When potato pulp was mixed with Indonesian starter ragi tapé and incubated, both lactic acid and ethanol were gradually formed and attained certain concentrations during 2 days of fermentation. Viable counts of fungi in fresh weight matter, yeasts and lactic acid bacteria after fermentation were 105, 107 and 105 CFU/g, respectively. Denaturing gradient gel electrophoresis of the PCR-amplified internal transcribed spacer of 18S–28S rRNA genes detected Amylomyces rouxii-Rhizopus oryzae, Mucor indicus, Candida tropicalis and Saccharomycopsis fibuligera and revealed that Amylomyces rouxii-Rhizopus oryzae dominated throughout the fermentation period. Amylomyces rouxii cannot be discriminated from the lactic acid-accumulating group of Rhizopus oryzae because the amplified sequences of these fungi were shown to be identical. Morphological characteristics were then studied for Rhizopus-like fungi isolated from fermented potato pulp. Those strains that had produced an enormous number of chlamydospores in the aerial and substrate mycelium were identified as Amylomyces rouxii. The microflora of fermented potato pulp was similar to that made from glutinous rice, namely tapé ketan

Ayumi Abe

I-Nengah Sujaya

Kozo Asano

Teruo Sone

Yuji Oda

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Microflora and Selected Metabolites of Potato Pulp Fermented with an Indonesian Starter Ragi Tapé

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UDC 663.532.1:579.864.1 original scientific paperISSN 1330-9862(FTB-1308)Microflora and Selected Metabolites of Potato PulpFermented with an Indonesian Starter Ragi TapéAyumi Abe1, I-Nengah Sujaya2, Teruo Sone1, Kozo Asano1 and Yuji Oda3*1Department of Bioscience and Chemistry, Graduate School of Agriculture, Hokkaido University,Sapporo, 060-8589, Hokkaido, Japan2 The Study Program of Public Health Science, Udayana University, Bukit Jimbaran Campus,Badung, Bali, Indonesia3Department of Upland Agriculture, National Agricultural Research Center for Hokkaido Region,Memuro, Kasai, 082-0071, Hokkaido, JapanReceived: February 19, 2004Accepted: July 6, 2004SummaryWhen potato pulp was mixed with Indonesian starter ragi tapé and incubated, bothlactic acid and ethanol were gradually formed and attained certain concentrations during2 days of fermentation. Viable counts of fungi in fresh weight matter, yeasts and lacticacid bacteria after fermentation were 105, 107 and 105 CFU/g, respectively. Denaturing gra-dient gel electrophoresis of the PCR-amplified internal transcribed spacer of 18S–28SrRNA genes detected Amylomyces rouxii-Rhizopus oryzae, Mucor indicus, Candida tropicalisand Saccharomycopsis fibuligera and revealed that Amylomyces rouxii-Rhizopus oryzae domi-nated throughout the fermentation period. Amylomyces rouxii cannot be discriminated fromthe lactic acid-accumulating group of Rhizopus oryzae because the amplified sequences ofthese fungi were shown to be identical. Morphological characteristics were then studiedfor Rhizopus-like fungi isolated from fermented potato pulp. Those strains that had pro-duced an enormous number of chlamydospores in the aerial and substrate mycelium wereidentified as Amylomyces rouxii. The microflora of fermented potato pulp was similar tothat made from glutinous rice, namely tapé ketan.Key words: Amylomyces rouxii, lactic acid fermentation, ragi tapé169A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)* Corresponding author; Phone: ++81 155 62 92 80; Fax: ++81 155 62 92 81; E-mail: yujioda@affrc.go.jpIntroductionIn Asian countries, dried powders, flat cakes or hardballs are often used as inocula for the production of fer-mented foods and alcoholic beverages from starchy ma-terials (1). These starters contain fungi, yeasts and lacticacid bacteria and are referred to by different names ineach area (2,3): ragi tapé in Indonesia, look-pang in Thai-land, chin-hueh in China, bubod in the Philippines, nurukin Korea and murcha in Himalayan regions. Lee and Fu-jio (4) have isolated some microorganisms from banhmen, a starter from Vietnam, and identified the fungalstrains as Amylomyces rouxii, Mucor circinelloides, Mucorindicus and Rhizopus oryzae, and the yeast strains as Can-dida pelliculosa, Pichia burtonii, Pichia anomala, Saccharo-myces cerevisiae and Saccharomycopsis fibuligera, based onthe morphological and biochemical characteristics. Simi-lar microbial composition has been reported for otherfermentation starters (1,3).Conventional Indonesian tapé is fermented glutinousrice produced by using ragi tapé and consumed as asweet dessert or snack (5–7). Among the microorganismspresent in ragi tapé, the fungus Amylomyces rouxii plays acrucial role in tapé fermentation. A. rouxii degrades starchto glucose, which sustains its growth and that of someyeasts and bacteria, and simultaneously synthesizes bothlactic acid and ethanol. Development of the characte-ristic and acceptable flavor of tapé further requires thegrowth of certain yeasts in addition to A. rouxii.Raw materials for tapé are glutinous rice, cassavaand other crops containing a high amount of starch. Po-tatoes (Solanum tuberosum) are rarely used in Indonesia(2). Through the research into the efficient utilization ofagricultural by-products, we suspected that potato pulpcould be substituted for an ingredient of tapé. Potatopulp containing starch, cellulose, hemicellulose and pec-tic substances emerges in the starch industry as the resi-due extracted from potato tubers (8). The pulp is crum-bled and usually composted or used as cattle feed afterbeing dried or ensiled. Recently, R. oryzae, which is taxo-nomically related to A. rouxii, has been used as an alter-native inoculant for ensiling potato pulp (9). The finalgoal of our research project is to convert potato pulp topalatable foodstuff by microbial fermentation. In thepresent study, microflora of tapé made with potato pulphas been compared to tapé ketan, the conventional tapémade with glutinous rice.Materials and MethodsFermentation testsPotato pulp (dry matter 20.8 %) was provided by alocal plant that manufactures starch from potato tubers.A commercial ragi tapé (Na Kok Liong, Solo, Indonesia)was purchased from a market in Yogjakarta, Indonesiain September 2002. The potato pulp (400 g of fresh mat-ter) was cooled to room temperature after being auto-claved at 121 °C for 15 min, then it was mixed with 4 gof ragi tapé and put on Petri dishes in the amount of 50g for fermentation at 27 °C for 4 days. Portions of thesamples were weighed and extracted with saline solu-tion (0.85 % NaCl) to analyze organic acids and ethanolusing high-performance liquid chromatography with themethod described elsewhere (10). Glutinous rice was auto-claved at 121 °C for 15 min after soaking at room tem-perature for 2 h and used instead of potato pulp as areference.Enumeration of microorganismsFive grams of the fermented pulp were suspendedin 30 mL of sterilized saline solution (0.85 % NaCl) andhomogenized by repeating three times in a Multi-beadShocker (Yasui Kikai Co., Osaka, Japan) for 60 s with in-tervals of 1 min. The suspension was diluted seriallyand spread on the defined media for the enumeration ofmicroorganisms. The media and incubation conditionswere as follows: MRS agar containing 10 µg/L of cyclo-heximide for lactic acid bacteria, 30 °C for 2 days in ananaerobic chamber; potato dextrose agar for yeasts, 27°C for 2 days; malt extract agar for fungi, 30 °C for 1day. Yeasts and fungi were discriminated based on theirappearance.DNA extractionThe suspension containing 5 g of the fermented po-tato pulp in 30 mL of TE (10 mM Tris, 1 mM EDTA,pH=8.0) was supplemented with lysozyme (0.1 mg/mL),labiase (0.1 mg/mL) and N-acetylmuramidase (30 µg/mL)and was incubated at 37 °C for 30 min. After centrifu-gation, DNA was extracted and purified from the pre-cipitate by UltraClean Soil DNA Isolation Mega Prep Kit(Mo Bio Laboratories, Inc., Carlsbad, CA).PCR amplificationThe entire region of the internal transcribed spacerbetween 18S and 28S rRNA genes (ITS) was first amplifiedby using the primers ITS4 (5’-TCCTCCGCTTATTGAT-ATGC-3’) and ITS5 (5’-GGAAGTAAAAGTCGTAACAA-GG-3’) targeted to 28S and 18S rRNA genes, respectively(11). The product was passed through a MicroSpin S-300HR Column (Amersham Bioscience) to remove residualprimers. A second PCR was conducted to attach a GC--clamp by using the primers ITS5-GC (5’-CGCCCGCCG-GGCGCGGCGGGCGGGGCGGGGGCACGGGGGAAGT-AAAAGTCGTAACAAGG-3’) and ITS6 (5’-CGTTCTTC-ATCGATGCGAGA-3’). The reaction mixture contained1 µL of the product of the first PCR, 10 pM of each pri-mer, 1.5 mM MgCl2, 0.2 mM dNTP and 2.5 U AmpliTaqGold (PE Applied Biosystems). These components weremixed in 1 × AmpliTaq Gold Buffer (PE Applied Biosys-tems) to obtain a final volume of 50 µL. Initial denatur-ation was done at 94 °C for 5 min, followed by 25 cyclesof denaturation at 94 °C for 30 s, annealing at 70 °C for1 min and elongation at 72 °C for 1 min, with the finalelongation at 72 °C for 5 min. The annealing tempera-ture was reduced by 1 °C per cycle from 70 to 65 °C andadjusted to 65 °C after the fifth cycle. After each PCR re-action, 10 µL was electrophoresed on 1.5 % agarose gelto check the amplification. The products in the remain-der of the reaction mixture (40 µL) were concentrated to10 µL by ethanol precipitation and used for the follow-ing experiments.Denaturing gradient gel electrophoresis(DGGE) analysisDGGE was performed with a DCode Universal Mu-tation Detection System (Bio-Rad Laboratories). A PCRsample was applied directly to 8 % polyacrylamide gelsin a 1 × TAE buffer (40 mM Tris, 20 mM acetic acid and1 mM EDTA, pH=8.3) with a denaturing gradient rang-ing from 20 to 50 %. One hundred percent of denaturantcorresponds to 7 M urea and 40 % formamide. The gra-dient gel was cast using the Gradient Delivery System,Model 475 (Bio-Rad Laboratories, Hercules, CA). Elec-trophoresis was run at a constant voltage of 65 V and at60 °C. After the electrophoresis was run for 16 h, the gelwas stained with SYBR Green I (Molecular Probes, Eu-gene, OR) at 10 000 × dilution in 1 × TAE for 30 minand photographed on a UV illuminator. DGGE bandswere excised from the denaturing gels, re-amplifiedwith primers ITS5 and ITS6, as described above, and se-quenced by each primer. A search of the GenBank data-base was conducted using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).170 A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)Results and DiscussionThe data in this study are representative results ofexperiments performed in three replicates.The metabolic production of lactic acid and ethanolis shown in Fig. 1 along with the development of themicrobial population. Lactic acid was formed in accor-dance with pH reduction and was accompanied by theproduction of ethanol. Concentrations of lactic acid andethanol decreased after 2 and 3 days of incubation, re-spectively. The viable count of all microorganisms in-creased within one day of fermentation except for lacticacid bacteria. The replacement of glutinous rice with po-tato pulp stimulated the growth of yeasts and lactic acidbacteria, resulting in high amounts of ethanol and lacticacid. In both cases, the population of fungi seemed to bemuch lower than that of yeasts, but the former valuesmay have been underestimated. A single colony appear-ing as a fungus is not derived from a single cell such asyeasts because of mycelial growth (12). The contributionof fungi to the fermentation may be more significantthan expected based on the evaluation of the colony-for-ming units (CFU) (6). However, this assumption cannotbe proved by the classical microbiological approach.Fig. 2 shows DGGE profiles of DNA harvested fromfermented potato pulp and glutinous rice. The diagramsinclude five minor bands that are hardly visible in thephotographs. When individual bands appearing in DGGEprofiles were sequenced, four of seven bands were affili-ated to Amylomyces rouxii-Rhizopus oryzae, indicating theirdominance throughout the fermentation period. One ofthree major bands was derived from Mucor indicus. It isunknown why multiple bands originated from A. rou-xii-R. oryzae on DGGE gels even though these bands hadidentical sequences. We suppose that the appearance ofthese minor bands is the result of non-uniformed disso-ciation depending on their nucleotide sequences. Whenlarge amounts of a single PCR product are loaded onDGGE gels, minor bands may emerge in addition to themajor band. Among yeasts, Candida tropicalis disap-peared just after the fermentation, and Saccharomycopsisfibuligera emerged on the third day. Further growth of S.fibuligera in the fermented glutinous rice, as judged froman additional band (h) in Fig. 2, is related to vigorousproduction of ethanol after 3 days (Fig. 1).In DGGE gels, the yeast DNA band was faint,whereas A. rouxii-R. oryzae DNA was clearly detected, incontrast to the population detected with the plating me-thod (Fig. 1). This result indicates that, in terms of DNA,A. rouxii dominated in the culture. This discrepancymight be due to the fact that yeasts, unicellular organ-isms, can easily dominate in the plate-counting method,whereas the fungi will develop a single colony frommultiple cells. Thus, the method we established herewill be useful to identify mixed populations of fungi intraditional fermented foods in Asia.171A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)0 1 2 3 4345670 1 2 3 4 0 1 2 3 4345670 1 2 3 4010203023456780 1 2 3 423456780 1 2 3 4Potato pulp Glutinous riceCulture period / dayViablecounts/(log[cfu/g])Massfraction/(mg/g)pH0102030Fig. 1. Biochemical and microbiological parameters monitored during the fermentation of potato pulp and glutinous ricePotato pulp and glutinous rice were mixed with the starter ragi tapé and incubated at 27 °C for 4 days. After the extraction with sa-line solution (0.85 % NaCl), pH and metabolites were analyzed. Microorganisms in the fermented products were enumerated on thedefined mediaSymbols:  pH,  lactic acid,  ethanol,  fungi,  yeasts,  lactic acid bacteriaBacterial communities of fermented foods (13,14) andcompost (15) have been assessed by DGGE for the V3region of rDNA. DGGE analysis of the 16S–26S rDNAspacer region from bacteria has been useful for discrimi-nating closely related species (16) or individual strainsin the same species (17). For eukaryotic organisms, a li-mited number of DGGE based studies was conductedusing partially amplified 18S rDNA (18) and 28S rDNA(19,20) but not the ITS region.Amylomyces is a monotypic genus containing thesingle variable species A. rouxii, which is closely relatedto R. oryzae, as identifiable from the formation of rhi-zoids, stolons and black-pigmented sporangia (12). Adistinct morphological characteristic of A. rouxii is theenormous number of chlamydospores produced in theaerial and substrate mycelium. Recently, R. oryzae strainshave been classified into two groups which principallyaccumulate either lactic acid or fumaric acid in a me-dium containing relatively high amounts of carbonsources. However, the ITS1 sequence cannot discrimi-nate among A. rouxii and lactic acid-accumulatingstrains of R. oryzae because these sequences are identical(11). We isolated twenty colonies of fungi from the fer-mented potato pulp, which were identified as Mucorindicus and A. rouxii based on morphological character-istics. The appearance of R. oryzae was not shown. Thishas led to the conclusion that A. rouxii dominated in theculture of the fermented potato pulp.From the DGGE pattern (Fig. 2), A. rouxii was alsofound to dominate in the fermented rice, tapé ketan, indi-cating that potato pulp is unlikely to stimulate the growthof undesirable fungi existing in ragi tapé. In Indonesia,peeled, diced and steamed cassava tubers are alterna-tively used for the production of tapé ketella. Potato pulpfermented by ragi tapé may be consumed as other tapéproducts.AcknowledgementsWe thank K. Saito for his helpful advice. This workwas supported in part by Special Coordination Fundsfor Promoting Science and Technology (Leading Re-search Utilizing Potential of Regional Science and Tech-nology) of the Japanese Ministry of Education, Culture,Sports, Science and Technology.References1. C. W. Hesseltine, Annu. Rev. Microbiol. 37 (1983) 575–601.2. W. H. Holzapfel, Int. J. Food Microbiol. 75 (2002) 197–212.3. C. W. Hesseltine, R. Rogers, F. G. Winarno, Mycopatholo-gia, 101 (1988) 141–155.4. A. C. Lee, Y. Fujio, World J. Microbiol. Biotechnol. 15 (1999)57–62.5. T. C. Cronk, K. H. Steinkraus, L. R Hackler, L. R. Mattick,Appl. Environ. Microbiol. 33 (1977) 1067–1073.6. M. M. Ardhana, G. H. Fleet, Int. J. Food Microbiol. 9 (1989)157–165.7. S. Siebenhandl, L. N. Lestario, D. Trimmel, E. Berghofer,Int. J. Food Sci. Nutr. 52 (2001) 347–357.8. F. Mayer, J. O. Hillebrandt, Appl. Microbiol. Biotechnol. 48(1997) 435–440.9. Y. Oda, K. Saito, H. Yamauchi, M. Mori, Curr. Microbiol. 45(2002) 1–4.10. Y. Oda, Y. Yajima, M. Kinoshita, M. Ohnishi, Food Micro-biol. 20 (2003) 371–375.11. A. Abe, T. Sone, I. N. Sujaya, K. Saito, Y. Oda, K. Asano,F. Tomita, Biosci. Biotechnol. Biochem. 67 (2003) 1725–1731.12. J. J. Ellis, L. J. Rhodes, C. W. Hesseltine, Mycologia, 68(1976) 131–143.13. F. Ampe, A. Sirvent, N. Zakhia, Int. J. Food Microbiol. 65(2001) 45–54.14. S. Fasoli, M. Marzotto, L. Rizzotti, F. Rossi, F. Dellaglio, S.Torriani, Int. J. Food Microbiol. 82 (2003) 59–70.172 A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)Fig. 2. Denaturing gradient gel electrophoresis (DGGE) profiles of the PCR-amplified ITS1 region of the DNA extracted from fer-mented potato pulp and glutinous riceDGGE analysis was conducted for the fermented potato pulp and glutinous rice used in Fig. 1.Shaded column in the diagram indicates the band invisible in the corresponding photograph. Each band was excised, re-amplified,sequenced and affiliated as follows: (a), (d), (e) and (f), Amylomyces rouxii-Rhizopus oryzae; (b) and (h), Saccharomycopsis fibuligera; (c),Mucor indicus; (g), Candida tropicalis15. Y. Ueno, S. Haruta, M. Ishii, Y. Igarashi, Appl. Microbiol.Biotechnol. 57 (2001) 555–562.16. N. D. Crosbie, M. Pockl, T. Weisse, J. Microbiol. Methods,55 (2003) 361–370.17. A. Buchan, M. Alber, R. E. Hodson, FEMS Microbiol. Ecol.35 (2001) 313–321.18. P. Möhlenhoff, L. Müller, A. A. Gorbushina, K. Petersen,FEMS Microbiol. Lett. 195 (2001) 169–173.19. G. A. Kowalchuk, F. A. de Souza, J. A. van Veen, Mol.Ecol. 11 (2002) 571–581.20. C. Schabereiter-Gurtner, G. Piñar, W. Lubitz, S. Rölleke, J.Microbiol. Methods, 47 (2001) 345–354.Mikroflora i odabrani metaboliti krumpirove pulpe fermentirane sdodatkom indonezijske starter kulture ragi tapéSa`etakKada se krumpirova pulpa pomije{a s indonezijskom starter kulturom ragi tapé i in-kubira, postupno se stvaraju mlije~na kiselina i etanol koji nakon dva dana fermentacijedosti`u odre|ene koncentracije. Nakon fermentacije utvr|eno je u svje`oj tvari 105 CFU/ggljivica, 107 CFU/g kvasca i 105 CFU/g bakterija mlije~ne kiseline. Denaturiraju}om gradi-jentnom gel-elektroforezom PCR-amplificiranih i transkribiranih internih razdjelnih zona(spacer region) iz 18S–28S rDNA gena utvr|eni su Amylomyces rouxii-Rhizopus oryzae, Mu-cor indicus, Candida tropicalis i Saccharomycopsis fibuligera, a ujedno je ustanovljeno da suAmylomyces rouxii-Rhizopus oryzae prevladavali tijekom fermentacije. Amylomyces rouxii nemo`e se razlikovati od Rhizopus oryzae, koja proizvodi mlije~nu kiselinu, jer su amplifi-cirane sekvencije tih gljivica identi~ne. Stoga su prou~avane morfolo{ke karakteristike vrstiRhizopus sli~ne gljivice izolirane iz fermentirane krumpirove pulpe. Oni sojevi koji su pro-izveli golem broj klamidospora u okoli{u, i u miceliju supstrata, utvr|eni su kao Amy-lomyces rouxii. Mikroflora fermentirane pulpe krumpira bila je sli~na kao kod ljepljive ri`e(tapé ketan).173A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)

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UDC 663.532.1:579.864.1 original scientific paperISSN 1330-9862(FTB-1308)Microflora and Selected Metabolites of Potato PulpFermented with an Indonesian Starter Ragi TapéAyumi Abe1, I-Nengah Sujaya2, Teruo Sone1, Kozo Asano1 and Yuji Oda3*1Department of Bioscience and Chemistry, Graduate School of Agriculture, Hokkaido University,Sapporo, 060-8589, Hokkaido, Japan2 The Study Program of Public Health Science, Udayana University, Bukit Jimbaran Campus,Badung, Bali, Indonesia3Department of Upland Agriculture, National Agricultural Research Center for Hokkaido Region,Memuro, Kasai, 082-0071, Hokkaido, JapanReceived: February 19, 2004Accepted: July 6, 2004SummaryWhen potato pulp was mixed with Indonesian starter ragi tapé and incubated, bothlactic acid and ethanol were gradually formed and attained certain concentrations during2 days of fermentation. Viable counts of fungi in fresh weight matter, yeasts and lacticacid bacteria after fermentation were 105, 107 and 105 CFU/g, respectively. Denaturing gra-dient gel electrophoresis of the PCR-amplified internal transcribed spacer of 18S–28SrRNA genes detected Amylomyces rouxii-Rhizopus oryzae, Mucor indicus, Candida tropicalisand Saccharomycopsis fibuligera and revealed that Amylomyces rouxii-Rhizopus oryzae domi-nated throughout the fermentation period. Amylomyces rouxii cannot be discriminated fromthe lactic acid-accumulating group of Rhizopus oryzae because the amplified sequences ofthese fungi were shown to be identical. Morphological characteristics were then studiedfor Rhizopus-like fungi isolated from fermented potato pulp. Those strains that had pro-duced an enormous number of chlamydospores in the aerial and substrate mycelium wereidentified as Amylomyces rouxii. The microflora of fermented potato pulp was similar tothat made from glutinous rice, namely tapé ketan.Key words: Amylomyces rouxii, lactic acid fermentation, ragi tapé169A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)* Corresponding author; Phone: ++81 155 62 92 80; Fax: ++81 155 62 92 81; E-mail: yujioda@affrc.go.jpIntroductionIn Asian countries, dried powders, flat cakes or hardballs are often used as inocula for the production of fer-mented foods and alcoholic beverages from starchy ma-terials (1). These starters contain fungi, yeasts and lacticacid bacteria and are referred to by different names ineach area (2,3): ragi tapé in Indonesia, look-pang in Thai-land, chin-hueh in China, bubod in the Philippines, nurukin Korea and murcha in Himalayan regions. Lee and Fu-jio (4) have isolated some microorganisms from banhmen, a starter from Vietnam, and identified the fungalstrains as Amylomyces rouxii, Mucor circinelloides, Mucorindicus and Rhizopus oryzae, and the yeast strains as Can-dida pelliculosa, Pichia burtonii, Pichia anomala, Saccharo-myces cerevisiae and Saccharomycopsis fibuligera, based onthe morphological and biochemical characteristics. Simi-lar microbial composition has been reported for otherfermentation starters (1,3).Conventional Indonesian tapé is fermented glutinousrice produced by using ragi tapé and consumed as asweet dessert or snack (5–7). Among the microorganismspresent in ragi tapé, the fungus Amylomyces rouxii plays acrucial role in tapé fermentation. A. rouxii degrades starchto glucose, which sustains its growth and that of someyeasts and bacteria, and simultaneously synthesizes bothlactic acid and ethanol. Development of the characte-ristic and acceptable flavor of tapé further requires thegrowth of certain yeasts in addition to A. rouxii.Raw materials for tapé are glutinous rice, cassavaand other crops containing a high amount of starch. Po-tatoes (Solanum tuberosum) are rarely used in Indonesia(2). Through the research into the efficient utilization ofagricultural by-products, we suspected that potato pulpcould be substituted for an ingredient of tapé. Potatopulp containing starch, cellulose, hemicellulose and pec-tic substances emerges in the starch industry as the resi-due extracted from potato tubers (8). The pulp is crum-bled and usually composted or used as cattle feed afterbeing dried or ensiled. Recently, R. oryzae, which is taxo-nomically related to A. rouxii, has been used as an alter-native inoculant for ensiling potato pulp (9). The finalgoal of our research project is to convert potato pulp topalatable foodstuff by microbial fermentation. In thepresent study, microflora of tapé made with potato pulphas been compared to tapé ketan, the conventional tapémade with glutinous rice.Materials and MethodsFermentation testsPotato pulp (dry matter 20.8 %) was provided by alocal plant that manufactures starch from potato tubers.A commercial ragi tapé (Na Kok Liong, Solo, Indonesia)was purchased from a market in Yogjakarta, Indonesiain September 2002. The potato pulp (400 g of fresh mat-ter) was cooled to room temperature after being auto-claved at 121 °C for 15 min, then it was mixed with 4 gof ragi tapé and put on Petri dishes in the amount of 50g for fermentation at 27 °C for 4 days. Portions of thesamples were weighed and extracted with saline solu-tion (0.85 % NaCl) to analyze organic acids and ethanolusing high-performance liquid chromatography with themethod described elsewhere (10). Glutinous rice was auto-claved at 121 °C for 15 min after soaking at room tem-perature for 2 h and used instead of potato pulp as areference.Enumeration of microorganismsFive grams of the fermented pulp were suspendedin 30 mL of sterilized saline solution (0.85 % NaCl) andhomogenized by repeating three times in a Multi-beadShocker (Yasui Kikai Co., Osaka, Japan) for 60 s with in-tervals of 1 min. The suspension was diluted seriallyand spread on the defined media for the enumeration ofmicroorganisms. The media and incubation conditionswere as follows: MRS agar containing 10 µg/L of cyclo-heximide for lactic acid bacteria, 30 °C for 2 days in ananaerobic chamber; potato dextrose agar for yeasts, 27°C for 2 days; malt extract agar for fungi, 30 °C for 1day. Yeasts and fungi were discriminated based on theirappearance.DNA extractionThe suspension containing 5 g of the fermented po-tato pulp in 30 mL of TE (10 mM Tris, 1 mM EDTA,pH=8.0) was supplemented with lysozyme (0.1 mg/mL),labiase (0.1 mg/mL) and N-acetylmuramidase (30 µg/mL)and was incubated at 37 °C for 30 min. After centrifu-gation, DNA was extracted and purified from the pre-cipitate by UltraClean Soil DNA Isolation Mega Prep Kit(Mo Bio Laboratories, Inc., Carlsbad, CA).PCR amplificationThe entire region of the internal transcribed spacerbetween 18S and 28S rRNA genes (ITS) was first amplifiedby using the primers ITS4 (5’-TCCTCCGCTTATTGAT-ATGC-3’) and ITS5 (5’-GGAAGTAAAAGTCGTAACAA-GG-3’) targeted to 28S and 18S rRNA genes, respectively(11). The product was passed through a MicroSpin S-300HR Column (Amersham Bioscience) to remove residualprimers. A second PCR was conducted to attach a GC--clamp by using the primers ITS5-GC (5’-CGCCCGCCG-GGCGCGGCGGGCGGGGCGGGGGCACGGGGGAAGT-AAAAGTCGTAACAAGG-3’) and ITS6 (5’-CGTTCTTC-ATCGATGCGAGA-3’). The reaction mixture contained1 µL of the product of the first PCR, 10 pM of each pri-mer, 1.5 mM MgCl2, 0.2 mM dNTP and 2.5 U AmpliTaqGold (PE Applied Biosystems). These components weremixed in 1 × AmpliTaq Gold Buffer (PE Applied Biosys-tems) to obtain a final volume of 50 µL. Initial denatur-ation was done at 94 °C for 5 min, followed by 25 cyclesof denaturation at 94 °C for 30 s, annealing at 70 °C for1 min and elongation at 72 °C for 1 min, with the finalelongation at 72 °C for 5 min. The annealing tempera-ture was reduced by 1 °C per cycle from 70 to 65 °C andadjusted to 65 °C after the fifth cycle. After each PCR re-action, 10 µL was electrophoresed on 1.5 % agarose gelto check the amplification. The products in the remain-der of the reaction mixture (40 µL) were concentrated to10 µL by ethanol precipitation and used for the follow-ing experiments.Denaturing gradient gel electrophoresis(DGGE) analysisDGGE was performed with a DCode Universal Mu-tation Detection System (Bio-Rad Laboratories). A PCRsample was applied directly to 8 % polyacrylamide gelsin a 1 × TAE buffer (40 mM Tris, 20 mM acetic acid and1 mM EDTA, pH=8.3) with a denaturing gradient rang-ing from 20 to 50 %. One hundred percent of denaturantcorresponds to 7 M urea and 40 % formamide. The gra-dient gel was cast using the Gradient Delivery System,Model 475 (Bio-Rad Laboratories, Hercules, CA). Elec-trophoresis was run at a constant voltage of 65 V and at60 °C. After the electrophoresis was run for 16 h, the gelwas stained with SYBR Green I (Molecular Probes, Eu-gene, OR) at 10 000 × dilution in 1 × TAE for 30 minand photographed on a UV illuminator. DGGE bandswere excised from the denaturing gels, re-amplifiedwith primers ITS5 and ITS6, as described above, and se-quenced by each primer. A search of the GenBank data-base was conducted using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).170 A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)Results and DiscussionThe data in this study are representative results ofexperiments performed in three replicates.The metabolic production of lactic acid and ethanolis shown in Fig. 1 along with the development of themicrobial population. Lactic acid was formed in accor-dance with pH reduction and was accompanied by theproduction of ethanol. Concentrations of lactic acid andethanol decreased after 2 and 3 days of incubation, re-spectively. The viable count of all microorganisms in-creased within one day of fermentation except for lacticacid bacteria. The replacement of glutinous rice with po-tato pulp stimulated the growth of yeasts and lactic acidbacteria, resulting in high amounts of ethanol and lacticacid. In both cases, the population of fungi seemed to bemuch lower than that of yeasts, but the former valuesmay have been underestimated. A single colony appear-ing as a fungus is not derived from a single cell such asyeasts because of mycelial growth (12). The contributionof fungi to the fermentation may be more significantthan expected based on the evaluation of the colony-for-ming units (CFU) (6). However, this assumption cannotbe proved by the classical microbiological approach.Fig. 2 shows DGGE profiles of DNA harvested fromfermented potato pulp and glutinous rice. The diagramsinclude five minor bands that are hardly visible in thephotographs. When individual bands appearing in DGGEprofiles were sequenced, four of seven bands were affili-ated to Amylomyces rouxii-Rhizopus oryzae, indicating theirdominance throughout the fermentation period. One ofthree major bands was derived from Mucor indicus. It isunknown why multiple bands originated from A. rou-xii-R. oryzae on DGGE gels even though these bands hadidentical sequences. We suppose that the appearance ofthese minor bands is the result of non-uniformed disso-ciation depending on their nucleotide sequences. Whenlarge amounts of a single PCR product are loaded onDGGE gels, minor bands may emerge in addition to themajor band. Among yeasts, Candida tropicalis disap-peared just after the fermentation, and Saccharomycopsisfibuligera emerged on the third day. Further growth of S.fibuligera in the fermented glutinous rice, as judged froman additional band (h) in Fig. 2, is related to vigorousproduction of ethanol after 3 days (Fig. 1).In DGGE gels, the yeast DNA band was faint,whereas A. rouxii-R. oryzae DNA was clearly detected, incontrast to the population detected with the plating me-thod (Fig. 1). This result indicates that, in terms of DNA,A. rouxii dominated in the culture. This discrepancymight be due to the fact that yeasts, unicellular organ-isms, can easily dominate in the plate-counting method,whereas the fungi will develop a single colony frommultiple cells. Thus, the method we established herewill be useful to identify mixed populations of fungi intraditional fermented foods in Asia.171A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)0 1 2 3 4345670 1 2 3 4 0 1 2 3 4345670 1 2 3 4010203023456780 1 2 3 423456780 1 2 3 4Potato pulp Glutinous riceCulture period / dayViablecounts/(log[cfu/g])Massfraction/ (mg/g)pH0102030Fig. 1. Biochemical and microbiological parameters monitored during the fermentation of potato pulp and glutinous ricePotato pulp and glutinous rice were mixed with the starter ragi tapé and incubated at 27 °C for 4 days. After the extraction with sa-line solution (0.85 % NaCl), pH and metabolites were analyzed. Microorganisms in the fermented products were enumerated on thedefined mediaSymbols:  pH,  lactic acid,  ethanol,  fungi,  yeasts,  lactic acid bacteriaBacterial communities of fermented foods (13,14) andcompost (15) have been assessed by DGGE for the V3region of rDNA. DGGE analysis of the 16S–26S rDNAspacer region from bacteria has been useful for discrimi-nating closely related species (16) or individual strainsin the same species (17). For eukaryotic organisms, a li-mited number of DGGE based studies was conductedusing partially amplified 18S rDNA (18) and 28S rDNA(19,20) but not the ITS region.Amylomyces is a monotypic genus containing thesingle variable species A. rouxii, which is closely relatedto R. oryzae, as identifiable from the formation of rhi-zoids, stolons and black-pigmented sporangia (12). Adistinct morphological characteristic of A. rouxii is theenormous number of chlamydospores produced in theaerial and substrate mycelium. Recently, R. oryzae strainshave been classified into two groups which principallyaccumulate either lactic acid or fumaric acid in a me-dium containing relatively high amounts of carbonsources. However, the ITS1 sequence cannot discrimi-nate among A. rouxii and lactic acid-accumulatingstrains of R. oryzae because these sequences are identical(11). We isolated twenty colonies of fungi from the fer-mented potato pulp, which were identified as Mucorindicus and A. rouxii based on morphological character-istics. The appearance of R. oryzae was not shown. Thishas led to the conclusion that A. rouxii dominated in theculture of the fermented potato pulp.From the DGGE pattern (Fig. 2), A. rouxii was alsofound to dominate in the fermented rice, tapé ketan, indi-cating that potato pulp is unlikely to stimulate the growthof undesirable fungi existing in ragi tapé. In Indonesia,peeled, diced and steamed cassava tubers are alterna-tively used for the production of tapé ketella. Potato pulpfermented by ragi tapé may be consumed as other tapéproducts.AcknowledgementsWe thank K. Saito for his helpful advice. This workwas supported in part by Special Coordination Fundsfor Promoting Science and Technology (Leading Re-search Utilizing Potential of Regional Science and Tech-nology) of the Japanese Ministry of Education, Culture,Sports, Science and Technology.References1. C. W. Hesseltine, Annu. Rev. Microbiol. 37 (1983) 575–601.2. W. H. Holzapfel, Int. J. Food Microbiol. 75 (2002) 197–212.3. C. W. Hesseltine, R. Rogers, F. G. Winarno, Mycopatholo-gia, 101 (1988) 141–155.4. A. C. Lee, Y. Fujio, World J. Microbiol. Biotechnol. 15 (1999)57–62.5. T. C. Cronk, K. H. Steinkraus, L. R Hackler, L. R. Mattick,Appl. Environ. Microbiol. 33 (1977) 1067–1073.6. M. M. Ardhana, G. H. Fleet, Int. J. Food Microbiol. 9 (1989)157–165.7. S. Siebenhandl, L. N. Lestario, D. Trimmel, E. Berghofer,Int. J. Food Sci. Nutr. 52 (2001) 347–357.8. F. Mayer, J. O. Hillebrandt, Appl. Microbiol. Biotechnol. 48(1997) 435–440.9. Y. Oda, K. Saito, H. Yamauchi, M. Mori, Curr. Microbiol. 45(2002) 1–4.10. Y. Oda, Y. Yajima, M. Kinoshita, M. Ohnishi, Food Micro-biol. 20 (2003) 371–375.11. A. Abe, T. Sone, I. N. Sujaya, K. Saito, Y. Oda, K. Asano,F. Tomita, Biosci. Biotechnol. Biochem. 67 (2003) 1725–1731.12. J. J. Ellis, L. J. Rhodes, C. W. Hesseltine, Mycologia, 68(1976) 131–143.13. F. Ampe, A. Sirvent, N. Zakhia, Int. J. Food Microbiol. 65(2001) 45–54.14. S. Fasoli, M. Marzotto, L. Rizzotti, F. Rossi, F. Dellaglio, S.Torriani, Int. J. Food Microbiol. 82 (2003) 59–70.172 A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)Fig. 2. Denaturing gradient gel electrophoresis (DGGE) profiles of the PCR-amplified ITS1 region of the DNA extracted from fer-mented potato pulp and glutinous riceDGGE analysis was conducted for the fermented potato pulp and glutinous rice used in Fig. 1.Shaded column in the diagram indicates the band invisible in the corresponding photograph. Each band was excised, re-amplified,sequenced and affiliated as follows: (a), (d), (e) and (f), Amylomyces rouxii-Rhizopus oryzae; (b) and (h), Saccharomycopsis fibuligera; (c),Mucor indicus; (g), Candida tropicalis15. Y. Ueno, S. Haruta, M. Ishii, Y. Igarashi, Appl. Microbiol.Biotechnol. 57 (2001) 555–562.16. N. D. Crosbie, M. Pockl, T. Weisse, J. Microbiol. Methods,55 (2003) 361–370.17. A. Buchan, M. Alber, R. E. Hodson, FEMS Microbiol. Ecol.35 (2001) 313–321.18. P. Möhlenhoff, L. Müller, A. A. Gorbushina, K. Petersen,FEMS Microbiol. Lett. 195 (2001) 169–173.19. G. A. Kowalchuk, F. A. de Souza, J. A. van Veen, Mol.Ecol. 11 (2002) 571–581.20. C. Schabereiter-Gurtner, G. Piñar, W. Lubitz, S. Rölleke, J.Microbiol. Methods, 47 (2001) 345–354.Mikroflora i odabrani metaboliti krumpirove pulpe fermentirane sdodatkom indonezijske starter kulture ragi tapéSa`etakKada se krumpirova pulpa pomije{a s indonezijskom starter kulturom ragi tapé i in-kubira, postupno se stvaraju mlije~na kiselina i etanol koji nakon dva dana fermentacijedosti`u odre|ene koncentracije. Nakon fermentacije utvr|eno je u svje`oj tvari 105 CFU/ggljivica, 107 CFU/g kvasca i 105 CFU/g bakterija mlije~ne kiseline. Denaturiraju}om gradi-jentnom gel-elektroforezom PCR-amplificiranih i transkribiranih internih razdjelnih zona(spacer region) iz 18S–28S rDNA gena utvr|eni su Amylomyces rouxii-Rhizopus oryzae, Mu-cor indicus, Candida tropicalis i Saccharomycopsis fibuligera, a ujedno je ustanovljeno da suAmylomyces rouxii-Rhizopus oryzae prevladavali tijekom fermentacije. Amylomyces rouxii nemo`e se razlikovati od Rhizopus oryzae, koja proizvodi mlije~nu kiselinu, jer su amplifi-cirane sekvencije tih gljivica identi~ne. Stoga su prou~avane morfolo{ke karakteristike vrstiRhizopus sli~ne gljivice izolirane iz fermentirane krumpirove pulpe. Oni sojevi koji su pro-izveli golem broj klamidospora u okoli{u, i u miceliju supstrata, utvr|eni su kao Amy-lomyces rouxii. Mikroflora fermentirane pulpe krumpira bila je sli~na kao kod ljepljive ri`e(tapé ketan).173A. ABE et al.: Potato Pulp Fermented with Starter Ragi Tapé, Food Technol. Biotechnol. 42 (3) 169–173 (2004)

Microflora and Selected Metabolites of Potato Pulp Fermented with an Indonesian Starter Ragi Tapé

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