Accepted Manuscript

Title: Stimulatory effects of Coix lacryma-jobi oil on themycelial growth and metabolites biosynthesis by thesubmerged culture of Ganoderma lucidum

Author: Hailong Yang Weihong Min Pengyang Bi HuabinZhou Furu Huang

PII: S1369-703X(13)00116-2DOI: http://dx.doi.org/doi:10.1016/j.bej.2013.04.012Reference: BEJ 5711

To appear in: Biochemical Engineering Journal

Received date: 3-1-2013Revised date: 4-4-2013Accepted date: 11-4-2013

Please cite this article as: H. Yang, W. Min, P. Bi, H. Zhou, F. Huang, Stimulatory effectsof Coix lacryma-jobi oil on the mycelial growth and metabolites biosynthesis by thesubmerged culture of Ganoderma lucidum, Biochemical Engineering Journal (2013),http://dx.doi.org/10.1016/j.bej.2013.04.012

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Stimulatory effects of Coix lacryma-jobi oil on the mycelial growth and

metabolites biosynthesis by the submerged culture of Ganoderma lucidum

Hailong Yanga,b,*, Weihong Minc, Pengyang Bia,c, Huabin Zhoua, Furu Huanga,b

aSchool of Environmental & Life Sciences, Wenzhou University, Chashan University

Town, Wenzhou 325035, China

bInstitute of Bioprocess Engineering, Wenzhou University, Chashan University Town,

Wenzhou 325035, China

cSchool of Food Science and Engineering, Jilin Agriculture University, Changchun

130018, China

* Corresponding author, Tel: +86-577-86691013; Fax: +86-577-86689257; Email:

yanghl999@yahoo.com (H. Yang)

Abstract: Effects of Coix lacryma-jobi oil (CLO) addition on the mycelia growth and

production of bioactive metabolites, such as triterpenoids, exopolysaccharide (EPS)

and intracellular polysaccharide (IPS) in the submerged culture of Ganoderma

lucidum were studied. The results showed that when a level of 2% CLO was added at

the beginning of culture, the biomass, triterpenoids, EPS, and IPS productions reached

a maximum of 10.71 g/L, 92.94 mg/L, 0.33 g/L and 0.389 g/L, respectively, that were

3.34-fold, 2.76-fold, 2.2-fold and 2.23-fold compared to that of control. Analysis of

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fermentation kinetics of G. lucidum suggested that glucose concentration in the culture

of CLO-added group decreased more quickly as compared to the control group from

day 2 to day 7 of fermentation process, while the triterpenoids and polysaccharides

biosynthesis were promoted at the same culture period. However, the culture pH

profile was not affected by the addition of CLO. There were no new components in

the two types of polysaccharides obtained by the addition of CLO. Enzyme activities

analysis indicated CLO or its fatty acids affected the synthesis level of

phosphoglucose isomerase and α-phosphoglucomutase at different stage.

Keywords: Ganoderma lucidum; Polysaccharide; Biosynthesis; Enzyme Activity;

Fermentation; Submerged Culture

1. Introduction

Medicinal mushrooms have long been used in traditional oriental therapies, and

modern scientific and medical studies demonstrate the potent and unique properties of

mushroom-extracted compounds for the prevention and treatment of cancer [1].

Ganoderma lucidum (Fr.) Karst, one of the most popular mushrooms used in

traditional Chinese medicine, has been used to prevent and treat various human

diseases such as hepatitis, chronic bronchitis, hypertension, hypercholesterolemia and

gastric cancer for more than thousand years [2]. Pharmaceutically active compounds

from fruiting body and mycelium of G. lucidum include polysaccharides, proteins,

triterpenoids, proteopolysaccharides, sterols, alkaloids, and nucleotides [3, 4]. Among

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them, polysaccharides and triterpenoids are the two main functional components that

are considered to possess multiple biological activities for therapeutic use [5].

Because of its perceived nutritional and health values, G. lucidum has gained wide

popularity as a nutraceuticals and functional foods in China, Japan, Korea and other

regions. Normally, G. lucidum is available in the form of mature fruiting bodies and

spores by solid cultures using substrates such as grain, sawdust or wood. However,

the production of fruiting bodies and spores includes a long cultivation for about six

months [6]. In recent years, submerged culture of G. lucidum has been developed

because of the potential for higher mycelia and bioactive components production in a

compact space and in shorter time with fewer chances for contamination, in which

mycelial biomass, triterpenoids and polysaccharides are the desired products [7, 8].

Secondary metabolite production by G. lucidum is affected by several factors. To

accelerate mycelial growth and metabolite production by G. lucidum, the effects of

environmental conditions [9], medium composition [10], inoculation density [11], pH

[12, 13], two-stage culture process [14], oxygen supply [15], pH-shift and DOT-shift

integrated fed-batch fermentation [16], etc. have been studied. It is known that

designing an appropriate fermentation conditions, besides of a productive strain

construction, is crucial for optimization of the microbial fermentation processes [17].

To enhance the production efficiency, modification of media composition would be

vital in the submerged culture of G. lucidum [6].

Coix lacryma-jobi, a distant relative of maize in the Maydeae tribe of the grass

family Poaceae, is native to India, Burma, China, and Malaysia and grown

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extensively in South Asia before maize became popular as an agricultural crop [18].

The seed of C. Lacryma-jobi is full of starch, protein, oil, mineral elements, and

vitamins, and has been used as a food source for humans and livestock, in the

production of alcoholic beverages and as a medicinal plant over the years [18]. Our

previous work showed C. lacryma-jobi was a good media ingredient and could

improve the growth and bioactive metabolites production of G. lucidum [19]. It was

reported that mycelial growth and metabolite production of G. lucidum could be

induced by plant oils [20], The objective of this work was to examine the effects of C.

lacryma-jobi oil (CLO) on the mycelial growth, triterpenoids and polysaccharides

production by medicinal mushroom G. lucidum in submerged culture, and the

influences on polysaccharides components and related biosynthesis enzyme activities

were also explored.

2. Materials and methods

2.1 C. lacryma-jobi and C. lacryma-jobi oil (CLO) extraction

The seeds of C. lacryma-jobi were purchased from Yetongren Medicinal Co. Ltd.

(Wenzhou, China), dried and pound to powder (40 mesh), then stored at 4°C. CLO

extraction was conducted using Soxhlet equipment as follows: in batches, seeds (50 g)

were extracted with hexane (500 mL) for 5 h, and the solvent was then evaporated.

2.2 Microorganism and culture conditions

G. lucidum WZ06 was screened and collected by the Laboratory of fermentation,

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Wenzhou University (Wenzhou, People’s Republic of China) and maintained on

potato dextrose agar (PDA). The inocula were prepared in a 500 mL Erlenmeyer flask

containing 150 mL media (see below) at 30°C for 7 days with shaking at 160 rpm.

This was then inoculated at 10% (v/v) into the same medium now containing different

amount of CLO. The cultivation medium contained (g/L): glucose, 35; peptone, 3.5;

KH2PO4, 1.0; K2HPO4, 1.0; MgSO4, 0.75; vitamin B1, 0.01.

2.3 Determination of biomass

Biomass was obtained by centrifuging at 8,000 rpm for 20 min, washing the

precipitated cells for three times with distilled water, and drying at 60°C for a

sufficient time to a constant weight.

2.4 Measurements of extracellular and intracellular polysaccharides

For the determination of exopolysaccharides (EPS), after the removal of mycelia by

centrifugation, the crude polysaccharide was precipitated with adding 4 times of 95%

(v/v) ethanol. The precipitated polysaccharide was collected by centrifugation at

8,000 rpm for 20 min, and washed with 80% (v/v) ethanol three times, then dried to

remove residual ethanol at 60°C. Total polysaccharide in the culture medium was

determined by the phenol-sulfuric acid assay. For the analysis of intracellular

polysaccharides (IPS), the dried mycelia were extracted with 1 mol/L NaOH at 60°C

(1 h), and then the supernatant was assayed by phenol-sulfuric acid method [7].

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2.5 Assay of triterpenoids

Triterpenoids production was measured as described by Fang and Zhong [13] with

some modification. The dried mycelia (100 mg) were extracted by 50% (v/v) ethanol

(5 mL) for 1 week (twice). After removal of mycelia by centrifugation, the

supernatants were dried at 50 °C under vacuum. The residues were suspended by

water, and later extracted with chloroform. The triterpenoids in the chloroform extract

were further extracted with 5% (w/v) NaHCO3. The pH of NaHCO3 phase was

adjusted to 2.0- 3.0 by 3 mol/L HCl at 0°C, and then the triterpenoids in the NaHCO3

phase were again extracted with chloroform. After removal of chloroform by

evaporation, triterpenoids were dissolved in absolute ethanol, and its absorbency was

measured at 245 nm in a spectrophotometer (Puxi General Analytical Instrument

Factory, Beijing, China).

2.6 Analysis of polysaccharide components

The IPS and EPS Polysaccharides from the samples of containing 2% (v/v) CLO in

the media and control were respectively collected as the method mentioned above,

and then fractionated on ÄKTA Explorer (Sweden). 2 mL polysaccharide (about 2-2.5

mg/mL) of each sample was eluted on a column (HiPrep 16/10 DEAE) with H2O and

followed stepwise by 0.05, 0.2, and 0.5 mol/L NaCl at 2 mL/min. Fractions (5 mL)

were assayed by the phenol-sulfuric acid method. The main components of IPS and

EPS were re-fractionated on a column of Superdex 200 HR 10/30 with 0.1 mol/L

NaCl at 0.25 mL/min, respectively. Fractions (1mL) were assayed by the

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phenol-sulfuric acid method.

2.7 Enzyme assays

Mycelia were harvested by centrifugation at15,000 g for 30 min, washed twice with

0.9% NaCl, and suspended in 20 mmol/L phosphate buffer (pH 6.5) containing 50

mmol/L NaCl, 10 mmol/L MgCl2, and 1 mmol/L dithiothreitol. Mycelia were

disrupted ultrasonically at 0°C and cell debris was removed by centrifugation. The

protein content of the cell extract was determined by the method of Bradford [21].

Enzyme assays were performed at 30°C in a total volume of 1 mL with freshly

prepared cell extracts. The formation or consumption of NAD(P)H was determined by

measuring the change in the absorbance at 340 nm as described by Looijesteijn et al.

[22].

The α-phosphoglucomutase (EC 2.7.5.1) reaction mixture contained 50 mmol/L

triethanolamine buffer (pH 7.2), 5 mmol/L MgCl2, 0.4 mmol/L NADP, 50 μmol/L

glucose-1,6-diphosphate, 4 U of glucose-6-phosphate dehydrogenase, and cell extract.

The reaction was started by adding 1.4 mmol/L α-glucose-1-phosphate.

The phosphoglucose isomerase (EC 5.3.1.9) reverse reaction mixture contained 50

mmol/L potassium phosphate buffer (pH 6.8), 5 mmol/L MgCl2, 0.4 mmol/L NADP,

4 U of glucose-6-phosphate dehydrogenase, and cell extract. The reaction was started

by adding 5 mmol/L fructose-6-phosphate.

The UDP-glucose pyrophosphorylase (EC 2.7.7.9) reverse reaction mixture

contained 50 mmol/L Tris-HCl buffer (pH 7.8), 14 mmol/L MgCl2, 0.3 mmol/L

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NADP, 0.1 mmol/L UDP-glucose, 2.1 U of α-phosphoglucomutase, 4 U of

glucose-6-phosphate dehydrogenase, and cell extract. The reaction was started by

adding 4 mmol/L inorganic pyrophosphate.

The reaction mixture of the dTDP-glucose pyrophosphorylase (EC 2.7.7.24)

reverse reaction mixture contained 50 mmol/L Tris-HCl buffer (pH 7.8), 8 mmol/L

MgCl2, 0.3 mmol/L NADP, 0.1 mmol/L TDP-glucose, 2.1 U of

α-phosphoglucomutase, 4 U of glucose-6-phosphate dehydrogenase, and cell extract.

The reaction was started by adding 4.7 mmol/L inorganic pyrophosphate.

2.8 Statistical analysis

Cultures were performed in a triplicate and data were analyzed by using Statistics

Analysis System (SAS) 8.1 version (SAS Institute Inc., USA). The results were

expressed as the mean ± SD. The significance of the mean difference between the

control and each treatment groups was determined by Student’s t-test.

3. Results and discussion

3.1 Effect of C. lacryma-jobi oil (CLO) concentration on the mycelial growth and

metabolites production of G. lucidum

Different amounts of CLO were supplemented into the media of G. lucidum and the

results obtained at day 7 were recorded in Fig. 1. Mycelial biomass, triterpenoids,

EPS and IPS production of G. lucidum were found increased with the increase of

CLO concentration from 0.5 to 2%, whereas further increasing oil concentration to

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4% exerted a negative effect on the production compared to that of 2% CLO addition.

The maximum mycelial biomass, triterpenoids, and EPS productions obtained under

2% CLO reached 10.71 g/L, 92.94 mg/L, and 0.33 g/L, respectively. 0.5% CLO was

sufficient to enhance IPS production and the IPS reached 0.373 g/L. Increasing oil

concentration could not further enhance IPS production, and IPS was only 0.389 g/L

at 2% CLO group.

The production of mycelia and metabolites by some mushroom species in

submerged cultures has prospered in recent years. Vegetable oils have been selected to

accelerate mycelial growth and polysaccharide production of some mushroom species

and proved to have a stimulatory effect [6, 20, 23, 24]. Sunflower oil at the level of

2% led to a significant increase in exo-biopolymer of Cordyceps militaris from 2.3 to

7.5 g/L, while the addition of 4% olive oil dramatically increased its mycelial biomass

from 5.8 to 19.0 g/L [23]. Olive, safflower seed, soy and sunflower oil were favorable

plant oil sources to the mycelial growth of Grifola frondosa at 1% [24]. Chang et al.

[10] reported adding 2.5 g/L of safflower oil into the media of G. lucidum increased

the mycelium yield from 3.6 g/L to 5.4 g/L (1.5-fold), while adding 2.5 g/L of olive

oil enhanced EPS from 0.177 g/L to 0.186 g/L (1.05-fold). Another report [20]

showed 2% corn oil was optimal for the submerged culture of G. lucidum in 7 days.

The mycelial biomass and EPS reached 8.23 and 0.706 g/L, about 1.94- and 1.81-

times compared to that of control, respectively. The stimulatory mechanism of plant

oils would be modifying membrane composition and increasing permeability, or by

directly affecting the synthesis level of the enzymes involved in polysaccharide

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production of G. lucidum [6].

In this work, 2% CLO was optimal and the biomass, triterpenoids, EPS, and IPS

were 3.34-fold, 2.76-fold, 2.2-fold and 2.23-fold compared to that of control.

Compared with the previous works, CLO was more effective in the mycelial growth

and metabolite production of G. lucidum than other plant oils, such as corn oil, olive

oil, safflower oil, etc.

3.2 Effect of addition time on the mycelial growth and metabolites production of G.

lucidum

To better understand the effect of CLO on the mycelial growth and metabolites

production of G. lucidum, 2% CLO were added in 0-, 1-, 2-, and 4-day of cultivation,

which were termed the initial time of culture, the lag phase, the exponential growth

phase, and the stationary phase. The results (Fig. 2) revealed that CLO addition at

different stages was all beneficial for mycelial growth and metabolites production

compared to that of control (without oil addition), whereas the enhancement was

slight when adding CLO at day 4. Addition at day 1 was preferred for triterpenoids

production and the maximum triterpenoids reached 130.4 mg/L. When CLO was

added at day 0, biomass, EPS and IPS was 10.71 g/L, 0.331 g/L and 0.389 g/L,

respectively. When CLO was added at day 1, biomass, EPS and IPS was 11.08 g/L,

0.276 g/L and 0.360 g/L, respectively. When CLO was added at day 2, biomass, EPS

and IPS was 11.44 g/L, 0.364 g/L and 0.328 g/L, respectively. No significant (P<0.05)

differences in biomass, EPS and IPS production were observed when CLO was added

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at day 0-, 1- and 2-day. Previous work reported that corn oil addition at day 0 was

preferred for the submerged culture of G. lucidum [20]. Accordingly, 2% CLO added

at day 0 was selected for the subsequent studies.

3.3 Effect of CLO on fermentation kinetics of G. lucidum

Fig. 3 described the changes in time-course profiles of glucose consumption, pH,

mycelial growth, triterpenoids, EPS and IPS production in the submerged culture of G.

lucidum with and without CLO addition. As shown in Fig. 3a, glucose concentration

in the oil-added culture decreased more quickly as compared with the control group

from day 2 to day 7, and its concentration decreased to 8.43 g/L on day 7 against

10.20 g/L in the control. Consistently, the productions of biomass, triterpenoids, EPS

and IPS were evidently promoted and reached 10.27 g/L, 88.15 mg/L, 0.317 g/L and

0.406 g/L at day 7, respectively (Fig. b-e).

Fig. 3f showed that both the initial pH in the oil added culture and the control

decreased to 3.8 during the first 3 days of cultivation. After that, they remained

relatively constant for about 3 days. The pH profile in the oil added cultures was

almost the same as that of the control, suggesting the oil addition did not change pH

profiles in the fermentation process of G. lucidum. Previous studies showed that

culture pH had significant effect on the biomass, triterpenoids (ganoderic acids) and

polysaccharides productions of G. lucidum [12, 13]. However, in this study, no

significant difference in culture pH profile between oil added group and the control

indicated the stimulatory effects on the biomass, triterpenoids and polysaccharides

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production by the CLO were not contributed by the changes of pH value.

3.4 Effect of CLO on the polysaccharide components of G. lucidum

The IPS and EPS from cultures grown with and without 2% (v/v) CLO were

fractionated by the column chromatography (Fig. 4). Fig. 4A showed the effects of

CLO on the IPS components of G. lucidum. Four fractions, IPS-1, IPS-2, IPS-3 and

IPS-4, were isolated on the column of HiPrepTM 16/10 DEAE in each sample with

IPS-1 being the main component. IPS-1 was fractionated on the column of Superdex

200 HR 10/30 and three components were separated but there was no new

polysaccharide component produced by the addition of CLO.

Effects of CLO on the EPS components were shown in Fig. 4B. There were also

four fractions in each EPS sample, and fraction 1 (EPS-1) was the main component.

EPS-1 was further fractionated on the column of Superdex 200 HR 10/30, and two

components were separated but there was also no new polysaccharide component

obtained by the addition of CLO. These results suggested that CLO did not change the

biosynthetic pathways of the polysaccharides though they significantly enhanced the

polysaccharide production by G. lucidum.

3.5 Effect of CLO on the enzyme activities of polysaccharide biosynthesis

Vegetable oils and fatty acids promoted the production of fungal metabolites like

protease [25], tetracycline [26], pleuromutilin [27] and polysaccharides [6, 20, 23, 24,

28]. The mechanism of stimulatory effect had been proposed as oils or fatty acids

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work by modifying membrane composition and increase permeability, or by directly

affecting the synthesis level of the enzymes involved in polysaccharide production [6,

23, 28]. As described above, 2% CLO addition could enhance the IPS and EPS

production of G. lucidum, but did not change the polysaccharide biosynthetic pathway.

The results (Fig. 3a, 3d and 3e) from the time-course profiles of glucose consumption,

EPS and IPS production in the submerged culture of G. lucidum showed that glucose

consumption and polysaccharide production were primarily happened from day 2 to

day 6. To examine the enhancing mechanism, the activities of some key enzymes

involved in the Embden-Meyerhof-Parnas (EMP) pathway and the synthesis of sugar

nucleotides, i.e. α-phosphoglucomutase, phosphoglucose isomerase, UDP-glucose

pyrophosphorylase, dTDP-glucose pyrophosphorylase, were determined at day 2, day

4, and day 6. The results (Table 1) showed the activity of phosphoglucose isomerase,

which catalyzes glucose-6-phosphate to fructose-6-phosphate, in the oil addition

culture was low significantly (p<0.05) at day 4 compared to that of the control

(without oil addition). The activity of phosphoglucose isomerase was 7.46 nmol/mg

protein/min in the control, while only 2.30 nmol/mg protein/min in oil addition

culture. The activity of α-phosphoglucomutase, which bilaterally catalyzes

glucose-6-phosphate to glucose-1-phosphate, in the oil addition culture was much

lower (p<0.05) than that of the control at day 6. α-phosphoglucomutase,

phosphoglucose isomerase, UDP-glucose pyrophosphorylase and dTDP-glucose

pyrophosphorylase were some key enzymes in polysaccharide biosynthesis pathway

of G. lucidum [29]. The results indicated CLO or its fatty acids directly affected the

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synthesis level of the enzymes involved in polysaccharide production.

4. Conclusions

Triterpenoids and polysacchrides from G. lucidum show multiple biological

activities, such as immuno-modulating, antitumor, antioxidant, and hepatoprotective

activity, etc. The production of triterpenoids and polysaccharides by submerged

cultures of G. lucidum has prospered in recent years. To accelerate mycelial growth

and metabolite production efficiency, the control of environmental conditions or the

modification of media composition would be vital. Plant oil has been reported to be

favorable to the mycelial growth in several medicinal mushrooms, and to increase the

production of bioactive metabolites. In the present study, we investigated the effects

of CLO in an attempt to enhance both metabolite production and mycelial growth of

G. lucidum in shake flask culture. It was preferred to add 2% CLO into the media at

day 0. Polysaccharide components analysis confirmed that the polysaccharide

biosynthesis way had not been changed by the addition of CLO. Time-course

examination showed glucose was consumed more quickly to a lower level compared

to that of control. Results indicated that addition of CLO into the culture medium

could enhance mycelial growth, triterpoids EPS and IPS production. The stimulatory

mechanism of CLO on polysaccharide production would be oils or its fatty acids work

by directly affecting the synthesis level of phosphoglucose isomerase and

α-phosphoglucomutase at different stage. Compared with corn oil, olive oil, safflower

oil, etc., CLO was more effective on the mycelial growth and metabolite production in

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the submerged culture of G. lucidum.

Acknowledgements

The work was supported by Zhejiang Provincial Natural Science Foundation of

China under Grant No.Y3100711.

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Table 1 Activities of enzymes involved in the Embden-Meyerhof-Parnas (EMP) pathway and the synthesis of sugar nucleotides in the

cultivation of G. lucidum in the medium containing 2% C. lacryma-jobi oil and the control.

Day 2 Day 4 Day 6Enzyme

(nmol/mg protein/min) Control CLO Control CLO Control CLO

phosphoglucose isomerase 0.46 ± 0.22 0.73 ± 0.28 7.46 ± 1.52 2.30 ± 0.86* 3.11 ± 0.75 3.86 ± 1.09

α-phosphoglucomutase 11.39 ± 3.64 3.24 ± 1.23 16.57 ± 3.69 10.08 ± 2.57 31.05 ± 6.55 6.89 ± 2.58*

UDP-glucose

pyrophosphorylase

1.04 ± 0.58 0.22 ± 0.15 1.69 ± 0.61 0.32 ± 0.18 0.83 ± 0.42 1.01 ± 0.47

dTDP-glucose

pyrophosphorylase

0.04 ± 0.03 0.08 ± 0.05 1.24 ± 3.26 0.97 ± 0.66 0.83 ± 0.56 0.64 ± 0.62

*p<0.05

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Figure captions:

Fig.1 Effects of C. lacryma-jobi oil (CLO) concentrations on the mycelial growth and

metabolite production of G. lucidum. a, biomass and triterpenoids; b, EPS and IPS.

Each fermentation was carried out in triplicate at 30°C for 7 days with 10 % (v/v)

inoculation on a rotary shaker at 160 rpm.

Fig. 2 Effects of 2% C. lacryma-jobi oil (CLO) addition at different stage on the

mycelial growth and metabolite production of G. lucidum. a, biomass and

triterpenoids; b, EPS and IPS. Each fermentation was carried out in triplicate at 30°C

for 7 days with 10 % (v/v) inoculation on a rotary shaker at 160 rpm.

Fig. 3 Time courses of cultures containing 2% C. lacryma-jobi oil in the media (filled

square) and the control (open triangle). a, glucose; b, biomass; c, triterpenoids; d, EPS;

e, IPS; f pH. G. lucidum was cultivated at 30 °C for 8 days on a rotary shaker at 160

rpm.

Fig. 4. HiPrepTM 16/10 DEAE elution profile of the intracellular polysaccharide (A)

and exopolysaccharide (B). The column was eluted stepwise with H2O, 0.05, 0.2, 0.5

mol/L NaCl solutions. ▲, Sample of containing 2% (v/v) oil in medium; ○, control.

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(Fig. 1)

0

2

4

6

8

10

12

0 0.5 1.0 2.0 4.0

CLO conc. (%)

Bio

mas

s (g

/L)

0

20

40

60

80

100

120

Tri

terp

enoi

ds (

mg/

L)Biomass

Triterpenoids

a

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.5 1.0 2.0 4.0

CLO conc. (%)

Pol

ysac

char

ides

(g/

L) IPS

EPSb

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(Fig. 2)

0

3

6

9

12

15

day 0 day 1 day 2 day 4

CLO addition stage

Bio

mas

s (g

/L)

0

40

80

120

160

Tri

terp

enoi

ds (

mg/

L)

Biomass

Triterpenoidsa

0

0.1

0.2

0.3

0.4

0.5

0.6

day 0 day 1 day 2 day 4

CLO addition stage

Pol

ysac

char

ides

(g/

L)

EPS

IPS b

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(Fig. 3)

0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7 8

Time (d)

pH

f

0

10

20

30

40

0 1 2 3 4 5 6 7 8

Time (d)

Glu

cose

(g/

L) a

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8

Time (d)

Bio

mas

s (g

/L)

b

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8

Time (d)

Tri

terp

enoi

ds (

mg/

L)

c

0

0.1

0.2

0.3

0.4

0 1 2 3 4 5 6 7 8

Time (d)

EP

S (

g/L

)

d

0

0.1

0.2

0.3

0.4

0.5

0 1 2 3 4 5 6 7 8

Time (d)

IPS

(g/

L)

e

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(Fig. 4)

0

60

120

180

240

0 20 40 60 80

Fraction number

IPS

con

cent

ratio

n(μg

/mL

)

0

0.1

0.2

0.3

0.4

0.5

0.6

NaC

l(m

ol/L

)

IPS-1

IPS-2

IPS-3 IPS-4

A

0

100

200

300

400

500

0 20 40 60 80

Fraction number

EP

S c

once

ntra

tion(

μg/m

L)

0

0.1

0.2

0.3

0.4

0.5

0.6

NaC

l(m

ol/L

)

EPS-1

EPS-2EPS-3

EPS-4

B

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Highlights

Stimulation of Coix lacryma-jobi oil (CLO) on the biomass of Ganoderma lucidum.

Enhancement of CLO on polysaccharide biosynthesis of G. lucidum.

CLO does not change the biosynthesis pathway of polysaccharides.

CLO affects synthesis level of some enzymes related to polysaccharide production.