01. Rahayu.cdr Vol.15, No.3, September 2021, p 77-83 DOI: 10.5454/mi.15.3.1 Domestication and Formulation of as a Tempeh StarterRhizopodopsis javensis GAYUH RAHAYU *, EFRIWATI , SEPTINA VERONICA 1 2 1 AND 1 Department of Biology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor, Darmaga Campus, Bogor 16680, Indonesia; 2 The Biomedical and Basic Health Technology Research and Development Centre National Institute of Health Research and Development Ministry of Health Republic of Indonesia, Jakarta 10560, Indonesia. Domestication of wild fungal strains involved in the manufacture of traditional fermented foods often occurs spontaneously. ) which taxonomically close to is a fungus type Rhizopodopsis javensis (Rh. javensis Rhizopus naturally found in cool climates, and promisingly can be used as a tempeh starter in temperate regions. However before using it as starter, a wild strain needs to be domesticated in human-made niches. At present Rh. javensis study, this species was domesticated by subculture using rice flour media at optimum growth temperature for every five days. Some parameters such as spore's density, viability, and starter water content were used to determine the successfully of domestication. The result showed that grew optimally at 22 ℃. Rh. javensis Moreover, it has been reproducible for seven times which indicated by no changes in growing rate and colony appearance. However, compared to the commercial tempeh starter and var. wild type, R. microsporus oligosporus it has lower spore's viability with higher water content. Therefore, the carrier material and other parameters such as drying processes and starter's lifespan need to be modified to increase the spore viability and starter quality. Key words: food fermentation, var , spore's viability, starter quality, wild Rhizopus microsporus . oligosporus strain Domestikasi galur liar kapang yang terlibat dalam dalam pembuatan makanan fermentasi tradisional, sering terjadi secara spontan. ( ) merupakan salah satu galur liar kapang yang Rhizopodopsis javensis Rh. javensis memiliki hubungan taksonomi dekat dengan . Strain liar ini ditemukan di daerah beriklim sejuk, Rhizopus sehingga berpotensi untuk dikembangkan sebagai starter tempe untuk produksi di daerah beriklim sedang. Untuk mendapatkan kultur yang tumbuh subur di relung ( ) buatan manusia, strain liar perlu niches Rh. javensis didomestikasi terlebih dahulu. Penelitian ini bertujuan untuk mendomestikasi strain liar yang Rh. javensis dilanjutkan dengan memformulasikannya sebagai starter tempe. Domestikasi dilakukan dengan menumbuhkan strain liar pada media tepung beras pada suhu pertumbuhan optimum dan diulangi setiap lima hari. Rh. javensis Kerapatan dan viabilitas spora, serta kadar air starter digunakan sebagai penilaian keberhasilan starter. Hasil penelitian menemukan tumbuh optimal pada suhu 22 ℃. Domestikasi dengan cara subkultur koloni Rh. javensis Rh. javensis Rh. javensis pada media tepung beras selama 7 kali tidak mengubah kecepatan pertumbuhan dan penampakan koloni Laju pertumbuhan relatif sama dengan laju pertumbuhan starter tempe . Rh. javensis komersial dan var. s murni, pada suhu optimum pertumbuhan masing-masing. R. microsporus oligosporu Formulasi tepung beras sebagai media pembawa starter menghasilkan kerapatan spora yang relatif Rh. javensis, sama dengan starter tempe komersial, namun viabilitas sporanya rendah dan kadar airnya tinggi. Starter Rh. javensis belum dapat digunakan untuk membuat tempe. Substrat dan proses pengeringan masih perlu dimodifikasi untuk meningkatkan viabilitas spora dan kualitas starter tempe secara keseluruhan, termasuk umur simpan starter. Kata kunci: fermentasi makanan, kualitas starter, strain liar, Rhizopus microsporus var. oligosporus, viabilitas spora MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone 62-816-1133-725 ; : + Fax: +62- ; gayuhrahayu@gmail.com E-mail: stolonifer were often found in Indonesian traditionally tempeh-making processes (Tamam 2019, Putri et al. et al. R. microsporus oligosporus2021). Furthermore, var. and were often used as commercial tempeh R. oryzae starters (Ahnan-Winarno . 2021). With the et al increasing preference of the Indonesian people to use a specific commercial starter, the diversity of Rhizopus species in tempeh decreases (Hartanti 2015). et al. Rhizopus delemar, R. which previously reported as oryzae, was no longer found in all fresh tempeh from Java island (Hartanti 2015). In contrast, Barus et al. et Tempeh is made from soybean fermented by the microbial community. Although sp. is the Rhizopus only microbe that is deliberately given as the tempeh starter, Lactic Acid Bacteria (LAB) and other types of bacteria (Seumahu 2013, Efriwati 2013), and et al. et al. yeast (Efriwati 2013) were found during tempeh et al. making process. , Rhizopus microsporus R. rhizopodiformis R. chinensis R. arrhizus R. , , , and al. R. delemar (2019) stated that two strains of obtained from Waru ( leaves produced a better Hibiscus tiliaceus) tempeh taste and more preferred than usual tempeh. Therefore, it is necessary to preserve various Rhizopus species from tempeh, including exploring new Rhizopus s specie or their close relatives for obtainig new potential strains for tempeh production. Domestication of microbial agents for food fermentation is defined as human selection to obtain cultivated strains that grow well in man-made environments, but may behave sub optimally in nature (Gallone 2016)et al. . Tempeh starters should include only food-grade microbes. var Rhizopus microsporus . oligosporus et has not been found in nature (Dolatabali al. 2013) and did not produce harmful metabolite (Ahnan-Winarno 2021). Dolatabali (2013) et al. et al. suggseted that var might R. microsporus . oligosporus become from the domestication of . R. microsporus Domestication increases functionality R. microsporus for humans. It also ensures food quality, homogeneity, and food safety (Gibbons and Rinker 2015). This domestication process occurs due to cultivating the mold in an isolated environment from its ancestor population to increase specific function for a human being (Gallone 2020). In traditional fermented et al. food, domestication might be due to back slopping (Wijaya 2015, Gallone 2020). et al. et al. Rhizopus oligosporus are cultivated during tempeh production, and this mold grow optimum at 30-37 ℃ (Tahir 2018). Tempeh production in other et al. countries with the same climate as Indonesia (tropical) was easy to be done, but not in countries with a subtropical climate. Within their study, Ahnan-Winarno et al. (2021) described tempeh production in the subtropical region was more challenging. Hence, it is necessary to search for alternative molds for subtropical regions. Rhizopodopsis javensis is a Mucorales that is expected to be able to grow at low temperature (around 20 ℃) as that mold has been found in mountain areas (Cibodas, Cianjur, West Java). Thus, can Rh. javensis probably be developed as a starter to produce tempeh in the subtropical region. However, domestication of Rh. javensis as artificial selection and wild species culturing must be done prior to making it a tempeh starter. Domestication is needed to obtain variants that thrive in human-made niches to meet human or industrial needs (Steensels et al. 2019). According to Pramudito et al. (2021), rice is usually made as a carrier material in tempeh starter. This research aimed to domesticate the wild strain of and followed by the Rh. javensis formulation of domesticated strain as a tempeh starter. The domesticated strain is expected to meet the criteria for tempeh starter, which was determined based on spore density and viability, as well as starter physical characteristics such as water content. MATERIALS AND METHODS Microbial Strains and Media. Rhizopodopsis javensis Rhizopus microsporus . IPBCC 19.1478, var oligosporus IPBCC 13.1102, and commercial tempeh starter (Raprima®) were used in this research. Rice flour from rice var. IR 64 Setra Ramos was used as Rh. javensis domestication media. Potato Dextrose Agar (PDA) was used as media for mold sub-culturing. Working culture of and Rh. javensis R. microsporus var. were obtained by rejuvenating stock oligosporus culture on PDA media and incubated for 2-7 days in 19- 23 ℃ for and in 32 ℃ for . Rh. javensis R microsporus var. . oligosporus Prior to domestication, Microbial Domestication. Rh. javensis optimum growth temperature was determined. This experiment was done by growing two-day-old culture on PDA and incubating them at 20, 22, 24, 26, 28, and 30 ℃. The experiment was done in 3 replicates. The colony diameter was measured every 6 hours until the colony entirely covered the media in all the Petri dishes. The growth and the sporulation rate determined the optimum growth temperature. The optimum temperature obtained was then used for Rh. javensis domestication process on the tempeh starter carrier. Rhizopodopsis javensis was domesticated on 50 gr sterile rice flour that was wetted by 15 mL sterilized water. A few pieces of inocula (±5 mm Rh. javensis diameter) were cultured on rice flour media and incubated at optimum growth temperature Rh. javensis for five days. This sub-culturing was done up to seven times. The colonies that grew from the last sub- culturing were regrown on PDA to evaluate their domestication growth capability. The colony characteristics and the growth rate before and after domestication were then compared. The domesticated strain that showed Rh. javensis comparable characters were used as materials to make starters. The inoculum, for starters, was produced in a similar procedure to that of the domestication. After five days of incubation, that culture was made into powder and dried at 30 ℃ for two days. These processes were also done to var.R. microsporus oligosporus Rh. javensis and the wild strain of for 78 RAHAYU ET AL . Microbiol Indones Volume 15, 2021 Microbiol Indones 79 comparison. Spore density was determined by observing the suspension of diluted starter in a hemocytometer counting chamber. Spore viability was determined by using the spread plate method with three replicates. The water content was determined using the oven method, according to SNI 01-2891-1992. The density and viability of spore and the starter's Rh. javensis water content were compared with the density and viability and water content of var. R. microsporus oligosporus and commercial starters. The Kruskal-Wallis test analyzes the differences within optimum growth temperature and spore viability, with 0.05 probability. Colony growth data of Rh. javensis R. microsporus oligosporus, var. , and Rhizopus in the commercial starter, Raprima®, were analyzed using the Brown-Forsythe test; meanwhile, data on spore density and starter water content were analyzed one-way ANOVA with a 95% confidence level. The optimum growth temperature Rh. javensis determination with distinctly different results was analyzed using the Mann-Whitney test at α = 0.05. All data were analyzed using SPSS (Statistical Package for the Social Science) software 16.00 version. RESULT The temperature affected growth, as Rh. javensis seen from the colony's diameter (Fig 1 and 2). The optimum growth temperature for was Rh. javensis about 22 ℃. At this temperature, grew at Rh. javensis the maximum rate (Fig 2b). At 28 ℃, the growth was sustained, while at 30 ℃ there was no growth (Fig 1). The growth rate of domesticated was not Rh. javensis significantly different from that of the wild type (Fig 3). Further, growth rate was not significantly Rh. javensis different from var when R. microsporus . oligosporus all were grown at their optimum growth temperatures (Fig 3). Domestication did not change the characteristics of Rh. javensis. The spore density in 1 g starter on both the wild and domesticated starters were Rh. javensis relatively the same with spore concentration in commercial tempeh starter and varR. microsporus . oligosporus. This research found that all dried starters contained insignificantly different concentration of spore i.e about 10 spore/g (Table 1). However, spore 6 viability in all starters was low, only 10 cfu/g or around 4 0.13-2.03 % (Table 1). The water content of all starters made in this study was from 8 to 10.58 %. The water content of Rh. javensis R. microsporus var. oligosporus and starter was higher but insignificantly different from that of the commercial tempeh starter (Table 2). DISCUSSION In terms of growth in its optimum temperature, Rh. javensis produced comparable characters to the commercial starter. grew Rhizopodopsis javensis optimally and sporulated well at 22 ℃, thus classified as either facultative psychrophilic or psychrotolerant fungi. Psychrotolerant microbes are mesophilic microbes that are cold-tolerant and thrive in an extensive temperature range, with the fastest growth at temperatures above 20 ℃. The growth rate of domesticated , at its optimum temperature, Rh. javensis was not significantly different from its original culture (Fig 3). These showed that was able to Rh. javensis adapt to rice flour media. However, is Rh. javensis Starter type Spore density Number of colonies Spore viability Log spore/g Log cfu/g (%) Rh. javensis wild type 6.23±0.39 4.06±0.30 0.67a Rh. javensis domesticated type 6.75±0.22 3.88±0.30 0.13a R. microsporus var. oligosporus 6.89±0.15 4.38±0.43 0.31a Raprima 6.51±0.20 4.82±0.03 2.03a Starter Type Water Content (%) Rh. javensis wild type 10.58±1.08a Rh. javensis domesticated type 10.08±1.17a R. microsporus var. oligosporus 9.08±1.59a Raprima 8.00±0.29a Table 1 Density and viability of spores of various molds in the starter Table 2 Water content during fermentation from several type of tempeh starter 80 RAHAYU ET AL . Microbiol Indones Fig 1 growth curve at various temperatures during 48 hours incubation. Vertical lines on Rhizopodopsis Javensis every data show the standard error. Alphabets at the right show a significant difference of average colony diameter on each temperature according to the Mann-Whitney test at = 0.05. α Fig 2 growth after 48 h incubation on PDA media at (a) 20 ℃, (b) 22 ℃ (c) 24 ℃, (d) 26 ℃, Rhizopodopsis javensis (e) 28 ℃, and (f) 30 ℃. Fig 3 The growth rate of , var. , and in Raprima on PDA media Rh. javensis R. microsporus oligosporus Rhizopus during 60 h incubation at each optimum temperature. Volume 15, 2021 Microbiol Indones 81 naturally found on ripe and fruit Elaeagnus Ficus (Boedijn 1958; Rahayu , unpublished). et al. Further, growth was not significantly Rh. javensis different from and R. microsporus var. oligosporus commercial starter (Raprima®) at their Rhizopus optimum growth temperature. Thus, it is suggested that the starter made from could produce Rh. javensis tempeh by bringing up the hyphal mass in the same compactness and times as a commercial starter. The commercial tempeh starter requires a temperature of 32 ℃ to be applied, making it challenging to use in subtropical regions (Samson 2007). Therefore, et al. Rh. javensis that grow optimum at 22 ℃ has an opportunity to be developed as a tempeh starter for tempeh production in regions with a temperature of 22 ℃ or regions with a subtropical climate. Domestication would depend on the media used. Rh. javensis that was domesticated on rice flour media could grow well. Colonization can be observed after three days of incubation. Wijaya (2015) stated et al. that the starter is usually produced by inoculating starter powder from the previous batch to the new rice flour dough which is called as back-slopping (Gallon et al. 2020). Cultivation or sub-culturing that was done continuously in the human-related environment causes microbes to adapt and be more beneficial to humans. The adaptation process caused the morphological and physiological change; therefore, domesticated microbes were different from their wild type. Domestication through the fermentation food process c o u l d c h a n g e o r g a n i s m s g e n e t i c a l l y a n d phenotypically (Steensels 2019). However, et al. domesticated growth and colony color had Rh. javensis no difference from its original culture. that Rh. javensis had been sub-cultured continuously had a growth rate that was also relatively the same as its wild type culture (Fig 2). Besides being a medium for growth, rice flour has functioned as a carrier in starter formulation. Starter carriers could also come from a mixture of rice flour and cassava flour (Ahnan-Winarno 2021, et al. Pramudito 2021). The addition of spices such as et al. garlic or pepper traditionally used in some fermentation starter was not done in this research. The drying process in this research was not conditioned in a very sterile place. It was just prepared like a household scale fermentation. Spices as a natural anti-microbe need to be considered in household-scale starter production as garlic and pepper were known as having anti-microbial and anti-fungal properties (Liu et al. 2017). Non-domesticated tempeh starter had Rh. javensis moderate hyphae growth, mild alcohol aroma, white or white with black spot, and hyphae bound rice granules firmly (Maulana 2019). The starter must contain high spores or other propagules concentration. In this research, var. and R. microsporus oligosporus Rh. javensis in the starter had relatively the same spore density (Table 1). However, starter had Rh. javensis low spore viability. Thanh and Nout (2007) and Putri et al. (2021) stated that spores viability may reduce by long storage. Furthermore, Putri (2021) stated et al. that high viability spores in starter are needed to ensure successful tempeh fermentation. Low viability is presumed caused by the drying and flouring process. Spore harvesting time, drying, and crushing are factors that cause of spore viability loss (Thanh and Nout 2002). Rhizopodopsis javensis tempeh starter does not meet the quality of commercial starter. It has a lower concentration of viable spore compared to the commercial starter (Raprima®). Thus, the viable spore concentration does not meet the required amount of viable spore of a commercial starter in tempeh production from 1 kg of soybean. Further, the water content is also still high (Table 2). In a low water content starter, a fermentation agent's metabolic processes will slow down, thus maintaining cell structure and function during storage (Santivarangkna et al. Rhizopus 2008). Some starters containing inoculum for rice wine production must have a moisture content of 4-5% or 7.8% (Dung and Phong 2011), while the water content of starter tempeh is about 4-5. % to 7.5% (Thanh and Nout 2002). The water content of the starter having was still Rh. javensis high, indicating that the drying process was not optimal. The drying process at 30 ℃ for 48 hours in this study has not reached the appropriate moisture content. Therefore, the drying method still needs to be modified. These characteristics indicate that the Rh. javensis starter has low quality and thus cannot be used in the making tempeh trial. In conclusion, grew optimally at 22 Rh. javensis ℃. The domestication using rice flour media with seven times subcultures did not change Rh. javensis growth rate and its colony characteristics. In the formulation of starter using rice flour as a Rh. javensis carrier resulted in its spore density relatively the same as commercial tempeh starter, but with lower spore viability and higher water content. The quality of Rh. javensis starter could not be used for tempeh production in sub-tropical climate regions yet. 82 RAHAYU ET AL . Microbiol Indones Preliminary study on tempeh production using non- domesticated . starter indicated that Rh javensis Rh. javensis can be used for tempeh production (Maulana 2019). Tempeh production using non-domesticated Rh. javensis needs a longer incubation period comparing to that of commercial starter. This indicate that a robust and high concentration of viable spores is needed to s ho r te n the incu b a ti on per io d s. Th e re f or e , domestication of to carrier substrate has to Rh. javensis be studied further for commercialization. Substrates used, adaptation method and drying process in starter production need to be modified to increase spore viability and overall starter quality, including starter lifespan. ACKNOWLEDGEMENTS We are highly grateful to the Department of Biology IPB University and the Laboratory Chemicals and the Biomedical and Basic Health Technology Research and Development Centre for support throughout the research. REFERENCES Ahnan-Winarno AD, Cordeiro L, Winarno FG, Gibbons J, Xiao H. 2021. Tempeh: A semicentennial review on its health benefits, fermentation, safety, processing, sustainability, and affordability. Compr Rev Food Sci Food Saf. 20:1717–1767. doi: 10.1111/1541- 4337.12710. Barus T, Salim DP, Hartanti AT. 2019. Kualitas tempe menggunakan TB 26 dan Rhizopus delemar R.delemar TB 37 yang diisolasi dari inokulum tradisional tempe "daun waru". J Apl Teknol Pangan. 8 (4): 143-148. doi:10.17728/jatp. 2019.4449. Boedijn KB. 1958. Notes on the Mucorales of Indonesia. Sydowia. 12 (1-6): 321-362. Dolatabadi S, Walther G, Gerrits van de Ende AHG, de Hoog GS. (2013). Diversity and delimitation of Rhizopus microsporus. Fungal divers. doi: 10.1007/s13225-013- 0229-6. Dung NTP, Rombouts FM, Nout MJR. 2005. Development of defined mixed-culture fungal fermentation starter granulate for controlled production of rice wine. Innov Food Sc i E merg Te c hn ol. 6 (4 ): 429- 4 41 . Doi:10.1016/j.ifset.2005.04.007. Dung NTP, Phong HX. 2011. Application prospects for the innovation of defined fungal starter in rice wine fermentation. J Life Scie. 5 (4): 255-263. doi: 10.17265/1934-7391/2011.04.002. Gallone 2016. Domestication and divergence of et al. Saccharomyces cerevisiae beer yeasts. Cell 166: 1397–1410. doi: 10.1016/j.cell.2016.08.020 Gallone B, Steensels J, Verstrepen KJ. 2020. Moulded by humans: The domestication of blue-veined cheese f u n g i . M o l E c o l . 2 9 : 2 5 1 7 – 2 5 2 0 . d o i : 10.1111/mec.15525 Gibbons JG, Salichos L, Slot JC . 2012. The et al evolutionary imprint of domestication on genome variation and function of the filamentous fungus Aspergillus oryzae. Curr Biol. 22(15): 1403-1409. doi: 10.1016/j.cub.2012.05.033 Hartanti AT, Rahayu G, Hidayat I. 2015. species Rhizopus from fresh tempeh collected from several regions in Indonesia. Hayati J. Biosci. 22 (3): 136-142. d : oi 10.1016/j.hjb.2015.10.004 Liu Q, Meng X, Li Y, Zhao C, Tang G, Li H. 2017. Antibacterial and anti-fungal activities of spices. Int J Mol Sci. 18 (6): 1283. doi:10.3390/ijms18061283 Maulana AI. 2019. Pembuatan tempe kedelai menggunakan Rhizopodopsis javensis sebagai starter alternatif [skripsi]. [Making soya tempeh using Rhizopodopsis javensis as alternative starter]. Bogor (ID): Insitut Pertanian Bogor. Pramudito TE, Putri EGA, Paluphi E, Yogiara Y. 2021. The effect of starter culture on bacterial profile in soybean tempeh. Food Res 5 (1): 380 – 389. doi: 10.26656/fr.2017.5(1).436 Rossi M, Buzzini P, Cordisco L, Amaretti A, Sala M, Raimondi S, Ponzoni C, Pagnoni UM, Matteuzzi D. 2009. Growth, lipid accumulation, and fatty acid composition in obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts. FEMS Microbiol E c ol . 6 9 ( 3) : 3 63 - 3 7 2 . d oi: 10. 1111 /j. 15 7 4 - 6941.2009.00727.x Santivarangkna C, Kulozik U, Foerst P. 2008. Inactivation mechanism of lactic acid starter cultures preserved by drying processes. J Appl Microbiol. 105 (1): 1-13. doi:10.1111/j.1365-2672.2008.03744.x Seumahu CA, Suwanto A, Rusmana I and Solihin DD. 2013. Bacterial and fungal communities in tempeh as reveal by amplified ribosomal intergenic sequence analysis. H a y a t i J B i o s c i e . 2 0 ( 2 ) : 6 5 – 7 1 . d o i : 10.4308/hjb.20.2.65 [SNI] Standar Nasional Indonesia. 1992. Cara Uji Makanan dan Minuman (SNI 01-2891-1992) [ Test of food and beverage]. Jakarta (ID): Badan Standardisasi Indonesia. Steensels J, Gallone B, Voordeckers K, Verstrepen KJ. 2019. Domestication of industrial microbes. Curr Biol. 29 (10): 381-393. doi:10.1016/j.cub.2019.04.025 Tahir A, Anwar M, Mubeen H, Raza S. 2018. Evaluation of physicochemical and nutritional contents in soybean Volume 15, 2021 Microbiol Indones 83 fermented food tempeh by J Adv Rhizopus oligosporus. Biol Biotechnol 17(1): 1-9. doi: 10.9734/JABB/ 2018/26770. Tamam B, Syah D, Suhartono MT, Kusuma WA, Tachibana S, and Lioe HN. 2019. Proteomic study of bioactive peptides from tempe. J Bioscie Bioeng 128 (2): 241–248. Doi: 10.1016/j.ijfoodmicro.2003.09.008. Thanh NV, Nout MJR. 2002. biomass, Rhizopus oligosporus sporangiospores yield and viability as influenced by harvesting age and processing conditions. Food Microbiol. 19 (1): 91-96. doi:10.1006/fmic.2001.0450. Thanh, NV, Rombouts, FM, Nout MJR. 2007. Viability and physiological state transitions of Rhizopus oligosporus sporangiospores in tempe starter culture. Anton Leeuw Int JG 91(1): 35–44. doi: 10.1007/s10482-006-9093-7. Wijaya CH, Nurjanah S, Utama QD. 2015. Implementasi dan analisis keuntungan teknologi pada Back-Slopping pembuatan Tempe" skala industri rumah tangga "Quick [Implementation and profit analysis using Back- Slopping technology for “Quick Tempe” making at home industrial scale]. Pangan, 24 (1): 49-62. http://repository.ipb.ac.id/handle/123456789/75390.