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International Journal of Biomass & Renewables, 4(2) : 17 - 21, 2015 Production and Antioxidative Activity of Alcoholic Beverages
Made From Newly Isolated Vietnamese Men Yeast
Hao Pan1, Ryoichi Takeshita1, Noriaki Saigusa1, Ngo Thi Phuong Dung2, Aporn Wongwicharn3 and Yuji Teramoto1*1Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Nishi Ward, Kumamoto 860-0082, Japan.
2Biotechnology Research and Development Institute, Can Tho University Campus II, 3-2 Street, Can Tho City, Vietnam.
3Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, 126 Prachautid Road, Bangmod, Ratburana, Bangkok 10140, Thailand.
Fermentation yeast was newly isolated from a traditional Vietnamese microbial starter for brewing alcoholic beverages, called men. The isolated yeast was identified as a strain of the Saccharomyces cerevisiae and designated as S. cerevisiae Y3. The alcoholic beverage made with 3 yeast strains, Y3, NP01, and K7 from uncooked and cooked nonglutinous rice grains had an ethanol concentration of approximately 11.6 to 14.5% (v/v). Resulting alcoholic beverages made with Y3, NP01, and K7 yeasts had antioxidative activity. The DPPH radical scavenging activity of the alcoholic beverages made with 3 yeast strains is equivalent to approximately 500 to 600 µM Trolox. The DPPH radical scavenging activity of the alcoholic beverage made with Y3 yeast was higher than that of the alcoholic beverage made with NP01 and K7 yeasts. The inhibitory activity of lipid peroxidation of the alcoholic beverages made with Y3 and NP01 yeasts was higher than that of the alcoholic beverages made with K7 yeast.
Key words: men, fermentation yeast, antioxidative activity, uncooked fermentation.
We have been researching fermented foods worldwide and out in order to utilize the isolated yeast to modern brewing their microbial resources. We previously reported on the characteristics of a traditional Thai alcoholic beverage called ou, which was drunk through tubes, and its fermentation yeast [1]. We also reported on the characteristics of alcoholic beverages drunk through tubes in Uganda and Bahrain [2, 3].
Yeast strain
Food materials containing antioxidative compounds are known to contribute to prevention of diseases such as arteriosclerosis We attempted to isolate a fermentation yeast strain from and many studies were done [4, 5].
commercial men, Nam Hoa, used in the local microdistillery in the Cai Lay District in Vietnam, using plates of an agar-solidified In this study, we tried to isolate and identify the yeast strain YPD medium (yeast extract, 10 g; peptone, 20 g; glucose, 20 g; from a Vietnamese microbial starter called men and to utilize tap water, 1,000 ml) containing 50 μg of chloramphenicol/ml. the isolated yeast for ethanol fermentation. The characteristics The isolated strain of yeast was maintained on YPD agar slopes.
of the alcoholic beverage made with the isolated yeast were A taxonomical study concerning rDNA analysis of the isolated determined to develop the functional alcoholic beverage, yeast strain was carried out by Toyobo Biologics, Inc. (Tsuruga, which has antioxidative activity [6, 7]. For a comparative study, S. cerevisiae NP01, stocked in our laboratory, and the industrial sake brewing yeast S. cerevisiae K7 were used as well.
For a comparative study, S. cerevisiae NP01 isolated in Thailand and stocked in our laboratory [1] and the industrial sake yeast We are now trying hard to research the undeveloped microbial S. cerevisiae K7 purchased from the Brewing Society of Japan resources of tropical area under the project of Japan Society (Tokyo, Japan) were used.
for Promotion of Science (JSPS)–National Research Council of Thailand (NRCT). Comparative studies of newly isolated yeast of tropical area and industrial brewing yeast are also carried *Corresponding author. E-mail address : [email protected] Yuji Teramoto et al. / International Journal of Biomass & Renewables, 4(2) : 17 - 21, 2015 Rice grains
Acidity was measured by titrating 10 ml of alcoholic beverage with 0.1 N NaOH. Reducing sugar as glucose was determined Commercial nonglutinous rice grains (Oryza sativa var. according to the methods of Somogyi and Nelson [8, 9]. The Japonica cv. Hinohikari) were ground to particles of 2–3 mm amount of total phenolic compounds, expressed as gallic acid, in diameter with an electric grinder and were used for ethanol was determined according to the Folin-Ciocalteu method [10, 11]. The ethanol concentration of alcoholic beverage was determined with a gas chromatograph (model GC-14A; Shimadzu Co., Kyoto, Japan) equipped with a 3.1-m PEG-HT A glucoamylase preparation, Sumizyme, kindly donated by column (Gasukuro Kogyo, Inc., Tokyo, Japan).
Shin Nihon Kagaku Kogyo Co., Ltd. (Anjo, Japan), was used as the saccharifying agent. Determination of antioxidative activity
The DPPH radical scavenging activity as the Trolox equivalent was measured on the basis of the method of Yamaguchi et DPPH (1,1-diphenyl-2-picrylhydrazyl) was purchased from al. [12]. The lipid peroxidation inhibitory activity as the BHT Nacalai Tesque (Kyoto, Japan). Trolox (6-hydroxy-2,5,7,8- equivalent was determined using β-carotene [11].
tetramethylchroman-2-carboxylic acid) was purchased from Sigma-Aldrich, Inc. (St. Louis, Mo, USA). BHT (2,6-di-tert-butyl-p-cresol) was purchased from Tokyo Kasei Co., Ltd. (Tokyo, Results and Discussion
Efforts were made to research traditional Vietnamese alcoholic Ethanol fermentation procedure
beverages in November 2011. Research was done in Go Den District, Cai Lay District, Tra Vinh District, and O Mon District in Traditional ethanol fermentation with cooking was performed the Mekong Delta [13]. In Vietnam, alcoholic beverages have according to the procedure below. Thirty grams of uncooked traditionally been made using a special microbial starter, the unpolished nonglutinous rice grains and 50 ml of deionized native name of which is men.
water were dispensed into a 300-ml Erlenmeyer flask and autoclaved at 121ºC for 15 min. After cooling, the cooked rice A strain of yeast designated Y3 was isolated from the was mixed with 0.2 g of Sumizyme as the saccharifying agent, Vietnamese commercial men Nam Hoa. Y3 yeast was found 40 ml of deionized water, and 10 ml of a yeast suspension, to be fermentation yeast with globose or subglobose which readily brought the population of yeast in the initial cells. Pseudohyphae were not observed during cultivation. mash to 3.0×107 cells/ml. Ethanol fermentation was conducted D-Glucose, D-galactose, mannose, maltose, and sucrose were at 25 ºC in the dark. fermented, while lactose was not.
Ethanol fermentation without cooking was done as follows. rDNA analysis of Y3 yeast was carried out by Toyobo Biologics, Thirty grams of uncooked unpolished nonglutinous rice grains, Inc., and was identified as a strain of Saccharomyces cerevisiae. 90 ml of deionized water, 0.2 g of Sumizyme, and 10 ml of a According to these characteristics, the isolated yeast was yeast suspension were dispensed into a 300-ml Erlenmeyer identified as a strain of S. cerevisiae. flask. The population of yeast in the initial mash was adjusted to 3.0×107 cells/ml, and fermentation was conducted in the The fermentation curves of the mashes made from the 3 yeast same manner as for ethanol fermentation with cooking. In this strains, S. cerevisiae Y3, NP01, and K7 are shown in Fig. 1. A procedure, there is no cooking or steaming process.
larger amount of CO2 was generated from the mash with Y3 and NP01 yeasts than that with K7 yeast on day 1. Fermentation The decrease in weight of the Erlenmeyer flask and its contents was completed after 5 days. as a result of the evolution of CO2 gas was measured every 24 h.
General analytical methods
Fermented mash made from nonglutinous rice grains was centrifuged at 3,000 rpm for 15 min and filtered through No. 101 filter paper (Advantec Toyo Co., Ltd., Tokyo, Japan), and the resulting alcoholic beverage was analyzed.
International Journal Of biomass & renewables
Yuji Teramoto et al. / International Journal of Biomass & Renewables, 4(2) : 17 - 21, 2015 Figure 1 : Time courses of fermentation of mashes using 3 yeast strains and nonglutinous rice grains.
, fermentation with cooking; ○, fermentation without cooking. Values are the mean of 3 replicates. A, Y3 yeast; B, NP01 yeast; C, K7 yeast.
Y3 yeast is applicable to conventional ethanol fermentation only and capture aqueous vapor and volatile compounds such with cooking and ethanol fermentation without cooking as as higher alcohols and esters. Then, sometime, depends on the well. In the system of ethanol fermentation without cooking, fermentation condition, too much aqueous vapor and volatile material rice grain was directly applied to ethanol fermentation, compounds were captured by gas trap and the volume of then we can save energy for cooking or steaming.
resulting filtrate decrease and ethanol concentration increase. We are not determined but there is a possibility, instead The characteristics of alcoholic beverage made from the 3 of ethanol other metabolite such as organic acid might be yeasts are shown in Table 1. The ethanol concentration of the produced. We observed sometime amount of released CO2 and alcoholic beverages was approximately 11.6 to 14.5% (v/v). We amount of ethanol are not precisely parallel.
use gas trap containing concentrated H2SO4 to release CO2 Table 1 : Composition of initial mash and analysis of resulting alcoholic beverages
uncooked cooked uncooked cooked Polished rice (g) Deionized water (ml) Yeast suspension (ml)* Ethanol concentration (%, v/v) Total phenolic compound content (µg/ml) Reducing sugar content (µg/ml) *Cell number of the initial mash was adjusted to be 3.0×107 cells/ ml.
Acidity of the resulting alcoholic beverages was 1.8 to 2.6 and Reducing sugar of the resulting alcoholic beverages were pH of them was 3.0 to 4.2. Acidity and pH of resulting alcoholic around 600 to 700 µg/ml. This means ethanol fermentation beverages were almost similar to Japanese sake.
proceeded properly and there are not so many residual sugar.
International Journal Of biomass & renewables
Yuji Teramoto et al. / International Journal of Biomass & Renewables, 4(2) : 17 - 21, 2015 The total amount of phenolic compounds of alcoholic beverage made from uncooked rice grains was higher than that of the beverage made from cooked rice grains. In the case of fermentation without cooking, rice grain was not heated or steamed; the phenolic compounds might be not decomposed by heating. Alcoholic beverages made with Y3 yeast contained relatively higher amount of phenolic compounds. Y3 yeast might produce much phenolic compounds including protein and aromatic amino acids.
The antioxidative activity of the alcoholic beverage was determined. The DPPH radical scavenging activity of various alcoholic beverages is equivalent to approximately 500 to 600 µM Trolox (Fig. 2). The DPPH radical scavenging activity of the alcoholic beverage made with Y3 yeast was higher than that of the alcoholic beverage made with NP01 and K7 yeasts.
Figure 3 : Inhibitory activity of lipid peroxidation of
alcoholic beverages made from 3 yeast The inhibitory activity of lipid peroxidation of the alcoholic strains and nonglutinous rice grains. beverages is shown in Fig. 3. The inhibitory activity of lipid peroxidation of the alcoholic beverage made with Y3 and Closed bars, alcoholic beverage made by fermentation without NP01 yeasts was higher than that of the beverages made with cooking; open bars, fermentation with cooking.
Y3 and NP01 yeasts were screened from the warm region. Then it was considered that fermentation advanced quickly in a warm environment of 25 ºC. It has been suggested that Newly isolated yeast strain S. cerevisiae Y3 was applicable for compounds related to DPPH radical scavenging activity and ethanol fermentation. Comparative study with S. cerevisiae the inhibitory activity of lipid peroxidation are different. We NP01 isolated in Thailand and S. cerevisiae K7 industrial sake would like to determine the compounds related to DPPH yeast used in Japan was done. Y3 yeast had similar fermentation radical scavenging activity and the inhibitory activity of lipid ability compared with NP01 yeast isolated in Thailand and K7 industrial sake yeast used in Japan.Y3 yeast was applicable for traditional ethanol fermentation with cooking and ethanol fermentation without cooking.
The alcoholic beverages made from various yeasts and nonglutinous rice grains showed antioxidative activity. The DPPH radical scavenging activity of the alcoholic beverage made with Y3 yeast was higher than that of the alcoholic beverage made with NP01 and K7 yeasts. We would like to determine the compounds contained in the alcoholic beverages which have DPPH radical scavenging activity and the inhibitory activity of lipid peroxidation.
We would like to improve the taste and aroma of the alcoholic beverage and produce a fine alcoholic beverage that also has physiological advantages. We also apply the tropical microbial resources to modern brewing industry.
Figure 2 : DPPH radical scavenging activity of alcoholic
beverages made from 3 yeast strains and nonglutinous rice grains. Closed bars, alcoholic beverage made by fermentation without This work was partially funded by the Japan Society for cooking; open bars, fermentation with cooking. Promotion of Science (JSPS)–National Research Council of International Journal Of biomass & renewables
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International Journal Of biomass & renewables

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