Analysis of volatile compounds of Antrodia camphorata in submerged culture using headspace solid-phase microextraction

Food Chemistry 127 (2011) 662–668

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Analysis of volatile compounds of Antrodia camphorata in submerged culture using headspace solid-phase microextraction Zhen-Ming Lu a,b, Wen-Yi Tao b,⇑, Hong-Yu Xu a, Joanne Lim b, Xiao-Mei Zhang a, Li-Ping Wang c, Jing-Hua Chen a, Zheng-Hong Xu a,b,⇑ a b c

Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, PR China The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China

a r t i c l e

i n f o

Article history: Received 22 June 2009 Received in revised form 29 October 2010 Accepted 29 December 2010 Available online 8 January 2011 Keywords: Antrodia camphorata Gas chromatography–mass spectrometry Gas chromatography–olfactometry HS-SPME Submerged culture Volatile

a b s t r a c t In this work a headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography– mass spectrometry (GC–MS) and GC–olfactometry (GC–O) was developed to evaluate the profile of the volatile compounds that contribute to the aroma of Antrodia camphorata in submerged culture. For this purpose, the HS-SPME sampling method for the volatile compounds of A. camphorata in submerged culture was optimised by a D-optimal design. A HS extraction of the culture broth of A. camphorata followed by incubation on a carboxen/polydimethylsiloxane (CAR/PDMS) fibre during 31.8 min at 54.6 °C gave the most effective and accurate extraction of the volatile compounds. By the optimised method, a total of 49 volatile compounds were identified in culture broth of A. camphorata, while a total of 55 volatile compounds were identified in the mycelia. A series of C8 aliphatic compounds (mushroom-like odour), several lactones (fruity odour) and L-linalool (citrus-like odour) were the most potent key odourant in both the mycelia and culture broth. This combined technique is fast, simple, sensitive, inexpensive and useful to monitor volatile compounds associated to A. camphorata. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction As fungal metabolites represent a wide diversity of chemical species, the investigation of the secondary metabolism of fungi attracts great scientific interest (Wu, Zorn, Krings, & Berger, 2005). A. camphorata (Polyporaceae, Aphyllophorales) is a fungus that only grows in the inner cavity of Cinnamomum kanehirae Hay (Lauraceae) in Taiwan (Wu, Ryvarden, & Chang, 1997). The red to light cinnamon fruiting bodies of A. camphorata are bitter and have a mild camphor scent like the host woods (Chang & Chou, 1995). Primary investigations have revealed that A. camphorata has extensive biological activities, such as hepatoprotective effect, anti-hepatitis B virus effect, anticancer effect, antioxidant and anti-inflammation activities (Lu, Tao, Zou, Fu, & Ao, 2007; Rao, Fang, & Tzeng, 2007). These activities are related to the chemical ingredients, such as polysaccharides, triterpenoids, sesquiterpene lactones, steroids, phenol compounds, adenosine, cordycepin, ergosterol, etc. (Chang, Lue, & Pan, 2005; Lu, Cheng, Lai, Lin, & Huang, 2006). Currently, A. camphorata is widely used in Taiwan ⇑ Corresponding authors. Address: Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China (Z.-H. Xu). Tel./fax: +86 510 85918206 (W.-Y. Tao). E-mail addresses: [email protected] (W.-Y. Tao), [email protected] (Z.-H. Xu). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.111

as food additives or functional ingredients (Ao et al., 2009; Chen, Chyau, & Hseu, 2007). However, the fruiting bodies of A. camphorata are in great demand in Taiwan due to host specificity, rarity in nature, and the difficulty of artificial cultivation. Thus, artificial cultivation was developed as a substitute. Submerged cultures of A. camphorata are highly odiferous, suggesting that the fungus might serve as an important source of natural aroma compounds for the food and cosmetic industries (Liu, Jia, Zhang, & Pan, 2008). However, to the best of our knowledge, there is little literature reporting aroma characterisation of A. camphorata in submerged culture. Solvent extraction and steam distillation–solvent extraction were employed for extracting volatile compounds in the culture broth of A. camphorata. Unfortunately, the number and category of the volatile compounds extracted by the two methods were quite different (Chen et al., 2007; Liu et al., 2008), indicating the loss or degradation of some of the volatile compounds (Iglesias & Medina, 2008). Moreover, steam distillation and solvent extraction are time- and solventconsuming. Recently, headspace solid-phase microextraction (HS-SPME) was reported as a fast, simple, sensitive, highly reproducible and solvent-free method for the isolation and concentration of the volatile compounds present in the headspace without modifications of these compounds due to temperature or solvent effect (Harmon, 1997; Pawliszyn, 2001; Yan, Zhang, Tao, Wang, & Wu, 2008).

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HS-SPME was used for the analysis of flavour compounds in several foods, and more particularly in fermentation products during the production (Feng, Larsen, & Scunürer, 2007; Ndagijimana et al., 2008). In this paper, a method based on HS-SPME coupled with gas chromatography–mass spectrometry (GC–MS) is proposed for the rapid analysis of volatile compounds of A. camphorata in submerged culture. The suitability of different fibre coatings was determined, and the influence of the main factors affecting the microextraction was extensively studied using a D-optimal design consisting of 20 experimental runs. Moreover, HS-SPME coupled with GC–olfactometry (GC–O) was used to determine the contribution of volatile compounds to A. camphorata aroma.

data were fitted, was a second-order polynomial model, expressed by the following equation: R = b0 + b1T + b2t + b3F + b12T  t + b13T  F + b23t  F + b11T2 + b22t2, where R means the response; F is the type of fibre; T represents the extraction temperature; t represents the extraction time; b0, bi (i = 1, 2, 3), bij (i = 1, 2, 3; j = 1, 2, 3) and bii (i = 1, 2) are the regression coefficients for intercept, linear, interaction and quadratic terms, respectively. Multiple regression analyses, analysis of variance (ANOVA) and significance tests were realised using the software JMP IN 7.0.1 (SAS Institute, Inc., SAS Campus Drive, Cary, NC, USA). Design Expert software (version 7.1.0, Stat-Ease Inc., Minneapolis, MN, USA) was performed for graphical analysis of data obtained.

2. Materials and methods

2.4. Gas chromatography–mass spectrometry

2.1. Materials

GC–MS analysis was performed on a Finnigan TRACE GC–MS (Thermo Quest Finnigan Co., USA) equipped with a PEG 20 M capillary column (30 m  0.25 mm  0.25 lm). Helium (flow rate, 1.0 ml/min) was used as the carrier gas. The GC oven temperature was maintained initially at 40 °C for 3.5 min, followed by increases to 60 °C at a rate of 5 °C/min, and from 60 to 230 °C at a rate of 10 °C/min, and then this temperature was held constant for 8 min. The mass spectra were acquired with a source temperature of 200 °C, under a 70 eV ionisation potential. The ionisation mode was EI+, emission current was 200 lA, and detector voltage was 350 V. n-Alkanes were run under the same conditions as the samples to calculate the Kovats index (KI) of compounds.

Four different coating fibres for HS-SPME were tested: 100 lm polydimethylsiloxane (PDMS), 85 lm polyacrylate (PA), 75 lm carboxen/polydimethylsiloxane (CAR/PDMS) and 65 lm polydimethylsiloxane/divinylbenzene (PDMS/DVB) coating fibres. These fibres were selected according to the different polarities and molecular weights of the studied analytes and since they were tested for determination of volatile compounds in several fungi (Evans, Eyre, Rogers, Boddy, & Müller, 2008; Ndagijimana et al., 2008). The fibres, from Supelco (Bellefonte, PA, USA), were conditioned prior use according to supplier’s prescriptions, 0.5 h at 250 °C for PDMS and PDMS/DVB, 1 h at 280 °C for PA and 1 h at 300 °C for CAR/PDMS. n-Alkanes and 2-octanol were purchased from Sigma–Aldrich (MO, USA). All the other chemicals used were either of analytical grade or of the highest purity commercially available. 2.2. Strain and culture condition The standard strain, A. camphorata CCRC35396, was used in this study. The strain A. camphorata was maintained on potato dextrose agar (PDA) slant. The slant was incubated at 28 °C for 21 days, and then stored at 4 °C. A shaking flask culture was performed in a 500 ml Erlenmeyer flask with a cotton plug, which contained 100 ml of the medium. The culture medium was shown as following: glucose 2%, peptone 1%, MgSO4 0.15%, KH2PO4 0.3% and wheat bran extract 2%, pH 5.2. Media were sterilized at 121 °C for 15 min, and glucose was autoclaved separately. The flasks were incubated on a Firstek Scientific orbital rotary shaker (stroke 7 cm) under the conditions of 150 rpm and 28 °C for 10 days.

2.5. Gas chromatography–olfactometry GC–O analyses were conducted using a Finnigan TRACE GC (Thermo Quest Finnigan Co., USA) equipped with a sniffing port. Column type and analysis conditions were as described above, and the temperature of the sniffing port was 250 °C. Sniffing tests on volatile compounds from A. camphorata in combination with reference compounds were performed by two panellists in paired alternate chromatographic runs conducted at 15 min intervals. The panellists had more than 30 h of training in GC–O analysis, and have more than 5 years of sensory analytical experience in fermented foods. Each sample was sniffed trice by each panellist.

Table 1 Experimental design matrix and responses obtained in the D-optimal experimental design.

2.3. Headspace solid-phase microextraction 2.3.1. Extraction of volatile compounds Culture broth of A. camphorata (8 ml) and NaCl (2 g) were placed in a 20 mL HS vial. The vial was immediately sealed with a silicone septum and placed into a water-bath at the controlled temperature. The SPME fibre was exposed to the HS 1 cm above the solution surface to adsorb the analytes. The trapped volatile compounds were desorbed at 250 °C in the GC injection port for 1 min and flushed into the GC column. 2.3.2. Optimisation of HS-SPME condition A D-optimal design consisting of 20 experimental runs was used to determine the effects of two continuous variables (extraction time and temperature) and a qualitative variable (type of fibre) on the extraction of volatile compounds of A. camphorata in submerged culture. The symbols and levels of three variables are shown in Table 1. The proposed model, to which the experimental

a b

Standard ordera

Run orderb

T Temperature (°C)

t Time (min)

F Fibre

Response

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

7 14 17 11 6 16 4 12 8 20 18 10 19 1 15 2 5 3 13 9

50 40 60 50 60 60 40 40 40 60 60 60 40 40 50 55 50 45 60 50

40 30 30 20 40 20 40 40 40 20 40 40 20 20 30 25 25 30 40 40

PA PA PA PA CAR/PDMS CAR/PDMS CAR/PDMS PDMS PDMS/DVB PDMS PDMS/DVB PDMS PDMS/DVB CAR/PDMS PDMS PDMS/DVB CAR/PDMS PDMS/DVB PDMS PA

4.71E+8 2.79E+8 5.10E+8 3.82E+8 1.38E+9 1.35E+9 1.19E+9 3.38E+8 9.60E+8 4.70E+8 1.29E+9 5.81E+8 8.36E+8 9.09E+8 5.65E+8 1.31E+9 1.36E+9 1.15E+9 5.81E+8 4.91E+8

Nonrandomized. Randomized.

(0) ( 1) (+1) (0) (+1) (+1) ( 1) ( 1) ( 1) (+1) (+1) (+1) ( 1) ( 1) (0) (+0.5) (0) ( 0.5) (+1) (0)

(+1) (0) (0) ( 1) (+1) ( 1) (+1) (+1) (+1) ( 1) (+1) (+1) ( 1) ( 1) (0) ( 0.5) ( 0.5) (0) (+1) (+1)

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Effluent from the GC containing the separated compounds was diluted with humidified air, and odour evaluation was carried out for each analyte leaving the chromatographic column during the entire GC analysis. Odour characteristics were recorded when the panellists assigned the same aroma attribute, and any odour characteristic that was recorded by fewer than three of the six analyses was considered as noise. 2.6. Qualitative and quantitative analyses Data were analysed using Xcalibur system software (version 1.2). Identification of volatile compounds was confirmed by comparing their mass spectra with those contained in the National Institute for Standards and Technology (NIST, Search Version 1.6) and Wiley (NY, 320 k compounds, version 6.0) mass spectral library and by comparison of KI with those present in user generated (INRA Mass, Dijon, France) and some others from the literature. The identifications of some volatile compounds were only performed using mass spectrometry data because the retention indexes were unavailable. The amount of each compound was determined by the method of internal standard and calculated from the peak areas of gas chromatography. 2.7. Analysis of volatile compounds in culture broth and mycelia of A. camphorata in submerged culture After incubation at 28 °C for 10 days, mycelia of A. camphorata were filtrated and the culture broth was retained. The mycelia were washed with distiled water to remove the extracellular culture broth. Volatile compounds of the mycelia and culture broth were analysed by means of the optimised HS-SPME method described as follows: Total of 8 ml culture broth or 1 g mycelia was placed in a 20 ml HS vial. The HS vial was immediately sealed with a silicone septum. The CAR/PDMS fibre was exposed to the HS of extract by incubating to 55 °C during 30 min under magnetic stirring. The fibre was immediately desorbed in the GC injector to 250 °C during 1 min.

Table 3 presents the values of the estimated parameters obtained by the multiple linear regressions. As the values of the variables were expressed in their coded forms, information about the ranking of the adsorption activity of the fibre could be directly inferred from the parameter estimates. This ranking was as follows: CAR/PDMS (4.67  108) > PDMS/DVB (3.43  108) > PDMS (-3.77  108) > PA ( 4.33  108). Therefore, the CAR/PDMS fibre was selected as the most suitable fibre for HS-SPME of volatile compounds of A. camphorata in submerged culture. In order to determine the optimal conditions, the response surface graph using CAR/PDMS as the fibre was generated (Fig. 1). The response increased with the increase of extraction temperature at a fixed extraction time, and the response reached a maximum when temperature was up to 55 °C (Fig. 1). A similar quadratic increase in the response with the increase of extraction time was also observed. In terms of the CAR/PDMS fibre, the regression equation obtained using actual values was given by R = 3.91  109 + 1.46  108T + 7.73  107t 4.71  105T  t 1.16  106T2 7.69  105t2, where R means the response; T represents the extraction temperature; t represents the extraction time. The optimal operation conditions of CAR/PDMS fibre were estimated as follows: extraction temperature, 54.6 °C; and extraction time, 31.8 min. Usually, the type of fibre determines the specificity of the HSSPME. On the basis of the polarity of polymers, the solid coatings of the fibres can be classified into three types: the first type is the polar coat, such as PA and PEG, which is suitable for the extraction of polar compounds and has already been used for the analysis

Table 2 Analysis of variance (ANOVA) of the fitted quadratic polynomial model for optimisation of the response.

3. Results and discussion 3.1. HS-SPME optimisation The SPME technique is well known as a simple, rapid, sensitive, and high reproducible sampling method. However, in order to obtain significant data using this technique, it is important to set up sampling conditions that are the most suitable for an experiment system. Therefore, in this study the parameters affecting the HSSPME efficiency, including the type of fibre, extraction time and temperature, were optimised by a D-optimal design. The design arrangement and the experimental results obtained by D-optimal design are shown in Table 1. To validate the regression coefficient, analysis of variance (ANOVA) for response was performed (Table 2). The fact that the value of ‘‘Model Prob > F’’ less than 0.0001 implies the model is significant. In this case T, t, F, T  t, T2 and t2 were significant model terms. The lack-of-fit measures the failure of the model to represent data in the experimental domain at points which are not included in the regression (Rastogi & Rashmi, 1999). The ‘‘Lack of Fit Prob > F-value’’ of 0.1012 implied that the lack-of-fit is insignificant, indicating that the model could adequately fit the experimental data (Table 2). A typical regression model having an R2 value higher than 0.9 is considered as having a very high correlation, since the R2 value of this model was 0.9990, it was therefore reasonable to use the regression model to analyse the trends in the responses.

a

Source

Sum of squares

DFa

F value

Prob > F

Model T t F Tt TF tF T2 t2 Lack of fit

2.96E+18 2.48E+17 4.60E+16 2.49E+18 1.51E+16 8.03E+15 6.77E+15 3.93E+16 1.70E+16 2.71E+15

14 1 1 3 1 3 3 1 1 3

755.77 917.21 136.95 2920.85 30.79 9.66 4.51 148.84 64.52 3.92

<0.0001 <0.0001 0.0003 <0.0001 0.0038 0.0669 0.0894 0.0004 0.0029 0.1012

Degrees of freedom.

Table 3 Analysis of variance (ANOVA) for the estimated parameters of the predicted model. Term

Parameter

Prob > F

Intercept T t F1 F2 F3 F4 Tt T  F1 T  F2 T  F3 T  F4 t  F1 t  F2 t  F3 t  F4 T2 t2

9.53E+8 1.41E+8 3.83E+7 4.33E+8 3.77E+8 4.67E+8 3.43E+8 4.71E+7 4.39E+7 6.67E+6 1.64E+6 3.89E+7 1.45E+7 3.63E+7 1.23E+7 3.42E+7 1.16E+8 7.69E+7

<.0001 <.0001 0.0034 <.0001 <.0001 <.0001 <.0001 0.0038 0.0280 0.6391 0.8925 0.0257 0.3042 0.0459 0.3063 0.0468 0.0004 0.0029

Note: F1, PA; F2, PDMS; F3, CAR/PDMS; F4, PDMS/DVB.

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and determination of organic-phosphor pesticides, organic constituents in waste water, flavour components in foods, and so on. The second is the nonpolar coating, such as PDMS, which is suitable for

the extraction of nonpolar and less-polar compounds and has already been used for the analysis and determination of certain hazardous materials existing in foods and fabrics. The third type is the mixed coating of the medium polarity, including PEG/DVB, CAR/ DVB, CAR/PDMS, PDMS/DVB, and so on (Yan et al., 2008). As regards to the effects of the salting-out and stirring, the highest sensitivity for almost all target compounds was achieved by the extraction in ultrapure water saturated with NaCl and with stirring (Pawliszyn, 2001). The addition of salt increases the ionic strength of the water sample by lowering the solubility of analytes in the aqueous phase, and stirring enhances the extraction efficiency in nonequilibrium situations. Thus, the HS-SPME was carried out with culture broth saturated with NaCl and with stirring in this study.

1.46E+09

Response

1.32E+09 1.18E+09 1.04E+09

3.2. Identification of volatile compounds for A. camphorata in submerged culture

9.00E+08

40.00

Using the optimal HS-SPME conditions, the volatile compounds of culture broth and mycelia ofA. camphorata in submerged culture were determined. The GC–MS total ion chromatogram for the volatile compounds is shown in Fig. 2, and the peaks are identified in Table 4. A total of 49 (98.23% of the total) and 55 (99.36% of the total) volatile compounds including 22 alcohols, 8 ketones, 7 aldehydes, 23 esters, 5 terpene hydrocarbons, and 3 aromatics were identified in the culture broth and mycelia of A. camphorata in submerged culture, respectively. Hereinto, 1-octen-3-ol (21.49%), ethanol (12.46%), 3-octanone (10.46%), L-linalool (10.17%) and methyl

60.00 35.00

55.00 30.00

50.00 25.00

B: Time

45.00 20.00 40.00

A: Temperature

Fig. 1. Response surface showing the effects of extraction temperature and time on the response (total chromatographic peak areas) when using CAR/PDMS as the headspace fibre.

9

100 90

(a) Culture broth in submerged culture

80 70

12

60 50

4 21

40

IS

Relative Abundance

30 20

10

5 1

10

2 3

11

8 6

20 13 15 19 17

23 24

25

26

27

28

0 0

5

10

20

15

30

25

Time (min)

0 10

1

2

6

3

5

20

19

7

13 14

8

16 15 18

IS

30 40

21 22

20

24 23

26 27

28

25

50 60 10

70 80

(b) Mycelia in submerged culture

4

90 100

9

11

Fig. 2. GC–MS total ion chromatograms for the volatile compounds identified in culture broth (a) and mycelia (b) of Antrodia camphorata in submerged culture. Peaks are identified in Table 4. 1, Ethanol; 2, 3-cyclopropyl-1-butyne; 3, 3-methyl-1-butanol; 4, 3-octanone; 5, 3,5-dihydroxybenzamide; 6, 1-octen-3-one; 7, octen-1-ol, acetate; 8, 3octanol; 9, 1-octen-3-ol; 10, acetic acid, octyl ester; 11, 1-octen-3-yl acetate; 12, L-linalool; 13, 1-octanol; 14, d-guainene; 15, (E)-2-octen-1-ol; 16, 4-decanoic acid, methyl ester; 17, 4-(benzoyloxy)-2H-pyran-3-one; 18, (+)-sativen; 19, 2-propyl-cyclohexanone; 20, 3,7-dimethyl-2,6-octadien-1-ol, acetate; 21, methyl phenyl acetate; 22, cyclopropanenonanoic acid, methyl ester; 23, benzeneethanol; 24, 1,4-cycloctanedione; 25, nerolidol; 26, (Z)-8,10-dodecadienal; 27, c-dodecalactone; 28, (Z)-dihydro-5-(2octenyl)-2(3H)-furanone.

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Table 4 Volatile compounds identified in culture broth and mycelia of Antrodia camphorata in submerged culture. Compound

Alcohols Ethanol 2-Methyl-1-propanol (E,E)-2,4-Hexadien-1-ol 2-Methyl-6-hepten-3-ol 3-Methyl-1-butanol 1-Hexanol 3-Octanol 3-Hepten-1-ol 1-Octen-3-ol 2-Ethyl-1-hexanol

MIa

Aromab

L-Linalool

MS, MS, MS, MS MS, MS, MS, MS MS, MS, MS,

KI KI KI

Alcohol

KI KI KI

Apple-like Green Mushroom-like and buttery

KI KI KI

Mushroom-like Rose-like Citrus-like

1-Octanol (E)-2-Octen-1-ol Farnesol Benzeneethanol Nerolidol Benzenepropanol Cedrol a-Cadinol T-Muurolol T-Cadinol a-Terpineol

MS, KI MS MS MS,KI MS, KI MS, KI MS MS, KI MS MS, KI MS, KI

Ketones 3-Heptanone 3-Octanone 1-Octen-3-one 5-Methylene-3-heptanone 4-(Benzoyloxy)-2H-pyran-3-one 2-Propyl-cyclohexanone 2-(1-Cyclopent-1-enyl-1-methylethyl) cyclopentanone 1,4-Cycloctanedione

MS MS, KI MS, KI MS MS MS MS MS, KI

Aldehydes Hexanal 3-Methyl-2-butenal Heptanal Nonanal (E)-2-Octenal Benzaldehyde Z-8,10-Dodecadienal

MS MS MS, KI MS, KI MS MS, KI MS

Esters Ethyl acetate Linalyl acetate Octen-1-ol acetate Octanoic acid methyl ester Acetic acid octyl ester Nonanoic acid methyl ester 1-Octen-3-yl acetate Decanoic acid methyl ester 4-Decanoic acid methyl ester Nerolidyl acetate 10-Undecenoic acid methyl ester 3,7-Dimethyl-2,6-octadien-1-ol acetate Methyl phenyl acetate Acetic acid, 2-phenylethyl ester Cyclopropanenonanoic acid methyl ester c-Octalatone Benzenepropanol acetate Methyl 2-hydroxydecanonate 2-Hydroxy-10-undecenoic acid methyl ester c-Undecalactone Farnesyl acetate c-Dodecalactone (Z)-6-Dodecene-c-lactone

MS, MS, MS, MS, MS, MS, MS, MS, MS MS MS MS MS, MS, MS MS, MS, MS MS MS, MS, MS, MS,

Terpene hydrocarbons trans-Epoxy-ocimene d-Guainene Thujopsene (+)-Sativen trans-b-Bisabolene

MS MS MS MS MS

KI KI KI KI KI KI KI KI

Chemical and sweet Mushroom-like Floral and sweet Woody

Spicy Pine tree-like

Mushroom-like and buttery Mushroom-like

Cut grass-like

Fatty and soapy

Fruity Floral

Pear-like and sweet Mushroom-like

Floral KI KI KI KI

Coconut-like and sweet

KI KI KI KI

Peach-like and sweet Fruity Peach-like and sweet

Relative peak areac (%) Broth

Mycelia

12.46 ± 1.39 0.23 ± 0.02 0.18 ± 0.01 0.43 ± 0.03 0.95 ± 0.09 0.25 ± 0.02 1.29 ± 0.11 nd 21.49 ± 2.13 0.21 ± 0.02 10.17 ± 0.91

4.92 ± 0.41 ndd nd nd 1.03 ± 0.06 0.35 ± 0.03 2.01 ± 0.15 0.13 ± 0.01 18.13 ± 1.55 nd 1.28 ± 0.10

0.60 ± 0.05 0.21 ± 0.01 nd 0.27 ± 0.02 1.66 ± 0.19 0.28 ± 0.03 0.56 ± 0.07 0.31 ± 0.02 0.11 ± 0.01 0.23 ± 0.03 0.39 ± 0.03

1.34 ± 0.07 1.81 ± 0.13 0.12 ± 0.01 1.53 ± 0.06 2.53 ± 0.19 nd nd 0.22 ± 0.02 nd 0.15 ± 0.01 nd

nd 10.46 ± 0.68 0.58 ± 0.03 nd 1.15 ± 0.88 1.29 ± 0.13 nd 1.45 ± 0.15

0.12 ± 0.01 16.23 ± 1.17 0.50 ± 0.03 0.25 ± 0.02 0.17 ± 0.01 0.80 ± 0.07 0.14 ± 0.03 0.97 ± 0.09

0.19 ± 0.02 0.28 ± 0.03 0.25 ± 0.02 0.57 ± 0.05 nd 0.31 ± 0.02 4.83 ± 0.38

nd nd nd nd 0.27 ± 0.02 0.22 ± 0.02 1.07 ± 0.05

4.72 ± 0.41 0.20 ± 0.01 nd nd 0.56 ± 0.04 nd 1.31 ± 0.11 nd nd nd nd 0.31 ± 0.02 6.47 ± 0.48 nd nd 0.39 ± 0.03 0.54 ± 0.06 0.18 ± 0.01 0.61 ± 0.06 0.47 ± 0.03 nd 0.32 ± 0.03 0.34 ± 0.03

3.55 ± 0.29 nd 0.35 ± 0.01 0.76 ± 0.06 8.18 ± 0.57 0.18 ± 0.01 12.15 ± 0.79 0.66 ± 0.07 2.44 ± 0.14 0.12 ± 0.01 0.74 ± 0.05 0.67 ± 0.05 1.43 ± 0.12 0.27 ± 0.02 1.20 ± 0.09 0.60 ± 0.04 nd nd nd 0.26 ± 0.02 0.41 ± 0.03 0.44 ± 0.03 0.53 ± 0.05

nd nd nd nd nd

0.19 ± 0.01 1.04 ± 0.08 0.12 ± 0.01 3.07 ± 0.25 0.37 ± 0.03

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Z.-M. Lu et al. / Food Chemistry 127 (2011) 662–668 Table 4 (continued) Compound

MIa

Aromab

Relative peak areac (%) Broth

a b c d

Aromatics Benzene, methyl3,5-Dihydroxybenzamide Methylnaphthalene

MS, KI MS MS

nd 3.21 ± 0.24 nd

Others Undecane 3-Cyclopropyl-1-butyne 2,4-Dimethyl-3,5,6-trimethoxybenzyl alchohol Dimethyl 5-hydroxypyridine-3,4-dicarboxylate 3,4-Dihydrophenaleno[1,9-BC]furan-4-ol

MS, KI MS, KI MS MS MS

nd 3.01 ± 0.29 0.28 ± 0.03 1.01 ± 0.08 0.66 ± 0.04

Mycelia 0.12 ± 0.01 1.10 ± 0.08 0.39 ± 0.03 0.19 ± 0.02 1.25 ± 0.07 nd nd 0.29 ± 0.02

Method of identification: MS, mass spectrum comparison using Wiley and NIST libraries; KI, Kovats index in agreement with literature values. Aroma properties perceived at the sniffing port. Average of relative peak areas (n = 3) ± standard deviation. nd: not detected.

phenyl acetate (6.47%) were the main volatile compounds in the culture broth of A. camphorata. And 1-octen-3-ol (18.13%), 3-octanone (16.23%), 1-octen-3-yl acetate (12.15%), acetic acid octyl ester (8.18%), and ethanol (4.92%) were the main volatile compounds in the mycelia. It is notable that relative peak areas of 1-octen-3-yl acetate (12.15%) and acetic acid octyl ester (8.18%) in the mycelia were much higher than those in the culture broth, while relative peak area of L-linalool (10.17%) in the broth was much higher than that in the mycelia. Several terpene hydrocarbons in the mycelia, including trans-epoxy-ocimene, d-guainene, thujopsene, (+)-sativen and trans-b-bisabolene, were not founded in the culture broth. Previously, solvent extraction and steam distillation–solvent extraction were employed for determining volatile compounds of the culture broth of A. camphorata (Chen et al., 2007; Liu et al., 2008), and the results showed that the number and category of the volatile compounds extracted by the two methods were quite different. For example, using simultaneous steam distillation with solvent extraction, several terpenoids existed in the fruiting bodies of A. camphorata, including a-cadinol, T-muurolol, T-cadinol and a-terpineol, were not detected in the culture broth of A. camphorata (Chen et al., 2007). However, Liu et al. (2008) found T-cadinol and a-terpineol in the culture broth of A. camphorata using organic solvent extraction, while a-cadinol and T-muurolol were not detected. It is suggested that some of the volatile compounds might be lost or degradated during the extraction process. In this study, a-cadinol, T-muurolol, T-cadinol and a-terpineol were detected in the culture broth by the HS-SPME sampling technique. Therefore, the HS-SPME–GC–MS technique is proposed for the sensitive analysis of volatile compounds of A. camphorata in submerged culture. By this technique, fungal volatiles can be collected in situ and identified by GC–MS without disturbing the fermentation process. On the other hand, the profiles of volatile compounds often vary with species and varieties and can be influenced by the cultivating conditions (Wu et al., 2005). Thus, the HS-SPME–GC–MS can be applied to control the volatile production by A. camphorata during the fermentation process. The evaluation for the reproducibility of the method was performed under optimised HS-SPME condition. Each sample was performed thrice, to evaluate the reproducibility of HS-SPME for the analysis of volatile compounds in A. camphorata (Table 4). The maximum relative standard deviation (RSD) for relative peak areas of compounds was 21.4%, and the minimum RSD was 3.9%. The average RSD values for the compounds of culture broth and mycelia were 9.9% and 7.6%, respectively, which indicated that the reproducibility of the HS-SPME was good for the analysis of volatile compounds of A. camphorata in submerged culture.

3.3. Characterisation of aroma key compounds by HS-SPME–GC–O Not only did we investigate the total volatile profiles, but we also estimated the contribution of particular volatile compounds to the aroma of A. camphorata. In the present study, 23 key odourants were found to contribute significantly to the characteristic flavour of the culture broth of A. camphorata (Table 4). The overall flavour of the A. camphorata in submerged culture was dominated by mushroom-like, fruity and flowery impressions. The impression ‘mushroom-like’ was mainly attributed to a series of C8 aliphatic compounds, including 3-octanol, 1-octen-3-ol, (E)2-octen-1-ol, 3-octanone, 1-octen-3-one and 1-octen-3-yl acetate. Several lactones, such as c-octalatone, c-undecalactone, c-dodecalactone and (Z)-6-dodecene-c-lactone, were accounted for the peach-like fruity odour of submergedly cultured A. camphorata. Furthermore, several flower-like odourants were picked up in various intensities during the GC–O investigation of the culture broth, with L-linalool giving the strongest impact. Among the diverse volatile compounds, C8 aliphatic compounds, such as 1-octen-3-ol, 2-octen-1-ol, 3-octanol, 1-octanol, 1-octe-3-one and 3-octanone, have been reported to be the major contributors to the characteristic mushroom flavour (Combet, Henderson, Eastwood, & Burton, 2006). In particular, an unsaturated alcohol, 1-octen-3-ol, described as ‘‘mushroom-like odour’’, has been found in many mushroom species and, together with its oxidation product, 1-octen-3-one, is considered to be mainly responsible for the characteristic flavour of most edible species of mushroom (Cho, Kim, Choi, & Kim, 2006). The compound 1-octen-3-ol is produced from linoleic acid by lipoxygenase and hydroperoxide lyase, and can regulate the germination of conidia of fungi (Chitarra, Abee, Rombouts, Posthumus, & Dijksterhuis, 2004). However, none of the C8 alcohols was reported in the culture broth of A. camphorata when organic solvent was used to extract the volatile compounds (Liu et al., 2008), indicating the loss or degradation of some of the volatile compounds. Thus, HS-SPME–GC–O is proposed for the sensitive and fast analysis of volatile compounds that contribute to the aroma of A. camphorata in submerged culture.

4. Conclusion Under the experimental conditions used in this study, HSSPME–GC–MS technique was successfully developed for the determination of volatile compounds of A. camphorata in submerged culture. In total, 49 compounds in culture broth and 55 compounds in mycelia of A. camphorata were identified by the proposed method. The overall flavour of A. camphorata in submerged

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culture was dominated by mushroom-like, fruity and flowery impressions. A series of C8 aliphatic compounds (mushroom-like odour), several lactones (fruity odour) and L-linalool (citrus-like odour) were the most potent key odourant in both the mycelia and culture broth. This combined technique is fast, simple, sensitive, inexpensive and useful to monitor volatile compounds associated to A. camphorata. Future research will focus on the changes in the volatile compounds of A. camphorata during the fermentation process. Meanwhile, the relationship between the volatile compounds and other secondary metabolism, such as triterpenoids and polysaccharides, will be of importance to the main research topic. Acknowledgement This work was supported by a grant from Natural Science Foundation of Jiangsu Province, China (No. BK2010142), National High-Tech Program of China (No. 2007AA021506), National Basic Research Program (973 Program) (No. 2007CB707800) and the program for New Century Excellent Talents in the University of China (No. NCET-07-0380). References Ao, Z. H., Xu, Z. H., Lu, Z. M., Xu, H. Y., Zhang, X. M., & Dou, W. F. (2009). Niuchangchih (Antrodia camphorata) and its potential in treating liver diseases. Journal of Ethnopharmacology, 121, 194–212. Chang, C. Y., Lue, M. Y., & Pan, T. M. (2005). Determination of adenosine, cordycepin and ergosterol contents in cultivated Antrodia camphorata by HPLC method. Journal of Food and Drug Analysis, 13(4), 338–342. Chang, T. T., & Chou, W. N. (1995). Antrodia cinnamomea sp. nov. on Cinnamomum kanehirai in Taiwan. Mycological Research, 99, 756–758. Chen, C. C., Chyau, C. C., & Hseu, T. H. (2007). Production of a COX-2 inhibitor, 2,4,5trimethoxybenzaldehyde, with submerged cultured Antrodia camphorata. Letters in Applied Microbiology, 44(4), 387–392. Chitarra, G. S., Abee, T., Rombouts, F. M., Posthumus, M. A., & Dijksterhuis, J. (2004). Germination of Penicillium paneum conidia is regulated by 1-octen-3-ol, a volatile self-inhibitor. Applied and Environmental Microbiology, 70, 2823–2829. Cho, I. H., Kim, S. Y., Choi, H. K., & Kim, Y. S. (2006). Characterizaiton of aroma-active compounds in raw and cooked pine-mushrooms (Tricholoma matsutake Sing.). Journal of Agricultural and Food Chemistry, 54, 6332–6335.

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