J. Microbiol. Biotechnol. (2017), 27(10), 1744–1752 https://doi.org/10.4014/jmb.1702.02027 Research Article Review jmb Microbial Communities and Physicochemical Properties of Myeolchi Jeotgal (Anchovy Jeotgal) Prepared with Different Types of Salts S Jae Min Shim1, Kang Wook Lee1, Zhuang Yao1, Jeong A Kim1, Hyun-Jin Kim1,2 , and Jeong Hwan Kim1,2* 1Division of Applied Life Science (BK21 Plus), Graduate School, 2Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea Received: February 13, 2017 Revised: May 4, 2017 Myeolchi jeotgals (MJs) were prepared with purified salt (PS), solar salt aged for 1 year (SS), Accepted: July 28, 2017 and bamboo salt (BS) melted 3 times at 10% and 20% (w/w) concentrations, and fermented for o First published online 28 weeks at 15 C. BS MJ showed higher pH and lower titratable acidities than the other July 31, 2017 samples because of the alkalinity of bamboo salt. Lactic acid bacteria counts increased until 4- *Corresponding author 6 weeks and then decreased gradually, and were not detected after 20 weeks from MJs with Phone: +82- 55-772-1904; 10% salt. Yeast counts of PS MJs were higher than those of BS and SS MJs. Bacilli were detected Fax: +82-55-772-1909; E-mail: [email protected] in relatively higher numbers throughout the 28 weeks, like marine bacteria, but archae were detected in lower numbers during the first 10 weeks. When 16S rRNA genes were amplified from total DNA from PS MJ (10% salt) at 12 weeks, Tetragenococcus halophilus was the major species. However, Staphylococcus epidermidis was the dominant species for BS MJ at the same S upplementary data for this time point. In SS MJ, T. halophilus was the dominant species and S. epidermidis was the next paper are available on-line only at http://jmb.or.kr. dominant species. BS and SS MJs showed higher amino-type nitrogen, ammonia-type nitrogen, and volatile basic nitrogen contents than PS MJs. SS and BS were better than PS for pISSN 1017-7825, eISSN 1738-8872 the production of high-quality MJs. Copyright© 2017 by The Korean Society for Microbiology and Biotechnology Keywords: Myeolchi jeotgal, solar salt, bamboo salt, purified salt Introduction contribute to development of the unique flavor, taste, and texture of each jeotgal [3]. Solar salt (SS) contains significant Jeotgals are salted and fermented Korean traditional amounts of minerals such as Mg, K, Ca, Fe, Zn, and Cu in seafoods prepared from various fishes, shrimps, oysters, addition to Na and Cl, and these minerals are essential for fish eggs, and fish intestines [1]. Many different types of growth of many microorganisms, affecting the qualities of jeotgals are produced depending upon the raw materials, fermented foods [4]. Bamboo salt (BS) is obtained by regions, and personal preferences, but the basic procedures recrystallization of SS after it is melted at high temperature. are the same. Raw materials are mixed with salt in a SS is filled in a bamboo trunk, and the top and bottom are container and fermentation starts. Final salt concentrations sealed with clay, and then heated in a furnace. SS is melted are 10-30% (w/w). Fermentation continues for several at 800-1,500°C and then recrystallized as the temperature months, and several years for some jeotgals. During decreases [5, 6]. fermentation, the unique texture and flavor of each jeotgal SS has been widely used for fermented foods such as is developed through hydrolysis of proteins by proteases kimchi (Korean fermented vegetable) and doenjang (Korean originated from the raw materials and microorganisms [2]. fermented soybean), and use of BS for fermented foods is Salt is an essential ingredient for jeotgals, and only salt also in the rise because of its functionalities [6]. However, and raw material are used for preparation of some jeotgals. few studies have been done on the effects of different types The main role of salt is growth inhibition of spoilage of salts on the qualities of fermented foods. In particular, microorganisms during fermentation. Under high salt the effects of SS and BS on the growth of microorganisms concentrations, salt-tolerant microorganisms can grow and during fermentation are rarely studied. Growth of micro- J. Microbiol. Biotechnol. Myeolchi Jeotgal with Different Types of Salts 1745 organisms affects the types of metabolites produced in using the primer pair F1 (5’-TGACGGTACCTAACCAGAAAGCCA fermented foods, and eventually determines the quality of CGGCT-3’) and R1 (5’-GTTTGTCACCGGCAGTCACCTTAGAGT foods. Therefore, understanding the effects of different salt GC-3’). PCR was done under the following conditions: denaturation types on the growth of microorganisms is important for the at 94°C for 5 min and then 30 cycles of 30 sec at 94°C, 30 sec at production of high-quality and functional fermented foods. 62°C, and 1 min at 72°C. The amplified fragments were purified from an agarose gel using a PCR purification kit (FavorPrep PCR In this work, myeolchi (anchovy, Engraulis japonica) jeotgals purification kit; Favorgen, Taiwan) and then ligated with the (MJs) were prepared with three different types of salts: pGEM-T easy vector (Promega, USA). E. coli DH5α (Invitrogen, purified salt (PS), 1-year aged SS, and BS. Jeotgal samples USA) competent cells were transformed with the ligation mixture were fermented for 28 weeks at 15°C, and growth of and colonies were selected on LB plates with ampicillin (100 μg/ml), microorganisms was examined together with other properties isopropyl β-D-1-thiogalactopyranoside (500 μg/ml), and 5-bromo- of jeotgal samples during fermentation. 4-chloro-3-indolyl-β-D-galactopyranoside (80 μg/ml). Thirty well isolated colonies were selected from each MJ and their plasmids Materials and Methods were prepared for DNA sequencing. DNA sequences were determined at Cosmogenetech (Korea), and the BLAST program Preparation of Myeolchi Jeotgal provided by the National Center for Biotechnology Information Myeolchi (anchovy) was purchased from a local fish market was used to find homologous sequences in the data library (http:// (Tongyeong Suhyup, Korea) in December 2015. Immediately after www.ncbi.nlm.nih.gov/ BLAST). purchase, the myeolchi was washed three times under running tap water, and stood for 10 min to remove excess water. Each 20 kg pH and Titratable Acidity Measurements of myeolchi was then mixed with salt. The NaCl concentration of Ten gram of each homogenized MJ was mixed with 40 ml of the MJs was adjusted to 10% and 20% (w/w), respectively, by distilled water, and shaken for 1 h in a water bath (150 rpm, 30°C). adding different amount of each salt: 2,017 g (10%) and 4,033 g The supernatant was obtained after centrifugation (4,000 ×g, (20%) for PS (Hanju, Ulsan, Korea, 2015; NaCl 99.18%); 2,505 g 20 min). The pH of the supernatant was measured using a pH (10%) and 5,010 g (20%) for SS (Taepong salt farm, Sinan, Jeonnam, meter (DP-215M; DMS, Korea) and titratable acidity (TA) was Korea; aged for 1 year, NaCl 79.84%); and 2,116 g (10%) and 4,232 g calculated by titrating the supernatant with 0.1 N NaOH to pH (20%) for BS (Insanga, Hamyang, Gyeongnam, Korea; melted and 8.4. The amount of NaOH was used to calculate the amount of recrystallized 3 times, NaCl 94.54%). The MJs were fermented for lactic acid (%). 28 weeks at 15°C and analyzed every 2 weeks during fermentation. Amino-Type Nitrogen (ANN), Ammonia-Type Nitrogen (AMN) Viable Cell Counting and Volatile Basic Nitrogen (VBN) Measurements Ten gram of each MJ was mixed with 90 ml of peptone water Five gram of each homogenized MJ was mixed with 95 ml of (0.1% (w/v)) and homogenized using a stomacher (Stomacher 80; distilled water, and the mixture was shaken for 1 h in a water bath Seward, USA). The homogenate was filtered with a bag filter (150 rpm, 30°C). The ANN contents were measured by the formol (Interscience, France) and diluted serially with peptone water. titration method [7]. The AMN contents were measured by an Diluted samples were spread on de Man-Rogosa-Sharpe (MRS) indophenol blue method [8]. Five gram of each homogenized MJ agar (Acumedia, USA) plates for lactic acid bacteria (LAB) was mixed with 95 ml of distilled water, and the mixture was counting, marine agar (BD Difco, USA) plates for marine bacteria shaken for 1 h in a water bath (150 rpm, 30°C). The mixture was counting, LB (Acumedia) plates for bacilli counting, YM agar (BD centrifuged, and 1 ml of supernatant was mixed with 3 ml of Difco) plates for yeasts counting, and DSMZ954 agar (casamino solution A (phenol 10 g, sodium nitroprusside dehydrate 1 g, EDTA 4 g, per liter). After standing for 5 min at room temperature, 5 ml acids 5 g, yeast extract 5 g, Tris 12.1 g, KCl 2 g, MgCl2·6H2O 20 g, of solution B (Na HPO ·12H O 9 g, NaOH 6 g, and NaOCl 10 ml, CaCl2·2H2O 0.2 g, NaCl 200 g, agar 20 g, per liter, pH 7.4) plates 2 4 2 for archaea counting. The plates were incubated for 24 h at 37°C per 1 liter) was added and stood for 20 min at 37°C. Changes in (marine, LB, and DSMZ954 agar plates), and 48 h at 30°C (MRS absorbance were measured at 665 nm with a spectrophotometer and YM agar plates), respectively. (UV-1601, Shimadzu, Japan), and the AMN content was determined on the basis of the standard curve of (NH4)2SO4. For measurement Identification of Bacterial Species by a Culture-Independent of VBN, 10 g of each homogenized MJ was mixed with 30 ml of Method distilled water and 20ml of 20% trichloroacetic acid solution.