed the lowest activity among the detected three enzymes. The high activity of PAL and low activity of C4H also explained why a much higher accumulation of cinnamic acid than p-coumaric acid was found.

According to the summary data shown in Table 2 (data collected from experiments carried out at 30 g/L starch, 56 h, and 35 g/L polysaccharide, 56 h) and Table 3 (data collected from the peak time and peak value for each detected factor), mung bean starch yielded higher total amount of cell weight, Phe, cinnamic acid, and Pin production, and C4H and 4CL activities than mung bean polysaccharide. This also held true when the yields of per amount of these substrates were calculated. However, the opposite happened when the yields of these substances were divided by the weight of cells: the mung bean polysaccharide yielded higher total amounts. It should be mentioned that when polysaccharide was used, Pin production decreased and the resulting amount of Pin plus PMG was almost equal to that of Pin produced when starch

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Fig. 5 The accumulation of cinnamic acid, p-coumaric acid, Pin, PMG, and PDG during reaction in the resting cell system with mung bean polysaccharide as the substrate. The used condition is 35 g/L mung bean polysaccharide in a and reaction time of 56 h in b, respectively. The signals in the figures indicate cinnamic acid (inverted triangle), p-coumaric acid (diamond), Pin (triangle), PDG (square), and PMG (five-pointed star)

was used, indicating that mung bean polysaccharide induced the bioconversion from Pin to PMG. PDG production was almost equal when polysaccharide and starch parts were analyzed, indicating similar effect of these carbon sources on the bioconversion of PDG by Phomopsis sp. XP-8. It can be also seen from the data shown in Table 3 that the accumulation of p-coumaric acid and PDG and PAL activity were almost the same when the starch and polysaccharide parts were used. Compared with the results obtained in the starch experiments, when the polysaccharide component was analyzed, the peak times for the accumulations of Phe, p-coumaric acid, Pin, PDG and PAL activity were delayed about 8 h, whereas cell growth was shortened. This may be due to the fact that the mung

bean polysaccharide is a heteropolysaccharide and was more difficult to hydrolyze to monosaccharides than the starch, which only contained glucose. In addition, accumulation of PMG was only found in the polysaccharide experiments. This indicates that something in the composition of mung bean polysaccharide induced the biosynthesis of PMG. Bioconversion of PDG, PMG, and Pin from different monosaccharides In order to explore the major part of the mung bean polysaccharide that induces the bioconversion of PMG by Phomopsis

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Fig. 6 Change of enzyme activities during reaction in the resting cell system with mung bean polysaccharide as the substrate. The data were obtained at 35 g/L mung bean polysaccharide in a and after 56 h in b. The signals in the figures indicate enzymes 4CL (triangle), C4H (diamond), and PAL (square)

sp. XP-8, different monosaccharides were separately added in the bioconversion system containing Phomopsis sp. XP-8 cells, and the production of PDG, PMG, and Pin was detected and measured. As shown in Fig. 7, bioconversion of PMG was only found when arabinose and galactose were analyzed together with mung bean starch. Bioconversion of Pin, PDG, and PMG was not found when mannose or rhamnose were separately analyzed as the only substrate for bioconversion, indicating that these two kinds of monosaccharide could not be converted to these products through metabolism. Bioconversion of

PDG and PMG was only found when there was starch in the bioconversion system, indicating that mung bean starch was the essential part for bioconversion of these glycoside compounds. Bioconversion of Pin was found only when arabinose, xylose, galactose, and glucose were separately analyzed as the sole substrate. The total molarity of core pinoresinol and the percentage of different products in the core pinoresinol were calculated according to the data shown in Fig. 7, and the results are listed in Table 4. Compared with the results of the system with only mung bean starch (S, the first line in the table), the addition of

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Table 2 The dry cell weight and productions by Phomopsis sp. XP-8 cells using mung bean starch and mung bean polysaccharide Substrate

Starch (30 g/L,56 h)

Polysaccharide (35 g/L, 56 h)

Phe (mg/L) Cinnamic acid (mg/L) p-Coumaric acid (mg/L) Pin (mg/L) PDG (mg/L) PMG (mg/L) DCW (g/L) The output of per gram of material Phe (mg/g) Cinnamic acid (mg/g) p-Coumaric acid (mg/g) Pin (mg/g) PDG (mg/g) PMG (mg/g)

556.02 9.00 0.20 9.84 8.29 – 4.73

609.00 9.90 0.51 7.40 8.20 5.60 2.75

18.53 0.30 0.01 0.33 0.28 –

17.40 0.28 0.01 0.21 0.23 0.16

DCW (mg/g) The output of per gram of dry cell weight Phe (mg/g) Cinnamic acid (mg/g) p-Coumaric acid (mg/g)

0.16

0.08

117.55 1.90 0.04

221.45 3.60 0.18

2.08 1.75 –

2.69 2.98 2.03

Pin (mg/g) PDG (mg/g) PMG (mg/g)

Data are average and standard deviation of duplicates DCW dry cell weight, Pin pinoresinol, PDG pinoresinol diglucoside, PMG pinoresinol monoglucoside, Phe phenylalanine, − the results are not detectable

glucose and arabinose promoted the total core pinoresinol production, whereas the addition of other monosaccharides decreased it. Most significantly, the addition of rhamnose inhibited the bioconversion of all products, especially PDG, and thus relatively increased the proportion of Pin in the core pinoresinol. Among the monosaccharides decreasing core pinoresinol production, xylose or mannose promoted the mass flow from Pin to PDG and resulted in higher production and percentage of PDG in total product, while galactose inhibited the production of PDG and shifted the mass flow from Pin to PMG. In the monosaccharides increasing core pinoresinol production, glucose improved both Pin and PDG productions to similar extent and slightly changed their proportions, while arabinose increased Pin production, decreased PDG production, and induced production of PMG and thus increased the proportion of Pin in total core pinoresinol-containing products. Overall, xylose and mannose appeared as the most efficient factors to increase the bioconversion of PDG, while arabinose and galactose were the major monosaccharide

constituents induced the formation of PMG, and glucose and arabinose were most efficient in promoting Pin production. Base on the above results, it can be concluded that the effect of mung bean polysaccharide on the bioconversion of Pin, PMG, and PDG by Phomopsis sp. XP-8 cells is complicated and comprehensive due to it contains different monosaccharides that have diverse and even contrary effects on the bioconversion process. Therefore, it is possible to improve the bioconversion efficiency of certain products by using manually made polysaccharide mixtures. Therefore, we tested the bioconversion of Pin, PDG, and PMG using the combination of glucose, arabinose, galactose, xylose, and mannose. We tested two kinds of monosaccharide combinations in the study and showed the results in Fig. 7 and Table 4. As it was predicted, when five different monosaccharides were mixed and used for the bioconversion process, simultaneous bioconversion of Pin, PMG, and PDG was successfully achieved in the absent of mung bean starch. More importantly, the productions of Pin (14.9 mg/L), PMG (5.9 mg/L), and PDG (8.5 mg/L) were significantly improved when the optimal monosaccharide mixture (31 g/L) was used and much higher than that when 30 g/L mung bean starch was used together with 10 g/L monosaccharide. Production of total core pinoresinol and each product in the 10-g/L monosaccharide combination system (2 g/L of each monosaccharide) were also much higher than that in all 10-g/L single monosaccharide systems. Therefore, it was certified that the production of PDG, PMG, and Pin could be greatly improved by using manually made monosaccharide mixture in proper ratio. In addition, it could be determined that the addition of most of these monosaccharides, except rhamnose, could increase cell growth in the bioconversion system when compared to the data obtained for the control without mung bean starch (Fig. 7).

Discussion PDG, PMG, and Pin are important lignan components found in Eucommia ulmoides Oliv. (He et al. 2014). Phomopsis sp. XP-8 is an endophyte isolated from E. ulmoides Oliv. with the capability to produce PDG in vitro (Shi et al. 2012). In this study, Phomopsis sp. XP-8 was discovered to produce PMG and Pin as well. Many endophytic fungi have been found to synthesize some secondary metabolites usually found in plants. However, this is the first study to report Pinproducing fungi. In addition, the discovery of important secondary metabolites usually found in plants produced by endophytic fungi revealed the prospect of using these fungi as alternative sources of synthesizing metabolites only produced in plants (Aly et al. 2010). The capability of endophytes to produce plant metabolites was explained as a horizontal gene transfer at some stage during coevolution, thus importing the respective pathways

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Table 3 The highest values of dry cell weight, products, and enzyme activities and the occurrence time during the bioconversion using Phomopsis sp. XP-8 cells with mung bean starch or mung bean polysaccharide as the substrate Starch (30 g/L)

Polysaccharide (35 g/L)

Highest value

Bioconversion time (h)

Highest value

Bioconversion time (h)

DCW

4.75 g/L

48

2.75 g/L

40

Phe Cinnamic acid p-Coumaric acid Pin PDG PMG PAL C4H 4CL

886.50 mg/L 15.00 mg/L 0.60 mg/L 11.83 mg/L 8.29 mg/L – 383.3 U/mg 225.0 U/mg 269.0 U/mg

32 32 32 48 56 – 32 32 40

779.00 mg/L 12.70 mg/L 0.59 mg/L 7.80 mg/L 8.20 mg/L 5.60 mg/L 390.0 U/mg 176.2 U/mg 210.0 U/mg

40 32 40 56 56 56 40 32 40

Data are average and standard deviation of duplicates DCW dry cell weight, Pin pinoresinol, PDG pinoresinol diglucoside, PMG pinoresinol monoglucoside, PAL phenylalanine ammonia-lyase, C4H transcinnamate 4-hydroxylase, 4CL 4-coumarate-CoA ligase, − the results are not detectable

from fungi into the host plant (Kusari et al. 2008), or vice versa. In plants, the Pin, PMG, and PDG biosynthesis pathways are reported as the phenylpropanoid pathway, with

Fig. 7 Production of PDG, PMG, and Pin by Phomopsis sp. XP-8 cells using different monosaccharides. Data were obtained after bioconversion for 56 h. Pin pinoresinol, PDG pinoresinol diglucoside, PMG pinoresinol monoglucoside, S mung bean starch, Rha rhamnose, Ara arabinose, Xyl xylose, Man mannose, Gal galactose, Glu glucose. For the treatments, the amount of S was 30 g/L and the amount of each monosaccharide was 10 g/L. The amount of each monosaccharide in the treatment combination 1 was 2 g/L glucose, 2 g/L arabinose, 2 g/L galactose, 2 g/L xylose, and 2 g/L mannose, and that in the treatment combination 2 was 10 g/L glucose, 5 g/L arabinose, 6 g/L galactose, 8 g/L xylose, and 2 g/L mannose

phenylalanine, cinnamic acid, and p-coumaric acid as intermediates (Suzuki and Umezawa 2007). The accumulation of these intermediates and related enzyme activities were also found during bioconversion of Pin, PMG, and PDG using Phomopsis sp. XP-8 with mung bean starch and polysaccharide as substrates. This indicated that the biosynthesis pathways of Pin, PMG, and PDG in this fungus might be similar to that reported in plants. However, further study is still needed to reveal the shift of this biosynthesis pathway between this fungus and its host plant. Starch, water-soluble polysaccharide, and protein are the three major components of mung bean and account for 90.03 % composition of the mung bean. When these three components were recombined in their original ratio found in the mung bean, production of PDG, PMG, and Pin were almost same as that produced when whole mung bean powder was used. When the protein component was removed, production of PDG, PMG, and Pin were slightly influenced. Moreover, no production of PDG, PMG, and Pin was detected when only the protein component was used as the only substrate. Therefore, the bioconversion of PDG, PMG, and Pin by Phomopsis sp. XP-8 was mainly reliant on the metabolism of carbon sources, but not to that of nitrogen sources. In the study, bioconversion of Pin was found in most cases, whereas production of PMG and PDG was only found in some cases, and the mass shift from Pin to PMG and PDG was found to some extent. This indicated that Pin might be the precursor of PMG and PDG. However, the intrinsic relationship among the synthesis of these three compounds in Phomopsis sp. XP-8 still needs further research. The formation of PMG was found only when arabinose, galactose, and mung bean polysaccharide were analyzed, while

4642 Table 4

Appl Microbiol Biotechnol (2015) 99:4629–4643 The percentage production of PDG, PMG, and Pin by Phomopsis sp. XP-8 cells using mung bean starch and different monosaccharides

Substrate used in the Total molarity of core PDG PMG Pin bioconversion system pinoresinol (μmol/L) Production Percentage in the Production Percentage in the Production Percentage in the (μmol/L) core pinoresinol (%) (μmol/L) core pinoresinol (%) (μmol/L) core pinoresinol (%) S

41.0

9.6

23.45

–

Rha+S Ara+S Xyl+S Man+S Gal+S Glu+S CK Rha Ara Xyl Man Gal Glu Combination 1 Combination 2

14.9 44.0 37.1 35.9 31.9 48.6 – – 5.1 20.3 – 16.6 23.0 33.8 65.2

0.2 1.9 11.7 10.4 – 11.0 – – – – – – – 4.7 12.4

1.46 4.21 31.41 29.02 – 22.58 – – – – – – – 13.90 19.02

– 1.2 – – 8.8 – – – – – – – – 4.3 11.3

– – 2.71 – – 27.67 – – – – – – – 0 12.72 17.33

31.4

76.55

14.7 40.9 25.4 25.5 23.1 37.6 – – 5.1 20.3 – 16.6 23.0 24.8 41.5

98.54 93.08 68.59 70.98 72.33 77.42 – – 100 100 – 100 100 73.37 63.65

Data are average and standard deviation of duplicates. For the treatments, the amount of S was 30 g/L and the amount of each monosaccharide was 10 g/L. The amount of each monosaccharide in the treatment combination 1 was 2 g/L glucose, 2 g/L arabinose, 2 g/L galactose, 2 g/L xylose, and 2 g/L mannose, and that in the treatment combination 2 was 10 g/L glucose, 5 g/L arabinose, 6 g/L galactose, 8 g/L xylose, and 2 g/L mannose Pin pinoresinol, PDG pinoresinol diglucoside, PMG pinoresinol monoglucoside, S mung bean starch, Rha rhamnose, Ara arabinose, Xyl xylose, Man mannose, Gal galactose, Glu glucose, − the results are not detectable

there was no PMG production in the other situations. The PDG production decreased and/or disappeared when the production of PMG increased. This phenomenon indicated that PMG and PDG are converted by two different sets of glycosyltransferase induced by the presence of different sugars. According to our results, it seems that the glycosyltransferase converting PMG is induced by the presence of galactose and arabinose, while the glycosyltransferase converting PDG is induced by the presence of mung bean starch, xylose, or glucose at certain concentration level. This is also consistent with the previous reports that chemical conversion from Pin to PMG by a glucosyltransferase could be obtained without the presence of PDG (Satake et al. 2013). However, PMG might be not a precursor of PDG because the bioconversion of PDG was not found when we used PMG as the solely substrate in our previous studies (data not shown here). In addition, glucose was identified as the sole glycosyl donor for the formation of PMG and PDG because biosynthesis of PDG was found only when glucose was used, and PMG was found only in the presence of mung bean starch together with arabinose and galactose. In the reported Pin biosynthesis pathway in plants, Pin is converted from coniferyl alcohol by a dirigent protein (Suzuki and Umezawa 2007). A chloroperoxidase-containing microorganism, Caldariomyces fumago, was reported to have

capability to catalyze the dimerization of coniferyl alcohol to (±)-Pin (Sih et al. 1976). However, in this study, the enzymes involved in the biosynthesis of Pin in Phomopsis sp. XP-8 have not been identified and Phomopsis spp. have not been found to contain chloroperoxidase. Biosynthesis of lignans is of great interest to bioorganic chemistry and would provide a model for biomimetic chemistry and its extensive applications (Umezawa 2005). From this pathway, improvement of lignan biosynthesis has been successfully achieved by constructing a lignan biosynthesis pathway in genetically modified trees (Chiang 2006). However, the lignan biosynthesis pathway in microorganisms has not been reported before. More important, the study presented a method to produce Pin, PMG, and PDG by Phomopsis sp. XP-8 in a modified medium containing different compounds isolated from mung bean, instead of whole mung bean. This operation effectively eliminated the fluctuation of results caused by the variation of mung bean materials and thus made the results consistent in different batches. More important, we also developed a medium containing defined compounds in defined composition and contents. Therefore, this medium should be a kind of chemically modified medium, instead of normally mentioned natural mung bean medium that varied among batches.

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Finally, the study presented a completely new way to produce Pin, PMG, and PDG by bioconversion process and Phomopsis sp. XP-8 showed great potential in producing lignans and their derivatives by microbial fermentation and enzymatic reaction due to its high efficiency in bioconversion of Pin, PMG, and PDG from mung bean starch and polysaccharides. In this study, the highest production of Pin, PDG, and PMG by Phomopsis sp. XP-8 reached 14.9, 8.5, and 5.9 mg/L, respectively, within 56 h. Combination of 10 g/L glucose, 5 g/ L arabinose, 6 g/L galactose, 8 g/L xylose, and 2 g/L mannose had the highest production of Pin, PDG, PMG. The production still has potential in enhancement after the optimization of bioconversion condition and regulatory controls.

References Adlercreutz H (2002) Phyto-oestrogens and cancer. Lancet Oncol 3:364– 373 Akihisa T, Orido M, Akazawa H, Takahashi A, Yamamoto A, Ogihara E, Fukatsu M (2013) Melanogenesis-inhibitory activity of aromatic glycosides from the stem bark of Acer buergerianum. Chem Biodivers 10:167–176 Aly AH, Debbab A, Kjer J, Proksch P (2010) Fungal endophytes from higher plants: a prolific source of phytochemicals and other bioactive natural products. Fungal Divers 41:1–16 Charles JS, Ravikumt PR, Huang FC (1976) Isolation and synthesis of pinoresinol diglucoside, a major antihypertensive principle of Tu-Chung (Eucommia ulmoides Oliv.). J Am Chem Soc 98: 5412–5413 Chiang VL (2006) Monolignol biosynthesis and genetic engineering of lignin in trees, a review. Environ Chem Lett 4:143–146 Donnez D, Jeandet P, Clément C, Courot E (2009) Bioproduction of resveratrol and stilbene derivatives by plant cells and microorganisms. Trends Biotechnol 27:706–713 El-Adawy TA (2000) Functional properties and nutritional quality of acetylate and succinylated mung bean protein isolate. Food Chem 70:83–91 Elder T (2014) Bond dissociation enthalpies of a pinoresinol lignin model compound. Energy Fuel 28:1175–1182 Hwang B, Lee J, Liu QH, Woo ER, Lee DG (2010) Antifungal effect of (+)-pinoresinol isolated from Sambucus williamsii. Molecules 15(5):3507–3516 He XR, Wang JH, Li MX, Hao DJ, Yang Y, Zhang CL, He R, Tao R (2014) Eucommi aulmoides Oliv.: Ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine. J Ethnopharmacol 151:78–92 Jung HW, Mahesh R, Lee JG, Lee SH, Kim YS, Park YK (2010) Pinoresinol from the fruits of Forsythia koreana inhibits inflammatory responses in LPS-activated microglia. Neurosci Lett 480:215– 20 Kim HJ, Oh MS, Hong J, Jang YP (2011) Quantitative analysis of major dibenzocyclooctanelignans in schisandraefructusby online TLCDART-MS. Phytochem Anal 22:258–262 Kusari S, Lamshöft M, Zühlke S, Spiteller M (2008) An endophytic fungus from Hypericum perforatum that produces hypericin. J Nat Prod 71:159–162 Lai F, Wen Q, Li L, Wu H, Li X (2010) Antioxidant activities of watersoluble polysaccharide extracted from mung bean (Vigna radiata L.) hull with ultrasonic assisted treatment. Carbohyd Polym 81:323–329

4643 Landete JM (2012) Plant and mammalian lignans: a review of source, intake, metabolism, intestinal bacteria and health. Food Res Int 46(1):410–424 Luo LF, Wu WH, Zhou YJ, Yan J, Yang GP, Ouyang DS (2010) Antihypertensive effect of Eucommia ulmoides Oliv. extracts in spontaneously hypertensive rats. J Ethnopharmacol 129:238– 243 Maisa B, Xu XY, Si YP, Arine H, Miao WH, Wang LF (2013) Effect of oxidation and esterification on functional properties of mung bean (Vigna radiata (L.) Wilczek) starch. Eur Food Res Technol 236: 119–128 Mubarak AE (2005) Nutritional composition and antinutritional factors of mung bean seeds (Phaseolus aureus) as affected by some home traditional processes. Food Chem 89:489–495 Pietinen P, Stumpf K, Männistö S, Kataja V, Uusitupa M, Adlercreutz H (2001) Serum enterolactone and risk of breast cancer a case–control study in eastern Finland. Cancer Epidem Biomar 10:339–344 Randhir R, Lin YT, Shetty K (2004) Stimulation of phenolics, antioxidant and antimicrobial activities in dark germinated mung bean sprouts in response to peptide and phytochemical elicitors. Process Biochem 39:637–646 Randhir R, Shetty K (2007) Mung beans processed by solid-state bioconversion improves phenoliccontent and functionality relevant for diabetes and ulcer management. Innovat Food Sci Emerg Tech 8:197– 204 Satake H, Ono E, Murata J (2013) Recent advances in the metabolic engineering of lignan biosynthesis pathways for the production of transgenic plant-based foods and supplements. J Agric Food Chem 61:11721–11729 Shi JL, Liu C, Liu LP, Yang BW, Zhang YZ (2012) Structure identification and fermentation characteristics of pinoresinol diglucoside produced by Phomopsis sp. isolated from Eucommia ulmoides Oliv. Appl Microbiol Biotechnol 93:1475–1483 Sih CJ, Ravikumt PR, Huang FC (1976) Isolation and synthesis of pinoresinol diglucoside, a major antihypertensive principle of TuChung (Eucommia ulmoides Oliv.). J Am Chem Soc 98(17):5412– 5413 Suzuki S, Umezawa T (2007) Biosynthesis of lignans and norlignans. J Wood Sci 53:273–284 Tsukamoto H, Hisada S, Nishide S (1984) Lignans from bark of Fraxinus mandshurica var. japonica and F. japonica. Chem Pharm Bull 32: 4482–4489 Umezawa T (2005) Biosynthesis of lignans, lignins, and norlignans. Kagaku to Seibutsu 43:461–467 Vermerris W, Nicholson R (2006) Families of phenolic compounds and means of classification. Phenolic Compd Biochem 10:1–34 Xie LH, Akao T, Hamasaki K, Deyama T, Hattori M (2003) Biotransformation of pinoresinol diglucoside to mammalian lignans by human intestinal microflora, and isolation of Enterococcus faecalis strain PDG-1 responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol. Chem Pharm Bull 51:508–515 Xie J, Tworoger SS, Franke AA, Terry KL, Rice MS, Rosner BA, Willett WC, Hankinson SE, Eliassen AH (2013) Plasma enterolactone and breast cancer risk in the Nurses’ Health Study II. Breast Cancer Res Treat 139:801–809 Yang B, Jiang YM, Zhao MM, Shi J, Wang LZ (2008) Effects of ultrasonic extraction on the physical and chemical properties of polysaccharide from longan fruit pericarp. Polym Degrad Stabil 93:268– 272 Zhang JH, Shi JL, Liu YL (2013) Substrates and enzyme activities related to biotransformation of resveratrol from phenylalanine by Alternaria sp. MG1. Appl Microbiol Biotechnol 97: 9941–9954