Firmicutes Is the Predominant Bacteria in Tempeh

International Food Research Journal 25(6): 2313-2320 (December 2018) Journal homepage: http://www.ifrj.upm.edu.my Firmicutes is the predominant bacteria in tempeh 1Radita, R., 1*Suwanto, A., 2Kurosawa, N., 1Wahyudi, A. T. and 1Rusmana, I. 1Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Dramaga Campus, Bogor 16680, Indonesia 2Department of Science and Engineering for Sustainable Innovation, Faculty of Science and Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan Article history Abstract Received: 4 August 2017 Tempeh is an Indonesian fermented soybean inoculated with Rhizopus spp. A number of Received in revised form: culturable bacteria in tempeh had been documented. However, comprehensive study of bacteria 26 February 2018 population in tempeh has not been reported. This research aimed to analyze species of bacteria Accepted: 9 March 2018 in tempeh employing culturable method and metagenomics analysis based on 16S rRNA gene sequences and high-throughput sequencing. Samples were obtained from two tempeh producers in Bogor, Indonesia, i.e. EMP and WJB. Metagenomics analysis indicated that Firmicutes was Keywords the predominant phylum in both samples, with Lactobacilalles as the predominant order. The second majority phylum was Proteobacteria. Similarly, the results obtained from culturable Tempeh Metagenome technique also showed that Firmicutes was the predominant phylum. One-time boiling of Firmicutes soybean employed for EMP tempeh harbored the highest bacterial diversity than two-times Major phylum boiling soybean (WJB tempeh). Four orders were the predominant bacteria in EMP tempeh, i.e. Lactobacillales, Clostridiales, Enterobacteriales, and Sphingomonadales, while Lactobacillales and Rhodospirilalles were the only predominant bacteria orders in WJB tempeh. © All Rights Reserved Introduction rest employ one-time boiling soybean, such as EMP tempeh producer. These two types of tempeh has Tempeh is soybean fermented by Rhizopus spp. been used as a model for monitoring the microbial from Indonesia. As a staple food, tempeh contains community in tempeh fermentation, especially in nutrients advantageous as a daily diet due to its high Indonesia (Barus et al., 2008; Efriwati et al., 2013; content of protein, fatty acids, and vitamin B12 (Keuth Seumahu et al., 2013; Soka et al., 2014). and Bisping, 1993). Utilization of mold in tempeh Besides Rhizopus spp., other microorganisms fermentation changes the indigestible macromolecule were also reported present in fresh tempeh, such as in soybean into digestible molecules (Nout and Kiers, lactic acid bacteria (LAB) and Enterobacteriaceae 2005). Tempeh also contains variety of vitamins (Moreno et al., 2002; Barus et al., 2008; Efriwati including vitamin B12 in higher amount than its et al., 2013; Nurdini et al., 2015). The methods for raw ingredients. Therefore, this food is very suited determining the bacterial communities in tempeh for vegetarians who usually deficit of vitamin B12 samples so far limited only to culturable method (Barus diet (Watanabe et al., 2013). Moreover, the bacteria et al., 2008; Nurdini et al., 2015), T-RFLP (Terminal present in tempeh could act as immunostimulant Restriction Fragment Length Polymorphisms) from which can promote human health (Soka et al., 2014). culturable bacteria (Efriwati et al., 2013), and ARISA Basically, tempeh is produced locally through (Amplified Ribosomal Intergenic Sequence Analysis) several kinds of processing methods which include (Seumahu et al., 2013). Based on culturable methods, cooking the soybean, soaking, dehulling, inoculation the number of bacteria present in tempeh EMP and of starter culture, and incubation processes. But WJB were different from time to time (Barus et al., some tempeh producers employ modified methods 2008; Efriwati et al., 2013; Nurdini et al., 2015). which add another boiling step after soaking process Efriwati et al. (2013) reported that tempeh made with (Barus et al., 2008; Efriwati et al., 2013). Barus et al. one-time boiling of soybean, had higher number of (2008) reported that one out of five tempeh producers lactic acid bacteria than tempeh made with two-times in Bogor, Indonesia, does the two-times boiling of boiling of soybean, but Nurdini et al. (2015) reported soybean, such as WJB tempeh producer, while the the opposite. These could explain that tempeh was *Corresponding author. Email: [email protected] 2314 Radita et al./IFRJ 25(6): 2313-2320 a very dynamic fermented food with its bacterial sample were further analyzed for identification based community might change over times. Moreover, on 16S rRNA gene sequence. these also could be caused by limited amounts of information and unreliable methods of culturable Identification based on 16S rRNA gene. technique in analyzing the microbial community Representative colonies from each medium (Hugenholtz et al., 1998). were identified through their 16S rRNA gene. A 1.5 These limitations could be overcome by utilization mL of aliquot bacterial suspension which had been of metagenomics approach by targeting 16S rRNA grown for one night in their respective medium was gene as a phylogenetic marker for representing centrifuged for 3 mins at 10 000 x g. Pellet formed the metagenome technique. This technique could then extracted according to the procedure of Presto be used to examine unculturable bacteria without Mini gDNA Bacterial Kit (Geneaid, Taiwan). The creating a bias (Kakirde et al., 2010) since the data PCR reaction contained 25 µL of final solution produced using the culturable method is failed to consisting of: 12.5 µL of EmeraldAmp MAX PCR precisely quantify some predominant taxa yet still Master Mix (TaKaRa, Japan); 1 µL of 10 ng.µL-1 of underestimated biodiversity (Ampe et al., 1999). each universal primer: B27F and U1492R; and 1 µL Moreover, metagenome technique can also provide of DNA extraction. Samples were amplified in PCR wealthy information about the microbial community machine (Applied biosystem 2720 Thermal cycler) in fermented foods (Kim and Chun, 2005) by with the following condition: 94°C for 3 mins; 30 sequencing 16S rRNA gene sequences libraries cycles of 94°C for 30 seconds, 58°C for 30 seconds, using high-throughput sequencing (HTS). Recently, 72°C for 1 mins 40 seconds; and continued with 72°C the development of HTS has led to the large-scale for 2 mins. Sequencing was carried out by Eurofin metagenomics sequencing of fermented foods, such Genomics (Japan). Sequences from representative as Italian salami (Polka et al., 2015), wine (Bukolich colonies were submitted for BLASTN database et al., 2013), and artisan cheese (Bukolich and Millis, searching for 16S rRNA gene to identify individual 2013). The aim of the present study was to achieve a sequence (BLAST, http://www.ncbi.nlm.nih.gov/ fine characterization of bacterial diversity in tempeh. BLAST/). Samples were retrieved from two local producers in Bogor, Indonesia. Culturable technique and cloned DNA extraction for metagenomics analysis 16S rRNA gene were also carried out to compare the A hundred gram of tempeh from each tempeh result with HTS analysis. samples were homogenized separately in 300 mL PBS (phosphate buffer saline) for 1 mins. The Materials and Methods mixture later blended using blender (Maspion, Indonesia) for 5 mins. Then, centrifugation was Enumeration and microbiological analysis done for 1 mins at 1,000 rpm and the supernatant Samples for this study were fresh tempeh obtained were moved into new tubes and centrifuge again at from two tempeh industries, designated each as EMP 10,000rpm for 3 mins. Supernatant were discarded and WJB tempeh (Barus et al., 2008; Efriwati et and the pellet were washed with TE (pH 8.0) prior al., 2013; Soka et al., 2014). Sampling were taken to DNA extraction. Microbial DNA was extracted three times from different tempeh production and by using PowerFood Microbial DNA Isolation kit transported in ice box for direct processing. (MOBIO, USA) (Seumahu et al., 2012) according 25 gram of each samples were homogenized to the protocol described by the manufacturer. The in 225 mL of NaCl 0.85%. A 100 µL of aliquot DNA products were visualized by electrophoresis on was plated into modified MRSA (Man Rogosa- 1% (w/v) agarose gel before being used for next step Sharpe Agar) (Merck, US) with 1% of calcium of analysis. carbonate supplementation (Efriwati et al., 2013) for enumerating lactic acid bacteria, EMB (Eosin Cloned 16S rRNA gene Methylene Blue) (Merck, USA) for enumerating DNA extractions from previous section were Enterobacteriaceae, and PCA (Plate Count Agar) amplified employing universal primer B27F and (Merck, USA) for enumerating mesophilic aerobic U1492R (Lane, 1991). PCR products (approximately bacteria. Enumeration was done in replicate and the 1,500bp) were purified by using Illustra GFX PCR plates were incubated at 26-300C for two days for DNA and gel band purification kit (GE healthcare, all media. The total numbers of colonies on plates UK). The 16S rRNA gene fragments were cloned were expressed as number of cfu per gram sample. into pT7 Blue T-Vector (Novagen, Germany). Representative colonies from each medium and Recombinant plasmid were transformed into Radita et al./IFRJ 25(6): 2313-2320 2315 Escherichia coli DH5α (TaKaRa, Japan) plated onto the relative abundance of fragment i. D was measured Luria-Bertani (LB) plates supplemented with 100 based on equation: D = Σ ni(ni-1)/N(N-1), where ni µg. mL-1 ampicillin (Wako, Japan) and 40 µg.mL-1 was

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