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Volatile Flavour Compounds Analysis of Solid State Fermented Thai Rice Wine (Ou)

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Volatile Flavour Compounds Analysis of Solid State Fermented Thai Rice Wine (Ou) R ESEARCH ARTICLE ScienceAsia 34 (2008): 199–206 doi: 10.2306/scienceasia1513-1874.2008.34.199 Volatile flavour compounds analysis of solid state fermented Thai rice wine (Ou) Pumnat Chuenchomrat, Apinya Assavanig, and Sittiwat Lertsiri* Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. * Corresponding author, E-mail: [email protected] Received 11 Jul 2007 Accepted 2 Jan 2008 ABSTRACT: Solid-state fermented Thai rice wines (Ou) were analysed to determine their chemical components. Determined parameters were pH (4.5–5.5), protein (0.45–0.99 g/100g), ash (0.10–0.30 g/100g), total solid (1.72–14.34 g/100g), glucose (4.07–7.91 mg/ml) contents and volatile compounds. The ethanol concentration was in the range of 12.15 to 104.60 mg/ml. Profiles of volatile compounds were analysed by dynamic headspace coupled with gas chromatography mass spectrometry and gas chromatography olfactometry. The potent odours were alcoholic and solvent-like, sweet, fruity, buttery, and pungent aromas. The concentration in Ou of n-propanol, iso-butyl alcohol, iso-amyl alcohol, furfuryl alcohol, benzene ethanol, acetol, 2,3-butanediol, glycerol, ethyl lactate, acetoin, furfural, 5-methyl furfural, 5-hydroxymethyl-2-furfural, and acetic acid were also determined by a direct injection technique. From principal component analysis, Ou samples could be categorized into two groups based on the concentration of ethanol and their profiles of volatile compounds. KEYWORDS: Thai rice wine, Ou, flavour profile, dynamic headspace, gas chromatography-olfactometry, principal component analysis INTRODUCTION rice and washed rice husk are mixed together in a ratio of 1:1 and cooked by steaming. The rice husk Rice wines are widely produced in Asian is added to maintain the moisture during solid–state countries such as Japan (Sake), China (Jiu), Korea fermentation. The mixture is cooled, mixed with (Yakju), Philippines (Tapuy),Vietnam (Ruou nep than), Loog-paeng powder (a dry starter culture) and incubated Malaysia (Tapai), and Thailand (Sato, Krachae and at room temperature for 24 hours. This step is similar Ou)1. The varieties of rice wines depend on raw to Koji production in Japanese Sake fermentation materials, inocula and brewing processes. Generally, except for the strains of microorganisms used4. The rice wine fermentation can be categorised into dry starter culture consists of fungi (Aspergillus sp. submerged and solid state process. In the case and Rhizopus sp.), yeast (S. cerevisiae) and herbs5. of submerged process, barley and rice are used as The mixture is then transferred into an earthenware substrates for the fermentation2. The inocula for rice wine jar, covered and tightly sealed with either banana brewing are fungi, such as Aspergillus oryzae. A. sojae, leaves or a plastic sheet. The jar is left at room and Rhizopus spp., and yeasts, such as Saccharomyces temperature to ferment for more than a week. To drink cerevisiae, S. sake, S. fibuligera, Hansenula mrakii Ou, water is poured into the opened jar to elute the and Pichia polymorpha1. Traditionally, microbial fermented content. A narrow bamboo stem is then starters are used for saccharification. However, in inserted into the rice mixture to allow the sucking Japanese rice wine (Sake) sprouting rice is also of Ou from the jar. The elution can be made several used for this purpose3. Following saccharification of times in this manner. rice, the sugar liberated, especially glucose, is then In Thailand, all rice wines are produced by converted to ethanol by submerged fermentation of various traditional methods. This was practiced yeasts. illegally until the government relaxed its liquor Ou is a Thai rice wine produced from solid state production laws. This has resulted in an incentive for fermentation. This kind of rice wine is also produced legal mass production. This work is a report on chemical in other Southeast Asian countries such as Cambodia, analyses based on proximate analysis and volatile Laos and Vietnam1. In Ou fermentation, solid state flavour compounds of Ou. These data are expected to fermentation is employed for both saccharification be valuable for standard setting of this product and and ethanol production. To make Ou, glutinous rice other related products. is first soaked in water overnight. Then, the glutinous www.scienceasia.org 200 ScienceAsia 34 (2008) MATERIALS AND METHODS with a flow rate of 1 ml/min at 40 °C. A refractive index detector was used8. Samples Ou samples were obtained from three regions Determination of ethanol by static headspace in Thailand where the wines are traditionally pro- technique duced, namely, the northern, north-eastern, and cen- Determination of ethanol concentration was tral regions (Table 1). Three earthenware jars were conducted according to the method of Otero et al9 purchased from each manufacturer. One litre of deo- with modification. One ml of Ou saturated with sodi- dourised water6 was poured into the earthenware jar. um chloride was put into a 20-ml headspace vial with Ou was then extracted by a peristaltic pump equipped 50 µl of 1,4-dioxane added as an internal standard. with a silicone tube and kept at -20 °C until further The vial was sealed with PTFE-coated rubber septum, analysis. and placed in a headspace autosampler (HP 7694E, Agilent, USA). Headspace gas was analysed by GC- Chemicals flame ionization (HP 6890A, Agilent, CA, USA) All standard volatile compounds with purity equipped with an HP-5 capillary column (5% phenyl- grade higher than 98% were purchased from Aldrich methylpolysiloxane; 30 m × 0.32 mm i.d. × 25 µm Chemical (St.Louis, MO) and Fluka (Buchs, Switzer- film thickness; Hewlett-Packard). Sample injection land). Other chemicals were HPLC grade or of the was performed with a split ratio of 10:1. The initial best grade available from Merck (Darmstadt, Germa- oven temperature was 35 °C holding for 2 min, then ny). The helium gas was ultra high purity. programmed to 200 °C at a rate of 20 °C/min. Helium gas was used as mobile phase (1 ml/min). The flame Analysis for protein, total solid, and ash contents ionization temperature was 250 °C. Protein content of Ou samples was determined by Kjeldahl’s method following AOAC Official7 No. Dynamic headspace analysis 981.10 with a conversion factor of 6.25. Total solid Dynamic headspace analysis (DHA) was con- and ash content was determined according to AOAC ducted according to Wanakhachornkrai and Lertsiri6 Official7 No. 925.10 and 920.67, respectively. These with modification. One ml of Ou saturated with so- analyses were performed with triplicate measure- dium chloride was placed in a dynamic headspace ments. tube (15.2 ×1.6 cm i.d.) and then installed in a Tekmar Dohrmann 3100 purge and trap concentrator (Tek- Glucose content mar, OH,USA). Helium gas at flow rate of 40 ml/min One ml of Ou was centrifuged at 5,000 g for over the headspace for 30 min was used to purge a 10 min. (Sigma 202 centrifuge, Harz, Germany) Tenax TA trap (part no. 12-0083-303, Tekmar) and to precipitate insoluble particles. To deproteinize, the trap was dry-purged for 2 min to remove mois- 0.5 ml of supernatant was added to 0.5 ml of cold ture. Volatiles trapped were desorbed at 220 °C for methanol, which was then filtered through a cellulose 2 min and then directly introduced onto gas chroma- acetate membrane (0.45 µm). A high performance tography mass spectrometer (HP 6890A, Agilent, CA, liquid chromatograph (Water 2690 Separation USA) with split ratio of 10:1. The transfer line was module, MA, USA) equipped with an Hypersil maintained at 220 °C with a trap pressure of 4 psi. APS2 column (250 × 4.6 mm, 5 µm; Hypersil, UK) Volatile compounds were separated on an HP-FFAP was used to analyse the glucose of Ou samples. An capillary column (polyethylene glycol modified Protein Ash Total solid Glucose Ethanol pH isocratic elution was performed by using a mixture of nitroterephthalic acid, 25 m × 0.32 mm i.d. × 0.50 (g/100 g) (g/100 g) (g/100 g) (mg/ ) (mg/ ) 5.51 0.73 0.10 14.34 4.79 12.15 acetonitrile and water (80:20; v/v) as mobile phase µm film thickness, Hewlett-Packard). The initial A (0.01) (0.00) (0.00) (2.08) (0.12) (0.15) 4.50 0.71 0.26 6.76 4.07 99.57 B oven temperature was 50 °C holding for 1 min, then (0.00) (0.05) (0.11) (0.05) (0.32) (0.44) 4.53 0.45 0.21 2.71 4.71 37.01 programmed to 100 °C at a rate of 20 °C/min. The C (0.06) (0.00) (0.00) (0.31) (0.20) (0.54) Table 1. The origin of Ou samples 4.63 0.66 0.29 3.69 4.55 32.06 D carrier gas was helium gas at a constant flow rate of (0.06) (0.01) (0.00) (0.16) (0.09) (0.12) Regions of Thailand City, Province Sample 4.52 0.99 0.25 6.43 4.94 92.66 E Muang, Ratchaburi A 1.5 ml/min. The ionization energy of the mass spec- (0.03) (0.00) (0.00) (0.34) (0.25) (0.64) Middle 4.55 0.79 0.20 1.72 4.64 104.60 Muang, Lop Buri B F trometer detector (HP 5973 Mass Selective Detector, (0.05) (0.01) (0.00) (0.23) (0.05) (0.92) Weang Nong Long, Lamphun C 4.49 0.94 0.21 8.27 7.91 66.60 Northern G Muang, Nan D Agilent, CA, USA) was 70 eV, and the mass range (0.02) (0.03) (0.00) (2.62) (0.51) (0.18) 4.50 0.67 0.29 6.22 4.21 57.58 Ranu Nakhon, Nakhon Phanom* E, F, G was 20–350 a.m.u.
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