Food Sci. Biotechnol. 21(2): 453-460 (2012) DOI 10.1007/s10068-012-0057-6
RESEARCH ARTICLE
Effect of Nuruk and Fermentation Method on Organic Acid and Volatile Compounds in Brown Rice Vinegar Su-Won Lee, Sung-Ran Yoon, Gui-Ran Kim, Seung-Mi Woo, Yong-Jin Jeong, Soo-Hwan Yeo, Kyong-Su Kim, and Joong-Ho Kwon
Received: 30 September 2011 / Revised: 3 December 2011 / Accepted: 6 December 2011 / Published Online: 30 April 2012 © KoSFoST and Springer 2012
Abstract The effects of different nuruk contents and fermentation methods (AV, vinegar fermented in an agitated culture; SV, vinegar fermented in a static culture) on organic acids and volatile compounds in brown rice vinegars were investigated. In the SV, the contents of acetic, oxalic, tartaric, and malic acids increased with hipher contents of nuruk. Acetic, tartaric, and malic acid contents were higher in the SV than those in the AV. Volatile compounds that can affect vinegar quality, including acetic acid, isoamyl acetate, phenethyl acetate, and phenethyl alcohol were present at high concentrations in the AV. With the increase in nuruk contents in the AV, acetic acid content decreased and isoamyl acetate and phenethyl acetate content increased. No significant differences in sensory scores were observed regarding the amount of nuruk and the type of fermentation. However, electronic-nose analysis showed its potential to effectively differentiate different samples.
Su-Won Lee, Gui-Ran Kim, Joong-Ho Kwon ( ) Department of Food Science and Technology, Kyungpook National University, Daegu 702-701, Korea Tel: +82-53-950-5775; Fax: +82-53-950-6772 E-mail:
[email protected] Sung-Ran Yoon BioHealth Convergence Center, Daegu Technopark, Daegu 704-801, Korea Seung-Mi Woo, Yong-Jin Jeong, Department of Food Science and Technology, Keimyung University, Daegu 704-701, Korea Soo-Hwan Yeo Fermentation and Food Processing Division, Department of Agrofood Resources, NAAS, RDA, Suwon, Gyeonggi 411-853, Korea Kyong-Su Kim Department of Food and Nutrition, Chosun University, Gwangju 501-759, Korea
Keywords: brown rice vinegar, nuruk, volatiles, organic acid, electronic-nose
Introduction Different varieties of vinegars are being extensively used around the world due to their unique taste and health associated benefits (1). In northeastern regions of Asia, such as Korea, Japan, and China, vinegar is traditionally produced from various cereals, primarily rice. Rice vinegar is well known in Chinese oriental medicine (2,3). Recent studies have shown that rice vinegar not only has flavor components but also contains various physiologically active ingredients (4,5). In particular, unpolished rice vinegar is used as a health food in Japan, and its antioxidant activity is well reported (6,7). One of the Japanese traditional rice vinegars, kurosu, is unpolished black rice vinegar produced by a traditional static fermentation process. As kurosu is produced from unpolished rice, it is characterized by higher levels of amino acids and organic acids than those of other vinegars (8). Vinegar fermentation from cereals requires a saccharification step in addition to alcoholic fermentation and oxidation of ethanol to acetic acid, in which the latter 2 steps are common during vinegar fermentation from materials rich in sugar, e.g., fruit juices (9). Vinegar is derived from the bacterial conversion of ethanol to acetic acid. Therefore, it can be produced from any alcoholic material ranging from alcohol-water mixtures to different wines (10). In Korea, vinegar is primarily produced from rice wine residues and various fruits. Korean traditional rice wines and liquors have a long brewing history using nuruk (Korean traditional fermented agents) or koji (11). Nuruk is used to make Korean traditional alcohol, such as makgeolli (12), which
454
combines various tastes and flavors resulting from the activities of amylase, protease, and a variety of fermentation organisms (13). It is difficult to determine the exact composition of nuruk, because each community differs in its production method (14-16). The color and aroma of vinegar depends greatly on the initial substrate (10). The next important step is the acetic acid fermentation of the alcoholic extract. Traditional static surface culture fermentation (slow) or a modern submerged (agitated) fermentation (fast) process could be employed (10). Acetic acid is the main aroma compound in vinegar. However, other volatile compounds also have a significant impact on the overall aroma of vinegar. Vinegar flavor is influenced by the raw materials, the compounds formed during fermentation, chemical compounds inherent in the raw materials, and the type of fermentation process (17). Aroma, one of the most important determinants of food quality and acceptance, is the result of high quantities of volatile compounds. These compounds may come from the raw material or may be formed by the fermentation method. Various scientists have revealed the importance of the fermentation process for the organoleptic qualities of vinegars, particularly, their final aroma (18,19). The objective of this study was to determine the organic acid and volatile compounds in brown rice vinegar as influenced by different amounts of nuruk and using different fermentation conditions. The effect of the analyzed compounds on sensory characteristics of vinegars was also investigated.
Materials and Methods Materials and strains Japonica brown rice, cultivated in Sangju area of Gyeongbuk in 2009, was used in this study. Nuruk was purchased from Sangju Gokja, Co., Ltd., Sangju, Korea. The yeast used for the brown rice alcohol fermentation was Saccharomyces cerevisiae GRJ, which was stored in the Fermentation Technology Lab. of Keimyung University and subcultured for 24 h at 30oC on YPD (BD, Franklin Lakes, NJ, USA) agar medium (1% yeast extract, 2% peptone, 2% glucose, and 2% agar, pH 6.0). Acetobacter pomorum KJY 8 (KCTC 10173BP) was subcultured for 48 h at 30oC on solid medium (3% glucose, yeast 0.5%, extract 1% CaCO3, 3% ethanol, and 2% agar, pH 7.0) and stored in a refrigerator at 4oC. Seed mash and vinegar starter The seed mash was 500 g nuruk, which was added to 1,500 mL of purified water and saccharified for 6 h at 55oC then filtered at 10oBx. The saccharified liquid was sterilized for 15 min at 121oC and was inoculated with S. cerevisiae GRJ. A stationary culture was conducted for 24 h at 30oC with
Lee et al.
constant-temperature culture medium (HB-103-2H; Hanbaek Scientific, Bucheon, Korea), and 5%(v/w) of the material amount was used. Brown rice alcohol fermented liquid was used as the starter for acetic acid fermentation, and the alcoholic content was diluted to 6%. This liquid was inoculated with A. pomorum KJY 8 and was cultured for 10 days after mixing at 250 rpm and 30oC. Alcoholic fermentation Brown rice (1 kg) was soaked in water for 5 h and was steamed after removing the water. Nuruk (10, 20, 30, or 40%) was mixed with the steamed brown rice, which was also mixed with 50 mL of seed mast and 1,600 mL of distilled water. A stationary culture was conducted for 3 days at 30oC in constant-temperature culture medium (HB-103-2H; Hanbaek Scientific). Agitated and static acetic acid fermentation After adjusting the filtered liquid of the alcohol-fermented brown rice by adding nuruk to the mixture with an alcohol content of 6%(v/v), 10%(v/v) vinegar starter was inoculated and both agitated fermentation (AV) and stationary fermentation (SV) were conducted. The AV was conducted in a shaking incubator (HB-201SL; Hanbaek Scientific) for 6 days at 250 rpm and 30oC, and the stationary culture was performed in a constant-temperature incubator (HB-1032H; Hanbaek Scientific) for 16 days at 30oC. The supernatants of each of the AV brown rice vinegars, including AV10 (brown rice vinegar produced with 10% nuruk content), AV20 (brown rice vinegar produced with 20% nuruk content), AV30 (brown rice vinegar produced with 30% nuruk content), AV40 (brown rice vinegar produced with 40% nuruk content), stationary-culture brown rice vinegar, SV10 (brown rice vinegar produced with 10% nuruk content), SV20 (brown rice vinegar produced with 20% nuruk content), SV30 (brown rice vinegar produced with 30% nuruk content), and SV40 (brown rice vinegar produced with 40% nuruk content) were used for analysis after centrifugation and filtration. Organic acid analysis Vinegar samples (1 mL) and water (9 mL) were added to a 10-mL flask and mixed using vortex mixer (VXR B; Janko & Kunkel, Morgan Hill, CA, USA) for 2 min. The mixture was filtered through a 0.45µm filter. Organic acid content was determined by HPLC (Waters 1515; Waters Co., Milford, MA, USA), using an AtlantisTM dC18 column (3.9×150 mm, Waters Co.), coupled with a UV detector set at 210 nm. The mobile phase was 20 mM NaH2PO4 (pH 2.7) with a flow rate of 1.0 mL/min and an injection volume of 20 µL. A standard solution of oxalic, tartaric, malic, acetic, lactic, citric, and succinic acids (Sigma-Aldrich, St. Louis, MO, USA) of known concentration were used for the identification purpose.
455
Organic Acid and Volatile Compounds of Brown Rice Vinegar
Extraction of volatile compounds by solid-phase micro extraction (SPME) Each brown rice vinegar sample (5 mL) was added in a 20-mL amber glass vial fitted with screw cap and a PTFE-faced silicon septum. A 20 µL internal standard (0.054 g/mL 4-methyl-2-pentanol in MilliQ water containing 80 g/L of acetic acid) was added and then mixed (20). The sample was stirred magnetically for 20 min at 70oC. Aroma compounds were extracted from the headspace using a 50/30 µm divinylbenzene/carboxen/ polydimethylsiloxane SPME fiber (Supelco, Bellefonte, PA, USA) at 70oC for 60 min (20). The SPME fiber was conditioned prior to sampling by inserting it into a GC injector (250oC), according to the instructions provided by the manufacturer. Analysis of volatile compounds by GC-MS Volatile compounds were analyzed using an Agilent 6890N GC system coupled to an Agilent 5973N quadrupole mass spectrometer (GC 6890N; Agillent, Palo Alto, CA, USA). After head space sampling, the SPME fibers were injected into the SPME injector of the GC system injector port, and the injection was performed in the splitless mode for 2 min at an injection port temperature of 250oC. The column used was a fused silica capillary column (DB-WAX; 60 m× 0.25 mm i.d., 0.25 µm film thickness, J&W, Folsom, CA, USA). Helium was used as the carrier gas at a flow rate of 1.1 mL/min. Detector temperature was 250oC. The GC oven was programmed as follows: held at 35oC for 10 min,
and then ramped at 5oC/min to 100oC. Then, it was increased to 210oC at 3oC/min and held for 1 min. The MS interface temperature was set at 250oC, the ion source temperature was 230oC, the MS quadrupole temperature was 150oC, and the ionization voltage was 70 eV. The mass ranges of MS was set at 30-450 amu, the scan rate was 2.91 scan/s, and the electron multiplier voltage was 1,400 V. The signal was recorded and processed with Masslab software supplied with the Wiley 6.0 MS library (20). Peaks were identified by analogous mass spectra and confirmed by retention indices of standards of the same from the literature. Quantitative data from the identified compounds were obtained by measuring the relative peak area compared to that of the internal standard (4-methyl-2pentanol). Electronic nose analysis Vinegar samples were analyzed by a zNose (Electronic Sensor Technology, Newbury Park, CA, USA) equipped with a surface acoustic wave (SAW) sensor. Sample (2 mL) was added in a 40-mL vial (Supelco) fitted with Teflon septa (PTFE/silicone septa, Supelco), sealed, and placed at room temperature for 24 h to obtain headspace equilibrium. The headspace was extracted using helium as the carrier gas (purity; 99.9995%) and injected into a DB-5 capillary column (Supelco). The equipment was operated at a SAW sensor temperature of 30oC, column of 60oC, valve of 120oC, inlet of 150oC, and trap of 220oC. All samples were analyzed in triplicate. VaporPrintTM
Table 1. Organic acid content in brown rice vinegar by agitated and static fermentation with different nuruk content Organic acid content (mg%) Sample1)
1)
Oxalic acid
Tartaric acid
Malic acid
Acetic Acid
Lactic acid
Citric acid
Succinic acid
Total
AV10
22.19 ±1.25c2)
36.52 ±0.75f
30.08 ±0.15f
4,176.14 ±10.36c
328.65 ±8.15a
ND
2.78 ±0.08c
4,596.36 ±20.74c
AV20
28.89 ±2.31b
135.09 ±2.51d
39.33 ±1.25e
4,157.17 ±14.45c
144.68 ±6.45d
37.12 ±0.87f
17.85 ±1.45c
4,560.13 ±29.29c
AV30
30.58 ±2.45b
154.01 ±2.41c
49.37 ±2.95c
3,823.51 ±36.72d
67.84 ±3.75f
82.56 ±1.94b
23.66 ±2.25b
4,231.53 ±52.47d
AV40
48.52 ±1.25a
151.88 ±1.71c
47.77 ±2.45c
3,148.17 ±26.71f
122.51 ±6.15e
65.83 ±2.45d
16.44 ±1.26c
3,601.12 ±41.98f
SV10
13.54 ±1.35d
51.45 ±2.75e
27.38 ±1.62f
3,605.42 ±34.71e
252.18 ±3.75b
29.48 ±2.15g
ND
3,979.45 ±46.33e
SV20
15.42 ±2.21d
135.71 ±3.65d
43.56 ±3.25d
4,160.81 ±56.85c
147.52 ±6.45d
47.19 ±2.31e
10.14 ±1.26d
4,560.35 ±75.98c
SV30
24.87 ±1.74c
198.79 ±3.45b
84.08 ±2.14b
4,499.11 ±26.45b
138.52 ±3.78d
71.33 ±1.15c
21.84 ±0.64b
5,038.54 ±39.35b
SV40
28.98 ±1.25b
239.95 ±3.15a
97.66 ±2.48a
4,711.97 ±34.78a
194.28 ±6.21c
94.07 ±3.25a
27.66 ±0.45a
5,394.57 ±51.57a
AV, agitated fermented vinegar was cultured in a shaking incubator at 250 rpm and 30oC for 6 days. AV10, AV20, AV30, and AV40 were brown rice vinegars by agitated fermentation having 10, 20, 30, and 40% nuruk contents, respectively. SV, stationary fermented vinegar was cultured in a constant temperature incubator at 30oC for 16 days. SV10, SV20, SV30, and SV40 were brown rice vinegars by stationary fermentation having 10, 20, 30, and 40% nuruk contents, respectively. 2) Means±SD (n=3); a-fValues within a column followed by different superscripts are significantly different at p
