https://orcid.org/0000-0003-0281-2101
Figures
Abstract
DNA-sequencing was performed on the V3-V4 regions of 16S rRNA genes to investigate the microbial diversity of five samples of fermented freshwater fish (pla-ra) from three provinces in northeastern Thailand. The samples had salt concentrations ranging from 7 to 10%, pH values from 4.83 to 7.15, and D-/L-lactic acid concentrations of 90 to 450 mg/l. A total of 598 operational taxonomic units were annotated at various taxonomic ranks based on the SILVA Database. The lactic-acid and halophilic genera Tetragenococcus, Halanaerobium and Lactobacillus were among the dominant taxa of bacteria. The top 20 non-redundant taxa were considered in more detail. In two pla-ra samples, Tetragenococcus muriaticus was commonly identified. Halanaerobium fermentans was the most abundant species in a third sample and co-dominant in another sample. Lactobacillus rennini was dominant in the pla-ra sample from Roi Et Province. Additionally, other beneficial bacteria were detected including Staphylococcus nepalensis, Lactobacillus sakei, Lactobacillus pentosus, Weissella confusa, and Bifidobacterium bifidum. Differences between samples may be due to use of different raw materials, salt concentrations, recipes, processes and fermentation periods. The microbial communities in pla-ra provide a better understanding of the production outcomes of traditional products. Further optimization of the fermentation process, for example by using dominant bacterial taxa in starter cultures, may improve processes of food fermentation, food quality and flavor control, providing useful guidelines for industrial applications.
Citation: Rodpai R, Sanpool O, Thanchomnang T, Wangwiwatsin A, Sadaow L, Phupiewkham W, et al. (2021) Investigating the microbiota of fermented fish products (Pla-ra) from different communities of northeastern Thailand. PLoS ONE 16(1): e0245227. https://doi.org/10.1371/journal.pone.0245227
Editor: Suzanne L. Ishaq, University of Maine, UNITED STATES
Received: June 12, 2020; Accepted: December 27, 2020; Published: January 14, 2021
Copyright: © 2021 Rodpai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files. All sequence reads have been deposited at the NCBI Sequence Read Archive (SRA) under project accession number PRJNA657478. Please see: https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA657478.
Funding: This study was supported by the Thailand Research Fund (grant no. DPG6280002) awarded to WM and PMI, the Khon Kaen University, Thailand (grant nos. I62-00- 24-11 awarded to WM and PMI and PD2563-08 awarded to WM and RR) and the Faculty of Medicine, Khon Kaen University grant awarded to RR, OS and WM (DR63101). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Please modify these sentences in financial disclosure.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Fermented fish, known as pla-ra or pla-daek, is a traditional product used as a condiment and a main dish by people in Thailand and Lao PDR [1]. Pla-ra has been consumed daily by Thai people since ancient times [2]. It is a famous ingredient used as seasoning in the north-eastern (Issan) food of Thailand, such as “namprik pla-ra”, “kang lao”, “namya”, and “somtum pla-ra” (a green papaya salad) which is popular in every region of Thailand [3, 4]. Pla-ra is in very high demand, with 20,000–40,000 tons/year being produced for both domestic use and export (worth about 800 million baht/year) [5]. Given this, the National Bureau of Agricultural Commodity and Food Standards has set quality standards for pla-ra [6] and suggested that pla-ra has an impact on the national economy.
Pla-ra is produced by fermenting raw fish with rice bran or roasted rice flour and salt in a closed container for at least six months to a year. Pla-ra has a very strong smell, and its salty and sour flavors depend on the amount of salt added and of lactic acid resulting from the fermentation process. The fermented products are usually stored at ambient temperatures and can last for a year or longer in the tropical climates of Southeast Asia. It is considered that longer fermentation makes the product taste better [3]. Rice bran serves as a source of carbon for fermenting microorganisms [7]. The most abundant amino-acid in pla-ra is glutamic acid, which suggests that the preferred taste in these fermented fish products is related to umami substances [7, 8]. Pla-ra varies greatly in flavor from community to community and between regions, and customers often have strong preferences for particular sources [2, 4]. In the northeastern part of Thailand, pla-ra is usually produced at home for family consumption, with excess being sold at local markets [9].
The quality and flavor of pla-ra may be related to its microbial community [10]. For example, to improve the flavor of fermented products, a microbial starter containing species such as Tetragenococcus halophilus or Staphylococcus sp. SK1-1-5 has been used in fish sauce production [10–12]. Variation in the microbial community may lead to differences in the quality, nutritional value and health benefits of fermented products [9, 13]. However, no bacterial starter cultures use in pla-ra processing. Therefore, knowledge of the microbial diversity in pla-ra is important. Although several studies have investigated communities of microorganisms in fermented fish products including pla-ra, there is little information from studies that did not depend on culturing microorganisms for identification [1, 5]. Using culture-independent techniques and sequencing of 16S rRNA, various lactic acid bacteria and/or halophilic or salt-tolerant bacteria have been identified from pla-ra products [1, 5]. However, the microbial diversity specifically found in pla-ra samples from northeastern Thailand is still unknown. In this work, we have identified the native bacterial communities in conventional fermented pla-ra from different areas of northeastern Thailand based on analysis of DNA sequences from the V3-V4 region of 16S rRNA gene. Sequences were generated using next-generation sequencing technology without the need for isolation and cultivation of the microorganisms. The results should improve our understanding of the differences in bacterial diversity between northeastern communities, thereby helping us to select appropriate functional bacteria for manufacturing high-quality and distinctive pla-ra products.
Materials and methods
Fermented-fish samples
Samples of fermented fish (pla-ra) were obtained from markets in three provinces in northeastern Thailand; Khon Kaen (3 markets), Kalasin (1 market) and Roi Et (1 market). Information about the products was provided by the sellers. In each case, the three main ingredients were raw freshwater fish, salt, and rice bran for fermentation (Table 1). The samples were collected between January 1st and February 5th, 2019. After purchase, the samples were place in foam boxes for transport to the laboratory. Aliquots taken for DNA preparation were frozen at -20°C to minimize further microbial activity until use. In triplicate, a ten-fold dilution of each sample using deionized water was prepared to investigate salt concentration using a salt meter (DRETEC Co., LTD, Saitama, Japan). The pH of each pla-ra sample was directly measured with a pH meter (Mettler Toledo, Columbus, OH). Lactic acid concentrations were measured using a QuantiQuikTM D-/L-lactic acid test strip (BioAssay Systems, Hayward, CA) following the manufacturer's instructions. These measurements were performed on the day when the sample was first thawed.
In each case, a mixture of fish was used, and fermentation lasted >1 year.
Bacterial DNA extraction and sequencing
Total DNA was isolated directly from the pla-ra samples (200 mg per sample) using a QIAamp® DNA stool mini kit (Qiagen, Hilden, Germany) as recommended by the manufacturers. Briefly, the pla-ra samples were extracted in optimized buffers in combination with inhibitEX buffer (Qiagen) to remove PCR inhibitors. Proteinase K was subsequently added. DNA from lysates was isolated using a DNA-binding silica-gel membrane column. Remaining impurities were efficiently removed in two wash steps. Amplification-ready DNA was then eluted in low-salt buffer (Qiagen). DNA concentration and purity were monitored using a NanoDrop™ One Spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and electrophoresis through 1% agarose gels. Each DNA sample was then diluted to 1 ng/μl using sterile water.
The V3-V4 regions of the 16S rRNA gene were amplified using universal region-specific primers 341F (5’-CCT AYG GGR BGC ASC AG-3’) and 806R (5’-GGA CTA CNN GGG TAT CTA AT-3’) tagged with sample-identifying barcodes. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA). The PCR cycling conditions were as follows: initial denaturation at 98°C for 1 min, followed by 30 cycles at 98°C for 10 s, 50°C for 30 s, and 72°C for 30 s, and a final extension step at 72°C for 5 min. The PCR products (400–450 bp in length) were quantified, qualified and used for library preparation. The libraries were generated using an Ion Plus Fragment Library Kit (Thermo Fisher Scientific) and quantified using Qubit and qPCR, then sequenced on an IonS5TMXL instrument (Thermo Fisher Scientific). Single-end reads were generated.
Analysis and annotation of operational taxonomic units (OTUs)
Single-end reads were assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Quality filtering on the raw reads was carried out under specific filtering conditions in order to obtain the high-quality clean reads [14] using Qiime (V1.7.0, http://qiime.org/scripts/split_libraries_fastq.html) [15]. The reads were compared with the reference database (Gold database) using the UCHIME algorithm (http://www.drive5.com/usearch/manual/uchime_algo.html) [16] to detect chimeric sequences. Any such sequences were removed using the UCHIME algorithm [17]. From the effective reads (Table 2), sequences sharing ≥ 97% similarity were assigned to the same OTU using Uparse software (Uparse v7.0.1001) [18]. A representative sequence from each OTU was screened for further annotation. Mothur software was used to compare our sequences against the SSU rRNA database, SILVA (http://www.arb-silva.de/), for species annotation at each taxonomic rank [19].
Results
Physicochemical properties of pla-ra
Details of salinity, pH, and D-/L-lactic acid content of each pla-ra sample are presented in Table 1. Salt content ranged from 7% to 10%, while the pH value range was 4.83–7.15. The highest pH level (7.15) was in the sample from Ban Thum market in Khon Kaen (KK3). The highest level of total lactic acid was 450 mg/l in the samples from Nonmuang market in Khon Kaen (KK1) and Yang Talat, Kalasin (KS). In samples from Nong Ruea market of Khon Kaen (KK2) and Nai Mueang, Roi Et (RE), total lactic acid was approximately 337.5 mg/l with L-lactic acid level being higher than D-lactic acid level. The sample from KK3 contained the lowest total lactic acid level, at approximately 90 mg/l.
Microbial diversity in pla-ra
Amplicons from the V3-V4 regions of 16S rRNA gene were sequenced to generate raw reads, and chimeric reads were filtered out. The output statistics are shown in Table 2. Sequences sharing ≥ 97% similarity were assigned to the same OTU. A total of 598 OTUs in 17 phyla were identified from the five pla-ra samples (S1 Table). The top 10 phyla (in terms of numbers of sequences) were considered in more detail. Firmicutes (77.1–99.4%) was the most-represented phylum in all samples (Fig 1A), while the top 10 families and genera varied among samples (Fig 1B and 1C). For the sample KK1, reads from the genus Tetragenococcus (Enterococcaceae), were by far the most abundant. Families Enterococcaceae and Halanaerobiaceae (genera Tetragenococcus and Halanaerobium, respectively) were abundant in sample KK2. In sample KK3, Halanaerobium (Halanaerobiaceae) was predominant. The families Lactobacillaceae and Enterococcaceae were abundant in sample RE, with smaller contributions from Vibrionaceae and Halanaerobiaceae. Families Enterococcaceae (genus Tetragenococcus) and Vibrionaceae were most abundant in the KS sample.
A) Phylum, B) Family, C) Genus.
Forty-seven OTUs were present in all samples (Fig 2 and S2 Table). Each sample also contained some unique OTUs (Fig 2). The highest number of OTUs was detected in sample KK2 (354 OTUs, 116 being unique) (Table 2 and Fig 2). In contrast, the other samples had fewer unique OTUs—30, 29, 81 and 46 OTUs for the samples KK1, KK3, RE and KS, respectively. The unique OTUs are listed for each sample in S3 Table.
All OTUs at each taxonomic rank were annotated using comparisons against the SILVA database. The top 20 non-redundant taxa are specified in Fig 3. As illustrated in Figs 1 and 3, each pla-ra sample appeared to contain two to three dominant bacterial species. In particular, Tetragenococcus muriaticus, Halanaerobium fermentans and Lactobacillus rennini were prominent in one or more samples. The pla-ra samples KK1, KK2 and KS had T. muriaticus as the dominant species, whereas KK3 had H. fermentans and RE had L. rennini as the dominant species.
Numbers in the heatmap are reads per sample.
Discussion
The present study used the IonS5TMXL sequencing platform for a comprehensive study of the bacterial community in pla-ra from five different areas across three provinces of northeastern Thailand. The pH, salinity and L-/D-lactic acid content of each pla-ra sample were measured. Nevertheless, these varied between samples: there are no standardized contents and recipes for pla-ra products [1, 5]. The pH level was likely affected by the lactic acid concentration: sample KK3 had the highest pH (7.15) but the lowest lactic acid content (90 mg/l) and salt concentration (7%). The starting salt concentration may one of the factors that leads to varying levels of lactic acid bacteria growth and their products, which in turn affects other sensory parameters in pla-ra [3, 7].
Seventeen phyla of bacteria as well as many genera and species were found in the samples. Community composition varied between samples, and also in comparison with previous reports [1, 5]. However, twenty-two genera from 47 OTUs were present in all our pla-ra samples (S2 Table) and among these were both beneficial (such as Lactobacillus) and harmful species (such as Clostridium and Escherichia-Shigella) [20, 21]. The phylum Firmicutes dominated the bacterial community, and especially the genera Tetragenococcus, Halanaerobium, Lactobacillus. These three genera, as well as Weissella, Pediococcus, Enterococcus, Aerococcus, Clostridium, and Sphingomonas, have been previously isolated from fermented fish (pla-ra, pa-daek, pla-som, pla-chom). Lactic-acid bacteria such as Lactobacillus and Tetragenococcus, producing lactic acid as a by-product of their carbohydrate metabolism, are the main genera in fermented food [1, 22–24]. We found high levels of D-/L-lactic acid in the pla-ra samples (KK1, KK2, RE and KS) in which Tetragenococcus and Lactobacillus were dominant, whereas sample KK3 was dominated by Halanaerobium, a halophilic taxon, and contained a lower concentration of lactic acid. This result may be due to different compositions of starting materials and recipes for pla-ra [1, 3, 7].
Tetragenococcus muriaticus is a halophilic lactic-acid species found in various salted and fermented fish [10, 25, 26]. It was detected in all samples and was the principal species in pla-ra samples KK1 and KS. These samples contained 9% salt and 450 mg/l D-/L-lactic acid but differed in their pH (5.13 and 4.83, respectively). However, the optimal growth parameters of this species appear to be broad. According to a previous report investigating pla-ra from central Thailand and Lao PDR, T. muriaticus is viable in salinities ranging from 11.3 to 20.3%, D-/L-lactic acid concentrations 0.40–0.88 g/100 g (equivalent to 4000 mg/l—8800 mg/l) and pH values between 5.6 and 6.1 [1]. Satomi et al. [27] reported an optimal pH between 7.5 and 8.0 and salt concentration about 7 to 10%: but T. muriaticus was able to grow in a wide range of pH (4.2 to 8.5) and in 1% to 25% salt while producing L-lactic acid. Furthermore, as the fermentation period increases to 12 months or longer, Tetragenococcus can rapidly grow and replace other taxa to become the most abundant genus [10]. This genus also reduces harmful substances such as ammonia and amines in fish sauce: numbers of Tetragenococcus and trimethylamine levels during fish sauce fermentation are negatively correlated [10]. Tetragenococcus may be a good bacterium to be used in pla-ra production [10].
Halanaerobium fermentans was the dominant species in pla-ra sample KK3, and was co-dominant with T. muriaticus in pla-ra sample KK2. Halanaerobium fermentans, a strictly anaerobic and moderately halophilic bacteria, can be isolated from salted puffer fish ovaries and its optimum growth condition is at 10% (w/v) (7–25%) salt, 35°C (15–45°C) and pH 7.5 (6.0–9.0) [28]. Unsurprisingly, the pla-ra from KK3 had the highest pH and lowest D-/L-lactic acid level, making it more suitable for H. fermentans growth. Sample KK2 contained as a co-dominant species T. muriaticus. This sample had 10% salt, 337.5 mg/l D-/L-lactic acid, and pH 5.02. Halanaerobium appears to be the dominant taxon until the 12th month of fish sauce fermentation, after which it is replaced by other taxa (such as Tetragenococcus) [10]. Similarly, in another fermented seafood, traditional Korean salted shrimp, that is normally fermented for 4–5 months, Halanaerobium is one of the dominant taxa after 49 days of fermentation [29]. Jung et al. [29] suggested that the Halanaerobium may be responsible for spoilage during fermentation. The presence of Halanaerobium and its products could be an indicator of over-fermentation of seafood.
Interestingly, the pla-ra microbiome in the sample from RE consisted mostly of Lactobacillus rennini, and other Lactobacillus species such as L. acidipiscis, L. acetotolerans and L. crustorum. The first of these was also detected in pla-ra products from Thailand and Lao PDR using polymerase chain reaction-denaturing gradient gel electrophoresis [1]. The genus Lactobacillus occurs naturally in various food products. Members of Lactobacillus are considered to be harmless and providing health benefits to humans and animals [20]. Indeed, they are commonly used as probiotics in food [30]. The dominant species, L. rennini, was originally isolated from rennet and associated with cheese spoilage [31]. It is a homofermentative species that can produce both D- and L-lactic acid. It is also related to L. casei and L. plantarum, which are probiotics and extensively used in the food industry as a microbial starter. Lactobacillus rennini possesses higher resistance to salt than do the other species [31–33]. Its presence as a dominant species in the pla-ra product from RE may benefit human health and could lead to improvements in fermented fish industrial applications. Lactobacillus acidipiscis was originally found in fermented fish (pla-ra and pla-chom) in Thailand and is capable of producing L-lactic acid from glucose [34]. The pla-ra product from RE contained more L-lactic acid (225 mg/l) than D-lactic acid (112.5 mg/l), possibly produced by L. acidipiscis but also by other species that cannot be specified.
Staphylococcus nepalensis was commonly found in all pla-ra samples, although with low read counts in each case. The genus Staphylococcus is naturally found in the environment and also in fermented fish, fermented seafood and fish sauce [35–38]. Staphylococcus species (S. carnosus and S. xylosus) are often used as starter cultures for enhancing the flavor and color of sausage products, controlling the fermentation process and improving product safety [39, 40]. Moreover, there is some evidence that Staphylococcus (S. nepalensis and S. xylosus) are able to improve odor by affecting volatile compounds in the fish sauce [35, 36]. Their presence in our pla-ra samples might be related to odor and taste that is different in each region of Thailand.
Ignatzschineria ureiclastica is another species found in all pla-ra samples. This genus is commonly isolated from flesh flies [41]. It may have been acquired from the environment.
Other species of interest include Weissella paramesenteroides, Weissella confusa, Brochothrix thermosphacta, Lactobacillus sakei, Lactobacillus pentosus, Lentibacillus juripiscarius, Psychrobacter cibarius and Bifidobacterium bifidum. These are all lactic-acid and halophilic bacteria common in fermented foods. Some have been used as probiotics for humans and animals [34, 42–47]. Additionally, to our knowledge, many genera we found in this study have not been previously reported in pla-ra; these include Fusobacterium, Subdoligranulum, Ruminococcaceae_UCG-014, Erysipelotrichaceae_UCG-003 and Bifidobacterium (Fig 1 and S1 Table).
Pathogenic bacteria were identified in pla-ra such as Clostridium haemolyticum, Clostridium perfringens, and Vibrio fluvialis [21, 47–49]. These contaminants may cause foodborne disease when using pla-ra as an ingredient for uncooked food. In particular, the acceptable microbiological concentration of C. perfringens is not more than 100 cfu/g pla-ra according to food hygiene criteria produced by Thai agricultural standard committees [6]. However, there is no history of severe illness caused by pla-ra in Thai communities. It seems that the preparation process and recipe destroy and prevent the growth of pathogens [7, 50]. Moreover, infection by these pathogenic agents may result in no symptoms, or mild to moderate symptoms, depending on the pathogenic potential of organisms, the host’s immune system, and the microbiota of local people [51–53]. Next-generation sequencing technology could potentially be used for screening for food pathogens in the pla-ra and/or other fermented fish products as a strategy to improve food safety standards.
Many species (598 OTUs) were found in the five pla-ra samples and there was considerable diversity in the microbiota among these samples. This may be due to different raw materials used, salt concentrations, recipes, processes and fermentation periods. The knowledge of microbial communities in pla-ra samples provides better understanding of these traditional products. However, the presence of some unclassified OTUs were found in present study and these microorganisms involved initiate fermentation are still unknown. The experiment was done without replicates, variation in diversity was because of different raw materials used for fermentation. The lacking data and biological replicates need to be clarified in the further investigations. Optimization of the fermentation process, using some dominant bacterial taxa in starter cultures, may help improve processes of food fermentation, food quality and flavor control, providing beneficial outcomes for industrial applications.
Supporting information
S1 Table. Complete list of bacterial OTUs found across all pla-ra samples.
https://doi.org/10.1371/journal.pone.0245227.s001
(XLSX)
S2 Table. List of OTUs shared by all pla-ra samples.
https://doi.org/10.1371/journal.pone.0245227.s002
(XLSX)
S3 Table. List of unique OTUs in each pla-ra sample.
https://doi.org/10.1371/journal.pone.0245227.s003
(XLSX)
Acknowledgments
We would like to thank Professor David Blair through Khon Kaen University Publication Clinic (Thailand) for the English editing of this manuscript.
References
- 1. Marui J, Boulom S, Panthavee W, Momma M, Kusumoto KI, Nakahara K, et al. Culture-independent bacterial community analysis of the salty-fermented fish paste products of Thailand and Laos. Biosci Microbiota Food Health. 2015;34(2): 45–52. pmid:25918672
- 2.
Phithakpol B. Fish fermentation technology in Thailand. In: Lee CH, Steinkraus KH, Reilly PJA, editors. Fish Fermentation Technology. Tokyo: United Nations University Press; 1993. pp. 155–166.
- 3. Sangjindavong M, Chuapoehuk P, Runglerdkriangkrai J, Klaypradit W, Vareevanich D. Fermented Fish Product (Pla-ra) from Marine Fish and Preservation. Kasetsart J. (Nat. Sci.). 2008;42: 129–136.
- 4. Adams MR, Cooke RD, Rattagool P. Fermented fish products of South East Asia. Trop Sci. 1985;25: 61−73.
- 5. Phewpan A, Phuwaprisirisan P, Takahashi H, Ohshima C, Ngamchuachit P, Techaruvichit P, et al. Investigation of Kokumi substances and bacteria in Thai fermented freshwater fish (Pla-ra). J Agric Food Chem. 2019. pmid:31757121
- 6.
National Bureau of Agricultural Commodity and Food Standards, Ministry of Agriculture and Cooperatives. Thai agricultural standard, TAS 7023–2018 “PLA-RA”. 2018 April 17 [cited 2020 Aug 31]. Available from: http://web.acfs.go.th/eng/commodity_standard.php?pageid=7
- 7. Chotechuang N. Taste active components in Thai foods: A review of Thai traditional seasonings. J Nutr Food Sci. 2012;S10:004.
- 8. Yoshida Y. Umami taste and traditional seasonings. Food Rev Int. 1998;14(2–3):213–246.
- 9. Tamang JP, Cotter PD, Endo A, Han NS, Kort R, Liu SQ, et al. Fermented foods in a global age: East meets West. Compr Rev Food Sci Food Saf. 2020;19(1):184–217. pmid:33319517
- 10. Du F, Zhang X, Gu S, Song J, Gao X. Dynamic changes in the bacterial community during the fermentation of traditional Chinese fish sauce (TCFS) and their correlation with TCFS quality. Microorganisms. 2019;7(9): 371. pmid:31546947
- 11. Udomsil N, Rodtong S, Choi YJ, Hua Y, Yongsawatdigul J. Use of Tetragenococcus halophilus as a starter culture for flavor improvement in fish sauce fermentation. J Agric Food Chem. 2011;59(15):8401–8408. pmid:21710980
- 12. Yongsawatdigul J, Rodtong S, Raksakulthai N. Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures. J Food Sci. 2007;72(9):M382–390. pmid:18034732
- 13. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. The international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014;11(8):506–514. pmid:24912386
- 14. Magoč T, Salzberg SL. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011; 27(21): 2957–2963. pmid:21903629
- 15. Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, et al. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods. 2013;10(1): 57–59. pmid:23202435
- 16. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7(5): 335–336. pmid:20383131
- 17. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinforma Oxf Engl. 2011;27(16): 2194–2200. pmid:21700674
- 18. Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, et al. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res. 2011;21(3): 494–504. pmid:21212162
- 19. Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 2013;10(10): 996–998. pmid:23955772
- 20. Papizadeh M, Rohani M, Nahrevanian H, Javadi A, Pourshafie MR. Probiotic characters of Bifidobacterium and Lactobacillus are a result of the ongoing gene acquisition and genome minimization evolutionary trends. Microb Pathog. 2017;111:118–131. pmid:28826768
- 21. Bintsis T. Foodborne pathogens. AIMS Microbiol. 2017;3(3):529–563. pmid:31294175
- 22. Miyashita M, Yukphan P, Chaipitakchonlatarn W, Malimas T, Sugimoto M, Yoshino M, et al. 16S rRNA gene sequence analysis of lactic acid bacteria isolated from fermented foods in Thailand. Microbiol. Cult. Coll 2012;28(1): 1–9.
- 23. Tanasupawat S, Okada S, Komagata K. Lactic acid bacteria found in fermented fish in Thailand. J Gen Appl Microbiol. 1998;44(3): 193–200. pmid:12501428
- 24. Hayek SA, Ibrahim SA. Current limitations and challenges with lactic acid bacteria: A Review. Food Nutr Sci. 2013;04(11): 73–87.
- 25. An C, Takahashi H, Kimura B, Kuda T. Comparison of PCR-DGGE and PCR-SSCP analysis for bacterial flora of Japanese traditional fermented fish products, aji-narezushi and iwashi-nukazuke. J Sci Food Agric. 2010; 1796–1801. pmid:20572057
- 26. Thongsanit J, Tanasupawat S, Keeratipibul S, Jatikavanich S. Characterization and identification of Tetragenococcus halophilus and Tetragenococcus muriaticus strains from fish sauce (Nam-pla). Jpn J Lact Acid Bact. 2002;13(1): 46–52.
- 27. Satomi M, Kimufu B, Mizoi M, Sato T, Fujii T. Tetragenococcus muriaticus sp. nov., a new moderately halophilic lactic acid bacterium isolated from fermented squid liver sauce. Int J Syst Bacteriol. 1997;47(3):832–836. pmid:9226914
- 28. Kobayashi T, Kimura B, Fujii T. Haloanaerobium fermentans sp. nov., a strictly anaerobic, fermentative halophile isolated from fermented puffer fish ovaries. Int J Syst Evol Microbiol. 2000;50(4): 1621–1627. pmid:10939669
- 29. Jung JY, Lee SH, Lee HJ, Jeon CO. Microbial succession and metabolite changes during fermentation of saeu-jeot: Traditional Korean salted seafood. Food Microbiol. 2013;34(2): 360–368. pmid:23541203
- 30.
Song D, Ibrahim S, Hayek S. Recent Application of Probiotics in Food and Agricultural Science. In: Rigobelo E, editor. Probiotics [Internet]. InTech; 2012 [cited 2020 Apr 7]. Available from: http://www.intechopen.com/books/probiotics/recent-application-of-probiotics-in-food-and-agricultural-science
- 31. Chenoll E, Macián MC, Aznar R. Lactobacillus rennini sp. nov., isolated from rennin and associated with cheese spoilage. Int J Syst Evol Microbiol. 2006;56(2): 449–452. pmid:16449456
- 32. Behera SS, Ray RC, Zdolec N. Lactobacillus plantarum with functional properties: An approach to increase safety and shelf-life of fermented foods. BioMed Res Int. 2018; 1–18. pmid:29998137
- 33. Hill D, Sugrue I, Tobin C, Hill C, Stanton C, Ross RP. The Lactobacillus casei group: History and health related applications. Front Microbiol. 2018;9: 2107. pmid:30298055
- 34. Tanasupawat S, Shida O, Okada S, Komagata K. Lactobacillus acidipiscis sp. nov. and Weissella thailandensis sp. nov., isolated from fermented fish in Thailand. Int J Syst Evol Microbiol. 2000;50(4): 1479–1485. pmid:10939653
- 35. Fukami K, Satomi M, Funatsu Y, Kawasaki KI, Watabe S. Characterization and distribution of Staphylococcus sp. implicated for improvement of fish sauce odor. Fish Sci. 2004;70(5): 916–923.
- 36. Fukami K, Funatsu Y, Kawasaki K, Watabe S. Improvement of fish-sauce odor by treatment with bacteria isolated from the fish-sauce mush (Moromi) made from frigate mackerel. J Food Sci. 2004;69(2): fms45–fms49.
- 37. Wilaipun P, Zendo T, Okuda K, Nakayama J, Sonomoto K. Identification of the Nukacin KQU-131, a New Type-A(II) Lantibiotic Produced by Staphylococcus hominis KQU-131 isolated from Thai fermented fish product (Pla-ra). Biosci Biotechnol Biochem. 2008;72(8): 2232–2235. pmid:18685189
- 38. Zhang H, Li Y, Xu K, Wu J, Dai Z. Microbiological changes and biodiversity of cultivable indigenous bacteria in Sanbao Larger Yellow Croaker (Pseudosciaena crocea), a Chinese salted and fermented seafood: Microbiological changes and biodiversity. J Food Sci. 2015;80(4): M776–M781. pmid:25874648
- 39. Löfblom J, Rosenstein R, Nguyen M-T, Ståhl S, Götz F. Staphylococcus carnosus: from starter culture to protein engineering platform. Appl Microbiol Biotechnol. 2017;101: 8293–8307. pmid:28971248
- 40. Cardinali F, Milanović V, Osimani A, Aquilanti L, Taccari M, Garofalo C, et al. Microbial dynamics of model Fabriano-like fermented sausages as affected by starter cultures, nitrates and nitrites. Int J Food Microbiol. 2018;278: 61–72. pmid:29702317
- 41. Gupta AK, Dharne MS, Rangrez AY, Verma P, Ghate HV, Rohde M, et al. Ignatzschineria indica sp. nov. and Ignatzschineria ureiclastica sp. nov., isolated from adult flesh flies (Diptera: Sarcophagidae). Int J Syst Evol Microbiol. 2011;61(6): 1360–1369.
- 42. Ayeni FA, Sánchez B, Adeniyi BA, de Los Reyes-Gavilán CG, Margolles A, Ruas-Madiedo P. Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from Nigerian traditional fermented foods and cow’s intestine. Int J Food Microbiol. 2011;147(2): 97–104. pmid:21482440
- 43. Izquierdo FU, Ortíz IS, Olivera RP, Cruz AA. Lactobacillus pentosus for Animal Nutrition. Review article. 2017;9(1): 7–15.
- 44. Jung SY, Lee MH, Oh TK, Park YH, Yoon JH. Psychrobacter cibarius sp. nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol. 2005;55(2): 577–582. pmid:15774627
- 45. Namwong S, Tanasupawat S, Smitinont T, Visessanguan W, Kudo T, Itoh T. Isolation of Lentibacillus salicampi strains and Lentibacillus juripiscarius sp. nov. from fish sauce in Thailand. Int J Syst Evol Microbiol. 2005;55(1): 315–320. pmid:15653893
- 46. Nychas GJ, Skandamis PN, Tassou CC, Koutsoumanis KP. Meat spoilage during distribution. Meat Sci. 2008 Jan;78(1–2):77–89. pmid:22062098
- 47. Zagorec M, Champomier-Vergès MC. Lactobacillus sakei: A starter for sausage fermentation, a protective culture for meat products. Microorganisms. 2017;5(3): 56. pmid:28878171
- 48. Igbinosa E, Okoh AI. Vibrio Fluvialis: An unusual enteric pathogen of increasing public health concern. Int J Environ Res Public Health. 2010;7(10):3628–3643. pmid:21139853
- 49. Saeb AT, Abouelhoda M, Selvaraju M, Althawadi SI, Mutabagani M, Adil M, et al. The use of Next-Generation Sequencing in the identification of a fastidious pathogen: A lesson from a clinical setup. Evol Bioinforma. 2017;13. pmid:28469373
- 50. Quinto EJ, Caro I, Villalobos-Delgado LH, Mateo J, De-Mateo-Silleras B, Redondo-Del-Río MP. Food safety through natural antimicrobials. Antibiotics. 2019;8(4):208. pmid:31683578
- 51. Sarowska J, Futoma-Koloch B, Jama-Kmiecik A, Frej-Madrzak M, Ksiazczyk M, Bugla-Ploskonska G, et al. Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports. Gut Pathog. 2019;11:10. pmid:30828388
- 52. Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell. 2014;157(1):121–241. pmid:24679531
- 53. Kurilshikov A, Wijmenga C, Fu J, Zhernakova A. Host genetics and gut microbiome: challenges and perspectives. Trends Immunol. 2017;38(9):633–647. pmid:28669638