NUCLEAR TECHNIQUE IN THE IMPROVEMENT OF QUALITY OFTRADITIONAL FERMENTED CASSAVA IN INDONESIA

Lina, M.R.*, Susiana *, Aryanti *, and E.G. Siagian *.

ABSTRACT

NUCLEAR TECHNIQUE IN THE IMPROVEMENT OF QUALITY OF TRADITION­AL FERMENTED CASSAVA IN INDONESIA. A study to know the possibility of using gammairradiation to induce mutation on microorganisms isolated from .m.gi illm< was performed in an attemptto improve the quality of tape singkong, a traditional fermented cassava. As much as 25 samples ofragi tape, a s~rter for making tape singkong were used in this study. Irradiation was done by usinggamma rays ( Co) at the dose rate of 2.0 kGy per hour. From 25 samples of ragi tape collected, therewere 3 amylolytic mould strains which had higher amylase ability, Le. Mucor sp. two yeast strainsproducing higher ethanol were also obtained from the samples. Irradiation to induce mutation on oneselected mould strain using LD resulted 47 surviving strains. Two of them had a higher amylase abili­ty than the wild strain. The ghj~ose production of these strains were about 4 times compared with theglucose produced by the wild strains, at 48 hours incubation. There were 226 yeast strains obtainedfrom irradiation of one selected yeast strain at LD . One of them had the ability to produce ethanol55% more the wild strain. 50

ABSTRAK

PENGGUNAAN TEKNIK NUKLIR DALAM PERUAIKAN KUALITAS UIU KAYUYANG IHFE'RMENTASI SECARA TRAIHSIONAL IH INDONESIA. Penelitian untuk mengetahuikemungkinan penggunaan iradiasi gamma untuk menginduksi mutasi pada mikroorganisme yang di­isolasi dari ragi tape, tclah dilakukan dalam rangka usaha perbaikan mutu tape singkong. Ragi tapesebagai starter untuk membuat tape sinJjkong yang digunakan, bcrjumlah 25 contoh. Iradiasi dilakukandengan menggunakan sinar gamma ( Co) degan laju dosis 2,0 kGy per jam. Dari 25 contoh ragitape, telah didapatkan 3 strain kapang amilolitik yang mcmpunyai kemampuan yang tinggi dalammcnghasilkan amilase, yaitu Mucor sp. Dua strain khalJlir pcnghasil etanol tinggi juga diperolch daricontoh ragi tcrsebul. Jumlah strain kapang yang hidup hasil seleksi satu strain kapang yang diiradiasipada LD adalah 47 strain. Dua diantaranya lIIempunyai kemampuan menghasilkan amilase lebihtinggi dafr strain kontrol (strain yang tidak diiradiasi). Glukosa yang diproduksi olch strain-strain iniIcbih kurang 4 kali Icbih tinggi dari strain kontrol, dalam waktu inkubasi 48 jam. Dari iradiasi 1 strainkhamir penghasil etanol pad a LD didapatkan 226 strain yang hidup. Satu strain diantaranya mem­punyai kcmampuan menghasilkan e-t>anol55% lebih tinggi dari strain kontrol.

INTRODUCTION

Cassava tuber is one of carbohydrate sources that can be used for supporting

the shortage of calories. It can be prepared for foodstuff. In the poor fertility area,cassave tuber is often used as staple food. One of the efforts to alter the appearance,texture. ami nutritional value of cassave tuber, is to convert it to fermented food.

* Centre for Application of Isotopes and Radiation, NAEA

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According to TAN (I), vitamine B2 content of unfermented cassava root is 0.6

]lg/g and the fermented one is 2.7 ]lg/g.

Tape singkong/tape ketela is a popular Indonesian fermented food madefrom steamed peeled cassave tuber, spread with ragi tape powder as a starter. Thefermented cassava are ready to be consumed after 2 days. Many Indonesian peopleeat tape as a supplementary meal or snacks. The taste of ~ is sweet with a mildalcoholic flavor. Tape singkong is usually consumed,directly as a fresh fermentedfood, but it is also taken after frying or boiling in coconut milk and brown sugar.Cake made from tape is also delicious. Tape is produced as a home industry or justfor family consumption.

Ragi tape is made from rice flour mixed with spices and produced by tradi­tional manufacturers and home industries. The preparation method varies from placeto place depending on avalilability of ingredients. Rag:i tape as a starter containscertain microorganisms such as mould, yeast, and bacteria. The microorganismscome from the surroundings and equipments used by the traditional manufacturerthrough natural infection (2).

As a starter ragi tape has an important role in obtaining a good quality oftape singkong. Therefore, improvement of the microbiological activity in the ragitape, through induced mutation is promising. By this technique, it is expected thatthe microorganisms activity can be increased and the time of fermentation can bereduced. According to MEYRATH (3) more than 50% of Aspergillus oryzae colo­nies irradiated with gamma rays at doses of 0.7 - 1.1 kGy could produce higheramylase than the wild strain. A strain of Saccharomyces vini irradiated with gammarays at doses of 1.5 - 2.0 kGy could ferment grape juice faster than the unirradiatedstrain (4) The purpose of this investigation is to study the posibility of using gammairradiation to induce mutation on microorganisms in ragi tape in an attempt toimprove the quality of tape singkong a traditional fermented cassava.

MATERIALS AND METHODS

As much as 25 ragi tape samples were used in this experiment. The sampleswere collected from different cities in Indonesia.

Isolation of Microorganisms from Ragi Tape. Ten grams of ragi tape washomogenized in 90 ml of sterile distilled water. The suspension was then cultured onthree media namely Malt Extract Agar for mould, Sabouraud Dextrose Agar foryeast and Nutrient Agar for bacteria (Difco), using spread plate method. The cul­tures then purified on slant agar after two days incubation at room temperature(28°C ± 2°C).

Preparation of Sub.\'trate. The substrate used for glucose production was5% (w/v) cassava starch ("tapioca"). The substrate for ethanol production was 5%cassava starch added with 0.5% (v/w) thermamyl (alpha amylase) and 0.5% (v/w)

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amyloglucosidase (Novo, Denmark) producing glucose content of about 4.7% (w/v)in the substrate. All of substrates then were sterilized in autoclave at 120°C, 15 psi,for 15 minutes.

Preparation of Inoculum. The inoculum of mould was prepared by scrapingthe spores from pure culture after being added with sterile distilled water. Prepara­tion of yeast and bacteria inocula has been described in the previous report (5).

Selection of Amylolytic and Alcoholic Microbial Strains. Five percents(v/v) of mould spores, bacteria, and yeast solutions were inoculated into the sub­strate for glucose production, where as the selection of yeast strains producingethanol was done by inoculating 5% (v/v) yeast solution into the substrate. Theselection was based on the ability of the isolating microorganisms to produce thehighest glucose or ethanol. Comparative experiment was carried out using amyloly­tic yeast strain (Elldomycopsis sp.) and 12 alcoholic yeast strains available in thelaboratory.

Selection of Lactic Acid Bacteria. The selection was based on the growth ofbacteria on Rogosa medium (Oxoid).

Detennination of LDso of Selected Strains. The determination was carriedout on the selected amylolytic mould strains (RBM3, RSMz), and alcoholic yeaststrains (RCrgYz' RBdgYz). This experiment was also done on Endomycopsis sp. andthe selected alcoholic yeast strains from laboratory collection (Pd As and Stl). The

LDso of selected amylolytic mould strain was determined by harvesting the sporesand mycelia from pure culture after addition of sterile distilled water, stirring, andthen filtration through sterile cotton. The suspension was centrifuged at 10,000 rpmfor 10 minute. The supernatant was taken out and spores pricipitate resuspendedwith sterile distilled water to gain a certain concentration i.e. (+ 108 celis/mI). Thespores suspension was then irradiated with gamma rays at the dose rate of 2.0 kGyper hour. Irradiation was carried out at room temperature and doses of 0, 0.5, 1.0,2.0, 2.5, 3.0, and 3.5 kGy. As much as 0.1 ml of irradiated spores suspension wascultured on one plate of Sabouraud Dextrose Agar (SDA) by spread plate methodafter being diluted with a serial dilution. Mould colonies were counted after one day

and two days incuhation at room temperature. The LDso can be estimated from thesurvival curve.

Preparation of irradiation treatment to obtain the LDso on yeast strain wasdescribed in our previous investigation (6) using Sabouraud Dextrose Broth as culti­vation medium. The temperature, irradiation dose, and the treatment after irradiationwere similar with those for mould spores as mentioned earlier. Yeast colonies werecounted after 3 days.

Mutation Screening 7echnique. The screening technique could be done just

on one selected mould strain (RBM3) and one selected yeast strain (RCrgYz). Mouldspores suspension containing about 10 spores/O.l ml and yeast suspension containing

about 20 - 30 cells/D. I ml were irradiated at LDso of each strain. The volume of

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each irradiated suspension was 2 mI. After irradiation all of the suspensions were

cultured on SDA by streak plate method. The low concentrations of suspensionswere used in order to be able to separate the cultured spores of mould and yeastcells, individually. All of the survive colonies of mould and yeast were purifiedindividually on slant agar. The irradiation surviving mould and yeast strains wereinoculated into the respective substrates. Inoculation of irradiation surviving mouldstrain into 100 ml of substrate was accomplished by scraping the pure culture fromone slant agar after addition of 5 ml of sterile distilled water. Inoculation of irradia­tion surviving yeast strain was done as mentioned earlier. Screening was based onthe highest glucose or ethanol produced by the strains after 2 days.

Determination of Glucose Content. Glucose content 0 the substrate wasdetermined using a glucose specific reagent (GOD PODLK), obtained from NagaseSangyo Co. Ltd., Japan (7).

Determination of Ethanol Content. Two methods were used to determine theethanol content. In the first method, ethanol was determined using gas liquid chro­matograph. This method was described in our previous report (8). The secondmethod was carried out with UV-method using alcohol dehydrogenase (BoehringerMannheim) according to Bonichsen (1971), as described by FUJIMURA (Personalcommunication) (9).

Identification of Mould Strain. The identification was accomplished usingoptical microscope.

RESULTS AND DISCUSSION

Variety of Microorganism in Ragi Tape. Microorganisms isolated from the25 samples of ragi tape were 75 moulds, 39 bacteria, and 47 yeasts.

The Selected Microbial Strain. From the isolated 75 mould strains, onlythree (RBM3, RSM2, and RCbM2) produced high glucose with the concentrationsof 1.82%, 1.73%, and 1.46% (w/v) from 5% cassava starch substrate at 96 hoursincubation, therefore, the converted cassava starch to glucose were 36%,34%, and

29% (w/v), respectively.Table I reveals that the highest glucose content was 0.3971 % so that the

converted cassava starch to glucose was 7.9420%. It was produced by one of the 47

yeast strains isolated, i.e. RCjrY 2 at the incubation time of 96 hours.The glucose produced by Endomycopsis sp. from substrate, was 0.2288 %.

and the converted cassava strach to glucose was 4.5760%. The incubation time wassimilar with that for mould strain as mentioned above. In comparison with glucosecontent produced by the selected mould strain as mentioned earlier, the glucose

production from the activity of RCjr2 and Endomycopsis sp. were much lower. Theisolated hacteria are considered as contaminants in ragi tape considering their lowactivity in producing glucose.

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At the incubation time of 96 hours two of the 47 isolated yeast strains

(RCrgY2 and RBdgY2) could produce higher etanol concentration up to 0.71 % and

0.67% (v/v), respectively and convert 15.09% and 12.98% (v/w) of glucose sub­

strate to ethanol, respectively. As comparison, Pd As and Stl produced ethanol

from substrate with concentrations as higher 1.15% and 1.04% under the same

incubation period (96 hours). It means that 24.16% and 22.00% of glucose contain­

ing in substrate were converted to ethanol by Pd, As, and Stl strains, respectively.In connection with lactic acid production, the experiment to select the lactic

acid bacteria was also performed. Out of 39 bacteria strains isolated, only one strain

(RBdgB2) could produce lactic acid.

The Wso Selected Microbial Strains. The LDso of two selected amylolytic

mould strains (RBM3 and RSM2), were 0.975 kGy and 1.825 kGy, whereas the

LDso of Endomycopsis sp. as comparative amylolytic yeast strain was 0.625 kGy.

The selected alcoholic yeast strains (RCrgYz) and RBdgY2) had the LDso of 0.35

kGy and 0.375 kGy, while the LDso of Pd As and Stl were 0.60 kGy and 0.475kGy.

The Irradiation Surviving Amylolytic Mould Strain. In comparison with the

unirradiated amylolytic mould strain (wild strain) which produced glucose with the

concentration of 0.4880% (W/V), about 49% out of the 47 irradiation surviving

strains had ability to produce higher glucose at 48 hours incubation (Table 2). This

table also indicates that two strains i.e. RBM3 S23 and RBM3 S42 had the capability

to produce the highest concentration of glucose which were about 4 times higher

than the wild strain. Radiation induced mutation on mould strain in this study

seemed to increase the glucose production, significantly. The previous investigators

(10) stated that radiation induced mutation in microorganisms can after the fermenta­

tive ability, increase or decrease the capacity to produce some end products.

The Irradiation Surviving Alcoholic ~ast Strain. The irradiation surviving

yeast strains obtained in the experiment were 226 strains. From this amount about

87% of the strains had a little higher capability in producing ethanol, and the

remaining 13 % gave lower production of ethanol than the wild strain. The highest

ethanol concentration (0.3723 % = v/v) was obtained from the surviving strain no.

128 (RCrgY 2 S 128) as compared to the wild strain of the wild strain (CRrgY2) whichproduced ethanol consentration of 0.2394% (v/v). By comparing the ability of

producing ethanol, that particular surviving strain could increase the ethanol produc­tion as much as 55 %. However, it seems that irradiation to induce mutation on

alcohol ic yeast strain used in this experiment, did not enhance the ethanol produc­

tion, significantly. The induced synthesis of a certain enzyme produced by yeast

cells, can be inhibited by ionizing radiation (II). PATRICK and HAYNES (12)

reported that although the biochemical mechanism of the recovery process in irradi­

ated yeast is unknown, but this microorganism possesses enzymatic repair mecha-I

nlsm.

127

Variety of the Selected Mould Strain. All of the selected mould strains

(RBMJ' RSM2, and RCbM), belonged to one genus, namely Mucor.

CONCLUSSIONS

Microorganisms obtained from 25 samples of ragi tape consisted of 75mould, 39 bacteria, and 47 yeast isolates/strains.

Out of 75 mould strains isolated, three strains i.e. RBMJ' RSM2, andRCbM2 belonging to genus Mucor, had the ability to produce glucose higher thanthe others. Among 47 isolated yeast strains, one of them (RCjrY2) gave a higherproduction of glucose and two of them (RCrgY 2) and RBdgY 2) produced moreethanol. Either RCjrY 2 strain or Endomycopsis sp. as a comparative amylolyticstrain, had the ability to produce glucose much less than the selected mould strains.

Among 37 bacteria strains isolated, one strain (RBdgB2) has been selected asa lactic acid bacteria. About 49% of the 47 surviving irradiated mould strains, had ahigher ability of producing glucose than the wild strain and the remaining 51 % were

on the contrary. Two strains (RBMJ SJ and RBMJ S42 produced the highest glucosecontent which is four times greater than that produced by the wild strain.

Out of the 226 surviving irradiated yeast strains, about 87 % of them showeda slight increase in ethanol production as compared with the wild strain. The surviv­

ing strain no. 123(RCrgY 2 S 128) could provide the highest ethanol production whichwas a production enhancement of about 55% as compared to the wild strain.

Futher study is required to obtain stable mutant strains which have higherability to convert cassava starch.

ACKNOWLEDGEMENT

This study is partly supported by the IAEA under Research Contract No.481l/RB. The authors are grateful to Mrs. Almaida, Mrs. Anasthasia S.D., andMr. Djuswardi for their technical assistance.

REFERENCES

1. TAN, T.H., Fermentative processing of cassava root, Ph.D. Thesis, Universityof Philippines (1971)

2. KO, S.D., Tape fermentation, Applied Microbiology 22 5 (1972) 976.

3. MEYRATH, J., BAHN, M., HAN, H.E., and ALTMANN, H., "Induction of

amylase-producing mutants in Aspergillus oryzae by different irradiation",Radiation and Radioisotopes for Industrial Microorganisms (Proc. Simp.Vienna, 1971) IAEA, Vienna (1971) 154.

128

4. TAUPOVA , S.H., GULYAMOVA, N., and BALASANYAN, LA., Effect of

gamma rays 60Co on the fermentation activity of Saccharomyces vini, Uzb.Bio. Zh. 1 (1978) 14.

5. UNA, M.R., SUSIANA, ARYANTI, and SIAGIAN, E.G., "Nuclear tech­

nique in the improvement of the quality from the traditional fermented cassavain Indonesia" Annual Report to IAEA (September I 1987 - August 31 1988),

Centre for the ApllicatlOn of Isotopes and Radiation, National AtomicEnergy Agency, Jakarta, Indonesia (1988).

6. UNA, M.R., SUSIANA, and GANDJAR, I., The effect of gamma irradia­tion on alcohol ic fermentation of cassava by Saccharomyces cerevisiae andKluyveromyces marxianus. Atom Indonesia 111 (1986) I.

7. KUMAKURA, M., and KAETSU, I., Enzymatic hydrolysis of chaff usingimmobilized growing cells. Agricultural Wastes II (1984) 259.

8. UNA, M.R., SUSIANA, and SIAGIAN, E.G., "The effects of H2S04 andNaOH solutions on irradiated sawdust for ethanol production", Applicationof Isotopes and Radiation (Proc. Third Symp. Jakarta, 1986 NAEA, Jakarta(1988) 441.

9. FUJIMURA, T., (1988) Personal communication

10. PELCZAR, M.J., and REID, R.D., Microbiology, International Student Edi­tion, Mc Graw-Hill Book Company, Inc., New York, Toronto, London,Kogakusha, Company, Ltd., Tokyo, 115.

II. KIEFER, J., GOCKE, E., and KOESTER, K., Synthesis of inducible enzymesin irradiated yeast cells: Inhibition by ionizing radiation, Int. J. Radiat. BioI.383 (1980) 267.

12. PATRICK, M.H., and HAYNES, R.H., Repair-induced changes in yeast radio­sensitivity, Journal of Bacteriology 95 (1968) 1350.

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Tabel 1.Glucose content(% )produced by theisolating yeaststrains

Yeast

Incubation time (hourNo. strain

24487296

1

RBY1 0,01260,01370,01270,01002

RBY2 0,01210,01300,01320,01303

RSY1 0,02580,07000,10350,13404

RSY2 0,01160,01670,01390,01005

RSY3 0,01260,01420,01390,01076

RsmgY1 0,03540,05460,10000,14747

RsmgY2 0,01210,01180,01300,01038

RPwtY1 0,03190,04710,07580,07739

RPwtY2 0,01210,01160,01250,010310

RKP1Y 0,04100,07000,09400,075011

RKP2Y1 0,01210,01220,01250,010512 RKP~Y2

0,03970,04890,07300,087213

RTa 0,04680,05250,11300,045014

RBdgY1 0,01090,01220,01210,012115

RBdgY2 0,01040,01150,01220,010116

RCrbY 0,03150,04220,07400,052417

RKamY1 0,03690,05360,08150,093618

RKamY2 0,01180,03400,02250,020019

RMgY 0,06050,07150,10140,120920

ROpY 1 0,03160,06460,10790,110721

ROpY2 0,02970,05380,07030,079222

ROpY 0,02780,04060,05280,066623

RCbY~l 0,01200,01150,01110,0122

24RCbY2 0,01080,01170,01300,0108

25

RSIY Q,04600,07590,09300,129626

RKdY 0,02550,04890,07980,112927

RKmY 0,04320,05660,08750,123328

RcrgY1 0,04690,03510,05140,079929

RcrgY2 0,01530,01100,01220,013530

RIrY1 0,01890,01130,01220,011331

RIrY 0,01370,01200,01670,010832

RCjr~l 0,01040,01100,01330,0122

33RCjrY2 0,04620,10720,15920,3971

34RSkbY1 0,05180,08890,14840,1593

35RSkbY2 0,03440,04260,13200,1097

36RPSaY1 0,01050,01090,01190,0119

37RPSaY2 0,01090,01230,01150,0122

38RPsaY3 0,01290,01240,01330,0114

39RCakY1 0,04580,07520,10260,1515

40RCakY2 0,01260,01270,01320,0107

41RTp 0,01270,01110,01250,0105

42RGedY1 0,04490,04660,05660,0652

43RGedY2 0,01210,01240,01420,0127

44RBrY 0,06560,08340,12090,1975

45RPaY1 0,03870,01600,11300,0720

46RPaY2 0,03380,03710,04230,0407

47RPaY3 0,02570,02930,03740,0311

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Tabel 2. Glucose content produced by the surviving strains ofirradiated mould strain (RBM3) at the incubation timeof 48 hours

No. MouldStrain

Glucose con­centration(%)

0,24460,82580,25800,39230,25800,24460,25800,51710,27140,38200,87740,94971,63090,25800,24461,34190,25800,86710,25801,59640,36130,59872,05410,7737

No. Mouldstrain

Glucose con­

centration(%)

0,89070,29941,96120,38201,24900,44390,19091,11480,84650,20650,51620,25800,65040,37170,88800,34441,37932,05350,41950,37110,29381,71070,7051

I31