Volatile organic compounds from a Tuber melanosporum fermentation system

Volatile organic compounds from a Tuber melanosporum fermentation system

Yuan-Yuan Li a ,b ,Guan Wang a ,Hong-Mei Li a ,Jian-Jiang Zhong c ,Ya-Jie Tang a ,?

a

Key Laboratory of Fermentation Engineering (Ministry of Education),Hubei University of Technology,Wuhan 430068,China

b

Research Group for Bioactive Products,Department of Biology and Chemistry,City University of Hong Kong,Kowloon,Hong Kong SAR,China c

Key Laboratory of Microbial Metabolism (Ministry of Education),School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China

a r t i c l e i n f o Article history:

Received 18January 2012

Received in revised form 24May 2012Accepted 2July 2012

Available online 14July 2012Keywords:

Tuber melanosporum Tuberaceae Fermentation Flavor volatiles

Volatile organic compounds Qualitative analyses

a b s t r a c t

A total of 59volatile organic compounds (VOCs)were identi?ed from Tuber melanosporum fermentation:53from its fermented mycelia and 32from the fermentation broth.Alcohol-derived compounds were predominant in both the fermentation mycelia and the broth,although long chain fatty acids and isopre-noids were,for the ?rst time,also found in the mycelia.The intense wine bouquet properties of the broth arose from several speci?c ?avor substances,including sulfur compounds,pyrazines,furans and https://www.360docs.net/doc/24589840.html,paring the VOCs identi?ed in this work with those previously reported,our results are more similar to the composition of the Tuber fruiting-body than previous Tuber fermentations.The composition and accumulation of ?avor volatiles (e.g.,pyrazines,sulfur compounds,and esters)and major constitu-ents (e.g.,3-methyl-1-butanol and 2-phenylethanol)in this fermentation were signi?cantly in?uenced by the sucrose concentration in the medium.The obtained information could therefore be useful in appli-cations to convert the ?avors of truf?e mycelia similar to those of the fruiting-body by optimising the fer-mentation process.

ó2012Elsevier Ltd.All rights reserved.

1.Introduction

Because of their unique and characteristic aromas,truf?es are widely used in French and Italian cuisines,particularly the French ‘‘black diamond’’truf?e Tuber melanosporum ,the notorious white Italian truf?e Tuber magnatum and the rare summer truf?e Tuber aestivum (Mello,Murat,&Bonfante,2006).Since the 1980s,an increasing number of scientists have focused on studying the vol-atile organic compounds (VOCs)produced by truf?es to discover the secret of truf?e aromas (Claus,Hoppen,&Karg,1981;Ney &Freytag,1980).Thus far,more than 200VOCs have been identi?ed from truf?e fruiting -bodies of various species around the world (Díaz,Ibá?ez,Se?oráns,&Reglero,2003;March,Richards,&Ryan,2006;Mauriello,Marino,D’Auria,Cerone,&Rana,2004;Pelusio et al.,1995;Splivallo,Bossi,Maffei,&Bonfante,2007).Alcohols,esters,ketones,aromatics and sulfur compounds are the most common constituents in the truf?e fruiting-body,and the sulfur compounds (i.e.,dimethyl sul?de,dimethyl disul?de,dimethyl tri-sul?de,and bis(methylthio)methane)are viewed as key odorants (Bellesia,Pinetti,Bianchi,&Tirillini,1996;Bellesia,Pinetti,Bianchi,&Tirillini,1998a,1998b;Bellesia et al.,2002;Talou,Delmas,&Gaset,1989;Talou,Gaset,Delmas,Kulifaj,&Montant,1990).More-over,the composition of VOCs is altered by genetic characteristics,geographical origin,and maturity (Gioacchini et al.,2009;Splivallo et al.,2007).Some special truf?es also possess distinct VOCs,such as 5H-furan-2-one and 2-methyl-4,5-dihydrothiophene for T.borchii (Splivallo et al.,2007).

Because of the great demand for truf?es in the market as well as a natural shortage of this wild resource,a new approach is urgently needed for truf?e production on a large https://www.360docs.net/doc/24589840.html,pared to natural ?eld collection and semi-arti?cial cultivation,the submerged fermentation of truf?es is a promising method for their ef?cient production (Tang,Zhu,Li,&Li,2007).In a previous study,a novel truf?e fermentation process was developed for producing T.melanosporum truf?e mycelia and polysaccharides (Liu,Li,Li,&Tang,2008;Liu et al.,2009;Tang,Zhu,Li,Mi,&Li,2008a;Tang et al.,2008b ).The presence of androstenol,a bioactive volatile compound in the T.melanosporum fruiting-body (Claus et al.,1981)found in truf?e fermentation,was also reported (Wang,Li,Li,&Tang,2008).Additionally,we have con?rmed that the fermen-tation conditions outweigh the truf?e species in affecting the aroma of the fermented mycelia and,furthermore,that this aroma can be adjusted by controlling the fermentation process (Tang,Wang,Li,&Zhong,2009).

Splivallo et al.and Tirillini et al.have reported the composition of VOCs from T.borchii cultured mycelia,but their Tuber species and fermentation process differed from ours (Splivallo et al.,2007;Tirillini,Verdelli,Paolocci,Ciccioli,&Frattoni,2000).Fur-thermore,our previous research con?rmed that the VOC composi-tion of the mycelia was signi?cantly in?uenced by fermentation conditions (Tang et al.,2009),so it was necessary to elucidate

0308-8146/$-see front matter ó2012Elsevier Ltd.All rights reserved.https://www.360docs.net/doc/24589840.html,/10.1016/j.foodchem.2012.07.013

Corresponding author.Tel./fax:+862788015108.

E-mail address:yajietang@https://www.360docs.net/doc/24589840.html, (Y.-J.Tang).

the composition of VOCs from our T.melanosporum fermentation system.

In this work,VOCs from T.melanosporum fermentation mycelia and broth were qualitatively studied by gas chromatography and mass spectrometry(GC–MS)for the?rst time.The composition of VOCs identi?ed in our fermentation was compared to that in the literature.Interestingly,the VOC composition by fermentation varied under different sucrose concentrations in the medium.

2.Materials and methods

2.1.Chemicals and reagents

The following authentic standards were purchased from Sigma–Aldrich China Inc.(Beijing,China):3-methyl-1-butanol, 2-methyl-1-butanol,3-methylbutanal,2-methylbutanal,2-methylbutanal,3-methyl butanoic acid,2-methyl butanoic acid, oleic acid,palmitic acid,linoleic acid,butylated hydroxy toluene,toluene,ethylbenzene,o-xylene,p-xylene,benzaldehyde, 2-phenylethanol,dimethylsulfane,and geranylacetone.

2.2.Tuber mycelia,broth and culture conditions

A strain of Tuber melanosporum was provided by the Mianyang Institute of Edible Fungi(Sichuan,China).The details of the basic culture medium and procedure have been described elsewhere (Wang et al.,2008).

To study the medium’s in?uence on volatile organic compound (VOC)production in truf?e fermentation,two medium types with different initial sucrose concentrations were used(Tang et al., 2009).Medium1was composed of sucrose(35g là1),peptone (5g là1),yeast extract(5g là1),MgSO4á7H2O(1g Là1),KH2PO4 (0.5g là1),and Vitamin B1(0.05g là1).Medium2was composed of the same ingredients as medium1except that the initial sucrose concentration was80g là1.

The truf?e fermentation mycelia and broth were harvested when the maximum dry cell weight(DCW)was obtained.After being centrifuged at9391.0?g for30min,both fermentation mycelia and broth were obtained and then stored at4°C.The fer-mentation mycelia(cultured by medium1)were dried at60°C in an oven for5days to obtain dried mycelia.

2.3.Mycelial VOCs extraction

Fermentation mycelia(200g)and de-ionized H2O(2L)were placed in a round-bottom?ask(5L)connected to a Clevenger vol-atile oil trap.To concentrate the VOCs from the vapour,ethyl ace-tate(4ml)was added to the top of the water in the volatile oil trap. After3-h distillation,the VOCs collected in the ethyl acetate layer were removed and dried using anhydrous sodium sulfate,and the ?nal volume was adjusted to5ml in an EP vial by addition of ethyl acetate.Dried fermentation mycelia(200g)and fermentation broth(500ml)were individually subjected to the same method. The liquid medium(without mycelia)was processed using the same method as the blank control.

2.4.GC/MS(FID)conditions

An Agilent7890A GC system equipped with a5975C quadru-pole mass spectrometer(MS)detector(CA,USA)was used.Separa-tions were carried out on a HP-5ms capillary column (30m?0.25mm i.d.,0.25l m?lm thickness)from Agilent Tech-nologies Inc.(CA,USA).The system was operated in the constant ?ow mode(1.0ml minà1)using He as carrier gas,and the sample was injected in split mode with the split ratio of10.The GC oven temperature was maintained at35°C for the?rst6min and then ramped to200°C at a rate of3°C minà1,held for2min,and?nally ramped to300°C at30°C minà1for1min.The injection port, transfer line and ion source temperatures were maintained at 200,250and200°C,respectively.The MS was run in an electron impact(EI)mode with electron energy of70eV and the total ion chromatogram(TIC)mode.The peaks,of which the area contrib-uted more than0.05%of the total peak area,were taken into fur-ther analysis(Tang et al.,2009).The relative percentage of each identi?ed peak was calculated with the area normalization method.

Some volatiles with very low boiling points,like3-methylbut-anal,2-methylbutanal,and dimethyl sul?de,were directly indenti-?ed using Shimadzu2010plus GC-FID system(Tokyo,Japan)by comparison of the retention times with those of standard com-pounds and by the spiked-in experiments respectively.The GC–FID temperature program was beginning at30°C for15min,then ramped by3°C minà1to50°C,held for an additional2min,then ramped by30°C minà1to300°C,and?nally held for5min.

2.5.Statistical analysis

The total ion chromatogram was processed by the Agilent MSD workstation.VOC identi?cation was performed in three ways: comparison of spectra with mass spectra databases(NIST05ò), comparison of Kovàts retention indices with data in NIST05,and comparison of retention times with authentic standards using GC–MS/FID directly.Three biological replicates(n=3)were per-formed for each experiment with deviations evaluated using the R.S.D(%)of retention times and the peak areas in the TIC(Tang et al.,2009).The relative amounts of volatiles in different media were statistically compared using the paired t-test(SPSS software).

2.6.Precision and repeatability

The injection precision was assessed by repetitive injections of the same sample solution six times in one day.The R.S.D.(%)of retention times and peak areas were lower than0.26and2.73 for mycelia and0.27and2.75for broth,respectively.

The reproducibility of the analysis method was determined by analyzing six independently prepared fermentation mycelia sam-ples using the same method.It was found that the R.S.D.(%)of retention time and peak areas were no more than0.29and3.12 for mycelia and0.32and3.12for broth,respectively.

The reproducibility of the fermentation method was deter-mined by analyzing six mycelial samples fermented by the same medium in parallel,where it was established that the R.S.D.(%) of retention times and peak areas were no more than0.29and 5.85for mycelia and0.40and7.83for broth,respectively.

3.Results and discussion

3.1.VOCs from Tuber melanosporum fermentation mycelia

A total of59VOCs were identi?ed(without chirality determina-tion)from T.melanosporum fermentation mycelia and broth, including53from the broth and32from the fresh mycelia.The number of VOCs identi?ed was more than those reported from T. borchii mycelia(Splivallo et al.,2007;Tirillini et al.,2000).Table1 shows the identi?ed VOCs and their relative percentages from fresh mycelia,broth,and dried mycelia.Fig.1shows the percent-age of each VOC compared to the total.The total ion current (TIC)chromatograms of the fresh mycelia and broth are shown in Fig.2.

Y.-Y.Li et al./Food Chemistry135(2012)2628–26372629

Table1

Identi?ed compounds and their relative percentage by area normalization method in Tuber melanosporum fermentation broth,fresh mycelia,and dried mycelia.(n=3).

No.RI Compound Mode of

identi?cation S.I.

(%)

Fermentation broth

(%)

Fresh mycelia(%)Dried mycelia

(%)

Medium

1

Medium

2

Medium

1

Medium

2

Alcohols

86833-Methylbut-3-en-1-ol MS,RI880.15____ 106983-Methyl-1-butanol MS,RI,std9439.18a58.02a13.5017.42_ 116982-Methyl-1-butanol MS,RI,std96 4.58 3.54 2.12 2.15_ 147553-Methylbut-2-en-1-ol MS,RI89_0.18___ 269002,4-Dimethylpentan-3-ol MS,RI770.39a0.07a___ 3710782-Ethylhexan-1-ol MS,RI880.39_0.46_ 1.32 4011152,3,6-Trimethyloct-7-en-3-ol MS,RI930.080.230.120.05_ 4311961-Isopropylcyclohexane-1,2-diol MS,RI70 3.31 2.6318.8011.24_ 4512112-Methylundecan-1-ol MS,RI850.980.59 2.84 2.34_ 4812273-Isopropyl-4-methyldec-1-en-4-ol MS,RI900.150.13 4.99 2.10_ 501240a-Santoline alcohol MS,RI76 5.97 6.7925.1332.91 1.47 Aldehydes

25473-Methylbutanal RI,std970.23_0.13__ 35892-Methylbutanal RI,std880.20_0.09__ 5647Hexanal MS,RI87____0.67 3910892-Isopropyl-5-methylhex-2-enal MS,RI640.030.040.270.100.16

Ketones

461212Hexahydro-2H-naphtho[1-b]oxiren-8(8a H)-one MS,RI880.400.20 1.51 1.52_ 5112475,5-Dimethyl-3-(pro-1-penyl)cyclohex-2-enone MS700.47a0.14a__0.29 5513714-(3-Methylbut-2-enyl)cyclopent-4-ene-1,3-dione MS,RI780.29a0.09a0.300.09 1.27 5716013-Methyl-2-(pent-2-enyl)cyclopent-2-enone(jasmone)MS730.730.37___ 5916053-Methyl-2-(penta-1,3-dien-2-yl)cyclopenten-2-enone MS770.710.75___ 6116993-Methyl-2-(penta-2,4-dienyl)cyclopent-2-enone MS790.080.07___ 6217083-Methyl-2-(penta-1,3-dienyl)cyclopent-2-enone MS750.260.15___ 6317184a-Methyl-4a,5,8,8a-tetrahydronaphthale-2(1H)-one MS860.230.11___

Esters and acid

6652Ethyl propionate MS,RI,std97 1.01a0.25a 1.55 1.69_ 7673Propyl acetate MS,RI930.140.050.180.45_ 13737Isobutyl acetate MS,RI900.16_0.07_0.16 18809Butyl acetate MS,RI94 1.25_ 1.63_ 5.05 24880Ethyl but-2-enoate MS,RI83_0.07_0.14_ 30933Isopentyl acetate MS,RI82 1.28a0.32a___ 742559Methyl palmitate MS89____ 1.32 752720Ethyl palmitate MS84____0.45 762909Methyl octadeca-9,12-dienoate(methyl linoleate)MS91____ 1.68 772910Methyl octadec-10-enoate(methyl oleate)MS85____ 1.19 782988Ethyl octadeca-9,12-dienoate(ethyl linoleate)MS84____0.59 792993Ethyl octadec-10-enoate(ethyl oleate)MS84____0.71 5613984a-Methyl-3,4,4a,5,6,7-hexahydro-2H-chromen-2-one MS800.960.33 4.93 4.40_ 329553-Methyl butanoic acid MS,RI,std84 1.88a0.77a___ 349802-Methyl butanoic acid MS,RI,std84 4.18a0.54a___ 671916Hexadec-9-enoic acid(oleic acid)MS,RI,std81__0.080.04_ 681923Hexadecanoic acid(palmitic acid)MS,RI,std90__ 2.78 3.13_ 691987Octadeca-9,12-dienoic acid(linoleic acid)MS,RI,std87__ 4.00a 1.32a_

Aromatic compounds

12717Toluene MS,RI,std850.420.410.220.900.80 25896Ethylbenzene MS,RI,std950.56a0.09a0.760.32 2.80 31936o-Xylene MS,RI,std910.58a0.07a0.560.28 2.20 27910p-Xylene MS,RI,std89 1.05_0.170.150.79 33976Paredrine MS,RI84_0.08___ 361021Benzaldehyde MS,RI,std850.070.07__ 6.49 381093Benzeneacetaldehyde MS,RI880.270.20__ 5.02 4111352-Phenylethanol MS,RI,std9423.0519.32 4.06a11.38a 3.45 421186Naphthalene MS,RI80____0.12 441197Phenethyl acetate MS,RI80_0.14__ 1.68 5413755-Methyl-2-phenylhex-2-enal MS,RI70____ 3.68 6016682,6-Di-tert-butyl-4-methylphenol(Butylated Hydroxy

toluene)

MS,RI,std94____45.41 661889Diisobutyl phthalate4MS,RI970.050.050.070.40_ 7326452-Methoxyphenazin-1-amine MS65____0.32 712542Butylhexyl phthalate4MS70____0.18 702120Bis(6-methylheptyl)phthalate4MS940.450.050.600.05 1.99 Sulfur

1471Dimethylsul?de RI,std80_ 1.26___ 17796Dimethyl trisul?de MS,RI72____0.77 6518842-(Methylthio)benzo thiazole MS,RI70____0.16 Nitrogen

2630Y.-Y.Li et al./Food Chemistry135(2012)2628–2637

Table1shows that VOCs from fresh fermentation mycelia are composed of a series of compounds including alcohols,aldehydes, ketones,esters,aromatic compounds,and ethers.Alcohols were the predominant VOCs and corresponded to about60%of the total VOCs(Fig.1C and D).The most abundant VOCs were a-santoline alcohol,3-methyl-1-butanol and1-isopropylcyclohexane-1,2-diol. Among the8alcohols identi?ed,3-methylbut-2-en-1-ol,2,3,6-trimethyloct-7-en-3-ol,1-isopropylcyclohexane-1,2-diol,2-meth-ylundecan-1-ol,and a-santoline alcohol were found in the truf?e genus for the?rst time.In this regard,linear chain or branched alcohols were probably biosynthesized by either a lipid oxidation process or Strecker degradation of amino acids(Bellesia,Pinetti, Tirillini,&Bianchi,2001).For the aromatic compounds,2-phenyl-ethanol,which was also detected in T.borchii mycelia(Splivallo et al.,2007)and the fruiting-body of most Tuber species(Díaz et al.,2003;Piloni,Tat,Tonizzo,&Battistutta,2005;Splivallo et al.,2007),was identi?ed to be the major constituent of this work.This rose perfume compound is generally considered to be the biotransformation product of L-phenylalanine by fungi(Lomas-colo,Stentelaire,Asther,&Lesage-Meessen,1999).A series of homologous esters were also identi?ed from fresh mycelia,includ-ing ethyl propionate,propyl acetate,and butyl acetate.It was rea-sonable to assume that these esters are created from a series of homologous alcohols that are oxidized to the corresponding acids that,in turn,react with the initial alcohols and to produce a series of homologous esters(March et al.,2006).Some long chain fatty acids with high boiling temperature,such as palmitic,oleic and lin-oleic acids,were detected in our samples,which is to be expected from the typical volatile oil extraction method used.These fatty acids were reported as precursors for producing VOC constituents, such as alkanes,ketones,alcohols and so on,and they play an important role in truf?e maturation(Zeppa et al.,2004).Addition-ally,several isoprenoid compounds,mainly farnesol and geranylac-etone derivatives,were present in the fermentation mycelia.It was proposed that a-farnesene,which was considered to be a sex pher-omone,was speci?c to the mature fruiting-body and that gerany-lacetone was related to the increased expression of isoprenoid biosynthetic genes in the mature fruiting-body(Splivallo et al., 2007;Zeppa et al.,2004).However,the appearance of the a-farne-sene and geranylacetone analogues in the vegetative mycelia may oppose this theory,and further investigation of the biological func-tion of these compounds in truf?e is required.

3.2.VOCs from T.melanosporum fermentation broth

By comparing the VOC pro?les of broth and mycelia cultured under the same medium(Fig.1A versus C,B versus D),it was found that both pro?les were composed of groups of alcohols,aro-matic compounds,esters,and ethers;alcohols were the predomi-nant group,comprising no less than50%of the total VOCs. Despite these similarities,there were conspicuous differences be-tween the broth and mycelia pro?les.In the VOC pro?les of the fer-mentation broth and mycelia(Fig.1A and C),the total percentage of alcohols in the broth was about12%lower than that in mycelia, and the total percentage of ethers in the broth was only one-sev-enth of that in mycelia.However,the amount of aromatic com-pounds in the broth was about four times higher than those found in mycelia.The same phenomenon was also observed in the broth and mycelia cultured under medium1(Table1).The rel-ative percentage of2-phenylethanol in the broth was six times greater than that in mycelia and was responsible for the higher percentage of aromatic compounds in the broth.Of the three major

Table1(continued)

No.RI Compound Mode of

identi?cation S.I.

(%)

Fermentation broth

(%)

Fresh mycelia(%)Dried mycelia

(%)

Medium

1

Medium

2

Medium

1

Medium

2

45932-Methylpyrazine MS82____0.06 157682,5-Dimethylprazine MS91_0.18___

167702,6-Dimethylprazine MS85_0.22___

208302-Ethyl-6-methylpyrazine MS,RI83_0.10__0.11 228402-Ethyl-5-methylpyrazine MS,RI81_0.10__0.06 218332,3,5-Trimethylpyrazine MS,RI81_0.20__0.16 299303-Ethyl-2,5-dimethylpyrazine MS80_0.17__0.18 289102-Ethyl-3,5-dimethylpyrazine MS85____ 1.03 359952-Ethyl-3,5,6-trimethypyrazine MS87____0.42 4712202-Isopentyl-3,5-dimethylpyrazine MS87____0.49

Isoprenoids

6417103,7,11-Trimethyldodeca-2,6,10-trien-1-ol(farnesol)MS,RI91__0.040.07_

4912282-Methyl-6,6-dimethylbicyclo[3.1.1]heptan-3-one

(isopinocamphone)

MS,RI74 1.33____

5313636,10-Dimethyl-5,9-undecadien-2-one(geranylacetone)MS,RI,std87____ 2.85 7225596,10,14-Trimethylpentadeca-5,9,13-trien-2-one MS88__0.030.08 1.50 Ethers

96861,1-Diethoxyethane,MS,RI840.28a0.06a0.050.09_

5815991-(3-Ethoxyprop-1-enyl)cyclohex-1-ene MS0.92a0.12a7.33 4.61_

Furanes and furanones

198102-Methyldilhydrofuran-3(2H)-one MS,RI85____0.21 23846Furan-2-carbaldehyde MS,RI920.720.26___

Indenes

5212481-Ethylidene-7a-methyloctahydro-1H-indene MS,RI790.230.09___

Three replicates were performed for VOCs identi?cation from fresh mycelia obtained in two different media,broth obtained in two different media,and dried mycelia, respectively.The initial sucrose concentration was35and80g Là1for media1and2,respectively.The relative percentage of each constituent was the average of three replicates(n=3)by area normalization method,and the standard deviation(R.S.D)of three replicates was around8%.The‘No.’of compounds from1to81was according to the Kováts indices(RI)from low to high,and each identi?ed compound was reported with their Kováts indices(RI),matching index(S.I)to the NIST‘05database(MS). Volatiles were identi?ed by one or more method mentioned under column‘Mode of identi?cation’as‘RI’(comparing their Kováts-indices with literature data),‘MS’(comparing their MS data to the NIST‘05database),and‘std’(using authentic standards under similar GC–MS conditions for comparison).Letter‘‘a’’indicated statistically signi?cant difference of relative amount in VOCs of broth or mycelia under two different medium(P60.05;paired t-test).4=the exotic contaminant..

Y.-Y.Li et al./Food Chemistry135(2012)2628–26372631

alcoholic compounds,the relative percentage of 3-methyl-1-buta-nol in the broth was higher,but the amount of 1-isopropylcyclo-hexane-1,2-diol and a -santoline was not more than one-?fth of the amount in mycelia.As described above,the total amount of alcohols in the broth was less than in the mycelia.Another differ-ence was that several groups of compounds (i.e.,sulfur,

pyrazine,

compounds (VOCs)pro?les from Tuber melanosporum fermentation.(A)broth obtained in medium 1,(B)broth obtained in medium (D)fresh mycelia obtained in medium 2,(E)the dried mycelia obtained in medium 1in oven at 60°C for 5days.Medium 1à1),yeast extract (5g l à1),MgSO 4á7H 2O (1g l à1),KH 2PO 4(0.5g),and Vitamin B 1(0.05g l à1).The composition of medium 2was concentration was at 80g l à1.The identi?ed compounds are grouped basis on their chemical structures (i.e.,ketones,esters,sulfur,each group are calculated as the total percentage of all compounds in that group (data shown in Table 1).‘‘Others’’,the value percentages of all compound groups from 100%,means the unidenti?ed compounds or compounds whose relative signal intensity calculated peak.

furan,and jasmone derivates)were only detected in the broth.The pyrazine series,whose welcome roasted ?avor is always present in fermented soybeans,cocoa,and cheese,(Maga,1992;Maga &Sizer,1973;Rizzi,1988)was identi?ed in truf?e genus for the ?rst time and was probably biosynthesized from amino acids (Belitz,Grosch,&Schieberle,2008).Of the volatile sulfur compounds,only

trace

(TIC)chromatogram of (A)fermentation broth obtained in medium 1,(B)fermentation broth obtained in medium 2,and (D)fermentation mycelia obtained in medium 2.Peak assignments are given in Table 1.

amounts of dimethyl sul?de and dimethyl trisul?de were detected in broth.These seem to be derived from methionine and are the major contributors to the?nal aroma of truf?e(Splivallo et al., 2007;Zeppa et al.,2004).Of the ketones,jasmone derivates were reported in the truf?e genus for the?rst time.These jasmone derivatives,with an enthralling jasmine fragrance,are best known as the constituents of the essential oil from plants,and the phyto-hormone plays a key role in inducible defence systems(Pickett et al.,2007).Due to low?avor threshold compounds like sulfur, pyrazine,furan and jasmone,the broth smells like an intense wine bouquet.In contrast,the fermentation mycelia smell much lighter, somewhat like the fresh mushroom.

3.3.VOCs variation during the oven drying process

To investigate the mycelia aroma variation during the drying process,the discrimination of VOCs between fresh and dried myce-lia was examined.In total,41VOCs were identi?ed in dried myce-lia,and16VOCs were produced during drying.Signi?cant differences were observed in Fig.1C and E.For example,the VOC pro?le of dried mycelia was dominated by aromatic com-pounds,whereas the predominant compounds in fresh mycelia VOCs were alcohols.As noted in Table1,only two alcoholic com-pounds(i.e.,2-ethylhexan-1-ol and a-santoline alcohol)were identi?ed in the dried mycelia,and the total relative percentage of alcohols decreased sharply from more than55%in fresh mycelia to no more than3%in dried mycelia.The disappearance of the alco-hols was mainly due to their volatilization and oxidation during oven drying.However,VOCs that have high boiling points and are stable with respect to temperature and oxygen were detected in dried mycelia,such as palmitate,oleate,linoleate,and gerany-lacetone and its derivatives.Although these compounds were spe-ci?c to dried mycelia,they are probably transformed from palmitic, oleic,and linoleic acids,as well as farnesol precursors,all of which were present in fresh mycelia.Of the predominant aromatic com-pounds,butylated hydroxyl toluene(BHT)had the highest percent-age of the total VOC amount(45%).This compound was previously reported in the fruiting-body of T.aestivum(Díaz et al.,2003),and its analogue was reported in T.borchii mycelia as well(Tirillini et al.,2000).However,contradictory results led to the assumption that BHT was a contaminant released from plastic vials or contam-inated analytical samples(Davoli,Bellesia,&Pinetti,2003).In our work,while the biosynthetic pathway is unclear,it was con?rmed that BHT was produced in the drying process because the com-pound was found in dried mycelia and undetected in the control. Additionally,its high percentage of total VOCs also suggests that it could not have been formed from degradation.The pyrazine and sul?de series,which are important?avor substances,were also identi?ed in dried mycelia.Differing from the formation in broth, pyrazine and sul?de compounds were probably the products of a Strecker and Maillard reaction between amino acids and carbohy-drates during the oven heating process,and the toasty aroma of dried mycelia is mainly attributable to these types of compounds (Belitz et al.,2008).Signi?cant differences were observed in the pro?les of VOCs from fresh and dried mycelia,and this result indi-cates that it is inadvisable to process aromatic mushrooms using oven drying because of the destruction of the original aroma.

https://www.360docs.net/doc/24589840.html,parison of VOCs identi?ed in T.melanosporum fermentation with those reported in the Tuber genus

As reported,VOC analysis of the truf?e fruiting-body was in?u-enced by species,geographical origin,intra-genetic variation, maturity,storage conditions,and analytical methods(Gioacchini et al.,2009;Splivallo et al.,2007).Due to the high inter-speci?c and intra-speci?c variation in the VOC pro?les of the natural truf?e,it was dif?cult to directly de?ne similarities or differences between the fruiting-body and fermentation by comparing the VOC spectra with inadequate sample quantities.However,through comparison of the VOCs between fermentation and the fruiting-body reported in the literature,it is possible to understand how many common VOCs exist in the fermentation and fruiting-body and their possible relationships to one another.Table2compares the VOCs identi?ed in the T.melanosporum fermentation with those found in fruiting bodies of T.melanosporum,T.borchii, T.magnatum,and T.indicum.A total of20VOCs produced by T.melanosporum fermentation were previously identi?ed in the Tuber genus.The analogues of farnesol and furan-2-carbaldehyde detected in this work were also found in the aforementioned fruiting-body.3-Methyl-1-butanol,2-methyl-1-butanol,3-methyl-butanal,2-methylbutanal,and2-phenylethanol were the most popular constituents for both the Tuber fermentation and fruiting-body VOCs.Additionally,among the aforedescribed fruit-ing-bodies,the VOC pro?le of our fermentation was more similar to that of T.melanosporum fruiting-body than the other species, because more common volatiles were found between them (Table2).However,the VOC pro?le of T.borchii mycelia was quite different:it was dominated by the aromatic compounds(Splivallo et al.,2007)or hydrocarbons(Tirillini et al.,2000),while the VOC pro?le of most truf?e fruiting bodies was dominated by alcohols. As shown in Table2,only eight common VOCs were found between T.borchii and the fruiting-bodies mentioned above.It was con-cluded that VOCs emitted from T.melanosporum fermentation were more similar to those emitted from the Tuber fruiting-body than VOCs emitted from T.borchii.

Although some common VOCs exist in both T.melanosporum fermentation and fruiting-body,they do not necessarily have the same aroma.Even though the truf?e aroma was produced by the ‘‘synergistic’’function of various VOCs,some distinctive com-pounds may have a decisive in?uence on the aroma.The discrim-ination in aroma between T.melanosporum fermentation and fruiting-body was primarily attributed to some key volatiles.Sulfur compounds were deemed responsible for the earthy aroma of truf-?e fruiting-body.However,only a trace amount of dimethyl sul?de and dimethyl trisul?de was detected in the fermentation broth, and none were found in the mycelia.Pyrazine compounds were speci?c to the fermentation broth,but were not detected in the fruiting-body.Due to its low?avor threshold,the presence of pyrazine would signi?cantly affect the fragrance of the broth, making it more like a wine bouquet than the truf?es’earthy aroma. 1-Octen-3-ol and3-octanone,C8alcohols and ketones,are responsible for the typical mushroom smell(Venkateshwarlu, Chandravadana,&Tewari,1999;Wnouk,Kinastowski,&Kaminski, 1983)and were previously identi?ed in various Tuber species but not found in our fermentation system.Therefore,through adjusting the fermentation strategy,if some?avor constituents, including sulfur compounds,1-occten-3-ol and their analogue, could be increased,meanwhile,pyrazine and its derivatives could be reduced or removed,the aroma produced by T.melanosporum fermentation would be much more similar to its fruiting bodies.

3.5.Effect of fermentation medium on the VOCs accumulation

From previous work,it was demonstrated that the VOCs from fresh mycelia were in?uenced signi?cantly by fermentation condi-tions(Tang et al.,2009).However,what types of VOCs were in?u-enced by the fermentation condition was unknown at the time.To study the in?uence of initial sucrose concentration on the VOC pro-?le,TIC chromatograms of both mycelia and broth in the fermen-tation system at an initial sucrose concentration of35(medium 1)and80g là1(medium2)were compared(Fig.2).For the broth VOCs,the discrimination of GC chromatograms was observed

2634Y.-Y.Li et al./Food Chemistry135(2012)2628–2637

between6to15min(Fig.2A and2B).Peak8(3-methylbut-3-en-1-ol),13(isobutyl acetate),and18(butyl acetate)only appeared in the broth in medium1,while peak14(3-methylbut-2-en-1-ol), 15(2,5-dimethylpyrazine),16(2,6-dimethylpyrazine),17(di-methyl trisul?de),20(2-ethyl-6-methylpyrazine),22(2-ethyl-5-methylpyrazine),and24(ethyl but-2-enoate)only appeared in the broth in the medium2.As shown in Fig.1A and B,the broth VOC pro?les in medium1and medium2were quite different. One difference is the relative percentage of alcohol compounds, which increased by nearly20%when the initial sucrose concentra-tion was increased from35to80g là1.Another difference is that the relative percentage of esters markedly decreased with increase in sucrose concentration.As shown in Table1,when the sucrose concentration increased from35to80g là1,the relative amounts of12VOCs signi?cantly changed.For example,the relative amount of3-methyl-1-butanol increased from39.18%to58.02%,while that of2-methyl butanoic acid,2,4-dimethylpentan-3-ol and2-methyl-butanal decreased from4.18%to0.54%,0.39%to0.07%,and1.01% to0.25%,respectively.These results indicate that3-methyl-1-buta-nol was the sole VOC in the fermentation broth,whose relative amount increased with the increase of sucrose concentration in the medium.For the mycelia VOCs,discrimination of the GC chro-matograms was also observed between6–19min(Fig.2C and2D). Peak13(isobutyl acetate),18(butyl acetate)and37(2-ethylhex-an-1-ol)were only observed in the mycelia cultured under med-ium1,while peak24(ethyl but-2-enoate)was speci?c to the mycelia cultured under medium2.As shown in Fig.1C and1D, no signi?cant difference was observed in the VOC pro?les of myce-lia cultured under medium1and2.The only change was observed in the aromatic compounds,whose relative percentage increased

Table2

Common VOCs in Tuber melanosporum fermentation and in the fruiting-body of T.melanosporum,T.borchii,T.magnatum,T.indicum,and T.aestivum.

Compound Truf?e fermentation system VOCs reported in fruiting-body(c)

Fresh mycelia of T .mel.(a)Broth of T

.mel.(a)

Mycelia of T.

borc.(b)

T.mel.T.

borc.

T.magn.T

.ind..

T.

aest.

Alcohols

3-Methyl-1-butanol p p p

71,3,6,77,1327,104,6,

9

2-Methyl-1-butanol p p p

71,3,4,6132,12,15–4,6,

9

2-Ethylhexan-1-ol p p

–47––4,9

1-Octen-3-ol––p

72,4,77,8,

13

2,1474,9

Aldehydes

3-Methylbutanal p p

–1,2,4,5,6,7132,15104,6,

9

Ethyl propionate p p

–1,2,3,4,5,

6,7

132,15104,6,

9

2-Isopropyl-5-methylhex-2-enal p p

–7––––

Esters and acid Ethyl propionate p p

–67––6

Ethyl but-2-enoate p p

––7–––

Isopentyl acetate–p

––7–––

Aromatic compounds Toluene p p

––––104

Ethylbenzene p p

–4–––4

o-Xylene p p

–4–––4,9

p-Xylene p p

–4–––4

Benzaldehyde–p p

74,77–7,104,9

Benzeneacetaldehyde–p

–7––74,9

2-Phenylethanol p p p

74,7712,1477

Phenylmethanol––p

777–7–

Sulfur

Dimethylsulfane–p

–1,2,3,4–2,12,14104,9

Methyl disul?de–p

–2,3,472,12,14,

15

–4

Dimethyl trisul?de––p

114,7–2,15–4,9

Furanes and furanones

Furan-2-carbaldehyde–p

–––12?,14?–4

Isoprenoids

2-Methyl-6,6-dimethylbicyclo[3.1.1]heptan-3-one (isopinocamphone)–

p

––8––

3,7,11-Trimethyldodeca-2,6,10-trien-1-ol(farnesol)p

–––8?–––

others

3-Octanone––p

7,112,4,772,1474,9

Compounds marked with‘p

’and‘

p

+numbers’in the Columns(a)and(b)were the common VOCs in the truf?e fermentation and the fruiting-body of T.melanosporum,T.

borchii,T.magnatum,T.indicum,and T.aestivum.

The marked compounds in Column(a)were the VOCs emitted from T.melanosporum culture mycelia or broth in either of two media.

The marked compounds in Column(b)were the VOCs emitted from T.borchii mycelia,and the number in Columns(b)and(c)indicated the corresponding literature which reported the compound.

Literature:(1)Talou et al.,1987;(2)Pelusio et al.,1995;(3)Bellesia et al.,1998b;(4)Díaz et al.,2003;(5)Mauriello,Marino,D’Auria,Cerone,&Rana,2004;(6)March et al., 2006;(7)Splivallo et al.,2007;(8)Zeppa et al.,2004;(9)Díaz,Ibá?ez,Reglero,&Se?oráns,2009;(10)Bellesia et al.,2002;(11)Tirillini et al.,2000;(12)Piloni et al.,2005;(13) Bellesia et al.,2001;(14)Aprea et al.,2007;(15)Bellesia et al.,1996.

Numbers marked with‘?’in Column(c)means the analogue of the corresponding compounds identi?ed in the literature.

Y.-Y.Li et al./Food Chemistry135(2012)2628–26372635

twofold when the initial sucrose concentration increased from35 to80g là1.As shown in Table1,only two compounds(2-phenyl-ethanol and linoleic acid)presented a signi?cant difference when the sucrose concentration was altered.The change in aromatic compounds(Fig.1C and D)was mainly due to an increase in 2-phenylethanol.All these variations in VOCs demonstrate that volatiles in T.melanosporum,such as pyrazine,sulfur,ester,and the predominant constituents,3-methyl-1-butanol and2-phenyl-ethanol,were signi?cantly in?uenced by the initial sucrose https://www.360docs.net/doc/24589840.html,pared to the VOCs of mycelia,the VOCs of broth were more strongly affected by the medium sucrose concentration. Furthermore,due to the different culture methods,the VOCs from T.borchii mycelia produced by two research groups were signi?-cantly different(Splivallo et al.,2007;Tirillini et al.,2000).Such a difference in T.borchii mycelia also con?rmed our theory that fermentation conditions were a key factor in affecting the VOC pro?le and accumulation.Therefore,it was possible to optimize the truf?e mycelia?avor and make its VOC chromatogram more like that of the fruiting-body through fermentation process control.

4.Conclusions

For the?rst time,a total of59VOCs were identi?ed from a Tu-ber melanosporum fermentation system.The VOCs in both mycelia and broth are composed of a series of compounds including alco-hols,aldehydes,ketones,esters,aromatic compounds,and ethers. Long chain fatty acids(i.e.,palmitic,oleic and linoleic acids)and isoprenoids(i.e.,farnesol and geranylacetone derivates)were found in T.melanosporum mycelia for the?rst time.The intense wine bouquet of the broth was mainly due to the presence of sev-eral speci?c?avor substances,such as sulfur compound,pyrazine, furan,and jasmone.Signi?cant variation in mycelia VOCs after oven drying demonstrated that drying procedure was not suitable to process aromatic mushroom by heating,and material pretreat-ment should be very careful to keep the preferable VOCs.By com-paring the VOCs identi?ed in this work to those in the literature, it was clear that our VOCs from T.melanosporum fermentation system were closer in composition to the Tuber fruiting-body than previously reported in Tuber fermentation mycelia.The su-crose concentration in the medium was found to have a signi?-cant in?uence on?avor volatiles,such as pyrazine,sulfur and esters,as well as3-methyl-1-butanol https://www.360docs.net/doc/24589840.html,-pared to the VOCs of mycelia,the VOCs from fermentation were more easily affected by the medium initial sucrose concentration. This discovery could be of special interest to optimize truf?e mycelia?avor to make it more similar to that of fruiting-body through bioprocess control and optimization.This work also pro-vides a good method for producing mushroom?avor substances through large scale fermentation.

Acknowledgements

Financial supports from the National Natural Science Foundation of China(NSFC,Project Nos.20976038and21176059),the Key Project of Chinese Ministry of Education(Project No.210132),Hubei Provincial Natural Science Foundation for Agriculture,Scienti?c Research Key Project of Hubei Provincial Department of Education (Project No.Z20101401),the Open Project Programs for the Key Laboratory of Fermentation Engineering(Ministry of Education), the National Key Laboratory of Biochemical Engineering(Project No.2010KF-06),and the State Key Laboratory of Bioreactor Engineering are gratefully acknowledged.Ya-Jie Tang also thanks the Chutian Scholar Program(Hubei Provincial Department of Education,China)(2006)and Program for New Century Excellent Talents in University(NCET-11-0961).Appendix A.Supplementary data

Supplementary data associated with this article can be found,in the online version,at https://www.360docs.net/doc/24589840.html,/10.1016/j.foodchem.2012.

07.013.

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