03. Barus.cdr Vol.14, No.3, September 2020, p 101-107 DOI: 10.5454/mi.14.3.3 Genotypic Characterization of Rhizopus species from Tempeh and Usar: Traditional Inoculum of Tempeh in Indonesia 1 2 2 TATI BARUS *, JASON WIRANATA SANJAYA , DAVID TANDJUNG , ANASTASIA TATIK 3 2 2 HARTANTI , ADI YULANDI , AND VIVITRI DEWI PRASASTY 1 Master of Biotechnology,Faculty of Biotechnology, Universitas Katolik Atma Jaya, Jakarta 12930, Indonesia; 2 Department of Biology, Faculty of Biotechnology, Universitas Katolik Atma Jaya, Jakarta 12930, Indonesia; 3 Food Technology, Faculty of Biotechnology, Universitas Katolik Atma Jaya, Jakarta 12930, Indonesia. Soybeans tempeh (tempeh) is processed by fermentation using Rhizopus spp. Tempeh is an important source of protein in Indonesia. The traditional inoculum in tempeh fermentation locally is known as Usar, which is made from the leaves of Hibiscus tiliaceus. However, Rhizopus information from Usar is still limited. Therefore, this study aims to identify and investigate the genetic diversity of Rhizopus species from Usar and tempeh based on the Internal Transcribed Spacer (ITS) sequences and the Random Amplified Polymorphic DNA (RAPD) markers. Twenty-three Rhizopus strains were isolated from Usar and ten Rhizopus strains were isolated from tempeh. Based on ITS sequences, the isolates were similar to Rhizopus microsporus (30 isolates) and Rhizopus delemar (3 isolates) with 98-99% similarity. The genetics of R. microsporus and R. delemar are varied and different from the genetics of R. microsporus from tempeh. The growth temperature of R. microsporus varies from 33°C to 48°C and R. delemar can grow to a maximum at 33°C. This research needs to be continued to obtain information about the role of Rhizopus from this study in determining the quality of tempeh. Key words: diversity, ITS, RAPD, Rhizopus, tempeh Tempe kedelai (tempe) diolah melalui fermentasi menggunakan oleh Rhizopus spp. Tempe adalah salah satu sumber protein penting di Indonesia. Inokulum tradisional dalam fermentasi tempe dikenal sebagai Usar yang terbuat dari daun Hibiscus tiliaceus. Namun, informasi Rhizopus dari Usar masih terbatas. Oleh karena itu, penelitian ini bertujuan untuk mengidentifikasi dan mengkaji keragaman genetik spesies Rhizopus dari Usar dan tempe berdasarkan urutan sekuen Internal Transcribed Spacer (ITS) dan penanda Random Amplified Polymorphic DNA (RAPD). Dua puluh tiga strain Rhizopus diisolasi dari Usar dan sepuluh strain Rhizopus diisolasi dari tempe. Berdasarkan sekuens ITS maka semua strain tersebut terdiri atas Rhizopus microsporus (30 isolat) dan Rhizopus delemar (3 isolat) dengan kemiripan 98-99%. Genetik R. microsporus dan R. delemar bervariasi dan berbeda dari genetic R. microsporus dari tempe. Suhu pertumbuhan R. microsporus bervariasi dari 33°C hingga 48°C dan R. delemar dapat tumbuh hingga maksimum pada 33°C. Penelitian ini perlu dilanjutkan untuk mendapatkan informasi tentang peran R. microsporus dan R. delemar dari penelitian ini dalam menentukan kualitas tempe. Kata kunci: ITS, keragaman, RAPD, Rhizopus, tempe MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone: +62-21- ; Email: 5727615 tati.barus@atmajaya.ac.id Tempeh is made through the fermentation of soybean, mainly by Rhizopus spp. Therefore, Rhizopus spp. is known as an economically important mold in Indonesia. Many species of Rhizopus spp. such as R. oligosporus, R. oryzae, R. arrhizus, and R. stolonifer were previously identified from Indonesian tempeh (Dwidjoseputro and Wolf 1970). In the current taxonomic system, R. arrhizus is considered as a synonym of R. oryzae (Abe et al. 2010), R. oligosporusas as synonym of R. microspores (Dolatabadi et al. 2014), and R. oryzae as synonym of R. delemar (Abe et al. 2007). At present, tempeh producers rarely use Usar (traditional inoculum) (Fig 1) because they generally use commercial inoculum. Usar is made by growing Rhizopus spp. on Hibiscus tiliaceus leaves for 24 hours and then dried. After that, Soybeans tempeh (tempeh) is a traditional fermented food from Indonesia. It has been consumed as main source protein by Indonesian for years. It contains essential compounds such as vitamin B12 (Keuth and Bisping 1994), isoflavon and essential fatty acids. It has also been reported that tempeh have many health benefits such as in preventing free radicals (Esaki et al. 1996). Tempeh can prevent diarrhea (Sudigbia 1999) and anemia (Astuti 1999) because of increased iron availability during fermentation. Tempeh can also stimulate the formation of good bacteria populations in the intestines (Stephanie et al. 2019). it is ready to be used as an inoculum in tempeh fermentation. However, information about Rhizopus spp. from Usar was not yet available. Many molecular techniques are available to study genotypic characterization of Rhizopus (Abe et al. 2007). To identify of Rhizopus spp. often done based on ITS sequences (Iwen et al. 2002; Lott et al. 1998). Abe et al. (2007) reported the classification of three Rhizopus species, i.e. R. microsporus, R. stolonifer, and R. oryzae. Therefore, in this study ITS sequences were used to assess the genetic diversity of Rhizopus isolates from Usar (Fig 1) and tempeh. Beside ITS sequences, various molecular methods have been developed to get more accurate data on the genetics of an organism. One of them is random amplified polymorphic DNA (RAPD) marker using polymerase chain reaction (PCR). RAPD markers have been successfully used in assessing differentiating fungal genetic diversity, such as the genetic diversity of Colletotrichum spp. (Mahmodi et al. 2014) and Rhizopus stolonifera (Vágvölgyi et al. 2004). Therefore, this study aims to identify and investigate the genetic diversity of Rhizopus species from Usar and tempeh based on the Internal Transcribed Spacer (ITS) sequence and the Random Amplified Polymorphic DNA (RAPD) markers. MATERIALS AND METHODS Rhizopus Isolation. Rhizopus isolates have been isolated from Usar were taken from Yogyakarta, Central Java-Indonesia. Usar sampling was carried out from Yogyakarta because to our knowledge that only in this area Usar was still used as an inoculum in tempeh production. A total 10 tempeh is collected from Yogyakarta and Solo, Central Java-Indonesia. All these tempeh are produced using commercial inoculums. A total of 23 pieces of Usar that grow Rhizopus has been cut off and homogenized in sterile 0.85% w/v NaCl by the use of a Stomacherlab-blender 400 (Seward Medical, London, UK) for 1 minute. All isolates were grown on potato dextrose agar (PDA) and incubated at o 28 C. All suspected Rhizopus isolates were stored at o 4 C for further analysis. DNA Extraction. Genomic DNAs of Rhizopus isolates were extracted from four days-old mycelia grown on PDA using the Phytopure™ DNA Extraction Kit (GE Healthcare, UK) according to the manufacturer's protocol. DNAs were visualized on 1% electrophoresis agarose gel (Promega, Madison, USA) then stained with ethidium bromide (Sigma-Aldrich, USA). Amplification of Its Region and Sequencing. Amplification of ITS region was performed using the GeneAmp® PCR System 2700 (Applied Biosystems, Carlsbad, CA, USA) and the primer pair ITS4 (5′–TCCTCCGCTTATTGATATGC–3′) and ITS5 (5′–GGAAGTAAAAGTCGTAACAAGG–3′) (White et al. 1990). A total 50 µL of reaction mixtures were used, containing 1 µL DNA template, 10 µL 5X KAPA (Sigma-Aldrich, USA), Taq EXtra Buffer, 2.5 µL of each primer, 1.5 µL 10 mM dNTPmix, 3.5 µL MgCl2, 28.5 µL nuclease-free water (NFW), and 0.5 µL KAPA Taq EXtra HotStart DNA Polymerase. PCR conditions were set as follow: initial denaturation at 94 °C for 2 minutes, followed by 35 cycles of denaturation at 94°C for 15 seconds, annealing at a temperature of 55°C for 30 seconds, and extension at 72°C for 1 minute. Final elongation was set at 72°C for 5 minutes. PCR products were visualized on 1% agarose gel and stained with ethidium bromide. PCR products were then partially sequenced at Macrogen Inc., Republic of Korea. The DNA sequencing results were compared to the GenBank database using BLASTN (http://blast.ncbi .nlm.nih.gov/Blast.cgi). Phylogenetic tree was constructed using MEGA7. The branch support was analyzed by 1000x bootstrap analysis. Growth of Rhizopus Isolates on Various Temperature. All isolates were grown on potato dextrose agar (PDA). To determine the optimal growth temperature, all Rhizopus isolates obtained in this study were grown on various temperature, i.e. 33°C, 102 BARUS ET AL. Microbiol Indones Fig 1 Usar: traditional inoculum of tempeh . Volume 14, 2020 Microbiol Indones 103 42°C, 45°C, and 48°C. Amplification of Random Amplification of Polymorphic DNA. Amplifications of RAPD markers were conducted using single six primers (Table 1) through GeneAmp® PCR System 2700 (Applied Biosystems, Carlsbad, CA, USA). Amplification of RAPD was carried out with a total of 25 μL of the reaction mixture with the same composition carried out with Barus et al. (2019). Amplifications of RAPD markers were performed with the same condition also with Barus et al. (2019). Annealing conditions were done based on the melting temperature of each primer (Table 1). Products of amplification were separated by electrophoresis in agarose gel (1% w/v). The agarose gel was stained with ethidium bromide and UV transilluminator was used to visualize the PCR products in agarose gel. The 1 kb ladder (Fermentas) was used as weight marker. Clearly resolved each band was manually scored for the presence (1) or absence (0) to make binary data. Dendrogram analysis among all the Rhizopus was computed using Roderic D.M. Page software. The unweighted pair group method analysis (UPGMA) was used for clustering and Tree View software was used for interactive visualization of the dendrogram. RESULTS A total of twenty-three Rhizopus isolates had been isolated from Usar (TB23-TB45) and ten Rhizopus isolates had been isolated from tempeh (TB46-TB55) (Fig 2). ITS sequences were successfully amplified and each PCR amplification showed DNA fragments with single band at 700 bp (Fig 2). BLASTN results of ITS sequence (± 600 nucleotides) showed that 30 isolates (TB23-TB25, TB27, TB29-TB36, TB38-TB55) were Rhizopus microporus with similarity about 98-100%. Only three isolates (TB26,TB28, TB37) were hizopus R delemar with similarity about 98-100%. All the ITS sequences have been submitted to GenBank with accession numbers MF445258 - Mf445290. The phylogenetic tree based on the ITS sequences showed that the thirty three of Rhizopus isolates were divided into two clusters (Fig 3). The first cluster consisted of R. microsporus (30 isolates) and the second cluster consisted of R. delemar (3 isolates). All Rhizopus spp. isolates were grown at 33°C, 42°C, 45°C, and 48°C. The growth temperature for all isolates R. microsporus varied . Eight strains (Table 2) of R. microspores could grow up to 48 , thirteen °C strains could grow up to 45 , seven strains could grow °C up to 42 , and two strains could grow up to 33 . °C °C Conversely, three R. delemar isolates could only grow up to 33°C. � Genomic DNAs isolated from 33 Rhizopus isolates were subjected to obtain RAPD-PCR markers using six primers (Table 1), but only 9 out of 33 Rhizopus isolates produced distinc and reproducible band RAPD marker using these primers. The dendogram (Fig 4) describes the genetic similarity R. microsporus and R. delemar was successfully created. UPGMA dendrogram based on RAPD – PCR separated the R. microsporus and R. Delemar in two main clusters. Among all R microporus, the smallest genetic similarity (GS) (35%) was found between TB34 and TB35 and the largest GS (63%) was found between TB32 and TB33. R. microporus from tempeh (TB49) is most similar to TB32 with GC 54% and most different with TB34 with GC 38%. R. delemar TB26 and R. delemar TB37 have genetic similarity 64%. DISCUSSION A study by Bressa et al. (2017) showed that lifestyle enhanced health-promoting bacteria. A previous Table 1 Primers to amplify RAPD markers of Rhizopus isolates. Primer Sequence Melting temperature Sources OPQ6 GAGCGCCTTG 34°C Vagvolgyi et al. 2004 OPA9 GGGTAACGCC 34°C Mahmodi et al. 2014 OPJ20 AAGCGGCCTC 34°C Mahmodi et al. 2014 R108 GTATTGCCCT 30°C Vagvolgyi et al. 2004 OPA11 CAATCGCCGT 32°C Mahmodi et al. 2014 OPA1 CAGGCCCTTC 34°C Mahmodi et al. 2014 1 104 BARUS ET AL. Microbiol Indones Source Isolate code Species Growth temperature (oC) 33 42 45 48 Usar TB24 R. microsporus ✔ ✔ ✔ ✔ Usar TB25 R. microsporus ✔ ✔ ✔ ✔ Usar TB31 R. microsporus ✔ ✔ ✔ ✔ Usar TB32 R. microsporus ✔ ✔ ✔ ✔ Usar TB33 R. microsporus ✔ ✔ ✔ ✔ Usar TB39 R. microsporus ✔ ✔ ✔ ✔ tempeh TB48 R. microsporus ✔ ✔ ✔ ✔ tempeh TB54 R. microsporus ✔ ✔ ✔ ✔ Usar TB23 R. microsporus ✔ ✔ ✔ - Usar TB27 R. microsporus ✔ ✔ ✔ - Usar TB30 R. microsporus ✔ ✔ ✔ - Usar TB35 R. microsporus ✔ ✔ ✔ - Usar TB36 R. microsporus ✔ ✔ ✔ - Usar TB38 R. microsporus ✔ ✔ ✔ - Usar TB40 R. microsporus ✔ ✔ ✔ - Usar TB43 R. microsporus ✔ ✔ ✔ - Usar TB45 R. microsporus ✔ ✔ ✔ - tempeh TB46 R. microsporus ✔ ✔ ✔ - tempeh TB52 R. microsporus ✔ ✔ ✔ - tempeh TB53 R. microsporus ✔ ✔ ✔ - tempeh TB49 R. microsporus ✔ ✔ ✔ - Usar TB29 R. microsporus ✔ ✔ - - Usar TB34 R. microsporus ✔ ✔ - - Usar TB41 R. microsporus ✔ ✔ - - Usar TB42 R. microsporus ✔ ✔ - - Usar TB44 R. microsporus ✔ ✔ - - tempeh TB47 R. microsporus ✔ ✔ - - tempeh TB51 R. microsporus ✔ ✔ - - tempeh TB50 R. microsporus ✔ - - - tempeh TB55 R. microsporus ✔ - - - Usar TB26 R. delemar ✔ - - - Usar TB28 R. delemar ✔ - - - Usar TB37 R. delemar ✔ - - - 1 Table 2 Growth on various temperatures of thirty-three hizopus isolates isolated fromUsar and tempeh.R reported that gut microbiota in obese subjects and/or with Type-2 Diabetes were different from lean and non- diabetic subjects (Patterson et al. 2016). To get beneficial gut microbiota population, probiotics consumption and dietary fibers are strongly recommended. Holscher (2017) reported that low fiber intake is associated with increased chronic diseases, such as obesity, cardiovascular disease, type 2 diabetes, and colon cancer. Tempeh, a popular fermented food in Indonesia, is one source of fiber-rich food. The main microorganism in fermentation of tempeh is Rhizopus spp. At present, many molecular techniques are available for identification of Rhizopus spp. However, internal transcribed spacer (ITS) is often used (Abe et al. 2003. Based on ITS sequence showed that 30 isolates (TB23-TB25, TB27, TB29-TB36, TB38-TB55) were Rhizopus microporus with similarity about 98-100% and three isolates (TB26, TB28, TB37) were hizopus delemar with similarity R about 98-100%.The ITS regions have become an important molecular target for fungal taxonomy and identification (Iwen et al. 2002). Due to greater sequence variations, the ITS domains are more suitable for species identification (Iwen et al. 2002; Lott et al. 1998). Therefore ITS sequences are widely used to identify and assess fungal genetic diversity. Volume 14, 2020 Microbiol Indones 105 Fig 2 Results of PCR amplification sequences of internal transcribed spacer (ITS) sequence of Rhizopus isolates. M: Marker 1-kb lambda ladder. TB23-TB45: Rhizopus isolates from Usar. TB46-TB55: Rhizopus isolates from tempeh. Fig 3 Phylogenetic tree generated from the internal transcribed spacer (ITS) sequences of 33 isolates of hizopus R spesies isolated from Usar and tempeh. 106 BARUS ET AL. Microbiol Indones Fig 4 UPGMA dendrogram of Rhizopus strains isolated from Usar and tempeh based on RAPD – PCR. In the past, it was reported that various Rhizopus species were used to make tempeh (Dwidjoseputro and Wolf 1970). In this study it was shown that only R. microspores were found in tempeh samples. This finding is similar to the report by Hartanti et al. (2015), where tempeh collected from 28 locations throughout Indonesia only contained R. microspores. This is caused by the use of commercial inoculums that only contain the R. microsporus. Surprisingly, in this study found three isolates of R. delemar from Usar. Based on the RAPD marker (Fig 4), TB26 and TB27 are in the same cluster, but they are different types represented by different RAPD markers. Information on R. delemar in tempeh is still limited. Therefore, the role of R. delemar in determining the quality of tempeh needs to be further investigated. The species of Rhizopus may have an important contribution to the variety of tempeh flavor and nutritional value. The different species of Rhizopus have different metabolic activities. Moreover, it has been reported that R. delemar produced fumaric acid and malic acid (Abe et al. 2007). Figure 3 showed that ITS sequences were not sufficent to distinguish R. microsporusup to the variety level. This can be seen from the phylogenetic tree which R. microsporus var. azygosporus, R. microsporus var. chinensis, R. microsporus var. oligosporus, R. microsporus var. rhizopodiformis, and R. microsporus var. tuberosus were all grouped as one cluster (Cluster 1). This indicated that the ITS sequences were not sufficent to distinguish R. microsporusup to the variety level. This report is in line with Hartanti et al. (2015) which ITS sequences were not sufficent to distinguish R. microsporus spesies from tempeh up to the variety. Rhizopus is the main microorganism in making tempeh. Information about the growth of Rhizopus isolates on various temperature is important as a basis for selecting isolates to be used as inoculums in tempeh fermentation. The growth temperature for all isolates R. microsporus (Table 2) This was found strains varied . that Rhizopus microsporus (TB32) can grow up to 48°C and Rhizopus microsporus (TB55) can grow up to 32°C (Table 2). Barus et al. (2019) reported that Rhizopus microsporus (TB32) produced tempeh with higher antioxidant activity compared to Rhizopus microsporus (Tb55). Figure 4 showed that RAPD markers can show genetic variation in nine Rhizopus. Previously it has been reported that RAPD marker can be used as an important technique to investigate for the genetic variations of fungal (Dwivedi et al. 2018). These reported are in line with our result, where RAPD marker can also distinguish the genetic variations of nine R. microsporus and two R. delemar well. Genetic of all R. microsporus isolate from Usar (TB26, TB30, TB32-TB37) were different from the genetic R. microsporus from tempeh (TB49). Furthermore, the genetics of R. microsporus and R. delemar derived from Usar also varied. RAPD markers of 24 isolates (TB 23-TB25, TB27-TB29, TB31, TB38, TB39-TB48, TB50-TB55) have not been successfully amplified using several primers (Table 1) even though they have been repeated several times. This might be accessible using another primer. ACKNOWLEDGEMENTS This study was funded by the Competitive Grant Program of Atma Jaya Catholic University of Indonesia Volume 14, 2020 Microbiol Indones 107 REFERENCES Abe A, Asano K, Sone T. 2010. A molecular phylogeny- based taxonomy of the genus Rhizopus. Biosci Biotechnol Biochem 74(7): 1325-1331. doi: 10.1271/bbb.90718. Abe A, Oda Y, Asano K, Sone T. 2007. Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers. Mycologia 99(5): 714-722. doi: 10.3852/mycologia.99.5.714. Astuti M. 1999. Iron availability of tempe and uses in iron deficiency anemia. The Complete Handbook of Tempe: The Unique Fermented Soyfood of Indonesia 41-45. Barus T, Halim R, Hartanti AT, Saputra PK. 2019. 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