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And Myeolchi-Jeotgal by 16S Rrna Gene Sequencing, MALDI-TOF

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And Myeolchi-Jeotgal by 16S Rrna Gene Sequencing, MALDI-TOF J. Microbiol. Biotechnol. (2018), 28(7), 1112–1121 https://doi.org/10.4014/jmb.1803.03034 Research Article Review jmb Identification of Lactic Acid Bacteria in Galchi- and Myeolchi-Jeotgal by 16S rRNA Gene Sequencing, MALDI-TOF Mass Spectrometry, and PCR-DGGE S Yoonju Lee†, Youngjae Cho†, Eiseul Kim, Hyun-Joong Kim, and Hae-Yeong Kim* Institute of Life Sciences & Resources and Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea Received: March 27, 2018 Revised: May 30, 2018 Jeotgal is a Korean traditional fermented seafood with a high concentration of salt. In this Accepted: June 4, 2018 study, we isolated lactic acid bacteria (LAB) from galchi (Trichiurus lepturus, hairtail) and First published online myeolchi (Engraulis japonicas, anchovy) jeotgal on MRS agar and MRS agar containing 5% June 6, 2018 NaCl (MRS agar+5% NaCl), and identified them by using 16S rRNA gene sequencing and *Corresponding author matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF Phone: +82-31-201-2660; MS) as culture-dependent methods. We also performed polymerase chain reaction-denaturing Fax: +82-31-204-8116; E-mail: [email protected] gradient gel electrophoresis (PCR-DGGE) as a culture-independent method to identify bacterial communities. Five samples of galchi-jeotgal and seven samples of myeolchi-jeotgal † These authors contributed were collected from different regions in Korea. A total of 327 and 395 colonies were isolated equally to this work. from the galchi- and myeolchi-jeotgal samples, respectively. 16S rRNA gene sequencing and MALDI-TOF MS revealed that the genus Pediococcus was predominant on MRS agar, and Tetragenococcus halophilus on MRS agar+5% NaCl. PCR-DGGE revealed that T. halophilus, Tetragenococcus muriaticus, and Lactobacillus sakei were predominant in both types of jeotgal. T. halophilus was detected in all samples. Even though the same species were identified by both culture-dependent and -independent methods, many species identified by the culture- S upplementary data for this dependent methods were not in the bacterial list identified by the culture-independent paper are available on-line only at methods. The distribution of bacteria in galchi-jeotgal was more diverse than in myeolchi- http://jmb.or.kr. jeotgal. The diverse LAB in galchi- and myeolchi-jeotgals can be further studied as candidates pISSN 1017-7825, eISSN 1738-8872 for starter cultures to produce fermented foods. Copyright© 2018 by The Korean Society for Microbiology Keywords: Lactic acid bacteria, galchi-jeotgal, myeolchi-jeotgal, 16S rRNA gene sequencing, and Biotechnology MALDI-TOF MS, PCR-DGGE Introduction diverse raw materials, including whole meat, intestines, or eggs of fish and shellfish. There are approximately 150 Jeotgal or jeot is traditional fermented and salted seafood different kinds of jeotgal in Korea that differ by main widely consumed in regions that have rice-based diets, ingredient and/or processing method [1]. Jeotgal is also such as Korea, Japan, and Southeast Asia. This food made in other countries; it is known as pla-ra and pa-daek product is manufactured by adding salt to various types of in Thailand and Laos [2], and nuoc-man in Vietnam [3]. seafood to prevent spoilage and has a distinctive flavor as a Jeotgal is made by adding salt to a final concentration of a result of enzymatic reactions among the ingredients. The 20-30% to the raw material and allowing the mixture to various types of Korean traditional fermented and salted ferment for 2-3 months [1]. Jeotgal is used as an important seafood, such as jeotgal, aekjeot (fish sauce), and sikhae condiment in kimchi or for seasoning Korean-style stews to (fermented fish with grains) are distinguished by the enhance flavor and taste. Additionally, the bacterial manufacturing process. In Korea, jeotgal is made using communities in jeotgal are thought to improve appetite J. Microbiol. Biotechnol. Identification of Lactic Acid Bacteria in Jeotgal 1113 and digestion [1]. culture-independent method is polymerase chain reaction- In general, most bacteria in jeotgal originate from the denaturing gradient gel electrophoresis (PCR-DGGE). seafood used as the main ingredient or the environment of Genomic DNA extracted directly from each sample is that seafood. Owing to the high salt concentration of separated by the GC content of the DNA, with different jeotgal, halophilic or halotolerant bacteria can survive and bacteria showing specific band patterns in the gradient gel participate in the fermentation, whereas the growth of after electrophoresis. This method has been used to detect harmful bacteria is decreased [1]. Examples of bacterial diverse bacterial communities grown in culture as well as genera present in jeotgal include Halobacterium and Halomonas organisms that cannot be cultured. PCR-DGGE has been in addition to Bacillus, Brevibacterium, Flavobacterium, used in several studies to investigate microorganisms in Micrococcus, Pediococcus, Pseudomonas, and Staphylococcus various fermented foods, including jeotgal [2, 15-18]. [1]. The bacterial communities vary according to the Among the many kinds of jeotgals made using various ingredients and fermentation period. There has been seafoods, galchi (hairtail)- and myeolchi (anchovy)-jeotgal extensive research into the bacteria present in jeotgal are common kimchi ingredients that are widely consumed because they contribute to its fermentation and ripening. in Korea. However, few studies have reported the lactic acid bacteria Our aim in this study was to investigate the bacterial (LAB) communities present in jeotgal and their functions in communities, especially LAB, present in galchi- and the fermentation process [4]. This is mostly because LAB myeolchi-jeotgals using diverse identification methods. cannot survive in the high salt environment of jeotgal. In First, the bacterial communities from various jeotgal this study, we identified LAB communities present in samples purchased from different regions of South Korea different jeotgal samples using agars containing salt. were identified using 16S rRNA gene sequencing and Tools to investigate bacterial community composition MALDI-TOF MS of isolates grown on agar plates with or can be categorized as culture-dependent or -independent. without salt as culture-dependent methods. Additionally, Culture-dependent methods rely on the application of the bacterial communities were analyzed using PCR-DGGE molecular techniques such as 16S rRNA gene sequencing as a culture-independent method. and matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) to identify Materials and Methods isolates from cultured media [5-7]. 16S rRNA gene sequencing is one of the most commonly used techniques Jeotgal Sample Preparation and Measurement of pH and Salinity for bacterial identification [4, 8]. However, this technique is Five samples of galchi-jeotgal were collected from five markets time-consuming because genes from extracted DNA need (Se-1, Se-2, Se-3, So-1, and Y-1) in three regions (Seoul, Sokcho, to be amplified by the polymerase chain reaction (PCR) and and Yeosu) and seven samples of myeolchi-jeotgal were be purified for sequencing. In contrast, MALDI-TOF MS is purchased from seven markets (B-1, B-2, B-3, Se-1, Se-2, Se-3, and Y-1) in three regions (Busan, Seoul, and Yeosu) in Korea. To an effective and accurate identification technique that can measure the pH and salinity of each sample, 1 g of sample and be performed in a short time period using only a single 10 ml of sterilized water were mixed and centrifuged at 16,200 ×g colony, and a large number of isolates can be analyzed for 10 min. The pH of the collected supernatant was then measured simultaneously [9, 10]. This quick and accurate technique has using an Orion star a211 pH meter (Thermo Fisher Scientific, previously been used to characterize bacterial communities USA) and the salinity was measured using a PAL-03S refractometer in diverse fermented foods from various countries, including (ATAGO, Japan). fermented meat nem chua from Vietnam [11] and vegetable dua muoi and ca muoi [10], including mukeunji, a long- Isolation of Lactic Acid Bacteria term aged kimchi [12], as well as yogurt and probiotics Ten grams of each sample was mixed with 90 ml of sterile water [13]. Nevertheless, few studies have used MALDI-TOF MS in a Stomacher filter bag (Seward Limited, UK) and homogenized analysis to identify bacterial communities in jeotgal. using a Stomacher 400 (Seward Limited, UK) for 30 sec at Although the distribution of viable cells in each sample 230 rpm. After serial dilution of the homogenized samples with phosphate-buffered saline, 100 μl of highly diluted sample was can be determined and isolates acquired through culture- spread onto MRS agar (Acumedia, USA) and MRS agar containing dependent methods, bacteria present at very low levels or 5% NaCl (MRS agar+5% NaCl), and incubated anaerobically for dead bacteria cannot be cultured [14]. These hard-to- 72 h at 30°C to culture LAB. Single colonies that differed in culture microorganisms can be detected effectively by morphological characteristics were picked from each plate and culture-independent methods [15]. One conventional subcultured in MRS broth (Acumedia, USA) and MRS broth July 2018 ⎪ Vol. 28⎪ No. 7 1114 Lee et al. containing 5% NaCl (MRS broth+5% NaCl) for 24 h at 30°C. Analysis of Bacterial Communities by PCR-DGGE Enriched bacterial isolates in both broths were mixed with 80% Ten grams of each sample was mixed with 90 ml of sterile water glycerol at the ratio of 7:3 and stored at -80°C. in a Stomacher filter bag and homogenized using a Stomacher 400 at 230 rpm for 30 sec. The homogenized samples were filtered Comparison of Whole Cell Protein Patterns through SDS-PAGE through four layers of cheesecloth and centrifuged at 4,250 ×g, at Analysis 4°C for 15 min. The collected pellets were washed twice with Each isolate was cultured in MRS broth or MRS broth+5% NaCl sterile water, and genomic DNA was extracted using the P&C for 24 h at 30°C and centrifuged at 16,200 ×g, 4°C for 15 min.
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