Fermentation and Quality Evaluation of Makgeolli, Korean Rice Wine Supplemented with Alcohol-Tolerant Pediococcus Acidilactici K3
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Korean J. Microbiol. Biotechnol. (2014), 42(4), 367–376 http://dx.doi.org/10.4014/kjmb.1409.09002 pISSN 1598-642X eISSN 2234-7305 Korean Journal of Microbiology and Biotechnology Fermentation and Quality Evaluation of makgeolli, Korean Rice Wine Supplemented with Alcohol-tolerant Pediococcus acidilactici K3 Danbie Jang1, Hyunjoo Lee1, Sangeun Pyo1, Seong Woon Roh2, Jin-Kyu Rhee3*, and Han-Seung Lee1,4* 1Department of Bio-Food Materials, College of Medical and Life Sciences, Silla University, Busan 617-736, Republic of Korea 2Jeju Center, Korea Basic Science Institute, Jeju 690-140, Republic of Korea 3Western Seoul Center, Korea Basic Science Institute, Seoul 120-140, Republic of Korea 4Research Center for Extremophiles and Marine Microbiology, Silla University, Busan 617-736, Republic of Korea Received: September 12, 2014 / Revised: September 25, 2014 / Accepted: September 26, 2014 This study’s purpose was to investigate the characteristics of a traditional Korean rice wine containing lactic acid bacteria (LAB), called makgeolli. The makgeolli was brewed with the alcohol-tolerant Pediococcus acidilactici strain K3, and was analyzed for LAB cell counts, alcoholic content, turbidity, pH, total acidity, amino nitrogen, total sugars, reducing sugars, solid contents, and organic acids. The physicochemical properties of the makgeolli were mostly maintained during fermentation (9 d) and storage (15 d). We also monitored the properties of LAB-supplemented commercial makgeollies, after adding P. acidilactici K3 at a concentration of 107 CFU/ml makgeolli, for one month. Most of their properties, such as alcoholic content, turbidity, pH, total acidity, amino nitrogen, total sugars, reducing sugars, solid contents, and organic acids, were preserved during storage at 10oC, suggesting that makgeolli supplemented with live LAB can be produced. These results suggest that alcohol-tolerant P. acidilactici K3 can be used for makgeolli brewing either as a starter or as a supplement. Keywords: Pediococcus acidilactici, lactic acid bacteria, makgeolli, fermentation, alcohol-tolerance Introduction of the sweet-sour taste of lactic acid, the taste of amino acids (a protein breakdown product), the bitter taste of alco- Makgeolli is a traditional Korean rice wine with an alcohol hol, and the sweet taste of saccharides, which are a starch content of more than 3% (v/v). Makgeolli is brewed with rice breakdown product [7, 8]. In addition, jubak (rice wine resi- and nuruk (Korean fermentation starter). Makgeolli is also due) formed during the process of filtration during makgeolli called takju, for its turbidity, or nongju, because it is a popu- production contains many nutrients, not only starches and lar lunchtime and thirst-quenching drink among farmers proteins, but fiber, minerals, vitamins, alcoholic and organic [15]. Makgeolli is one of Korea’s most famous drinks, and it acids, enzymes, and yeast. Several studies of the health has a distinctive taste, which comes from the combination benefits of consuming jubak-containing noodles have been performed, and they have reported various biological func- *Corresponding authors tions, including anti-diabetes, anti-cancer, anti-hyperten- H.-S. L. Tel: +82-51-999-6308, Fax: +82-51-999-5458 sion, and anti-cardiovascular disorder activities. In addition, E-mail: [email protected] several studies aimed at applications for patents have J.-K. R. investigated the application of makgeolli concentrate as an Tel: +82-2-6908-6224, Fax: +82-2-6908-6215 E-mail: [email protected] antioxidant and whitening agent, makgeolli soap, and natu- © 2014, The Korean Society for Microbiology and Biotechnology ral cosmetics made from grain fermentation [18]. December 2014 | Vol. 42 | No. 4 368 Jang et al. Lactic acid bacteria (LAB) in makgeolli form acids at the lated into 5 ml of MRS liquid medium at various pH values initial stage of fermentation and lower the pH, preventing (pH 2.0, 2.5, and 3.0). The bacteria were incubated at 37oC contamination with various other microbes and producing for 2 h, sampled every hour, diluted using a serial dilution various organic acids that improve the taste of fermented method, stained, and the number of colonies was counted. wine; however, overgrowth of LAB can increase acidity, To measure bacterial resistance to bile acid, bacteria were impart a sour taste, and cause acidification that negatively cultivated in MRS liquid medium for 12 h and precipitated affect stable fermentation of makgeolli [12, 16]. Therefore, by centrifugation (7,000 rpm, 10 min). The precipitated bac- application of LAB as an additive in makgeolli needs optimi- teria were washed twice with phosphate-buffered saline zation to balance between advantages and disadvantages. (PBS) solution, and the diluted bacterial solution, at 1%, Since alcohol concentration of makgeolli brewed usually was inoculated into 5 ml of MRS liquid medium containing reaches above 12% (v/v), LAB cannot survive actively in 0.3% ox gall. The bacteria were incubated at 37oC for 4 h, such an environment with more than 5% of alcohol. When and sampled every 2 h, diluted using a serial dilution makgeolli is fermented using the traditional yeast, LAB are method, stained, and the number of colonies was counted. rapidly reduced as the alcohol concentration surpasses 10% [2, 19]. Pre- and post supplementation of LAB to makgeolli Recently we isolated and reported alcohol-tolerant Pedi- To produce LAB-containing makgeolli, 0.3% white-koji ococcus acidilactici K3 for makgeolli brewing [6]. In this mold (Choongmu fermentation Inc.) was added to 880 g of study, we added the strains to makgeolli before fermenta- hard-cooked rice and incubated at 30oC for 2 h to produce tion (starter addition) and after fermentation (post-fermenta- the starting culture. Eighty grams of this mixture and 150 ml tion supplementation) for the application of the alcohol- of water were mixed and 3% pre-cultivated yeast (Saccha- tolerant strains to makgeolli. We measured the physico- romyces cerevisiae) was injected to produce the mix; the chemical properties of the makgeollies and evaluated the resulting mixture was incubated at 25oC for 2 d. Eight hun- feasibility for production of LAB contained makgeolli. dred grams of the mixture and 1350 ml of water were added to a fermentation container and were subjected to an Materials and Methods initial soaking. After 2 d, 3.2 kg of hard-cooked rice, 4.8 L of water, and 1.5 g of P. acidilactici K3 was added to the initial Ethanol, acid, and bile tolerance soaking container, and were subject to primary soaking. To measure the viability of alcohol-tolerant P. acidilactici This mixture was fermented for 9 d and was sampled at K3, its growth at each alcohol concentration was compared 24-h intervals for chemical analysis and viable cells counts to that of the standard neotype strain P. acidilactici DSM (LAB). After filtering, the makgeolli was stored in a refrigera- 20284. Bacteria were cultivated at 37oC on MRS liquid tor at 10oC for 15 d, and sampled at 24-h intervals for the medium for 12 h and then cells were precipitated by centrif- first 2 d, and every 5 d from the third day for chemical qual- ugation (6,000 rpm, 10 min). The precipitated bacteria were ity analysis and viable LAB cell counts. For post-fermenta- suspended in a tenth volume of 0.1 M phosphoric acid buf- tion supplementation experiments, 2.5 g of P. acidilactici K3 fer (pH 7.0). The 1% (v/v) of suspended bacteria solution was added to 2 L of commercial makgeollies from Com- was inoculated into 5 ml of 0.1 M phosphoric acid buffer pany K. For the first two days, the makgeolli was sampled containing various concentrations of alcohol (0, 6, 12, and every day and the number of viable LAB cells was counted. 18% v/v). After incubation with shaking for 4 h at 37oC, the Afterwards, it was sampled every 5 d for chemical quality bacteria were diluted using a serial dilution method, analysis and to count the number of viable LAB cells. stained, and the number of colonies was counted. To mea- sure the resistance to low pH, bacteria were cultivated in Viable cell counts MRS liquid medium for 12 h and precipitated by centrifuga- One milliliter of sample was diluted using a serial dilution tion (7,000 rpm, 10 min). The precipitated bacteria were method and then smeared on MRS solid medium contain- washed twice with phosphate-buffered saline (PBS) solu- ing BCP-sample. After 24 h of incubation at 37oC, the num- tion, and the diluted bacteria solution, at 1%, was inocu- ber of bacterial colonies was counted. http://dx.doi.org/10.4014/kjmb.1409.09002 Quality Characteristics of Korean Rice Wine with Alcohol-tolerant LAB 369 Alcohol content and turbidity Acidity (%) = (NaOH Added × NaOH Strength × 0.006) / The makgeolli was sampled at 24-h intervals, and 10 ml Sample Volume × 100 of sample was quantified in a mass cylinder, and it was To measure the optimum amount of amino nitrogen, 1 ml then transferred to a distillation flask. The cylinder used to of sample was added to a 50-ml volumetric flask, and quantify the sample was washed twice with distilled water, diluted with distilled water. Afterwards, it was separated into and the rinsing water was added to the distillation flask. a 25-ml conical flask and mixed with 20 ml of formalin solu- Then, the sample-containing flask was heated and the liq- tion (Sigma, USA) and 20 ml of water. Then, 3-5 drops of uid was distilled by connecting the flask to the cooler. When 1% phenolphthalein was added, and optimized by addition 70 ml of distilled solution was obtained, the distillation was of 0.05 N NaOH. The amino nitrogen content was deter- stopped and distilled water was added to raise the volume mined by inserting the amount of NaOH added into the fol- to 100 ml.