Antimicrobial Efficacy of Fermented Dark Vinegar from Unpolished Rice
Antimicrobial Efficacy of Fermented Dark Vinegar from Unpolished Rice
Microbiology and Biotechnology Letters. 2015. Jun, 43(2): 97-104
Copyright © 2015, The Korean Society for Microbiology and Biotechnology
  • Received : April 28, 2015
  • Accepted : May 13, 2015
  • Published : June 28, 2015
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About the Authors
학준, 최
경자, 곽
다빈, 최
재영, 박
현숙, 정

Vinegar is a widely used acidic seasoning and can be manufactured using various methods and bases, including cereals, wheat, and fruits. Most studies on vinegar have been conducted to evaluate its antioxidant activity. In the present study, fermented dark vinegar (FDV) produced from unpolished rice was examined for its antimicrobial activity, biochemical content, including the amounts of sugar, total soluble sugar, organic acid, and free amino acids, and pH and physiological activity. The antimicrobial efficiency of FDV was assessed using the paper disc-agar diffusion method. FDV exhibited strong antimicrobial activity against the pathogenic bacteria and yeast strains that were tested. In fact, the activity of FDV was shown to be higher than that of the commercial antibiotics carbenicillin (50 µg/ml) and tetracycline (50 µg/ml) against Staphylococcus aureus , Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Salmonella typhimurium , Yersinia enterocolitica , and Lodderomyces elongisporus . The antioxidant activity of FDV and ascorbic acid was evaluated. Using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, we found that FDV has the highest activity of the antioxidants. After spreading FDV onto tryptic soy broth and yeast extract-peptone-dextrose agar media, the microbial strains were isolated and characterized through physiological and biochemical analysis. Based on 16S ribosomal DNA sequence analysis, the isolated microorganisms exhibited a close similarity to Acetobacter papayae , Acetobacter pasteurianus , and Acetobacter peroxidans .
Unpolished rice (UR) is composed of external thin layers (bran) that enclose the embryo (germ) and endosperm. The nutritional components in UR mainly exist in the germ and bran layers, which are mostly removed as a consequence of the milling or polishing process [3] . For this reason, UR has higher nutritional quality than polished rice. Recently, human and animal studies have shown that consumption of UR reduces the risk of type-2 diabetes, cardiovascular disease, and cancer, and these protective health effects have been linked to the presence of bioactive compounds such as polyphenols, GABA, acylated steryl b-glucoside, and c-oryzanol [3 , 5 , 7 , 11 , 17 , 29] . Organic acids and their salts have been used as feed additives, functioning as acidifiers of animal feed. Such organic acids, including acetic, butyric, citric, formic, lactic, malic, propionic, and sorbic acids have been shown to improve health and growth performance in livestock and poultry by altering gastrointestinal tract function and energy metabolism, increasing the availability of nutrients and inhibiting the growth of pathogenic bacteria [4] . Vinegar is a widely used acidic seasoning and can be manufactured using various methods and base materials, including cereals, wheat, and fruits [20] . It is produced via a fermentation process carried out by several microorganisms including molds, yeasts, lactic acid bacteria, and acetic acid bacteria (AAB). These organisms produce not only acetic acid, but also various metabolic compounds that modify the taste and flavor of the product. Moreover, some types of vinegar have been shown to contain antioxidants, antitumor compounds, and other bioactive metabolites, which may be responsible for its beneficial health effects [8 , 10 , 18 , 25] . Kurozu, a traditional jar-fermented Japanese black vinegar, is made from unpolished rice and has been reported to inhibit tumor growth, lipid peroxidation, and inflammation [6 , 18 , 19 , 21 - 24] .
In this study, we investigated the antimicrobial activity of fermented dark vinegar (FDV) against various pathogenic bacteria and a yeast strain. Further, the antioxidant activity and the organic components of FDV were analyzed, and culturable microbial analysis was carried out during the fermentation process.
Materials and Methods
- Preparation of FDV
Mother brew (Mitsul) was prepared as follows: 4 kg polished rice was rinsed 3 times to remove impurities, followed by soaking in water for 4 h until saturation of water adsorption occurred. Excess water was removed, and the soaked rice was then steamed for around 40 min to allow full gelatinization. The steamed rice was cooled to 25℃, then mixed with 2 kg yeast leavening agent (Nuruk powder), and incubated at 32℃ for 2−3 days to allow saccharification.
Preparation of steamed rice cakes (Baekseolgi) was as follows: 20 kg UR was rinsed 3 times to remove impurities, followed by soaking in water for 8 h until saturation of water adsorption occurred, and water was the drained for 1 h. For the instant rice cakes, the crushed powder was steamed to Baekseolgi.
Twenty kilograms of Mitsul, 20 kg Baekseolgi, 2 kg Nuruk, and 50 L water were mixed and then incubated at 32℃ to reach an alcohol content of 12%. After that the mixture was incubated at 25℃ for 1 month to allow fermentation. FDV was then available as the supernatant of the fermented broth.
- Alcohol, pH, and total soluble solid content
One hundred milliliters of the supernatant sample was run through a distiller until around 70 ml had been collected. Distilled water was added to the collected sample to a total volume of 100 ml, and the alcohol content (%) was measured using a vinometer. The alcohol-temperature correction table was used with the sample’s alcohol content and a temperature. The pH was measured using an Orion 420A pH meter (Thermo Fisher Scientific, Inc., MA, USA). Total soluble solid content was measured using a Brix Refractometer HI 96811 (Hanna Instruments, RI, USA) [28] .
- Free amino acid and componential analysis of FDV
Sugar, organic acid, moisture, ash, and crude protein contents of FDV were determined by proximate composition analysis. The sugars investigated were fructose, glucose, and sucrose. The organic acids investigated were oxalic acid, lactic acid, acetic acid, and propionic acid. Free amino acid analysis was done using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI), operated in selected ion monitoring (SIM) mode. This analysis was performed at Biotechnology Industrialization Center (BIC; Dongshin University, Korea).
- Culture conditions and isolation of microbial strains
To isolate microbial strains from FDV, 100 µl of FDV was spread onto yeast extract-peptone-dextrose (YPD) agar medium (20.0 g/l peptone, 10.0 g/l yeast extract, 20.0 g/l glucose, and 20.0 g/l agar) and tryptic soy broth (TSB) agar medium (BD 211825; Becton, Dickinson and Co., NJ, USA). The plates were incubated at 30℃ for 2 days. Single colonies were purified by transferring onto fresh plates, followed by re-incubation. To classify morphological and biochemical characteristics, gram staining was performed using a gram staining kit (Fluka-77730, Sigma-Aldrich, St Louis, MO). Catalase activity was examined by measuring the production of oxygen bubbles in aqueous hydrogen peroxide solution. To identify the carbon sources used by the bacteria, they were grown on basal salt media (BSM) [2] supplemented with maltose, mannitol, cellobiose, Dmannose, D-glucose, lactose, fructose, and D-arabinose, respectively, to a final concentration of 2% as a carbon source [2] .
- Polymerase chain reaction (PCR) amplification and sequencing of 16S ribosomal DNA (rDNA)
The 16S rDNA was PCR amplified using the 27F primer 5'-AGAGTTTGATCMTGG-CTCAG-3' and the 1492R primer 5'-TACGGYTACCTTGTTACGACTT-3' [1 , 27] . 16S rDNA PCR amplification was performed using an MJ Research Tetrad PTC 225 thermal cycler (Bio-Rad Laboratories, Inc., CA, USA) with the sample in a final volume of 50 µl, containing 10 mM Tris-HCl pH 7.4, 50 mM KCl, 1.5 mM MgCl 2 , 0.2 µM of each deoxynucleotide (dNTP), 0.2 µM of each primer, 1.25 U of Taq DNA polymerase (Roche Diagnostics, Basel, Switzerland), and 3 µl of extracted DNA. The PCR product was purified using a QIAquick PCR purification kit (Qiagen, Limburg, Netherlands), according to the manufacturer’s instructions. 16S rDNA sequence analysis was performed using an ABI PRISM BigDye Terminator Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Inc., CA, USA) and ABI 310 DNA sequencer (Applied Biosystems, Inc.) following the manufacturer’s protocol.
- Free radical scavenging activity of FDV
The overall antioxidant activity of the prepared sample was assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method [12 , 13] . DPPH is a stable free radical that shows decreased absorbance at 517 nm when it is reduced by antioxidants. DPPH stock was prepared by dissolving 0.4 mM of DPPH in 100 ml absolute ethanol. Twenty microliters of FDV sample was added to 180 µl of DPPH solution. The mixture was shaken vigorously, and the absorbance was measured at 517 nm using a microplate spectrophotometer (Eon, BioTek, VT, USA) for 30 min. As a positive control, ascorbic acid (1 mg/ml, 5 mM) was used. The percentage of inhibition, which represents the scavenging ability of the sample were DPPH radicals, was calculated as follows [13] :
Antioxidant Activity (AOA) = 100 − [(absorbance increase of sample / absorbance increase of control) × 100]
- Antimicrobial activity of FDV
Determination of FDV antimicrobial activity was performed using the agar diffusion method [14 , 16] on solid media, employing common pathogenic bacteria including Staphylococcus aureus , Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Salmonella typhimurium , Yersinia enterocolitica , Lactobacillus casei , Gluconacetobacter intermedius , and Lodderomyces elongisporus . Lodderomyces elongisporus was plated onto a YPD agar plate and all other test strains were plated onto TSB agar plates. Paper discs (8 mm diameter) were impregnated with FDV or four organic acids (latic acid, oxalic acid, acetic acid and propionic acid; 25 µg/ml, Sigma-Aldrich, St Louis, MO) and were placed onto the surface of the inoculated agar plates. These were then incubated at 37℃ for 18 h. The total diameter (mm) of the inhibition zone was measured for each test microorganism [16]. Tetracycline (50 µg/ml) and carbenicillin (50 µg/ml) were used as positive controls. The antimicrobial activity of 3-year FDV was further measured according to changes in its acidity (pH). As a control, 100 mM KCl buffer (pH 3.0) was used.
- Alcohol, pH, and total soluble solid content, and componential analysis of FDV
Values for the alcohol, pH, and total soluble solid content of FDV increased during fermentation, while moisture content decreased. Componential analysis of FDV was carried out for three sugars and four organic acids. The three sugars were fructose, glucose, and sucrose. Fructose was not present in FDV; however, glucose and sucrose content increased as fermentation progressed. The four organic acids were oxalic, lactic, acetic, and propionic acid. Lactic acid levels did not increase during fermentation, but oxalic acid, acetic acid, and propionic acid content increased as the fermentation process went on. These results are shown in Table 1 .
Biochemical component analysis of fermented dark vinegar (FDV)
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aFDV fermented for 1 year, bFDV fermented for 3 years.
- Analysis of the free amino acid content of FDV
The free amino acid contents of FDV were analyzed and the results are shown in Table 2 . A total of 18 amino acids were assessed, of which 15 amino acids showed increasing levels as fermentation progressed. The total free amino acid content for the 1-year-FDV was 1398 ± 409.1 mg/l and one for 3-year-FDV was 1841.1 ± 202.4 mg/l. These results are higher than those reported by Kim et al . [10] . In particular, alanine, valine, threonine, leucine, isoleucine, arginine, tyrosine, and GABA were present at higher levels in FDV than by Kim et al . [10] .
Amino acid analysis of fermented dark vinegar (FDV).
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aFDV fermented for 1 year, bFDV fermented for 3 years.
- Identification of microorganisms isolated from FDV
The reason for isolated the microorganisms of FDV was to investigate the influence of strains appearing during fermentation to vinegar and standardization of FDV. The three microorganisms isolated from FDV were named as fermented dark vinegar strain (FDVS)-1, -2, and -3. All strains were gram-negative and did not sporulate. Peptidoglycan typing and 16S rDNA sequence analysis revealed that FDVS-1 was 98% homologous to Acetobacter pasteurianus , FDVS-2 was 100% homologous to Acetobacter peroxidans , and FDVS-3 was 96% homologous to Acetobacter senegalensis ( Fig. 1 ). All strains FDVS-1, -2, and -3 represent the names of each identified bacterial strain. Carbon source usage tests for the three strains were conducted using BSM ( Table 3 ).
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Phylogenic tree based on 16S ribosomal RNA (rRNA) gene sequences of three microorganisms isolated from fermented dark vinegar (FDV). The tree was based on an alignment of 1,318 bp of 16S rRNA gene sequences, and constructed by the neighbor-joining method, Acetobacter sp. FDVS-1, -2, and -3; fermented dark vinegar strains.
Carbon source usage of three microorganisms isolated from fermented dark vinegar (FDV).
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Carbon source usage tests were carried out using basal salt media. a+, growth; -, no growth.
- Free radical scavenging activity of FDV
Antioxidants reduce the oxidative stress in cells and are therefore useful in the treatment of human diseases. Plantderived antioxidants have been widely studied due to their relative safety for consumption compared to synthetic antioxidants [18] . The radical scavenging activity of FDV was determined using DPPH systems. The activities of FDV after 1 year- and 3 years of fermentation were found to be 82.07 ± 1.90% and 86.76 ± 1.14%, respectively. The antioxidant activities of FDV determined by the DPPH method were similar to those of ascorbic acid (1 mg/ml) ( Fig. 2 ).
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Antioxidant activity of fermented dark vinegar (FDV). The overall antioxidant activity of FDV was assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. Ascorbic acid (1 mg/ml, 5 mM) was used as a positive control. FDV1, FDV fermented for 1 year; FDV3, FDV fermented for 3 years.
- Antimicrobial activity of FDV
The antibacterial activity of the organic acids studied and FDV was examined using the paper disc diffusion method against three strains of gram-positive bacteria ( Staphylococcus aureus , Pseudomonas aeruginosa , and Lactobacillus casei ), six gram-negative bacterial strains ( Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Salmonella typhimurium , Yersinia enterocolitica , and Gluconacetobacter intermedius ), and one strain of fungus ( Lodderomyces elongisporus ). The results are shown in Table 4 . FDV effectively inhibited the growth of all three gram-positive strains, five gram-negative bacterial strains ( Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Salmonella typhimurium , Yersinia enterocolitica , and Gluconacetobacter intermedius ), and the fungus strain. In these cases, FDV produced a zone of inhibition ranging from 12 to 22 mm in diameter.
Screening for antimicrobial activity of fermented dark vinegar (FDV) and variety of other antibiotics and control substance by paper disc assay.
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Loaded with each sample (50 µl) was placed on the agar plate which was seeded with each test microorganism. Tetracycline was used as a positive control. K; KCl Buffer pH 3, C; Carbenicillin (50 µg/ml), T; Tetracycline (50 µg/ml), F1; FDV 1 year, F3; FDV 3 year.
Most of the organic acids tested showed no inhibitory activity against any strains. However, propionic acid showed reasonably strong activity against all strains (15-28 mm zones of inhibition). At basic pH ranges, FDV did showed no antimicrobial activity, as shown in Table 5 .
Screening for antimicrobial activity according to the acidity of 3-year fermented dark vinegar using paper disc assay.
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Acidity was adjusted using 0.1 N NaOH. Units: mm, values represent diameter of the inhibition zone.
The purpose of this study was to evaluate the antimicrobial activity of FDV and to determine its antioxidant activity, pH-, sugar-, total soluble solid-, total acid-, and free amino acid content. Three-year-FDV had an organic acid content of 62.946 ± 0.245 mg/l, free amino acid content of 1841.1 ± 202.4 mg/l, and sugar content of 1.18 ± 0.127 mg/l. These results represent the highest organic acid and free amino acid contents compared to previous report [10] . And the organic acid and total free amino acid content of FDV increased during the fermentation processes. Three strains of AAB, Acetobacter sp. FDVS-1, -2, and -3, were isolated from FDV. AAB are an important group of bacteria in the food and beverage industry, mainly due to their ability to oxidize ethanol to acetic acid. These bacteria represent the key microorganism in vinegar production [15] .
In the DPPH system, the radical scavenging activity of FDV in the 1-year and 3-year fermented vinegar was found to be 82.07 ± 1.90% and 86.76 ± 1.14%, respectively. FDV showed antioxidant activity comparable to that of ascorbic acid.
Regarding its antimicrobial activity, FDV inhibited the growth of Staphylococcus aureus , Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Salmonella typhimurium , Yersinia enterocolitica , and Lodderomyces elongisporus . This inhibitory activity of FDV was greater than that of the commercial antibiotics carbenicillin (50 µg/ ml) and tetracycline (50 µg/ml). In particular, FDV strongly inhibited the growth of Salmonella typhimurium and Yersinia enterocolitica . Salmonella typhimurium , which infects a variety of animals including humans, is widely distributed throughout the world and causes enteritis and septicemia [9] . Yersiniosis is the third most reported food-borne bacterial zoonoses in humans, and Yersinia enterocolitica is the most commonly reported bacterial species to cause this disease in humans [26] . But, propionic acid in the FDV may have affected the antimicrobial activity. In conclusion, FDV exhibits strong antioxidant and antimicrobial activity against human pathogenic microorganisms.
This research was supported by the IPET (Korea Institue of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries) funded by the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Thechnology Commercialization) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2012R1A1A3019667).
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