Characterization of volatile compounds from Daqu-a traditional

Original articleCharacterization of volatile compounds from Daqu-a traditional Chinese liquor fermentation starterChunlin Zhang,1Zonghua Ao,2Weiqiang Chui,1Caihong Shen,2Wenyi Tao 1*&Suyi Zhang 21School of Biotechnology,Jiangnan University,Wuxi 214122,China 2Luzhou Laojiao Co.Ltd,Luzhou 646000,China(Received 15December 2010;Accepted in revised form 5April 2011)SummaryDaqu is a fermentation starter and substrate complex that is used to initiate the solid fermentations for the production of Chinese liquor.The aroma of Daqu is one of the most important factors that influence the flavour of Chinese liquor.To further study the aroma of Daqu,volatile compounds in ten Daqu samples were extracted by headspace solid-phase microextraction (HS-SPME)and analysed by gas chromatography–mass spectrum (GC-MS).The samples were pre-equilibrated at 50°C for 10min and extracted with stirring at the same temperature for 30min prior to injection into a GC-MS.A total of 75volatile compounds were characterised by GC-MS,including twelve alcohols,eleven esters,eight acids,nine aromatic compounds,four phenols,two furans,fourteen nitrogen-containing compounds and fifteen aldehydes and ketones.By a principal component analysis (PCA),the ten Daqu samples could be classified into three groups according to their origins and,in particular,the production technologies.KeywordsChinese liquor,daqu,gas chromatography–mass spectrum,principal component analysis,volatile.IntroductionChinese liquor is a popular traditional alcoholic beverage with a long history in China.The invention and development of its manufacturing technique is considered as one of the progress in the technological history of ancient China (Shen,2002).Compared with other spirits,such as vodka,whisky and brandy,Chinese liquor is typically fermented from sorghum with Daqu and microorganisms.While producing Chi-nese liquor,the grain is ground and cooked,mixed with Daqu powder and then fermented for several months.After fermentation,the liquor is distilled out with distillatory.The distillate is aged for several years to develop the bouquet aroma (Zhang &Lai,2009).In the process of brewing Chinese liquor,Daqu is a source of microorganisms and crude enzymes,serves as a portion of materials and provides flavour substances for Chinese liquor (Zhang &Lai,2009;Ao et al.,2010).Daqu is a moulded cereal,which is prepared by natural inoculation of moulds,bacteria and yeasts (Ao et al.,2010).During Daqu-making process,the raw materials are typically milled,mixed with water,pressed into a brick-shaped block and then incubated under nonsterileconditions (Shen,2002;Zhang &Lai,2009).As a result of fermentation,Daqu is rich in a wide variety of microor-ganisms such as Bacillus ,Acetobacter ,Aspergillus ,Mu-cor ,Saccharomyces and Hansenula and various enzymes including amylases,glucoamylase and proteases (Wang et al.,2008).Many substances including amino acids and carbohydrates are also accumulated in Daqu during fermentation (Zhang &Lai,2009).Therefore,as a source of microorganisms and crude enzymes,as well as a portion of brewing materials,Daqu is important in the production of Chinese liquor.On the other hand,Daqu also plays a key role in forming the aroma of Chinese liquor.It has been reported that when commercial enzymes were used instead of Daqu for Chinese liquor fermentation,the aroma of the wine was significantly different from that of traditional Chinese liquor (Shen,2002).This indicates that the aroma of Chinese liquor is contributed by the aroma from Daqu during Chinese liquor brewing process;without it,the wine loses its characteristic aroma and flavour.Up to now,most of the studies have focused on enzymes and microorganisms of Daqu (Zhang et al.,2008;Zhang &Lai,2009),but the volatile compounds of Daqu have not yet been studied by gas chromatography–mass spectrum (GC-MS),and the aroma of Daqu is evaluated only by its simple sensory description with the term ‘normal or no odour of Daqu’.The aroma compounds of Daqu are the most important*Correspondent:Fax:+8651085918309;e-mail:wytao1946@International Journal ofFood Science and Technology 2011,46,1591–15991591doi:10.1111/j.1365-2621.2011.02660.xÓ2011The Authors.International Journal of Food Science and Technology Ó2011Institute of Food Science and Technologyfactors that influenced the aroma of Chinese liquor(Fan &Xu,2007),so it is important to characterise the volatile compounds of Daqu.According to theflavour characteristics of the liquor obtained,several types of Daqu can be distinguished, such as light-flavour,strong-flavour,and sauce-flavour Daqu.The primary reason for the differences is the different formulation of ingredients caused by the fermentation conditions,which leads to the formations of metabolites and reaction products(Wu et al.,2009). Therefore,the aroma profiles of various Daqu are quite different because of the differences in manufacturing technique.Many extraction techniques had been used in the analysis of volatiles,such as solid-phase extraction (SPE)(Wada&Shibamoto,1997),purge and trap (P&T)(Carmen et al.,1995)and simultaneous distilla-tion–extraction(SDE)(Bicalho et al.,2000).Solvent-assistedflavour evaporation(SAFE)is a good technique for volatile extraction,and it can allow careful isolation of volatile compounds from complex matrices(Engel et al.,1999).Solid-phase microextraction(SPME)is another good technique for the extraction of volatiles. The main advantages of this method are simplicity,high sensitivity and small sample volume,and it can also extract a wide range of aroma compounds(Fan&Qian, 2005).The SPME method has been employed for many diverse disciplines including the analysis of aroma compounds in red grape cultivars(Gonza lez Mas et al., 2009),volatiles from apple cider(Wang et al.,2004;Xu et al.,2007),white and red wine(Weingart et al.,2010), Chinese liquor(Fan&Qian,2006a,b)and papaya wine (Lee et al.,2010).The objective of this study was to investigate the flavour compounds in Daqu samples from two major types of Daqu.To achieve this goal,ten Daqu samples were collected and used to analyse the volatileflavour compounds by using headspace solid-phase microex-traction(HS-SPME)and GC-MS.Principal component analysis(PCA),a powerful statistical tool,was further employed to analyse and identify the characteristic flavour compounds of Daqu samples.Materials and methodsDaqu samplesTypical brick-shaped Daqu representing theflavour types was judged by experienced technical personnel. Ten Daqu samples were selected,which were produced by Luzhou Laojiao Company located in Luzhou of China,includingfive Daqu samples fermented by middle temperature procedure were labelled as ZY1, ZY2,ZY3,ZP1and ZP2andfive samples made by high temperature procedure were labelled as GY1,GY2, GY3,GP1and GP2.A whole block of Daqu sample was put into a sterile bag,and then,each Daqu sample was ground and stored at)20°C before analysis.All of these Daqu samples were made in2010.ChemicalsMethanol(special grade reagent)was purchased from Hanbon Sci.&Tech.Co.,Ltd(Jiangsu,China),and2-octanol from Sigma-Aldrich China Co.(Shanghai, China)was used as the internal standard.Others were also obtained from Sigma-Aldrich China Co. Headspace solid-phase microextraction methodThe50⁄30-l m divinylbenzene⁄carboxen on poly-dimethylsiloxane(DVB⁄CAR on PDMS)-coatedfibres (Supelco,Inc.,Bellefonte,PA,USA)were used for volatile compound extraction.Each Daqu sample (0.5g)was added in deionised water and placed in a 15-mL vial.The total5mL solution volume,in which 50l L of the internal standard2-octanol solution at 0.5mg L)1in absolute methanol was added,was saturated with sodium chloride.Then,the vial was tightly capped with a teflon⁄silicone septum,stirred and left to equilibrate for10min and then incubated for 30min at50°C.After extraction,thefibre was intro-duced into the injection port of the GC-MS system (250°C)for5min to desorb the analytes.All analyses were carried out in triplicate.GC-MS analysisThe analyses were carried out on a GC-MS(Trace MS⁄GC;Thermo Quest Finnigan Co.,Silicon Valley, CA,USA).The separations were performed using DB-WAX capillary column(30m·0.25mm i.d·0.25l m film thickness;J&W Scientific,Folsom,CA,USA).The oven and injector temperatures were250°C.Helium at a constantflow rate of0.8mL min)1was used as the column carrier gas.The oven temperature was held at 50°C for2min and then raised to230°C at a rate of 6°C min)1and held at230°C for15min.Mass was used for the identification of the unknown compounds. The electron impact(EI)energy was70eV.The ion source and transfer line temperatures were set at230°C and280°C,respectively.EI mass spectra ranged from 30to550amu.Identification and semi-quantification of unknowns Unknown compounds were identified by comparison with those in the Wiley6Library(Finnigan Co.,Silicon Valley,CA,USA)and NIST05library(Finnigan Co.) mass spectral database.RIs were calculated in accor-dance with a modified Kovats method(Cates&Meloan, 1963).A standard mixture of paraffin homologues C6-Characterization of volatile compounds C.Zhang et al.1592International Journal of Food Science and Technology2011Ó2011The AuthorsInternational Journal of Food Science and TechnologyÓ2011Institute of Food Science and TechnologyC26was prepared.The sample and the hydrocarbon standard mixture were co-injected into the GC,and the retention times were used to calculate RIs.Semi-quantification of the volatile compounds was performed using2-octanol as the internal standard.Data were collected in total ion mode for all mixed standards and samples.Preparation of standards and SPMEfibre calibration Each standard stock solution of the volatile compounds was prepared by dissolving compound in absolute methanol.Working solution was made from the stock solutions by mixing them with the solution which was saturated with sodium chloride.The concentrations of theflavour compounds in the working solution were as follows:2-methylpropanol(143.66l g L)1),2-pentanol (1278.57l g L)1),3-methylbutanol(920.95l g L)1),ethyl acetate(2832.15l g L)1),methyl butyrate(975.29l g L)1),hexanal(485.35l g L)1),2-pentanone(347.29l g L)1),acetic acid(544.87l g L)1),hexanoic acid(220.59 l g L)1),2-methylpyrazine(98.73l g L)1),2,3,5,6-tetramethylpyrazine(1182.33l g L)1),4-ethyl guaiacol (488.62l g L)1),benzaldehyde(523.10l g L)1),phen-ethyl alcohol(1328.79l g L)1)and furfuryl alcohol (507.75l g L)1).This solution was used as the highest concentration calibration standard,and more calibra-tion solutions were prepared by using serial dilution with the solution which was saturated with sodium chloride.Statistical analysisThe SPSS18.0software(SPSS Inc.,Chicago,IL,USA) was used for the statistical analysis of the volatile flavour compounds and the parameters.Statistical differences among the Daqu samples were determined using the one-way anova analysis.For all the statistical analyses performed,differences were considered signif-icant at P£0.05.Factor analysis was performed to reduce the dimensionality offlavour compound data and to identify underlying variables.Results and discussionHeadspace solid-phase microextraction parametersHS-SPME is based on the equilibrium of analytes among sample matrix,the headspace and thefibre coating,that is,once equilibrium has been reached,the concentration of the analytes can be considered constant in the three phases,and precision of the method will be improved(Zhang&Pawliszyn,1993).Fourfibres coated with85-l m polyacrylate(PA),100-l m PDMS,75-l m CAR⁄PDMS and50⁄30-l m DVB⁄CAR on PDMS were used to detect the volatile compounds in Daqu.The results showed that theDVB⁄CAR on PDMSfibre extracted more compoundswith higher intensities,while the PAfibre extracted the fewest compounds(Fig.1).So,the50⁄30-mmCAR⁄DVB on PDMSfibre was used.It is well knownthat the extraction was strongly influenced by temper-ature in the HS-SPME analysis.The extraction temper-atures(30,40,50and60°C)were evaluated in theHS-SPME parameter screening experiment.The results shown in Fig.2reveal that the extraction efficiency for volatile compounds was increased with the extraction temperature up to50°C and that when the extraction temperature was raised to60°C,the extraction effi-ciency has no distinct improvement.Extraction time wasalso an important factor for extraction efficiency.The Daqu samples were extracted for10,20,30,40andCharacterization of volatile compounds C.Zhang et al.1593Ó2011The Authors International Journal of Food Science and Technology2011 International Journal of Food Science and TechnologyÓ2011Institute of Food Science and Technology50min.As shown in Fig.3,the total peak area was clearly increased when the extraction time was 30min,while there were few changes when the extraction time was prolonged to 50min.Based on these observations,the volatiles were extracted by the 50⁄30-mm CAR ⁄DVB on PDMS fibre at 50°C for 30min.Volatile compounds in DaquThe results in Table 1show that seventy-five volatile compounds were identified by GC-MS from the ten Daqu samples.Chemical functionalities included alco-hols,acids,esters,aldehydes,ketones,aromatic com-pounds,phenols,furans and nitrogen-containingcompounds.Nitrogen-containing compounds that included four-teen compounds were the largest group in the Daqu samples.The relative concentration of nitrogen-contain-ing compounds ranged from 255.33to 1362.14l g kg )1.Among these samples,GP1Daqu had the highest concentration.Four chemicals that were 2,5-dimethyl-pyrazine,2,6-dimethylpyrazine,2,3,5-trimethylpyrazine and 2,3,5,6-tetramethylpyrazine were detected in all Daqu samples,while 2-methylpyrazine,2,3-dimethylpyr-azine,2-ethyl-6-methyl pyrazine,5-ethyl-2,3-dimethyl pyrazine,2-vinyl-6-methyl pyrazine and 2-acetylpyrrole were identified in most Daqu samples.These alkylpyr-azines often impart nutty,baked and roasted notes and can be brought into Chinese liquor to form its aroma (Fan et al.,2007).Daqu is not only the starter,but also the fermentation material because it typically accounts for 25%of the total amount of grains used in the fermentation.Because pyrazines are mostly formed through the Maillard reaction between saccharides and amino residues (Adams et al.,2005)and a high temper-ature would be benefit to the Maillard reaction and produce more pyrazines.Furthermore,alcohols were the second largest group identified in the Daqu,with concentrations between 854.74and 1519.90l g kg )1.Among which 1-hexanol and 2-ethyl hexanol contributed to floral and green aroma notes,3-methylbutanol imparted a nail polish aroma,1-heptanol imparted a green and fruity odour,1-pentanol and 2-pentanol contributed to fruity odours and 1-octanol and 3-octanol provided green and floral notes.In addition,1-octen-3-ol was detected in all Daqu samples and gave mushroom aroma.Higher alcohols could be formed during the fermentation,under aerobic conditions from sugar or anaerobic conditions from amino acids (Mauricio et al.,1997).Because the raw materials (wheat)are rich in amino acid content,higher alcohols can be converted from amino acids by yeast via the Ehrlich metabolic path-way (Wondra &Berovic,2001).Small amounts of higher alcohols could also be formed by yeast,through reduction in corresponding aldehydes (Fan &Qian,2006a,b).From the result of fermentation,as many as eleven esters and nine aromatic compounds were detected from the extracts.The ZY1and GY2Daqu samples had the highest concentration of esters and aromatic com-pounds,respectively.Ethyl acetate,methyl butyrate,hexadecanoic acid methyl ester,9-octadecenoic acid methyl ester and linoleate were identified in all Daqu samples.These compounds often contributed rosy,honey,floral,and fruity odours and have been identified in Chinese liquors previously (Fan &Qian,2006a,b)and could be also important contributors to the aroma of Daqu samples.Yeast and other microorganisms can synthesise esters during fermentation and ageing pro-cesses (Chen &Xu,2010).Among all the aromatic compounds,which produced by Saccharomyces cerevi-siae ,phenethyl alcohol had the highest concentration.Benzaldehyde,phenylacetaldehyde,benzyl alcohol and phenethyl alcohol were detected in all Daqu samples.In addition,methyl benzoate,methyl phenylacetate,phenyl ethyl acetate,methyl 3-phenylpropionate and naphtha-lene were also detected in the Daqu samples.Phenethyl alcohol contributes to rosy and honey aromas,while phenyl ethyl acetate and benzyl alcohol impact floral and fruity notes (Xu et al.,2007).These compounds could be important contributors to the aroma of Daqu samples.A few aldehydes and ketones were identified in this study,and the concentration of these compounds was not very high.Hexanal and 2-pentanone were identified in all Daqu samples.Aldehydes and ketones were probably formed by the yeast (Tao et al.,2008).Volatile acids are important to the flavour characteristics of Daqu samples.Hexanoic acid was probably the most important compound among the fatty acids and wasCharacterization of volatile compounds C.Zhang et al.1594International Journal of Food Science and Technology 2011Ó2011The AuthorsInternational Journal of Food Science and Technology Ó2011Institute of Food Science and TechnologyTable1Volatile compounds identified in Daqu samplesCode RI Basis ofidentification a CompoundsConcentration b(l g kg)1)ZY1ZY1ZY3ZP1ZP2GY1GY2GY3GP1GP2AlcoholsV11106MS,RI2-Methylpropanol44.7632.86n.d.c32.22n.d.n.d.n.d.n.d.n.d.33.02V21116MS,RI2-Pentanol120.95627.63483.37740.06224.4660.08445.37515.36308.32606.11V31152MS,RI1-Butanol12.3829.0526.1118.3315.43n.d.20.7711.9227.06n.d.V41229MS,RI3-Methylbutanol262.86159.53188.90226.68102.12426.25249.99330.76382.20355.17V51240MS,RI2-Hexanol8.0930.0132.2237.7822.34n.d.20.7637.30n.d.37.00V61255MS,RI1-Pentanol81.4366.6765.5655.0172.3446.2548.46n.d.n.d.n.d.V71345MS,RI1-Hexanol251.43146.66150.56125.01138.82195.33130.7662.30184.13139.71V81390MS,RIL3-Octanol n.d.n.d.n.d.n.d.n.d.31.2552.30528.82n.d.41.22V91439MS,RI1-Octen-3-ol24.2826.6631.1132.7832.4440.2853.0716.9241.4553.51V101457MS,RIL1-Heptanol16.6616.1921.1116.1816.48n.d.n.d.n.d.n.d.n.d.V111476MS,RI2-Ethyl hexanol22.3820.4724.4424.1515.9542.5527.699.9932.1344.41V121548MS,RI1-Octanol9.5212.8521.6617.7717.5527.5821.53 6.53n.d.n.d.P854.741168.581045.041325.97657.93869.571070.701519.90975.291310.15EstersV13892MS,RI Ethyl acetate556.20441.43385.03567.2674.462178.75267.6835.76877.4360.63V14948MS,RI Methyl propionate n.d.n.d.n.d.n.d.14.89n.d.n.d.n.d.n.d.n.d.V15982MS,RI Methyl butyrate125.7186.6663.3385.56475.51240.82236.91319.21196.49125.02V161118MS,RI Methyl pentanoate n.d.n.d.n.d.n.d.n.d.n.d.n.d.33.46n.d.n.d.V171204MS,RI Methyl hexanoate19.5218.57n.d.n.d.63.2947.5632.3014.2360.7153.80V181225MS,RI Ethyl hexanoate n.d.n.d.n.d.n.d.n.d.n.d.24.61n.d.n.d.n.d.V192093MS,RIL Pentadecanoic acid methyl ester n.d.n.d.n.d.n.d.17.55n.d.n.d.n.d.n.d.n.d.V202198MS,RIL Hexadecanoic acid methyl ester182.38161.43118.89137.78482.43143.75253.8366.92225.92391.42V212405MS,RI Octadecanoic acid,methyl ester25.71n.d.63.33n.d.n.d.n.d.n.d.n.d.n.d.44.99V222424MS,RI9-Octadecenoic acid methylester298.5764.28172.2350.04129.2547.5964.6118.4661.47527.18V232479MS,RI Linoleate268.10100.08160.5693.89214.35106.25111.5339.61148.39513.10P1476.19872.45963.37934.531471.732764.72991.47527.651570.411716.14 AldehydesV24952MS,RI3-Methyl butyraldehyde n.d.n.d.469.48281.68n.d.3217.50819.19219.991128.22941.61V25960MS,RI Pentanal n.d.n.d.40.70n.d.n.d.n.d.n.d.n.d.46.23n.d.V261069MS,RI Hexanal118.09164.76186.1286.11286.69197.53250.7523.07358.28165.01V271201MS,RI Heptanal n.d.n.d.18.33n.d.n.d.n.d.n.d.n.d.n.d.n.d.V281270MS,RI Octanal n.d.8.09n.d.8.3312.7658.7517.69n.d.n.d.n.d.V291369MS,RI Nonanal23.8117.6123.8921.1114.36136.2559.22n.d.n.d.60.51V301911MS,RIL2-Phenyl-2-butenal n.d.n.d.n.d.n.d.n.d.30.0020.76n.d.n.d.n.d.V312052MS,RIL5-Methyl-2-phenyl-2-hexenal n.d.n.d.n.d.n.d.n.d.81.2536.15n.d.n.d.n.d.P141.9190.46738.52397.23313.813721.281203.76243.061532.731167.13 KetonesV32968MS,RI2-Pentanone50.0079.5256.6729.44126.06101.2550.7653.45139.4746.92V33991MS,RIL2-Methyl-3-pentanone n.d.n.d.n.d.145.01n.d.n.d.n.d.n.d.n.d.n.d.V341185MS,RI2-Heptanone20.0917.1414.4415.0113.29n.d.n.d.n.d.n.d.n.d.V351193MS,RIL5-Methyl-2-hexanone n.d.n.d.n.d.n.d.n.d.n.d.22.30n.d.n.d.n.d.V361250MS,RIL5-Methyl-3-heptanone n.d.n.d.n.d.n.d.n.d.n.d.n.d.57.69105.0147.02V371317MS,RIL6-Methyl-5-hepten-2-one12.8515.2318.3322.7715.4226.2544.61n.d.58.2850.01V382071MS,RIL(Z)-Oxacycloheptadec-8-en-2-one44.2838.57n.d.44.4454.78n.d.44.6171.5358.06282.86P127.22150.4689.44256.67209.55127.50162.28182.67360.82426.81AcidsV391433MS,RI Acetic acid n.d.n.d.n.d.n.d.n.d.123.7579.22n.d.47.92113.63V401606MS,RI2-Methyl propioric acid n.d.n.d.n.d.n.d.12.7647.5833.079.9956.8195.92V411642MS,RI Butyric acid n.d.12.25n.d.n.d.n.d.n.d.38.468.07n.d.n.d.V421679MS,RI Pentanoic acid n.d.n.d.n.d.n.d.n.d.356.25413.82101.53483.72706.95V431665MS,RI3-Methyl-butanoic acid12.8527.1427.2220.2079.78n.d.n.d.n.d.n.d.n.d.V441824MS,RIL4-Methyl-pentanoic acid n.d.n.d.n.d.n.d.n.d.n.d.22.388.4321.0332.92V451844MS,RIL2,3-dimethyl-2-pentenoicacid n.d.n.d.n.d.19.4439.36n.d.45.3843.84n.d.n.d.V461858MS,RI Hexanoic acid10.478.09n.d.9.4425.53132.5841.5314.9947.8157.29Characterization of volatile compounds C.Zhang et al.1595Ó2011The Authors International Journal of Food Science and Technology2011 International Journal of Food Science and TechnologyÓ2011Institute of Food Science and Technologydetected in all Daqu samples.Most of the fatty acids in Daqu were produced by microbial fermentation.Four phenols were detected in the Daqu samples among which 4-ethylphenol was detected in all -pared with the nitrogen-containing compounds and aromatic compounds,the concentration of phenols was not very higher.These could be formed from lignin degradation of raw materials (Tuomela et al.,2000).Two kinds of furans including furfural and furfuryl alcohol were identified in the Daqu samples.Similar topyrazines,a high fermentation temperature also facili-tates furan formation through nonenzymic browning of sugars.Comparison of ten Daqu samplesIn this study,ten samples made by two different procedures were investigated.A principal component analysis was carried out to show relationships among different Daqu samples (Fig.4).PCA is a mathematicalTable 1ContinuedCode RI Basis of identification a CompoundsConcentration b (l g kg )1)ZY1ZY1ZY3ZP1ZP2GY1GY2GY3GP1GP2P23.3235.2327.2249.08157.43660.16673.86186.85657.291006.71Nitrogen-containing compounds V471265MS,RI 2-Methylpyrazine 9.0414.7613.8915.55n.d.38.7527.6917.6936.8139.73V481321MS,RI 2,5-Dimethylpyrazine 55.2347.6241.1139.4444.67117.5942.3025.38157.5184.99V491330MS,RI 2,6-Dimethylpyrazine 34.2861.4250.7066.1138.29121.25124.6130.76121.21155.77V501334MS,RIL 2-Ethyl Pyrazine n.d.8.09n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.V511342MS,RI 2,3-Dimethylpyrazine 26.6625.2315.5520.5519.68n.d.29.228.8435.8639.06V521375MS,RIL 2-Ethyl-6-methyl pyrazine 11.9030.4723.3336.6626.59107.5966.15n.d.112.4377.19V531388MS,RIL 4-Dimethylaminopyridine n.d.13.3310.5515.5526.59n.d.n.d.n.d.96.80n.d.V541402MS,RI 2,3,5-Trimethylpyrazine 177.62144.76103.34127.78130.84147.54140.7636.53205.38137.96V551415MS,RI 2,6-Diethylpyrazine n.d.n.d.n.d.14.44n.d.n.d.n.d.n.d.n.d.n.d.V561433MS,RIL 5-Ethyl-2,3-dimethyl pyrazine 48.0950.4757.7851.1157.9770.3977.68n.d.105.44121.97V571460MS,RI 2,3,5,6-Tetramethylpyrazine 427.62234.76273.35269.46251.58195.88286.9189.22311.52138.84V581489MS,RIL 2-Vinyl-6-methy pyrazine 61.4267.6263.3373.3367.01100.06n.d.27.30115.84112.64V591514MS,RIL 2,5,6-Trimethyl-4-ethyl pyridine 10.4712.3810.5513.8914.89n.d.20.76n.d.n.d.n.d.V601950MS,RI 2-Acetylpyrrole 7.1413.33n.d.17.7716.4873.7573.8419.6163.3465.24P869.47724.24663.48761.64694.59972.80889.92255.331362.14973.39Phenols V611862MS,RI Guaiacol n.d.n.d.n.d.n.d.n.d.40.7515.38n.d.n.d.26.42V621989MS,RI Phenol23.8123.81n.d.23.8951.0643.75303.8333.8466.68104.78V632010MS,RI 4-Ethyl guaiacol43.8123.8124.4429.9426.5980.8268.4516.5380.91103.42V642217MS,RIL2,4-Ditertbutyl phenol22.3914.2813.8311.11n.d.n.d.n.d.n.d.n.d.n.d.P90.0161.9038.2764.9477.65165.32387.6650.37147.59234.62Aromatic compounds V651506MS,RI Benzaldehyde 44.7654.7662.7870.5653.72320.25206.9134.22156.88253.81V661611MS,RIL Methyl benzoate n.d.n.d.n.d.n.d.n.d.36.25n.d.n.d.23.2855.42V671626MS,RI Phenylacetaldehyde 17.6130.9546.1147.2226.06243.75159.2235.76139.95156.33V681701MS,RI Naphthalene16.1973.3376.6762.7844.14108.7147.6919.23n.d.39.31V691722MS,RI Methyl phenylacetate n.d.n.d.n.d.n.d.n.d.n.d.54.6111.9227.8234.19V701801MS,RIL Phenyl ethyl acetate8.09n.d.n.d.11.11n.d.50.1729.22n.d.n.d.n.d.V711830MS,RIL Methyl 3-phenylpropionate n.d.n.d.n.d.n.d.n.d.n.d.46.15 6.5323.47n.d.V721858MS,RI Benzyl alcohol 37.1420.4828.8922.7821.2861.2583.0713.0878.2749.42V731902MS,RI Phenethyl alcohol260.48498.10618.93638.38481.90398.75608.44108.46648.26441.07P384.27677.62833.38852.83627.101219.131235.31229.201097.931029.55FuransV741493MS,RI Furfuraln.d.n.d.n.d.n.d.n.d.126.2540.7711.1537.0369.34V751698MS,RI Furfuryl alcohol20.0018.0931.6725.5627.66262.53159.9926.54169.21144.10P20.1218.0931.6725.5627.66388.78200.7637.69206.24213.44aMS,compounds were identified by MS spectra.RI,compounds were identified by comparison with pure standard.RIL,compounds were identified by comparison with RI from the literatures Xu et al.(2007);Fan &Qian (2006a,b);Tao et al.(2008);Kumazawa &Masuda (2002);Yu et al.(2004).bAll the results are the average of three parallel experiments,and the RSD values in the quantification of flavour compounds did not exceed ±10%.cn.d.,not detected.Characterization of volatile compounds C.Zhang et al.1596International Journal of Food Science and Technology 2011Ó2011The AuthorsInternational Journal of Food Science and Technology Ó2011Institute of Food Science and Technologyprocedure for resolving sets of data into orthogonal components,whose linear combinations (principal components)approximate the original data to any desired degree of accuracy (Delgado et al.,2010).In most cases,two principal components are sufficient to explain a great proportion of the variation in the original variables,thus resulting in a considerable compression of the data.In this case,35%and 14%of the variability was explained by principal compo-nents PC 1and PC 2,respectively.Positive loading of PC1was related to 2-methyl propioric acid,penta-noic acid,2,6-dimethylpyrazine,4-ethyl guaiacol and 2-methylpyrazine,whereas 1-pentanol,1-heptanol,2-heptanone and 2,4-ditertbutyl phenol showedtheFigure 4Principal component analysis of volatile flavour compounds in Daqusamples.Figure 5Principal component analysis score plot according to volatile compounds in Daqu samples.Characterization of volatile compounds C.Zhang et al.1597Ó2011The AuthorsInternational Journal of Food Science and Technology 2011International Journal of Food Science and Technology Ó2011Institute of Food Science and Technologynegative loadings.For PC2,loadings were character-ised by octanal,5-methyl-2-phenyl-2-hexenal,naphtha-lene and phenyl ethyl acetate with positive values, whereas2,3-dimethylpyrazine,2-pentanol,2-hexanol and2-methylpropanol showed negative values.The PCA showed a good separation of the different Daqu samples and three groups were clearly defined(Fig.5). Daqu samples fermented by middle temperature pro-cedure formed a clear group,which was associated with 2,3,5,6-tetramethylpyrazine,2,5,6-trimethyl-4-ethyl pyridine and3-methyl-butanoic acid.Daqu samples fermented by high temperature procedure formed two different groups,one group including GY1,GY2 and GY3was associated with octanal,2-phenyl-2-butenal,5-methyl-2-phenyl-2-hexenal and phenyl ethyl acetate,another group including GP1and GP2was associated with6-methyl-5-hepten-2-one,(z)-oxacyclo-heptadec-8-en-2-one,5-ethyl-2,3-dimethyl pyrazine and 4-methyl-pentanoic acid.ConclusionsIn conclusion,HS-SPME using50⁄30-l m DVB⁄CAR⁄P DMSfibre combined with GC-MS was a very simple and fast technique for analysing volatile compounds in Daqu samples.The original Daqu samples were extracted by HS-SPME.The samples were pre-equili-brated at50°C for10min and extracted with stirring at the same temperature for30min prior to injection into GC-MS.A total of seventy-five volatile compounds were identified from ten typical Daqu samples,including twelve alcohols,eleven esters,eight acids,fifteen alde-hydes and ketones,nine aromatic compounds,four phenols,two furans and fourteen nitrogen-containing compounds.In particular,the elaboration of data has shown a good discrimination among Daqu samples made by two different procedures.By means of the PCA analysis,Daqu samples can be classified into three groups according to the production technologies.This research indicates that Daqu samples made by different production techniques have obvious differences;further research including sensory evaluation and GC-olfac-tometry should be carried out.Daqu is not only a source of inoculum but also plays an important role during the fermentation as a substrate.At present,knowledge of the microbiota of Daqu is still far from complete and is the subject of current research.To this day,the manufacturing process of Daqu still relies on workers experience. The Daqu quality cannot be kept stable even for the same batch of products.Daqu making involves several complex fermentation stages.We believe that the microbiota would change according to the factory locations and therefore influenceflavour profile.How-ever,we tried to extract the common information for eachflavour type of Daqu in this paper.Further study of microbial ecology during the production of Daqu and the ensuing alcoholic fermentation will be neces-sary.The analysis of metabolites based on GC-MS could be useful in the control offlavour development during Daqu production.AcknowledgmentsFinancial support from the Science and Technology support program,Sichuan province,P.R.China,under No.2010SZ0032and open project from Liquor Making Bio-technology Application of Key Laboratory of Sichuan province,P.R.China,under No.NJ2009-01 are gratefully acknowledged.ReferencesAdams,A.,Borrelli,R.C.&Fogliano,V.(2005).Thermal degradation studies of food melanoidins.Journal of Agricultural and Food Chemistry,53,4136–4142.Ao,Z.H.,Shan,X.H.,Shen,C.H.&Xu,D.F.(2010).Domestic related quality standards of Daqu and its research progress.Liquor-making Science and Technology,188,104–108.Bicalho,B.,Pereira,A.S.,Neto,F.,Pinto,A.C.&Rezende,C.M.(2000). 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