The 16S Microbiota of Budu, the Malaysian Fermented Anchovy Sauce
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bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.986513; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The 16S microbiota of Budu, the Malaysian 2 fermented anchovy sauce 3 Muhammad Zarul Hanifah Md Zoqratt1,2, Han Ming Gan3, 4 4 1 Genomics Facility, Tropical Medicine and Biology Platform, Monash University Malaysia, Petaling 5 Jaya, Selangor, Malaysia 6 2 School of Science, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia 7 3 Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, 8 Geelong, Victoria, Australia 9 4 GeneSEQ Sdn Bhd, Bandar Bukit Beruntung, 48300, Rawang, Selangor, Malaysia 10 11 Corresponding author: 12 Muhammad Zarul Hanifah bin Md Zoqratt, 13 [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.986513; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 14 ABSTRACT 15 Budu is a Malaysian fermented anchovy sauce produced by immersing small fishes into a brine 16 solution for 6 to 18 months. Fermentation of the anchovy sauce is contributed partly by microbial 17 enzymes, but little is known about the microbial community in Budu. Therefore, a better 18 understanding of the Budu microbiome is necessary to better control the quality, consistency and 19 safety of the product. In this study, we collected 60 samples from twenty bottles of Budu produced by 20 seven different manufacturers. We analyzed their microbiota based on V3-V4 16S rRNA amplicon 21 sequencing at the time of opening the bottle as well as 3- and 7-months post-opening. 22 Tetragenococcus was the dominant genus in many samples, reaching a maximum proportion of 23 98.62%, but was found in low abundance, or absent, in other samples. When Budu samples were not 24 dominated by a dominant taxa, we observed a wider genera diversity such as Staphylococcus, 25 Acinetobacter, Halanaerobium and Bacillus. While the taxonomic composition was relatively stable 26 across sampling periods, samples from two brands showed a sudden increase in relative abundance of 27 the genus Chromobacterium in the 7th month. Based on prediction of metagenome functions, non- 28 Tetragenococcus-dominated samples were predicted to have enriched functional pathways related to 29 amino acid metabolism and purine metabolism compared to Tetragenococcus-dominated microbiome; 30 these two pathways are fundamental fermented quality and health attributes of fish sauce. Within the 31 non-Tetragenococcus-dominated group, contributions towards amino acid metabolism and purine 32 metabolism were biased towards the dominant taxa when species evenness is low, while in samples 33 with higher species evenness, the contributions towards the two pathways were predicted to be evenly 34 distributed between taxa. 35 36 Keywords: Microbiome, Fermented food, Fish sauce, Tetragenococcus, prediction of metagenome 37 functions 38 39 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.986513; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 40 1 INTRODUCTION 41 Budu is a Malaysian fermented anchovy sauce which is prepared by immersing anchovies in brine 42 solution (salt concentration 21.5 – 25.7 %) containing natural flavor-enhancing ingredients and left to 43 ferment in earthen containers for 6 to 12 months (Rosma et al., 2009). Compared to other Southeast 44 Asian fish sauces such as Nuoc Mam from Vietnam and Nampla from Thailand which are transparent, 45 Budu is turbid and heterogenous (Lopetcharat et al., 2001). Fish sauce such as Budu is as food 46 condiment because of its pleasant umami flavor. It is also nutritious because it is rich in antioxidants, 47 vitamins and fibrin-clotting inhibitors (Shivanne Gowda et al., 2016). However, certain metabolites 48 in Budu might present as a potential health risk. For instance, Budu has a high amount of purine (Li et 49 al., 2019) and histamine (Rosma et al., 2009), metabolites that are associated with gout (Paul and 50 James, 2017) and scombroid poisoning (Tortorella et al., 2014), respectively. 51 52 Fish sauce microbes are responsible towards health and gastronomic properties of the fish sauce by 53 altering the metabolite content of the fish sauce. For instance, Tetragenococcus muriaticus was known 54 to produce histamine, while Staphylococcus, Bacillus and Lactobacillus were known to express 55 histamine oxidase which can degrade histamine (Shivanne Gowda et al., 2016). Therefore, a better 56 understanding of the Budu microbial community can elevate the Budu organoleptic and health value 57 by harnessing its microbiome. So far, all microbiological studies of Budu to date were done based on 58 culture-based methods. For example, Yuen et al. (2009) discovered bacterial succession takes place 59 in Budu fermentation from Micrococcus- to Staphylococcus arlettae-dominated community, and 60 uncovered the presence of yeast species such as Candida glabrata and Saccharomyces cerevisiae. 61 Subsequent microbiological studies on Budu revealed a few other cultivable bacteria such as Bacillus 62 amyloliquefaciens FS-05 and Lactobacillus planatarum which were able to produce glutamic acid 63 which is associated with the the umami taste (Zaman et al., 2010; Zareian et al., 2012). A recent study 64 on Malaysian fermented foods also displayed the potential of Bacillus sp. in producing biosurfactants 65 that inhibit pathogenic bacterial growth (Mohd Isa et al., 2020). While culture-dependent methods are 66 useful in gaining the first insights of role of microbes in Budu fermentation, they are biased towards 67 microbes that can grow optimally under culture conditions. To date, there has yet any culture- 68 independent methods to provide us an overview of the Budu microbial diversity. 69 70 In this study, we surveyed the microbial community of 60 samples from twenty bottles of Budu 71 purchased of multiple brands. We investigated their microbial community structure at different 72 sampling periods. We then find association between the microbial composition and diversity found in 73 Budu with its predicted metabolic pathways. Lastly, we explored the use of microbial interaction 74 networks modeled on 16S abundance information. 75 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.986513; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 76 2 MATERIALS AND METHODS 77 2.1 Sample collection 78 Twenty bottles of Budu from different brands were purchased from shops in the state of Kelantan, 79 Malaysia, at five different time points (July 2016, October 2016, November 2016, March 2017 and 80 April 2017). These twenty bottles were sampled at three different periods (24th April 2017, 24th July 81 2017 and 20th November 2017), amounting a total of 60 samples. The samples were stored at room 82 temperature to emulate typical retail storage condition. The sample metadata is shown in Table 1. 83 Samples were named according to the following format:<brand><bottle number>_<months since last 84 opened>. 85 86 2.2 DNA extraction and sequencing 87 200 µl of the sample was poured into 1.5 ml Eppendorf tubes and centrifuged at 14 800 rpm for 15 88 minutes to remove the supernatant. The samples were then resuspended in 400 µl of 20 mM Tris- 89 EDTA buffer and added with 100 μm silica beads (OPS Diagnostics LLC, Lebanon, NJ, USA). The 90 samples were later subjected to bead beating using Vortex Genie 2 Mo Bio (Carlsbad, CA, USA) at 91 3200 rpm for 1 hour. They were later added with 20 ul of 0.5% SDS and 20 ul of 50 mg/ml Proteinase 92 K and incubated at 55°C for 1 hour. The samples were added with 100 ul of saturated potassium 93 chloride solution and were later incubated on ice for 5 minutes. Total DNA was extracted using 94 chloroform-isopropanol precipitation and purified with Agencourt AMPure XP beads (Beckman 95 Coulter, Inc., Indianapolis, IN, USA). 96 97 The V3-V4 region of the 16S rRNA gene was amplified using the forward primer 5’- 98 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’ and reverse 99 primer 5’ 100 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’ 101 containing partial Illumina Nextera adapter sequence (Klindworth et al., 2013) following the Illumina 102 16S Metagenomic Sequencing Library Preparation protocol. To enable multiplexing, 16S amplicons 103 were barcoded using different pairs of index barcodes to prepare DNA libraries. DNA libraries were 104 normalised, pooled, denatured and sequenced on the Illumina MiSeq (Illumina, San Diego, CA, USA) 105 in Monash University Malaysia Genomics Facility, using either 2 × 250 bp or 2 × 300 bp run 106 configuration. 107 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.986513; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 108 2.3 Sequence data analysis, phylogenetic tree construction, taxonomic 109 assignment and generation of feature table 110 The forward and reverse PCR primer sequences were trimmed off using Cutadapt version 111 1.16 with default parameters (Martin, 2011).