BIOTROPIA Vol. 28 No. 1,2021: 11 - 20 DOI: 10.11598/btb.2021.28.1.820 DYNAMICS OF MICROBIAL COMMUNITY DURING TEMPEH FERMENTATION RAHMADINA RADITAl, ANTONIUS SUWANTOl*, NORIO I(UROSAWA2, ARIS TRI WAHYUDI' AND IMAN RUSMANA' 'Department ofBiolo8, Faculij ofMathematics and Natural Sciences, Institat Pertanian Bogor, Dramga, Bogor 16680, Indonesia 'Department o f Science and Engineeringfor Sustainable Innovation, Faculg ofscience and Engineerin& Soka Universig, Hachioji, Tokyo 192-8577, Japan Received 27 February 2017/Accepted 14 February 2020 ABSTRACT Tempeh is a sliceable soybean-cake fermented by the bacteria Rhixopzls olgo@orus. Various bacteria are detected during tempeh cultivation, yet limited information is available about their origin or sources. The present study aimed to examine the source/s of bacterial community in tempeh by combining metagenomics analysis and cultivation technique. Samples were obtained from a number of steps in the tempeh production using the double-boiling method for the soybean (WJB tempeh production). All samples were plated on Enterobacteriaceae and Lactic Acid Bacteria media. Total DNA were extracted directly from tempeh for metagenomics analysis, applying the High-Throughput Sequencing (HTS) and cloned 16s rRNA genes. Firmicutes and Proteobacteria were the predominant and second dominant bacteria that existed in the fresh tempeh (FT) obtained by metagenomics analysis. However, cultivation technique showed that Proteobacteria was the predominant phylum, suggesting that most of the Firmicutes were not culturable. FT was dominated by Lactobadus and Acetobacter. Both FT and water-soaked tempeh (SW) were dominated by the same species of Lactobacillus, i.e., L. delbreuckii and L mucosae, indicating that SW was probably the source of bacterial community that was established in the final fermentation product. Predominant bacteria in the starter culture (SC), Acinetobacter, was not detected in FT, indicating that the bacteria in SC did not play a significant role in the FT bacterial community development. Keywords: firmicutes, high-throughput sequencing, Proteobacteria, starter culture INTRODUCTION The Indonesian traditional food tempeh is made from soybean fermented by the mold lL3ippu.r olzg0spom.r. However, the microbial composition of tempeh also included bacteria, as the "contaminants" in addition to the mold (Mulyowidarso e t al. 1990; Barus e t al. 2008; Efriwati, e t al. 201 3; Nurdmi e t al. 201 5). Among the producers in Indonesia, the tempeh production procedures usually differ and depend on the area, experience, and resources. However, the general procedure for tempeh production has few similarities, such as boiling the soybean, dehulling, soaking, mixing with inoculum, and incubation for two days. The producers usually "Corresponding author, e-mail: asuwanto@indo.net.id &ffer in boiling the soybean for the second time after soaking (Barus e t al. 2008; Efriwati e t al. 2013; Nurdini e t al. 2015). Four out of five tempeh producers in Bogor, did not re-boil the soybean after soaking. However, WJB producers applied two-times or double-boiling of soybean (Barus e t al. 2008). Of all the methods used for tempeh processing, the initial source of the bacteria in the final product fermented has not yet been verified. Tempeh production in Indonesia is usually done in "open" fermentation using unsterile wooden equipments, where other microbes are probably involved in the production processes. Hence, various bacteria are found in tempeh, such as lactic acid bacteria (LAB) and Enterobacteriaceae (Barus e t al. 2008; Efriwati e t al. 2013; Nurdini e t al. 2015). Lactic acid bacteria BIOTROPIA Vol. 28 No. 1,2021 are present at the beginning of the fermentation process (water soaking of soybean) until the end of the fermentation process (Efriwati e t al. 2013). The population of lactic acid bacteria varies according to the stage of fermentation process. Its presence is usually shown by the decrease in pH as a consequence of natural acidification, especially during the soaking processes (Efriwati etal. 2013). However, recent studies showed that laru which waJ used as a starter culture in WJB productions, did not contain any Enterobac- teriaceae nor lactic acid bacteria (Nurdini e t al. 2015). Bacterial community determination in tempeh was only limited to the culture- dependent method (Barus e t al. 2008; Nurdini e t al. 2015), T-RFLP (Terminal Restriction Fragment Length Polymorphsms) from culturable bacteria (Efriwati e t al. 2013), and ARISA (Amplified Ribosomal Intergenic Sequence Analysis) (Seumahu e t al. 2013). For the starter culture, the method used was only culture-dependent (Nurdini e t al. 2015). These approaches however, provided only limited information and, therefore, could not reflect the composition of microbial communities. To overcome this limitation, the 16s rRNA gene was used to represent the microbial community in a metagenomics analysis. T h s study attempted to determine the origin of the bacterial community in tempeh ecosystem by applying the metagenomics approaches derived from cloned 16s rRNAs gene sequences from high-throughput sequencing using the total 16s rRNA gene. MATERIALS AND METHODS Sampling and Sample for Analysis The samples used in this research were obtained from WJB (Warung Jambu) Tempeh Industries (Barus e t al. 2008), namely raw soybean (RS), fresh water soybean (FW), water- soaked soybean (SW), pre-inoculated dehulled- soybean (BI), commercial lam as starter culture (SC), and fresh tempeh (FT). Each sample was taken three times from WJB tempeh production and transported in ice boxes for direct analysis. Microbiological Analysis One gram of SC was inoculated into 25 g sterile water-soaked soybean in a 250 mL Erlenmeyer. Incubation was done for 2 days before doing the serial dilution. Twenty-five grams each of RS, BI, SC, FT, or 25 mL of FW and SW, were added into 225 mL 0.85% (w/v) NaCl (Merck, USA) for serial dilution. A 100 yL aliquot of suspension from each dilution were spread on Eosin Methylene Blue Agar (EMB) (Merck, USA) to determine the total count of Enterobacteriaceae and on Man-Rogosa and Sharpe Agar (MRSA) (Merck, USA) with 0.2% of sodium azide (Plengvidhya e t al. 2007) for enumeration of lactic acid bacteria. Each analysis was done in two replicates and incubated at 30 "C for 3-5 days for mesophilic bacteria? and Enterobacteriaceae; and 5-7 days for lactic acid bacteria. DNA Extraction for Metagenornics Analysis Another one gram of SC was inoculated into 25 g sterile soaked soybean in a 250 mL Erlenmeyer and incubated in 30 "C for 48 days to make a sterile tempeh. A hundred gram of sterile tempeh, BI, and FT were homogenized separately in 300 mL of PBS (phosphate buffer saline) for 1 rnin, following the procedure described in Seumahu e t al. (2012). Twenty-five grams of RS sample were soaked overnight into 225 mL sterile water in the shaker. A hundred liters of FW and water-soaked RS; and 30 rnL of SW were extracted using Power Water Microbial DNA Isolation-IGt (MOBIO, USA) according to the protocol described by the manufacturers. DNA products were initially visualized by electrophoresis using 1% (w/v) agarose gel for further analysis. PCR Amplification for 16s rRNA Gene The predominant colonies from each medium and DNA extracted for metagenome analysis were then amplified by using B27F and U1492R (Sakai & Icurosawa 2016). The PCR reaction contained the 25 pL of final solution consisting of 12.5 yL of Emerald Amp MAX PCR Master MIX (TaICaRa, Japan), 1 yL of 10 pmol of each primer, and 1 yL of DNA extract. Samples were amplified in the PCR machine with the following conditions: 94 "C for 3 min; 30 cycles of 94 "C for 30 seconds, 58 "C for 30 seconds, 72 "C for 1 min 30 seconds; and continued with 72 "C for 2 min. The PCR products (about 1,500 bp) were purified by Dynamics of microbial community during tempeh fermentation - Radita e t al. using Illustra GFX PCR DNA and gel band 5 min. Sequencing of the PCR products was purification kit (GE healthcare, UI<). PCR done using the custom service provided by the products of predominant colonies were Bioengineering Lab (Japan) and Fastmac (Japan). sequenced by custom service provided by Eurofin Genomics (Japan). Data Analysis Cloned 16s rRNA Gene Analysis The 16s rRNA gene fragments obtained by PCR were cloned into pT7 Blue T-Vector (Novagen, Germany). The recombinant plasmid were transformed into Eschem'chia coli DH5a (TaI