Surface Film Formation in Static-Fermented Rice Vinegar: a Case Study
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MYCOBIOLOGY 2019, VOL. 47, NO. 2, 250–255 https://doi.org/10.1080/12298093.2019.1575585 RESEARCH ARTICLE Surface Film Formation in Static-Fermented Rice Vinegar: A Case Study Jeong Hyun Yuna, Jae Ho Kimb and Jang-Eun Leea,b aDepartment of Food Biotechnology, Korea University of Science and Technology, Daejeon, Republic of Korea; bResearch Group of Traditional Food, Korea Food Research Institute, Jeollabukdo, Republic of Korea ABSTRACT ARTICLE HISTORY In the present study, we aimed to determine the cause of surface film formation in three Received 27 April 2018 rice vinegars fermented using the traditional static fermentation method. The pH and total Revised 10 November 2018 acidity of vinegar were 3.0–3.3 and 3.0–8.7%, respectively, and acetic acid was the predomin- Accepted 29 December 2018 ant organic acid present. Colonies showing a clear halo on GYC medium were isolated from KEYWORDS the surface film of all vinegars. Via 16S rDNA sequencing, all of the isolates were identified Acetic acid bacteria; as Acetobacter pasteurianus. Furthermore, field-emission scanning electron microscopy ana- Acetobacter pasteurianus; lysis showed that the bacterial cells had a rough surface, were rod-shaped, and were pellicle; static fermentation; 1 Â 2 mm in size. Interestingly, cells of the isolate from one of the vinegars were sur- surface film; vinegar rounded with an extremely fine threadlike structure. Thus, our results suggest that formation of the surface film in rice vinegar was attributable not to external contamination, to the pro- duction of bacterial cellulose by A. pasteurianus to withstand the high concentrations of acetic acid generated during fermentation. However, because of the formation of a surface film in vinegar is undesirable from an industrial perspective, further studies should focus on devising a modified fermentation process to prevent surface film formation and consequent quality degradation. 1. Introduction sometimes leads to the formation of a pellicle-like film on the surface. Formation of such a film causes Vinegar is a liquid condiment that contains bio- overoxidation and turbidity in vinegar. Surface fer- active compounds such as organic acids, mainly mentation is also called the static method, because acetic acid, and phenolic compounds. Owing to the characteristic sourness of acetic acid and the health the AABs are fixed on the surface of the material to benefits, vinegar has been used not only as an essen- allow contact with atmospheric oxygen. Therefore, it tial condiment, but also as a pharmaceutical agent requires a long time to yield the final product, vin- since ancient times [1]. Vinegar is produced via a egar. In contrast, the submerged fermentation pro- two-stage fermentation process. The first stage is cess involves direct injection of oxygen into the anaerobic alcohol fermentation by yeast, in which substrate liquid using a generator or an acetator to fermentable sugars are converted to ethanol [2]. In increase the surface area, and therefore, the fermen- this stage, if the raw material for vinegar is cereals tation is faster. The surface fermentation method is such as rice, malt, or sorghum, saccharification of employed to produce traditional and selected vin- starch into fermentable sugars using mold is neces- egar, despite disadvantages such as a long fermenta- sary as the preceding step to alcohol fermentation tion period, a risk of contamination, and difficulty [3]. The second stage is aerobic acetification by of quality control. This is not only because volatile acetic acid bacteria (AABs), which involves oxida- aromatic compounds are retained, but also because tion of ethanol to acetic acid via acetaldehyde inter- the initial investment is more economical than that mediates [2]. for the submerged fermentation process [2,4]. Based on the processing technology employed, However, in static-fermented vinegar, surface film vinegar production can be broadly divided into two formation is often found, for example, Shanxi aged categories, surface fermentation and submerged [3], brown rice [5], wine [6], and strawberry vinegar fermentation processes. Although the surface fer- [7]. The surface film of vinegar was first reported in mentation method is preferred to the submerged 1886 to be made of cellulose produced by fermentation method because it is economical and it Gluconacetobacter xylinus (formerly Bacterium xyli- preserves some volatile aromatic compounds, it num and Acetobacter xylinum)[8]. In addition, CONTACT Jang-Eun Lee [email protected] Traditional alcoholic beverage research team, Korea Food Research Institute, Jeollabukdo 55365, Korea ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. MYCOBIOLOGY 251 other AABs such as A. tropicalis SKU1100 and A. 2.3. Organic acid analysis lovaniensis (formerly A. pasteurianus IFO3284) have Organic acids were analyzed using HPLC Agilent also been reported to produce cellulose [9,10]. It is 1200 series (Agilent, Santa Clara, CA) system. For known that the bacterial cellulose produced by G. separation, a Zorbax SB-AG column (250 Â 4.6 xylinus plays multiple roles such as protecting the mm, Agilent) was used at a setting of 35 C oven cell from desiccation, UV light, and coexisting com- temperature. The mobile phase was 20 mM phos- petitors, and aiding in adherence to surfaces to phate buffer adjusted to pH 2.0 using phosphoric allow maximal use of atmospheric oxygen [11]. acid, with a flow rate of 1.0 mL/min for all the However, in the vinegar industry, AABs and cellu- chromatographic separations. For chromatography, lose are targeted for removal because they cause 10 mL of a 50-fold diluted sample was injected and overoxidation and turbidity in vinegar [12]. analyzed for 7 min, after which the diode array Owing to increased awareness of the functional detector was used for detection at 210 nm. Organic and health properties of vinegar, recently, there has acid concentrations in the sample were determined been an increasing demand for high-quality vinegar using the calibration curve calculated from six naturally fermented using conventional starter cul- standard substances: acetic acid, citric acid, succinic ture and fermentation methods. Nonetheless, only a acid, lactic acid, malic acid, and oxalic acid. few studies have examined the contamination of, and cellulose film formation in, vinegar in Korea 2.4. Bacterial strain isolation and identification [12]. Therefore, the present study aimed to deter- mine the cause of the occurrence of surface in A proportion of the floating mass and the surface static-fermented rice vinegar by analyzing the char- film from the vinegar was spread on the culture acteristics of the vinegar and the responsible strains. medium plates for primary cell isolation. Single-col- ony isolation was carried out by consecutive pure isolation using a single isolated strain. Culture con- ditions were maintained at 27 C for 5 days. The 2. Materials and methods PCR primers used for identification were 27 F 50- (AGA GTT TGA TCM TGG CTC AG)-30 and 1492 2.1. Vinegar collection and culture media R50-(TAC GGY TAC CTT GTT ACG ACT T)-30. Three rice vinegars with surface film were obtained PCR was carried out using EF-Taq (SolGent, from a vinegar farmhouse. The vinegar was pro- Daejeon, Korea) with 20 ng of genomic DNA as the duced in the same brewery but not at the same time template in a 30 mL reaction mixture. First, Taq and processed by surface fermentation in a pot ino- polymerase was activated at 95 C for 2 min; fol- culated with A. pasteurianus. The selected strain was lowed by 35 cycles for 1 min at 95 C, 55 C, and isolated from farmhouse vinegar in Chungbuk prov- 72 C; and extension for 10 min at 72 C. The ince, Korea, because it has high acid-producing abil- amplified products were purified using a multiscreen ity. The collected vinegars were categorized as filter plate (Millipore Corp., Billerica, MA). vinegar 1 (V1), vinegar 2 (V2), and vinegar 3 (V3) Sequencing was carried out using the PRISM on the basis of the morphological characteristics of BigDye Terminator v3.1 Cycle Sequencing Kit. A their surface films. Glucose, yeast extract, and cal- DNA sample containing the extension products was cium carbonate (GYC) culture medium was used to mixed with Hi-Di formamide (Applied Biosystems, Foster City, CA), and the mixture was cultured at culture causative species of surface film formation isolated from the vinegar samples. The GYC 95 C for 5 min before it was incubated on ice for 5 medium was prepared using 10.0% glucose, 1.0% min. An ABI Prism 3730XLDNA analyzer (Applied Biosystems) was used for analysis. The phylogenetic yeast extract, 2.0% CaCO , and 1.5% agar with a pH 2 analysis was conducted in MEGA7 using Maximum of 6.8. Likelihood method. 2.5. Field-emission scanning electron 2.2. pH and total acidity microscopes The pH of the vinegar was measured using a pH To observe the morphology of AABs, isolated from meter (D-71G, Horiba, Kyoto, Japan). Total acidity surface film of vinegar, via scanning electron was calculated using the acetic acid coefficient microscopy (SEM), bacterial samples were fixed (0.006) and 20 mL of 20-fold diluted vinegar solu- with 2% paraformaldehyde and 2% glutaralde- tion titrated with a 0.1 N NaOH standard solution hyde in 0.05 M sodium cacodylate solution (pH (f = 1.000) to pH 8.3. 7.2) for 90 min at 4 C. The immobilized cells 252 J. H. YUN ET AL. were washed three times in 0.05 M sodium cacodylate buffer (pH 7.2), and fixed with 1% osmium tetroxide solution in 0.05 M sodium cacodylate buffer (pH 7.2) for 90 min at 4 C.