The effect of the use of commercial tempeh starter on the diversity of Rhizopus tempeh in Indonesia At present, only a single Rhizopus species, R. microsporus, can be found in fresh tempeh produced in Java, Indonesia. The loss of diversity of Rhizopus in tempeh has been associated with the widespread use of commercial tempeh starter in Indonesia since the 2000s. However, the identities of the previous Rhizopus strains associated with tempeh, which have been preserved in a culture collection in Indonesia, have not been verified. The present study aimed to verify the identities of 22 Rhizopus strains isolated from tempeh produced using the traditional tempeh starters from the 1960s to the 2000s. Phylogenetic analysis based on the ITS regions in the rRNA gene sequence data, revealed that the Rhizopus strains belonged to the species R. arrhizus (five strains); R. delemar (14 strains); and R. microsporus (three strains). Verification of the identities of these Rhizopus strains in the present study confirmed the loss of diversity of Rhizopus species in tempeh produced in Indonesia, particularly in Java. Our findings confirmed that the morphological changes in Rhizopus species isolated from tempeh as a result of centuries of domestication. Tempeh is a soybean-based fermented food that is popular worldwide. It is regarded as a good source of protein and is easily digestible food. Tempeh has been a very popular traditional fermented food in Indonesia for many centuries that reported by Nout and Kiers in 20051. It is a very important protein source in the Javanese diet2. The production of traditional tempeh is thought to have started in Indonesia in the early 1600s3. It originated from Central or East Java. English word tempeh comes from Indonesian ‘tempe’. The word ‘tempe’ originated from Central Java, Indonesia. Serat Centhini is the first known manuscript in Java to mention this word ‘tempe’3. As written in ‘The History of Tempeh’3, traditionally since at least 1875, the name for this food in Indonesia was written témpé, with various accents being used. Then in August 1972, when Indonesia modernized its language, the accents were dropped and the word came to be spelled tempe (still pronounced TEM-pay). In English and other European languages, the word has come to be spelled ‘tempeh,’ the final ‘h’ being added to prevent the word from being pronounced ‘temp’. Hendrik Coenraad Prinsen Geerligs was the first European man who use the spelling tempeh in German article in 18963,4. Van Veen and Schaefer in 19505 were the first scientists used term tempeh in an English language article. Then Steinkrauss et al.6 were the first in the US. Since then, the word has consistently been spelled tempeh in European languages.The taxonomy of the genus Rhizopus (Ehrenb. 1821) has undergone dramatic changes, especially in the last 40 years. It has been significantly changed from traditional7,8,9 to molecular approaches10,11,12,13,14. Since the description of R. arrhizus by Fischer in 1892 (Fischer 1892), hundreds of species have been described based on discrete morphological and physiological features7,8. In 1965, Inui et al.7 examined 449 Rhizopus species in their monographs. Almost 20 years later, Schipper8 and Schipper and Stalpers9 revised the classification of Rhizopus based on comprehensive morphological characters, temperature tolerance and mating. They separated the genus into three groups—R. microsporus, R. stolonifer, and R. arrhizus (= oryzae), with the re-integration of many species. Schipper8 synonymized 29 species with R. arrhizus (= oryzae). The group classification of Schipper8 and Schipper and Stalpers9 are widely accepted. In 1985, Ellis15 concluded that R. arrhizus, Amylomyces rouxii, and R. delemar are conspecific based on DNA renaturation experiments and proposed to accommodate them in three varieties.Abe et al.16 established the first molecular phylogeny of Rhizopus based on three molecules of the ribosomal RNA-encoding DNA (rDNA) and confirmed the same taxonomic grouping microsporus-group, stolonifer-group, and R. arrhizus. Liu et al.11 combined analyses of the ITS regions of rRNA and pyrG genes data and only allowed eight species to be distinguished: R. americanus, R. caespitosus, R. homothallicus, R. microsporus, R. reflexus, R. schipperae, R. sexualis, and R. stolonifer. The remaining two morphologically distinct species, R. arrhizus and R. niveus, formed an unresolved cluster. They considered A. rouxii as synonymous of R. arrhizus. In the same year, Zheng et al.12 revised the monograph of Rhizopus and organized the genus into 10 species and seven varieties by reanalyzed the data from Liu et al.11 along with morphological data. Abe et al.17 used rDNA ITS, actin-1, and translation elongation factor 1a (TEF-1a) sequences to confirm the eight-species division of Rhizopus. They showed that the R. microsporus complex consisted of a single species. Dolatabadi18 investigated the species boundaries of R. microsporus using ITS, ACT, and TEF markers in combination with mating tests, morphology, physiology, ecology, geography, and MALDI-TOF MS data, and reduced the six varieties of R. microsporus (vars microsporus, azygosporus, chinensis, oligosporus, rhizopodiformis, and tuberosus; Liu et al.11) to synonyms. The widely held suggestion that the strains with the morphology of R. oligosporus represent a separate species that can be found just in fermented food sources i.e. tempeh should thus be rejected because positive mating results have been found between all varieties of R. microsporus, therefore all strains concluded as a single species, R. microsporus.The species boundaries among R. arrhizus and R. delemar was studied by Abe et al.10 and Gryganskyi et al.19, they show clearly that R. arrhizus and R. delemar represent taxonomic entities that either deserves the rank of varieties or species. Dolatabadi et al.13 considered R. arrhizus consisted of two varieties, e.g. var. arrhizus and var. delemar, based on sequence data of multi-locus studies as well as amplified fragment length polymorphism (AFLP) and mating experiment. They found there is still zygospore formation between members of both varieties, although their number is reduced suggesting that the mating barrier is not complete yet. There is also a nomenclatural issue with arrhizus. Rhizopus arrhizus was described first, but R. oryzae has been used by most authors. Schipper8 treated R. arrhizus as a doubtful species. Ellis et al.15 took up the name R. arrhizus again by designating NRRL 1469 as ex-neotype strain of R. arrhizus. Zheng et al.12 in their monograph on Rhizopus preferred R. arrhizus over R. oryzae. Similarly, Dolatabadi et al.13 also use the name R. arrhizus based on the protologue of the first described R. arrhizus. Gryganskyi et al.14, also use the name R. arrhizus in their classification of the genus Rhizopus using phylogenomic approaches based on 192 orthologous genes. They classified Rhizopus strains into four species, e.g. R. microsporus, R. stolonifer, R. arrhizus, and R. delemar. In the present study, we followed this classification system (taxonomy of Rhizopus sensu Gryganskyi et al.14).Earlier studies on tempeh before the Second World War by Dutch microbiologists3 revealed that tempeh in Java was fermented with R. arrhizus. The first scientific report on tempeh was published in 1896 and was written by the Dutchman H.C. Prinsen Geerligs, who lived in Java3,4. He stated that tempeh was fermented by the mold R. arrhizus. The same species was also mentioned by van Veen and Schaefer5. Some reports around the 1960s20 also found that R. arrhizus, was the dominant species from highly preferred tempeh samples in Java, such as tempeh ‘Malang’ and tempeh ‘Purwokerto’.In the 1960s, the cottage-scale tempeh industry spread to all regions in Indonesia by using traditional methods for tempeh production and producing tempeh with various local tastes and flavors. The method for preparing the inoculum (tempeh starter) varied based on locality. In the traditional process, the previous batch of tempeh or the mold grown and dried on Hibiscus tiliaceus leaves (daun Waru) was used as the tempeh inoculum. These leaves are used to carry tempeh inoculum as natural starters (known as usar in Indonesia). Following this, beans were wrapped using banana or other large leaves and finally placed in a warm location to ferment for 1 or 2 days5,21 Tempeh has a pleasant odor and a slight cheese-like flavor6. In the earlier study of tempeh by a group of scientists from Cornell University, USA, around the 1960s, revealed that R. arrhizus to be the essential microorganism isolated from Indonesian tempeh scrapings6.The interest in tempeh produced in Indonesia rapidly increased among Indonesian scientists after the late 1960s. Several Rhizopus species associated with tempeh produced using the traditional process in Indonesia have been reported by Indonesian mycologists. Dwidjoseputro and Wolf22 reported R. arrhizus, R. microsporus, and R. stolonifer to be associated with tempeh and tempeh starters in Malang, Surakarta, and Jakarta.Extensive research on tempeh was also conducted in the USA since the 1960s by groups of microbiologists and food scientists2,5,6,23. An Indonesian microbiologist, Ko Swan Djien from the Bandung Institute of Technology, West Java, brought tempeh samples from Java to the laboratory of Dr. Hesseltine at NRRL, USA, in 1961 in order to study tempeh fermentation23. Forty Rhizopus strains were isolated from these tempeh samples. These strains belonged to species: R. achlamydosporus, R. arrhizus, R. formosaensis, R. microsporus (= R. oligosporus), and R. stolonifer23. Hesseltine23 stated that only R. arrhizus and R. microsporus (= R. oligosporus) were commonly used to produce tempeh. Wang and Hesseltine24 reported the best strain for producing tempeh from wheat and soybeans was R. microsporus (= R. oligosporus) NRRL 2710. Since they claimed that R. microsporus (= R. oligosporus) as the best tempeh mould, this species was then used by many Indonesian microbiologists for their study on tempeh (Gandjar and Santoso)20.Large-scale commercial tempeh production began in the 1980s with the aim of guaranteeing a good tempeh product. The first commercial inoculum for tempeh, which consisted of mixed cultures of R. arrhizus and R. microsporus, was developed by the Chemistry Institute-Indonesian Institute of Sciences (LKN-LIPI) and the Cooperative of Tempeh and Tofu Producers of Indonesia (KOPTI) in 1985; they then distributed it to tempeh producers20. The next generation of commercial tempeh starter developed by LIPI was Raprima, containing only a single species, R. microsporus. Raprima has been produced by PT. Aneka Fermentasi Industri, Bandung, Indonesia, since 2001, and is widely used in tempeh fermentation in Indonesia and abroad.Taxonomy of Rhizopus strains obtained from tempeh in Indonesia has been well studied by many scientists in abroad and those strains are well maintained at Centraalbureau voor Schimmelcultures KNAW (currently hosted by Westerdijk Institute) (The Netherlands), others in Mycothèque de l’Université catholique de Louvain (MUCL) (Belgium) and Northern Regional Research Laboratory (NRRL) (USA). On the other side, it is difficult to trace the genetic diversity of Rhizopus spp. previously used for tempeh production that preserved in culture collections in Indonesia, because Rhizopus cultures were rarely collected or were never preserved properly in culture collections in Indonesia. Their representation within sequence database is lacking and their molecular study has never been reported.One of the authors (I. G.), collected Rhizopus strains and accumulated hundreds of strains from almost all regions in Indonesia since the 1960s. These Rhizopus strains have been preserved in the Universitas Indonesia Culture Collection (UICC), Depok, Indonesia. It is the only culture collection in Indonesia that maintains the Rhizopus strains isolated from tempeh produced using the traditional tempeh starters. Because of a lack of budget, this collection of Rhizopus strains was originally maintained only as living cultures; therefore, many strains have been lost. Since 2012, the strains have been maintained using a long-term preservation method, the liquid drying (L-drying) method, after financial support was obtained from the Society for Applied Microbiology of the United Kingdom (SfAM UK) Endangered Collection Grant.At present, only 127 Rhizopus strains available from those isolated from tempeh produced using traditional starters (1960s–2000s) that are preserved in UICC. The molecular identification of these strains was not performed until 2017, when we sequenced 15 strains of Rhizopus from UICC based on the ITS regions of ribosomal RNA (rRNA) gene25,26,27. The present study aimed to sequence another 22 strains the Rhizopus strains from UICC based on the ITS regions of ribosomal RNA (rRNA) gene, to provide the accurate taxonomic identity of Rhizopus strains that were isolated from tempeh produced using traditional tempeh starters (1960s–2000s).All Rhizopus strains were obtained from UICC, Center of Excellence for Indigenous Biological Resources-Genome Studies, FMIPA Universitas Indonesia, Depok, Indonesia. UICC maintains 127 Rhizopus spp. strains that originated from various types of tempeh (e.g., tempe kedelai, tempe gembus, tempe kopra, tempe kedelai hitam, tempe koro, tempe koro wedus, tempe benguk, tempe kapok, and tempe lamtoro) and traditional tempeh starters (e.g. laru daun waru and laru daun pisang) and were isolated from the 1960s to the 2000s. The origin of the 22 strains used in the present study and their year of isolation are provided in Table 1. Tempeh and tempeh starter samples were obtained
https://www.nature.com/articles/s41598-021-03308-6
The effect of the use of commercial tempeh starter on the diversity of Rhizopus tempeh in Indonesia
