Nus Biosci | vol. 1 | no. 3 | pp. 105-158 | November 2009

FIRST PUBLISHED: 2009

ISSN: 2087-3948 (printed edition), 2087-3956 (electronic edition)

EDITORIAL BOARD: Abdulaziz M. Assaeed (King Saud University, Riyadh, Saudi Arabia), Alfiono (Sebelas Maret University, Surakarta), Edwi Mahajoeno

(Sebelas Maret University, Surakarta), Ehsan Kamrani (Hormozgan University, IR Iran), Eko Handayanto (Brawijaya University, Malang), Endang Sutariningsih (Gadjah Mada University, Yogyakarta), Faturochman (Gadjah Mada University, Yogyakarta), Iwan Yahya (Sebelas Maret University, Surakarta), Jamaluddin (R.D. University, Jabalpur, India), Lien A. Sutasurya (Bandung Institute of Technology, Bandung), Magdy Ibrahim El-Bana (Suez Canal University, Al-Arish, Egypt), Mahendra K. Rai (Amravati University,

India), Marsetyawan H.N. Ekandaru (Gadjah Mada University, Yogyakarta), Oemar Sri Hartanto (Sebelas Maret University, Surakarta), R. Wasito (Gadjah Mada University, Yogyakarta), Rugayah (Indonesian Institute of Science, Cibinong-Bogor), Sameer A. Masoud

(Philadelphia University, Amman, Jordan), Supriyadi (Balitbiogen, Bogor), Sri Margana (Gadjah Mada University, Yogyakarta), Suranto (Sebelas Maret University, Surakarta), Sutarno (Sebelas Maret University, Surakarta), Sutiman B. Sumitro (Brawijaya University, Malang), Taufikurrahman (Bandung Institut of Technology, Bandung), Wayan T. Artama (Gadjah Mada University, Yogyakarta)

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EXPERTISE OF THE EDITORIAL BOARD: AGRICULTURAL SCIENCES: Eko Handayanto ([email protected]), ANTHROPOLOGY: Sri Margana ([email protected]), APPLIED BIOLOGICAL SCIENCES: Suranto ([email protected]), BIOCHEMISTRY: Wayan T. Artama ([email protected]), NATURAL PRODUCT BIOCHEMISTRY: MAHENDRA K. RAI, BIOPHYSICS AND COMPUTATIONAL BIOLOGY: Iwan Yahya ([email protected]), CELL BIOLOGY: Sutiman B. Sumitro ([email protected]), DEVELOPMENTAL BIOLOGY: Lien A. Sutasurya ([email protected]), ECOLOGY: Magdy Ibrahim El-Bana ([email protected]), ENVIRONMENTAL SCIENCES: Abdulaziz M. Assaeed ([email protected]), EVOLUTION:

Taufikurrahman ([email protected]), GENETICS: Sutarno ([email protected]), IMMUNOLOGY: Marsetyawan H.N. Ekandaru ([email protected]), MEDICAL SCIENCES: Alfiono ([email protected]), ANIMAL AND VETERINARY SCIENCES: R. Wasito

([email protected]), MICROBIOLOGY: Endang Sutariningsih ([email protected]), NEUROSCIENCE: Oemar Sri Hartanto ([email protected]), PHARMACOLOGY: Supriyadi ([email protected]), PHYSIOLOGY: Sameer A. Masoud

([email protected]), PLANT BIOLOGY: Rugayah ([email protected]), POPULATION BIOLOGY: Ehsan Kamrani ([email protected]), PSYCHOLOGICAL AND COGNITIVE SCIENCES: Faturochman ([email protected]), SUSTAINABILITY SCIENCE:

Jamaluddin ([email protected]), SYSTEMS BIOLOGY: Edwi Mahajoeno ([email protected])

| Nus Biosci | vol. 1 | no. 3 | pp. 105‐158 | November 2009 |

ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)

I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

ISSN: 2087-3948 (print) Vol. 1, No. 3, Pp. 105-109 ISSN: 2087-3956 (electronic) November 2009

Activity of Zymomonas mobilis on ethanol products made of cashew nut apple (Anacardium occidentale) with different sources of nitrogen

AKHMAD MUSTOFA1,2,♥, SURANTO2 ¹ University of Slamet Riyadi (UNISRI), Jl. Sumpah Pemuda, No. 18, Joglo, Surakarta. Central Java, Indonesia, Tel. +62-271-7013199, 856879, Fax.:

+62-271-854670 ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia.

Manuscript received: 7 June 2009. Revision accepted: 6 August 2009.

Abstract. Mustofa A, Suranto. 2009. Activity of Zymomonas mobilis on ethanol production made of cashew nut apple (Anacardium occidentale) with different sources of nitrogen. Nusantara Bioscience 1: 105-109. This research is aimed at identifying Zymomonas mobilis in producing ethanol through batch fermentation process (in 24, 48 and 72 hours) using cashewnut apple extract (red, green and yellow variety) and urea, ammonium sulphate, extract of green peanut sprout and extract koro (Mucuna pruriens) as sources of nitrogen. The research showed that green cashewnut extract with ammonium sulphate in 24 hours of fermentation produced ethanol in optimum result. This treatment had pH of 5.87, 7.64 g/100 mL of sugar (with 48.44% of consumption), 8.0x107 amount of bacterium (µ = 0.154) and production of ethanol equal to 33.02 g/L (Ye = 90.19%).

Key words: Zymomonas mobilis, cashewnut apple extract, ethanol.

Abstrak. Mustofa A, Suranto. 2011. Aktivitas Zymomonas mobilis pada produk etanol dari buah semu jambu mete (Anacardium occidentale) dengan variasi sumber nitrogen. Nusantara Bioscience 1: 105-109. Penelitian ini bertujuan mengetahui kemampuan Zymomonas mobilis dalam memproduksi etanol melalui proses fermentasi batch (selama 24, 48 dan 72 jam), menggunakan sumber karbon sari buah jambu mete (varietas merah, hijau dan kuning) dan sumber nitrogen berupa urea, ammonium sulfat, ekstrak kecambah kacang hijau dan ekstrak kacang koro (Mucuna pruriens). Hasil penelitian menunjukkan bahwa varietas buah jambu mete hijau dengan sumber nitrogen ammonium sulfat dan lama fermentasi 24 jam memberikan hasil etanol yang paling optimal. Pada perlakuan tersebut diperoleh nilai pH 5,87, kadar gula reduksi 7,64 g/100 mL (tingkat konsumsi 48,44%), jumlah bakteri 8,0x107 (µ = 0,154) dan etanol sebesar 33,02 g/L (Ye = 90,19%).

Kata kunci: Zymomonas mobilis, sari buah jambu mete, etanol.

INTRODUCTION

There are 5,322 ha land in Yogyakarta used as an area to grow cashewnut apple and Gunung Kidul is the largest one (60.38%). Meanwhile in Central Java it covers 11,828.68 ha with Wonogiri covering equal to 7,059 ha. Both Gunung Kidul and Wonogiri produce 3,242.9 tons of nuts without its fruit (Darsono 2004). There are 200 nuts in 1 kg of the nuts of cashewnut apple, or 648.580.000 of nuts for 3,242.9 tons. It means there are about 194,574 tons of cashew fruit for that amount of nuts.

Cashew fruits are not widely utlilized in Indonesia . Meanwhile in Brazil from where it comes, People produce juices from the fruits. In India it is widely used to make alcoholic beverage called feni (Van Eijnatten 1991). Cashew fruit contains 16.3% of carbohidrat that can be fermented into ethanol (Thomas 1989; Van Eijnatten 1991). Some researches indicate that Saccharomyces cereviseae and Zymomonas mobilis ferment sugar from cashew fruit extract into ethanol (Hermawan et al. 2000; Sapariantin 2005). However, Z. mobilis is less frequently used than S. cerevisiae in ethanol products made of

cashew. Pinheiro et al. (2008) and Neelakandan and Usharani (2009) indicate that cashew apple juice is a suitable substrate for S. cerevisiae yeast growth and ethanol production. Pacheco et al. (2009) and Rodrigues et al. (2009) showed that cashew apple bagasse is an efficient support for cell immobilization aimed at ethanol production. While, Karuppaiya et al. (2009) showed the ability of Z. mobilis for alcohol production from cashew apple bagasse.

Zymomonas mobilis, a gram-negative bacterium, is considered as an alternative organism in large-scale fuel ethanol production (Gunasekaran and Raj 2002). Z. mobilis has some good characteristics for producing ethanol namely its higher sugar uptake and ethanol yield, lower biomass production, higher ethanol tolerance, not require controlled addition of oxygen during fermentation and its amenability to generic manipulations (Wijono 1988; Doelle 1990; Hobley and Pamment 1994; Nowak 2000).

Cashew fruit contains only 4.6% of protein (Thomas 1989; Van Eijnatten 1991), resulting in less nitrogen in which it is used for growth and metabolism production. For those reasons, it has been researched on producing ethanol from cashew apple extract (red, green and yellow variety)

1 (3): 105-109, November 2009

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with Z. mobilis in 24, 48 and 72 hours of fermentation. Urea, ammonium sulfat, green bean (Phaseolus radiates) sprout extract and koro (Mucuna pruriens) extract were added as sources of nitrogen.

MATERIALS AND METHODS

Materials Cashew apples with red, yellow and green variety that

have been used as the source of carbon were taken from Ngadirojo sub district, Wonogiri district, Central Java. The added carbon source was glucose 7 g /100 mL of fermented media. The nitrogen sources were from urea, ammonium sulfat, Phaseolus radiatus (green bean) sprout extract and Mucuna pruriens (koro) extract. Z. mobilis was taken from Food and Nutrient Microbiology Laboratory of Gadjah Mada University Yogyakarta. The chemicals needed in this research are MgSO4.7H2O 0.1% (b/v), anhydrate glucose, amonium molibdat, H2SO4, Na2H2SO4, Na2CO3, KNa, NaHCO3, Na2SO4, CuSO4.5H2SO4, MgSO4, and NaOH. The cashew extract was made from the blended juice and kept in a freezer (Hermawan et al. 2000).

Ethanol fermentation The fermentation media (100 mL)

consisted of cashew apple extract (red, yellow or green variety) with some amount of sugar about 7.44-7.82% (b/v), MgSO4.7H2O 0.1% (b/v) and glucose 7 g. The sources of nitrogen were urea 0.2 g/L, ammonium sulfat 0.443 g/L, Phaseolus radiatus sprout extract 7.73 mL/L and Mucuna pruriens extract 9.25 mL/L. NaOH was added to keep the pH around 6. The fermentation media was sterilized at 121oC for 10 minutes. 6.108 cell/mL of Z. mobilis were used in this research.

The fermentation media consisted of cashew apple extract and different source of nitrogen was fermented for 24, 48 and 72 hours. The pH, consumption of sugar (Nelson-Somogyi) (Nelson 1944; Somogyi 1952), the amount of bacterium (Standard Plate Count) and ethanol production (Conway microdiffusion) (Conway and O'Malley 1942) were analysed during the fermentation process.

RESULT AND DISCUSSION

Ph level The pH level determines the process of

fermentation due to the characteristic of the enzyme that only works in certain pH interval. This research showed that pH level decreased along the fermentation. The pH was set at 6 at the beginning of this research, as Worden et al. (1983) said that Z. mobilis

grows well at 5.6-7.5 of pH. The changes of pH at Table 1 below showed the decrease of pH in each treatment. The decrease of pH was caused by contamination of other bacteria especially lactose acid bacteria. Although fermentation media has been sterilized but this contamination can still posibly happen (Rahayu dan Rahayu 1988). The research showed insignificant decrease of pH; 0.19 (1.7%). It indicates that the acid level formed was not significant. Theoretically, the forming of ethanol by Z. mobilis will not produce another element in which 1 mol of glucose produce 2 mol of ethanol and 2 mol CO2.

Table 1 showed that the pH from urea and ammonium sulphate was lower compared to pH from P. radiatus sprout extract and extract of M. pruriens. It is because the nitrogen source of ammonium (urea dan amonium sulfat) was produced NH4

+ in the medium that then get into the cell as R-NH3

+ so that H+ remains in the media and reduce the pH. While in another nitrogen source namely nitrate or protein, the H+ is taken from the media to form R-NH3

+ so that the pH increase (Wang et al. 1979; Standbury and Whitaker 1984). The acid as the product of the fermentation will increase the pH in the medium with urea and ammonium sulphate compared to those in media with

Table 1. The data of pH changing in cashew fruit fermentation into ethanol by Zymomonas mobilis

Cashew variety

Nitrogen source Fermentation time

(hours) Urea Amonium Green bean sprout Koro

Red 5.90 5.88 5.95 5.94 24 5.87 5.84 5.91 5.89 48 5.84 5.80 5.87 5.86 72

Yellow 5.91 5.87 5.93 5.91 24 5.88 5.85 5.89 5.88 48 5.83 5.82 5.84 5.82 72

Green 5.89 5.87 5.92 5.90 24 5.86 5.85 5.86 5.86 48 5.83 5.81 5.81 5.84 72

Table 2. The data of sugar level (g/100 mL) changing in cashew fruit fermentation into ethanol by Zymomonas mobilis

Cashew variety


(hours) Urea Amonium Green bean sprout Koro

Red 9.29 8.33 9.50 9.42 24 8.77 6.73 8.73 8.62 48 8.11 5.39 8.41 8.31 72

Yellow 8.85 7.87 9.32 9.04 24 8.17 6.61 9.07 8.90 48 7.50 5.93 8.87 8.77 72

Green

8.61 7.64 9.61 9.06 24 8.07 6.92 9.07 8.82 48 7.10 5.17 8.93 8.81 72

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extract of P. radiatus sprout and M. pruriens extract . The statistic test with Tukey’s method showed that the

differences in the variety of cashew apples, nitrogen sources, and the duration of the fermentation influenced the pH of the media

The sugar level The glucose from cashew fruit extract and the added

sugar (7 g) were the sources of carbon for Z. mobilis that was then changed into ethanol and CO2. The amount of sugar before the fermentation was 7.44 g/100 mL for the red, 7.26 g/100 mL for the yellow and 7.82 g/100 mL for the green variety of cashew apple. Because of the lack of the sugar (under 10 g/100 mL) in cashew apple, 7 g of glucose was added to each treatment. Kirsop and Hilton (1981) suggested to get more economical fermentation by giving at least 10% of sugar fermentation media. The use of sugar by Z. mobilis was indicated in Table 2. The Table shows that there was a use of glucose as a carbon source. Up to the third day, hour 72, the highest use of sugar by Z. mobilis was in the treatment with ammonium sulphate. The high use was 65.12% for green variety, 62.67% for red and 58.42% for yellow.

The use of sugar was better compared to the similar research (The ethanol fermentation with cashew extract by Z. mobilis) by Sapariantin (2005) that only produced 33.83%, but compared to other research, the use of this sugar was low. Hermawan et al. (2000) reported of the use of sugar 80-90% in a research of ethanol fermentation with cashew fruit extract by Saccharomyces cerevisiae. Another research with only glucose media was reported to use 98.6% of Z. mobilis (Nowak 2000). The low usage of reduced sugar by Z. mobilis was because of the usage of standard bacteria (as used by Sapariantin (2005), while Nowak (2000) used modified Z. mobilisi namely strain 3881 dan 3883. It was also a possibility of a contamination by other bacteria consuming the glucose.

Table 2 shows that the use of glucose by Z. mobilis was optimum by using nitrogen of ammonium sulphate, 65.12% for green variety. Torres and Barrati (1988) stated that the best nitrogen source for Z. mobilisi is khamir extract, ammonium sulphate and the mixture of them. It produces different amount for those using Saccharomyces cerevisiae for ethanol fermentation. For this microbia, the best nitrogen source is urea compared to ammonium sulphate (Hermawan et al. 2000). Table 2 also shows that the highest decrease of the sugar occurred in 0-24 hours, while after 24 hours the decrease was not high. Doelle (1990) stated that the optimum time of ethanol fermentation for Z. mobilis occurs in 24 to 34 hours.

The nitrogen source of extract P.

radiatus sprout and extract of M. pruriens contain carbohydrate (4.1 g and 55 g respectively out of 100 g) meaning that both contribute sugar as carbon source for the bacteria. The big amount of the leftover of sugar from the media with extract of P. radiatus sprout and extract of M. pruriens could be caused by the bigger amount of the sugar in them compared to other media with different sources of nitrogen.

The statistic test of sugar changing with Tukey’s method shows that the differences of the cashew variety, nitrogen sources, and the duration of the fermentation impact the sugar level to the result of the fermentation

The amount of bacteria The amount of bacteria gives huge impact of the

success of a fermentation. The more amount of bacteria, the better fermentation will get. The amount of bacteria is also to cover up with the time needed fot the adaptation toward the new media.

This research used Z. mobilis FNCC 056 with 6.108 cell/mL for each treatment injected to 100 mL media to get 6.106 cell/mL bacteria. The changing of the amount of bacteria is shown in Table 3. Table 3 shows that the use of ammonium sulphate influenced the growth of the bacteria. The average amount of the bacteria with ammonium sulphate as the source of nitrogen showed the highest compared to other sources.

Table 3. The data of the amount of bacteria (x 107) changing in cashew fruit fermentation into ethanol by Zymomonas mobilis

Cashew variety


(hours) Urea Amonium P. radiatus sprout

M. pruriens

Red 6.60 7.30 6.40 6.50 24 26.7 34.7 26.7 27.3 48 46.0 69.3 44.3 45.0 72

Yellow 6.80 7.70 6.50 6.70 24 28.7 35.3 25.7 26.3 48 49.7 63.0 42.3 42.7 72

Green 7.10 8.00 6.30 6.70 24 29.0 33.7 25.7 26.3 48 52.7 72.3 42.0 42.3 72

Table 4. The growth of Zymomonas mobilis on media for each variety of cashew

Cashew variety


(hours) Urea Amonium P. radiatus sprout

M. pruriens

Red 0.146 0.150 0.144 0.145 24 0.102 0.107 0.102 0.102 48 0.076 0.081 0.075 0.075 72

Yellow 0.147 0.152 0.145 0.146 24 0.103 0.108 0.101 0.102 48 0.077 0.080 0.074 0.075 72

Green

0.149 0.154 0.144 0.146 24 0.104 0.107 0.101 0.102 48 0.077 0.082 0.074 0.074 72

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This research showed that the growth rate of specific µ indicating the speed of changing of a microorganism growth. The bigger µ value, the faster a microorganism grows. Counted every 24 hours from hour 0 to 72 the µ value was got as it is shown in Table 4. The highest growth of bacteria was reached in the fermentation with amonium sulphate as the nitrogen source and cashew of green variety. It showed µ of 0.082/hour, and then red variety with 0.081 and yellow with 0.08 with the same nitrogen source. The Table also showed that eventhough there was always increase of the amount of bacteria at every hour but it also showed the decrease of µ meaning that eventhough there was an increase of bacteria but there was also a decrease of the growth. This decrease was caused by the limited media so that in certain step the bacteria were competing in gaining nutrition causing the decrease of the bacterias growth.

The µ in this research was higher than that in the similar research by Sapariantin (2005). In her research, the µ is 0.062 after hour 72. It is also higher than that of Gunasekaran and Raj (2002), namely 0.03 after hour 12. That was in fermentation of 12 hours that may change in the next period. The statistic test shows that there was a significant impact causing by different variety, nitrogen sources, and the duration of the fermentation toward the amount of bacteria.

Ethanol level The different nitrogen sources gave different impact

toward the ethanol produced by the fermentation of cashew fruit extract with Z. mobilis. This research showed that the highest ethanol level gained in fermentation with ammonium sulphate as the nitrogen source and green variety namely 46.31 g/L and then red 43.24 g/L, yellow 38.57 g/L with the same nitrogen source. The result can be seen in Table 5.

The other nitrogen source producing high ethanol level is urea, and then M. pruriens extract and green bean sprout extract. It is similar with what is stated by Torres and Barrati (1990) that the best nitrogen source for Z. mobilis is khamir extract, amonium sulfat or the mixture of them.

The green variety showed a good performance in gaining ethanol in this research. It was caused by the usage of sugar of the bacteria. It was shown by the sweetness of the green variety compared with other varieties shown in

the reduced sugar level (5.1% higher than the red and 7.7% higher than the yellow)

Theoretically, the ethanol produced by Z. mobilis is 0.51 g for each gram of glucosa given (Gunasekaran and Raj 2000). Therefore, if it uses 9.65 g/100 mL (Table 1), for the green with ammonium sulphate as the nitrogen source, it will produce 4.922 g/100 mL or 49.22 g/L ethanol. If it produces the highest, 46.31 g/L, it will gain yield ethanol 94.09%. It can be seen in Table 5. Another research showed ethanol level to 98% (Gunasekaran and Raj 2000), while Nowak (2000) reported that fermentation by Z. mobilis with batch fermentation got 96% and 94,5% of yield ethanol with continuous fermentation.

Table 5 shows that during hour 0 to 24 the increase of ethanol level was higher than between hour 24 to 48 or 72. As it is stated by Doelle (1990) that the time needed to produce ethanol by Z. mobilis is between 24 and 34 hours to gain optimum production. Therefore. it is advisable to conduct only 24 hours to produce ethanol from cashew fruit extract. The statistic test with Tukey showed that the ethanol from green variety was much different with other variety. while those of red and yellow variety were not significantly different. The usage of nitrogen source of ammonium sulphate gave significant impact to the ethanol production than those of other sources. While the production of ethanol by green bean sprout extract is not much different with those by M. pruriens extract. The duration of the fermentation also gave significant impact to the production. This research showed that though the production of ethanol with natural resources (Phaseolus radiatus sprout and Mucuna pruriens) was not as high as those with urea and ammonium sulphate but they are potential to be used for its easy sources and the less residu and eco friendliness.

CONCLUSION

The green variety of cashew apple produced the highest ethanol than those of red and yellow variety. It also gave optimum production with ammonium sulphate in 24 hours. This treatment gave 33.02 g/L of ethanol or in other words gave 90.19% of ethanol’s yield.

Table 5. The data of ethanol level (g/L) and yield ethanol (%) changing in cashew fruit fermentation into ethanol by Z. mobilis.

Cashew variety

Nitrogen source and yield ethanol (Ye) Fermentation time (hour) Urea Ye Amonium Ye P. radiatus sprout Ye M. pruriens Ye

Red 23.05 87.77 28.11 90.27 21.64 85.97 22.31 87.11 24 25.78 89.13 36.50 92.73 25.50 87.62 26.15 88.07 48 29.03 89.86 43.24 93.70 26.83 87.21 27.32 87.40 72

Yellow 24.16 87.51 29.66 91.04 21.53 85.47 22.98 86.28 24 27.92 89.85 35.74 91.63 22.95 86.49 23.86 87.22 48 30.56 88.60 38.57 90.77 23.56 85.66 25.05 89.49 72

Green 27.70 87.42 33.02 90.19 22.90 86.21 25.45 86.71 24 31.03 90.11 37.70 93.62 26.00 88.69 27.26 89.09 48 35.44 90.04 46.31 94.09 27.12 90.21 27.70 90.42 72

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Linoleic and linolenic acids analysis of soybean tofu with Rhizopus oryzae and Rhizopus oligosporus as coagulant

CICIK SUDARYATININGSIH1,♥, SUPYANI² ¹ SMA Kristen Kalam Kudus. Jl. Diponegoro, Madegondo, Grogol, Sukoharjo 57552, Central Java, Indonesia. Tel. +92-271-21605.

² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia.

Manuscript received: 14 July 2009. Revision accepted: 15 October 2009.

Abstract. Sudaryatiningsih C, Supyani. 2009. Linoleic and linolenic acids analysis of soybean tofu with Rhizopus oryzae and Rhizopus oligosporus as coagulant. Nusantara Bioscience 1: 110-116. The aims of this research are to know the potency of Rhizopus oligosporus and Rhizopus oryzae as a coagulant in tofu processing for increasing the amount of linoleic and linolenic acids, and to know the time that needed by R. oligosporus and R. oryzae for increasing the amount of linoleic and linolenic acids. It uses PDA for inoculating fungi, and it is done at Sub-Lab Chemistry, Central Laboratory for Mathematics and Natural Sciences, Sebelas Maret University, Surakarta. The tofu making was done in “Dele Emas” Tofu Factory, Surakarta. Analysis of linoleic and linolenic acids were done by Gas Chromatography, in LPPT-UGM Yogyakarta. The conclusion of this research are R. oligosporus dan R. oryzae having a potency as a coagulant in tofu processing for increasing the amount of linoleic and linolenic acids. R. oryzae needs 18 hours to coagulate the tofu, and R. oligosporus needs 12 hours for the same process. The highest amount of linoleic and linolenic acids were obtained by R. oryzae at 6 hours of fermentation (0.26% and 0.14%), and 24 hours of fermentation by R. oligosporus (0.06% and 0.04%).

Key words: linoleic acid, linolenic acid, tofu, coagulant, Rhizopus oryzae, Rhizopus oligosporus.

Abstrak. Sudaryatiningsih C, Supiyani. 2009. Analisis kandungan asam linoleat dan linolenat tahu kedelai dengan Rhizopus oryzae dan Rhizopus oligosporus sebagai koagulan. Nusantara Bioscience 1: 110-116. Penelitian ini bertujuan untuk mengetahui potensi jamur R. oligosporus dan R. oryzae sebagai koagulan pada proses pembuatan tahu dalam meningkatkan kadar asam linoleat dan linolenat tahu dan mengetahui waktu yang diperlukan oleh keduanya untuk memfermentasi tahu sehingga menghasilkan asam linoleat dan linolenat yang tinggi. Inokulasi jamur menggunakan PDA,dilakukan di Sub-Lab Kimia, Laboratorium Pusat MIPA, Universitas Sebelas Maret, Surakarta. Pembuatan tahu dilakukan di pabrik tahu Dele Emas Surakarta. Analisis kandungan asam linoleat dan linolenat dilakukan dengan metode kromatografi gas, dan dikerjakan di LPPT-UGM Yogyakarta. Dari hasil penelitian ini diperoleh kesimpulan R. oryzae dan R. oligosporus memiliki potensi sebagai koagulan dalam pembuatan tahu, dan waktu yang diperlukan untuk melakukan koagulasi R. oryzae adalah 18 jam R.oligosporus adalah 12 jam. Asam linoleat dan linoleat tertinggi diperoleh. Pada 6 jam fermentasi R. oryzae (0,26% dan 0,14%), dan 24 jam fermentasi oleh R.oligosporus (0,14% dan 0,08%).

Kata kunci: asam linoleat, asam linolenat, tahu, koagulan, Rhizopus oryzae, Rhizopus oligosporus.

INTRODUCTION

Fat is a food substance that is always present in the daily diet. Various kinds of foods, ranging from fried bananas, fried potatoes, fried chicken, rendang, to pizza, are not apart from fat. The addition of fat in the form of oil is useful for the food to improve taste, texture, and improve the flavor (Muchtadi 2000). Fat is useful for the body as the source of energy, and also as a solvent to various vitamins (Campbell 1987). As a source of energy, fat has a high calorific value which is 9.3 kcal/g. This value is higher than carbohydrates and protein. In addition, during oxidation, fatty acid metabolic generates a lot of water, compared to carbohydrates and proteins (Harper et al. 1979). It is advantageous for organisms that live in dry areas.

Eating too much fat is dangerous for heart health (Forman and Bulwer, 2006; Muchtadi 2007), including the causes of obesity (Wilson et al. 2002; Panagiotakos et al. 2004) and hypertension (Houston et al. 2005). Foods that

have high fat content, can lead to the formation of plaque in arteries. The formation of plaque will lead to the inside of the artery wall thickening, and narrowing the cross section. This disease is called atherosclerosis, and can cause strokes and heart attacks (Campbell 1987). Cooking oil used to fry more than once is also dangerous for our health, because it can cause cancer (Nadesul 2007).

Fats can be hydrolyzed by lipase into fatty acids and glycerol. In the process of metabolism, fatty acids can be harmful to health, but some are really needed for health. Fatty acids which are harmful are saturated fatty acids, i.e. fatty acids with no double bond on carbon chain constituent. Such saturated fatty acids can be found a lot in the animal fat, lard, milk, eggs, and poultry skin (Nadesul 2007). Fatty acids which are not harmful to health are the ones which are not saturated (Sipayung 2003). Unsaturated fatty acid is a fatty acid having double bond on carbon chain constituent. Examples of unsaturated fatty acids that are important for the health of the body are linoleic and

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linolenic acids. Both types of these fatty acids are essential fatty acids, which are fatty acids that can not be produced by the body (Harper et al. 1979). Because they can not be produced by the body, it must exist in the consumed food. Linoleic acid plays an essential role in the prevention of coronary heart disease and healthy blood vessels (Nadesul 2007).

One source of linoleic and linolenic acids are soybean seeds (Harper et al. 1979). Indonesian people consume soy in various forms of processed foods such as soy sauce, tofu, tempe, tauco, soy milk, and others. In some processed foods, a change in the nutritional value of soybeans occurs, for example in the fermentation process of soybeans into tempe, there is a trend of increased fatty acid of linoleic and linolenic acids (Bisping et al. 1993; de Reu 1994; Naidoo and Hermana 1995). Tofu is one of the types of foods derived from soybeans widely consumed by the public. Tofu is made without the process of fermentation, so the content of linoleic and linolenic acids are not as high as tempe. That is why in this study the researchers tried to analyze the content of linoleic and linolenic acids on the tofu by using the fungus of Rhizopus oligosporus and Rhizopus oryzae as a coagulant.

This study aims to: (i) determine the potential for fungus of R. oligosporus and R. oryzae as a coagulant in the making process of tofu; (ii) determine the length of time needed by both to perform coagulation in the making process of tofu; (iii) determine the potential of both in increasing levels of linoleic and linolenic acids in the tofu; and (iv) determine the length of time needed by both to ferment the tofu so it produces high levels of linoleic and linolenic acids levels.


Time and place This research was conducted in June-July 2009. The

inoculum of Rhizopus oligosporus and Rhizopus oryzae was created at the Laboratory of Biology, Sebelas Maret University, Surakarta. The tofu production was done at the tofu factory "Dele Emas" Surakarta. Unsaturated fatty acid test was conducted at the Integrated Research and Development Laboratory (LPPT), Gadjah Mada University, Yogyakarta.

Material Soybean imported from the United States (Glycine max

(L.) Merr.), used as the material to make the tofu were obtained from soybean import wholesalers, in the market of Mojosongo, Surakarta. R. oligosporus and R. oryzae were obtained from the Laboratory of Microbiology, University of Setia Budi, Surakarta. The media of Potato Dextrose Agar (PDA) was obtained from the Laboratory of Biology, Sebelas Maret University, Surakarta, to breed R. oryzae and R. oligosporus

Procedures The making of inoculum. Media to make the pure

culture of inoculum is the PDA. In the rules of use, it is

said that to make 1 liter of gelatin requires 39 g PDA, so to make the culture of pure inoculum of 4 test tubes, 5 mL for each tube is needed {(4x5)/1000} x39 g = 0.78 g. How to the culture are as follows: as much as 20 mL of distilled water is inserted in a glass beaker, then added with 0.78 g of PDA (E. Merck) into it and stir it until well blended. PDA solution was poured into four test tubes 5 mL for each. The tubes were plugged with cotton, and inserted into the autoclave with a temperature of 121°C for 15 minutes. After it was placed on a board tilted up for the cold and dense, and then was left for 3 days. 1 ose pure culture of R. oligosporus was streaked on to tilt, and left for 48 hours to be ready for use. The same was done to make the pure culture of R. oryzae.

Calculation of the spores of Rhizopus sp. The surface and the lid of haemacytometer were cleaned by lens paper and distilled water. The cover glass of haemacytometer was placed on the surface of the counting machine. A total of 5 mL aquades was poured onto the culture of Rhizopus sp. By using a needle loop, Rhizopus sp. was scraped in order that all the spores are able to be lifted. Fungal suspension was poured into another tube, and shaken. Drawn 1 mL suspension of fungus with a pipette, and inserted into a V-shaped groove on the edge of the lid of haemacytometer. Haemacytometer was placed under the microscope, and the spores were counted. The result turned out that in 1 mL of suspense there were 8.3x106 R. oryzae spores; and to make tofu of 1 L of soymilk needed 107 spores (Purwoko 2001). Thus the number of suspension required is: 107/8.3 x106 = 1.2048 mL. While in 1 mL of suspense obtained 10.9 x106 R. oligosporus spores, so that for 1 L of material required 107/10.9x106 = 0.9174 mL.

The tofu making. One kilogram of soybean was washed, and then soaked for 6 hours. Next milled until smooth, strained and the juice was taken, the waste was removed. One liter of soymilk was cooked until boiling, then cooled, and when cool it was inoculated with 107 spores of R. oligosporus. The soybean which had been inoculated was made into 4 kinds of treatments, namely left for 6 hours, 12 hours, 18 hours, and 24 hours. The precipitate that had been formed was separated from the liquid and pumped by a fabricated pump layered with a coat. The same process was done to R. oryzae.

Determination of linoleic and linoleic acids. Determination of linoleic and linoleic acids was performed by the method of gas chromatography.

Preparation of materials. Each sample’s weigh was 10 g, then crushed/grinded to be homogenized. The sample was put into the flask of 50 ml and then added with 4 mL of concentrated HCl, homogenized, then added with 7 mL of concentrated HCl.

Hydrolysis. The materials that had been homogenized were put into the water heater, then heated at 70°C, then continued to boil, and left for 90 minutes. While heating, the container was covered with a plastic so it did not evaporate.

Extraction. The materials were cooled, then added with 7 mL of ethanol, boiled, and then added with 25 mL diethylether and boiled again. 25 mL of Petroleum benzene fractions was added into the materials at 40-60 degrees

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Celsius, then vortexted. The top layer that had been formed was separated from. The bottom layers were extracted again, using 15 mL of dietileter and 15 ml of petroleum benzene. The top layer was separated again, and then made into one with the top layer of the first. Next step is evaporation at a temperature of 50 degrees centigrade with the help of N2 gas, until it dried.

Saponification. 1 mL solution of 0.5 M NaOH-metanolic was added into the dry ingredients, heated to boil for 1 minute.

Esterification. Materials that had been saponificated were added with 2 mL of 20% BF3-methanol, and heated to boiling, for 1 minute. Then extracted with 1 mL of n-heptane and 1 mL of saturated NaCl, and until it formed 2 layers with the upper layer consisting of heptane and methyl esters. The top layer was taken and then injected into the GC. At the same conditions, the standard Methyl linoleic and linolenic methyl were also injected with a concentration of 0.125%.

Research variables Variables examined in this study include: (i)

Morphology of the tofu fermented by the fungus of R. oligosporus and R. oryzae. (ii) Changes in pH of the tofu during the fermentation process (iii) the time required for the fungus of R. oligosporus and R. oryzae to perform coagulation. (iv) The highest content of linoleic and linolenic acids formed during fermentation by the fungus R. oligosporus and R. oryzae. (v) the optimum time required by R. oligosporus and R. oryzae to produce the highest levels of linoleic and linolenic acids during the fermentation

RESULTS AND DISCUSSION

Morphology of tofu In the process of the tofu making, the fermentation was

done for some long times, namely 6 hours, 12 hours, 18 hours and 24 hours. It was from the treatment above it can be seen different morphological forms of tofu , as in Table 4. In the table it can be seen the morphological changes of the tofu out of the shape that was initially soymilk. The changes can be seen at: discoloration, formation of cavities, the change becoming acidic odor, density, and the increase of water content.

Changes of the tofu color In this event of tofu fermentation the change of color

happened in soy milk from white color turned into yellowish, and then became brown. These changes occurred slowly over a long period of fermentation. These color changes in both Rhizopus oligosporus and Rhizopus oryzae fermentation began after the fermentation lasted for 6 hours. Furthermore, the color changed to yellow after 12 hours fermentation, then changed to brownish yellow after 24 hours fermentation. According to Cook (1994), the yellow color is caused by a pigment consisting of alpha carotene and beta carotene. This pigment is a natural dye ingredient contained in the material consisting of oil, or producing oil, for example the groups of fungi of the order of Mucorales. Wiesel et al. (1996), reported that the yellow color formed is the result of β-carotene biosynthesis by Rhizopus sp. and indicate the fermentation process going well. Denter et al. (1998) and Bisping et al. (1993) write that Rhizopus is a type of mold that is capable of forming β-carotene.

In this study, color changes that occur indicate the existence of fermentation activity by the fungus Rhizopus sp. During the fermentation, Rhizopus sp. synthesized β-carotene and released it to the media, making media changes color from white to yellow. This event at R. oryzae fermentation occurs continuously up to 24 hours. But not so in the fermentation by R. oligosporus. After the fermentation lasted for 24 hours, the fermented tofu with R. oligosporus, the color was no longer yellow, but changed to brown. This showed the change of the protein into peptide bond hydrolysis by the enzyme protease, resulting amine group, which can react with aldehyde or ketone group and produce the brown color. This is consistent with reports Subagio et al. (2002), which is in addition to the β-carotene, color changes in the process of fermentation by Rhizopus sp. is also due to the process of protein into peptide bond hydrolysis by protease enzymes. The result of hydrolysis is amine group, which then reacts with aldehyde or ketone group and produce brown color. Brown color appears in the fermentation by R. oligosporus after 24 hours, and has not appeared on the fermentation by R. oryzae after 24 hours. This proves that R. oligosporus has the ability to hydrolyze proteins faster than R. oryzae, or in other words R. oligosporus is more active in fermentation compared to R. oryzae.

Table 4. Morphology of fermented tofu.

Time of fermention

(hours) R. oryzae R. oligosporus

6 White color, the smell of soy milk White color, the smell of soy milk 12 Soymilk was denser, yellowish-white color, smell

like tempe. Soymilk was denser (tofu was formed), yellowish color, smell like tempe, small cavities raised, some water was produced.

18 Soymilk was denser (tofu was formed), yellow color, smell of tempe mixed with sour, small cavities aroused, some water aroused.

Soymilk was denser, yellow color; smell like tempe mixed with a little sour, the cavities was larger, more water was produced.

24 Soymilk was solid, yellow, sour smell was stronger, the cavities grew more, water were produced more.

Soymilk was solid, brownish yellow color, acid smell was stronger, a lot of large cavities, water were produced more.


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Tofu cavities In addition to color changes, in the fermentation process

also arise out of the cavities. In the fermentation by R. oryzae cavities began to appear after 18 hours fermentation, and increased much after 24 hours fermentation. While in the fermentation by R. oligosporus small cavities already started to appear after 12 hours fermentation, and more and more in line with the length of fermentation. The cavities were formed because the proteins that were initially spread evenly on the liquid soymilk began to clot.

At first only a few proteins that were coagulated, so it only had a light molecular weight, and could still be spread on the fermentation container. As a result, cavities that were formed were only small. The incident occurred at the fermentation of R. oligosporus after 12 hours of fermentation. Further, after the fermentation by R. oligosporus lasted for 18 hours, the proteins that were formed got more and more, these proteins formed larger clumps, with large molecular weight, which tends to settle, and form greater cavities.

In the tofu production process carried out in the factory clotting proteins that occurred was because the soybeans were given an additional acid as coagulant. The given acid for example acetic acid, or the tofu liquid had been kept for one night. Furthermore, this acid reacts and binds to proteins contained in the material out, and together with lipids to form clots (Santoso, 1993). Coagulant material will determine the quality of the tofu. The better the coagulant, the more protein-bound, resulting more randemen (Suprapti 2005).

In this research, it was obtained that the coagulation by R. oligosporus were faster in forming the solid tofu than the fermentation by R. oryzae. R. oryzae took 18 hours to form thetofu, while R. oligosporus only took 12 hours, to agglomerate soybean into tofu. This is consistent with the reports of Sapuan and Sutrisno (1997), that R. oligosporus has a protein hydrolysis speed that is higher than that of R. oryzae. With the protein resulted from this hydrolysis, it will form clumps, and then it settles to form the tofu.

The increase of the water content and the tofu density As a result of clots formed during the fermentation

process there were areas that did not contain the protein mass. This area was not empty but filled with water. The existence of this water could not only be seen through the cavities that had appeared, but also through the condensation on the fermentation container lid. In the fermentation by R. oligosporus, the formation of water and water vapor on the container lid fermentation occurred after 12 hours fermentation, and the water content increased until 24 hours fermentation. Along with the presence of water and water vapor in the closed container, soya also turned into a solid, but not so with the fermentation by R. oryzae. Formation of water that was trapped in the cavities occurred after 18 hours fermentation, and continued to increase up to 24 hours of fermentation. The formation of solid soymilks was seen after 18 hours fermentation, showing fermentation by R. oligosporus turned out to be more quickly than that of R. oryzae. In the process of tofu

making carried out in a factory, high water content may be removed by it being compressed using a mold out of wood covered with calico cloth (Suprapti 2005).

Fermentation of tofu by R. oligosporus produced a lot of clumps of protein, more solid tofu, compared to the fermentation by R. oryzae. This is in accordance with the opinion of Suhaidi (2003), that the more the clot is formed, the more protein that is trapped inside, so it will generate a lot of randemen.

The smell of acid Tofu fermented by Rhizopus sp. has a sour smell. Sour

odor occurs due to decrease in soymilk pH. The smell of acid can be observed by the senses in 18 hours fermentation, both by R. oryzae, and R. oligosporus. Observations soymilk pH using a pH meter shown in Table 5 below Table 5. The degree of acidity (pH) of soymilk fermented by Rhizopus spp.

Time of fermentation

(hours)

pH

R. oryzae R. oligosporus

6 12 18 24

5.81 5.52 5.31 5.53

5.86 5.61 5.44 5.47

In Table 5 it can be seen that the fermentation by Rhizopus sp produced a product that has a pH of 5. According to the Sapuan and Sutrisno (1997), the decrease of pH in the fermentation media can occur until the pH of the media reached 4.5 to 5.3 and this pH change causes the mold to grow well, and there the process of fermentation occurred. Hidayat (2009), reported on the tempe fermentation process, the decrease of pH started at the immersion process of soybeans. The immersion process provided an opportunity for lactic acid bacteria to grow so that it decreased the pH of soybean seeds. This decrease in pH can inhibit the growth of bacterial contaminants that are decomposers. In addition, the decrease in pH caused the mold to be able to ferment properly.

Furthermore, when the fermentation is in progress, Rhizopus sp. will produce lactic acid, and cause the pH of soymilk to get decreased. This decrease of pH resulted in the soybean protein having clotting or coagulation (Gaman 1992). In the process of tofu fermentation, this clumping resulted in the protein which was originally spread out, becoming mutually bonded to one another, then forming the tofu. In this study, the sharpest decline in acidity occurred after 18 hours fermentation. The smell of acid can be observed even with the sense of smell. This shows the optimum coagulation activity in fermentation of Rhizopus sp. after 18 hours.

Analysis of chromatography gas The peaks of chromatogram

The test of linoleic and linolenic acids content was conducted by the gas chromatography, using ethanol as

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solvent, and Flame Ionization Detector (FID) as detector. The use of gas chromatography can be performed on samples that require high temperatures to evaporate, such as fatty acids. The gained results were in the form of chromatogram which had crests. From the observation of chromatogram analysis of the content of tofu fermented by Rhizopus sp., the gas chromatography could detect the three dominant peaks. Based on the comparison with the standard, it is certain the first peak was linoleic acid, and the second peak was linolenic acid. While the third peak was a compound formed from the oxidation of linolenic acid. Presumably that the third compound was arachidonic acid. This is in accordance with the opinion of fever (1997), that linoleic acid would be oxidized into the linolenic acid, and then the linolenic acid is oxidized into arachidonic acid. The oxidation process is strongly influenced by temperature and light intensity. The higher the temperatures and the greater the light intensity, the faster the oxidation will be.

High peaks that formed on the chromatogram of this experiment showed the concentration of a substance that was tested the samples, in this study it showed the levels of linoleic and linolenic acids. The levels of linoleic and linolenic acids can be seen in Table 6. The height of the peak from the bottom of the chromatogram showed retention time. According to Adnan (1997), the retention time is the time required to separate, and then evaporate, exit the column. A long retention time shows that those materials require high temperatures to separate. The high temperature is required to perform the separation of material that has a lot of double chains. So the longer retention time indicates that these substances have more double bonds.

Retention time can also indicate the speed of molecular motion. In this experiment the retention time was used to compare the standard molecular velocity with the velocity of the sample molecules. In this study, the standard retention time was 8.80 minutes for the linoleic acid, and 11.0 for the linolenic acid. While the retention time of the sample can be seen as Table 6.

Table 6. Retention time (Rf) fermented tofu by Rhizopus sp.

Time of fermentation

(hours) Peak of

R. oryzae R. oligoorus Rf

(Minutes)High peak

Rf (Minutes)

High peak

6

1 2 3

8.998 10.873 11.029

1487980 3561719 604055

8.983 10.847 10.907

86814842593481708088

12

1 2 3

8.996 10.885 11.031

1513297 4157405 646773

8.987 10.862 11.022

10773143169875466356

18

1 2 3

8.990 10.873 11.027

1390400 3711272 619996

9.009 10.928 11.051

217504557334751090065

24 1 2 3

8.991 10.873 11.026

1283842 3582183 573739

9.014 10.941 11.060

248447164734681268570

From Table 6 above it is shown the analysis by gas chromatography was going well, as indicated by the retention times close to the standard, and the visible differences distance of time with each other.

Linoleic and linolenic acids content The analysis results of linoleic and linolenic acids

content using gas chromatography is shown in Table 7, which the tofu of the control group, made by using vinegar as a coagulant, contained linoleic and linolenic acids respectively 0.05% and 0.03%. Meanwhile, linoleic and linolenic acids content in the tofu with Rhizopus sp. as coagulant was higher. Allegedly linoleic and linolenic acids existing in the tofu were the linolenic and linoleic acids which already existed in soybean. This is consistent with the reports of Iskandar (2004), that soybean is a source of linoleic acid. Table 7. Linoleic and linolenic acids content in the tofu.

Name of sample Content (%) Linoleic acid Linolenic acid

Tofu control R. oryzae 6 hours R. oryzae 12 hours R. oryzae 18 hours R. oryzae 24 hours R. oligosporus 6 hours R. oligosporus 12 hours R. oligosporus 18 hours R. oligosporus 24 hours

0.05 0.26 0.10 0.09 0.07 0.06 0.07 0.12 0.14

0.03 0.14 0.05 0.06 0.04 0.04 0.05 0.07 0.08

The process of making the tofu distributed in the market usually uses vinegar or the remaining water of the tofu from the previous day that has been kept overnight, and without undergoing fermentation. Acetic acid will react with proteins, resulting in the occurrence of clotting proteins, and forming the tofu. The change of soymilk into a tofu lasts for about 90 minutes. While the tofu making using Rhizopus sp. changes soymilk into tofu slowly. These changes occur enzymatically, to form new substances. For example, changes with the enzyme lipase, resulting in the formation of linoleic and linolenic fatty acids (Pawiroharsono 1997).

Tofu fermentation by Rhizopus oryzae For the first 6 hours of fermentation by R. oryzae, tofu

contained the highest linoleic and linolenic acids, and then there was the tendency to decrease. This happened because at the beginning of fermentation, R. oryzae would synthesize a high linoleic acid, after that this fatty acid was converted into the linolenic acid. Linoleic acid was derived from oleic acid which was the result of fat hydrolysis by lipase enzyme during the fermentation process. This is consistent with the theory of Teng et al. (2008), that Rhizopus produces lipase during the fermentation process. This lipase does hydrolysis activation. Initially the first acid that was formed was oleic acid, but then it was saturated acid into linoleic acid, and changed again into linolenic acid (Styme and Stobart 1986).


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Mushrooms of the class of Zigomycetes have the desaturase enzyme, an enzyme which can alter the results of oleic acid hydrolysis of fat by lipase into linoleic and linolenic acids (Suharyanto et al. 2006). In the early stages of the hydrolysis of linoleic, the fatty acids were formed; further these acids had an addition of double bonds becoming the linolenic acid (Harper et al. 1979). Desaturation reaction of fatty acids is a chain reaction. Oleic acid is converted into linoleic acid with the aid of Δ12 desaturase enzyme, further into linoleic was changed acid linolenic acid with the help of the enzyme Δ6 desaturase (Suharyanto et al. 2006). Briefly, the changes of linoleic acid into linolenic acid can be written as follows:

Oleic acid linoleic acid linolenic acid In the fermentation of R. oryzae for 12 hours and

beyond, linoleic acid was not formed anymore but was changed into linolenic acid, so that the number of linoleic gradually decreased, followed by a decrease in linolenic acid levels. The declining trend in linoleic and linolenic acids continued, in 18 hours fermentation, up to 24 hours fermentation. This shows the optimum time of the formation of linoleic and linolenic acids in the tofu fermented by R. oryzae is 6 hours, and then the formation of linoleic acid stopped, and the linoleic experienced hydrolysis and become linolenic acid. The formation of linoleic acid presumably was caused by several factors, including: temperature, moisture content, and oxygen.

Purwoko et al. (2001) reported the increase in temperature on soybean tempe fermentation by Rhizopus sp. According Suharyanto et al. (2006), the increase in temperature in the fermentation process can inhibit the desaturation of the enzyme activity, so the formation of unsaturated fatty acids gets decreased. De Man (1997) reported that the oxidation process of linoleic and linolenic acids is strongly influenced by temperature and light intensity. The higher the temperature, the formation of fatty acids could decrease. In this study, the fermentation by R. oryzae was performed at the room temperature (25-26oC). At this temperature the formation of fatty acids occurred normally. Furthermore, along with the occurrence of fermentation, the temperature got increased. As a result, the formation process of linoleic acid got decreased and the formation of linolenic acid also got decreased.

The water content will affect the action of the enzyme lipase. With high water content, enzyme lipase will hydrolyze fats into glycerol and fatty acids. While at low water content, alcohol or other esters will be formed. In this research, the media used were soybeans in the form of liquid, so that the water content was high, and the resulting lipase enzyme activity that hydrolyzed fat ran well. Suharyanto et al. (2006) also reported that too high water content will affect the solubility of oxygen. At the beginning of the fermentation process, the available water was the water contained in the media. This water was to accelerate the formation of fatty acids of linoleic, and

linolenic. Furthermore, the media got additional water from the fermentation process (Purwoko 2004). The increased water content in the tofu can be seen from the emergence of cavities in the tofu, and also from the existence of water vapor on the fermentation container lid. As a result the solubility of oxygen got reduced, and the formation of linoleic acid also got decreased.

Aerobic and facultative anaerobic microorganisms require oxygen in the process of fatty acid desaturation. Aeration can increase the solubility of oxygen thus increasing the degree of fatty acid’s unsaturatability (Suharyanto et al. 2006). In this study, at the beginning of fermentation, the oxygen content in the media was high, but after several times of fermentation, the oxygen content decreased, so the formation of linoleic acid also decreased.

Tofu fermentation by Rhizopus oligosporus In the tofu Fermentation using R. oligosporus, there

was a distinct tendency from the fermentation using R. oryzae. In the fermentation of 6 hours, the smallest amount of linoleic and linolenic acids were formed. This showed that the active fermentation has begun and R. oligosporus has been churning out a lipase enzyme in 6 hours of fermentation. Furthermore, in 12 hours fermentation, the content of linoleic acid was increased, and followed by linolenic acid. This proves, in 12 hours fermentation, the lipase enzyme was still actively working to produce linoleic acid. Besides the formation of linoleic acid, the hydrolysis change from linolenic into linoleic acid also occurred. This characteristic is very different from the fungal fermentation with R. oryzae, because the lipase activity in fermentation by R. oryzae after 12 hours they no longer produced linoleic acid.

If we have a look at Table 7, the fermentation by R. oligosporus for 18 hours, the fermentation activity looks stronger and more linoleic acid production. The speed of reaction of linoleic acid into linolenic was smaller than the speed of reaction formation of linoleic acid. As a result the increase in linoleic acid content was greater than linolenic acid. But this did not last long, because in the fermentation activity after 24 hours, although the formation of linoleic acid increased, but not as much as in 18 hours fermentation.

The increase of linoleic and linolenic acids occurred continuously throughout the time of fermentation. This is in accordance with the opinion of Sapuan and Sutrisno (1997) that R. oligosporus has higher lipase activity than R. oryzae, so as to produce more fatty acids. Also R. oligosporus can perform and produce the perfect fermented soy and tempe in less time. Steinkrauss et al. (1983) reported that R. oryzae required 48 hours to produce the perfect tempe, while R. oligosporus only took 24-36 hours to produce the perfect tempe. Meanwhile, Arias (2003) reported that R. oligosporus was actively doing fermentation until the 48th hour.

With the ability to perform activities of a long fermentation, then R. oligosporus was able to perform the metabolism of various substances, including linoleic acid, thus forming a high linoleic acid. Furthermore, while still active in fermentation, linoleic acid portion has been

desaturase Δ6 enzyme

desaturase Δ12 enzyme

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changed into linolenic acid. Consequently, in the fermentation using R. oligosporus linoleic acid content tended to increase, while linolenic acid was also increased.

CONCLUSION

Rhizopus oryzae and R. oligosporus have the potential to be used as a coagulant in tofu. The optimum time to perform coagulation for R. oryzae is 18 hours and for R. oligosporus is 12 hours. Tofu made using R. oryzae and R. oligosporus as a coagulant has higher content of linolenic and linoleic than in tofu using vinegar as a coagulant. The highest of linoleic and linolenic acids was obtained: (i) R. oryzae fermentation at 6 hours (0.26% and 0.14%), after that linoleic and linolenic acids tended to decrease, along with the length of fermentation. (ii) R. oligosporus fermentation of 24 hours (0.14% and 0.08%).

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Slaughter weight and carcass of male New Zealand White rabbits after rationing with koro bean (Mucuna pruriens var. utilis)

URIP SANTOSO1,♥, SUTARNO² ¹ VEDCA/PPPPTK Pertanian Cianjur. Jl. Jangari Km. 14 Sukajadi – Karangtengah, Cianjur 43202, West Java, Indonesia. PO Box 138. Tel. :+92-263-

285003, Fax.: +92-263-285026 ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Manuscript received: 5 July 2009. Revision accepted: 26 August 2009.

Abstract. Santoso U, Sutarno. 2009. Slaughter weight and carcass of male New Zealand White rabbits after rationing with koro bean (Mucuna pruriens var. utilis). Nusantara Bioscience 1: 117-122. The objectives of the research were to know the effects of koro bean (Mucuna pruriens var. utilis) present on slaughter weight and carcass of rabbits and to know the optimum dosage that resulted the best slaughter weight and carcass. The research used Randomized Block Design whereas 25 heads of six weeks old rabbits with 450-1270 g of body weight were devided into five groups according to the body weight. Each group were treated with different treatment. The treatment were unpresent of M. pruriens as a control (R0) and various percentage of M. pruriens as much as 21.5%, in the ration with treatment as follows: R1 (raw), R2 (heating), R3 (boiling), and R4 (fermentation). The parameters observed were slaughter weight, carcass weight, meat weight, bone weight, and adipose tissue weigth. The data analyzed by analysis of variance (ANOVA) followed with Duncan’s Multiple Range Test (DMRT). The present of processed M. pruriens could increase production of slaughter weight better than the present of unprocessed M. pruriens. The additional of 21.5% of fermented M. pruriens resulted in the best production of slaughter weight and carcass of rabbits.

Key words: koro bean, Mucuna pruriens, ration, rabbits, New Zealand white.

Abstrak. Santoso U, Sutarno. 2009. Bobot potong dan karkas kelinci New Zealand White jantan setelah pemberian ransum dengan kacang koro (Mucuna pruriens var. utilis). Nusantara Bioscience 1: 117-122. Penelitian ini bertujuan untuk mengetahui pengaruh pemberian tepung kacang koro (Mucuna pruriens var. utilis) terhadap produksi bobot potong dan karkas kelinci galur New Zealand White serta mengetahui dosis optimum pemberian tepung kacang koro yang memberikan bobot badan dan karkas terbaik. Metode penelitian digunakan adalah eksperimen dengan pola Rancangan Acak Kelompok. Sebanyak 25 ekor kelinci berumur sekitar 6 minggu yang memiliki kisaran bobot badan 450-1270 g dikelompokan menjadi lima kelompok menurut bobot badannya. Masing-masing kelompok mendapat perlakuan yang berbeda, perlakuan yang diberikan adalah ransum tanpa tepung kacang koro (R0) sebagai kontrol dan ransum dengan pemberian tepung kacang koro masing-masing sebanyak 21,5%, dengan perlakuan sebagai berikut: R1 (mentah), R2 (pemanasan), R3 (perebusan), dan R4 (fermentasi). Parameter yang diamati bobot potong, bobot karkas, bobot daging, bobot tulang, dan bobot lemak. Data yang diperoleh dianalisis ragam (ANAVA) dan dilanjutkan dengan uji jarak berganda Duncan (DMRT). Pemberian tepung kacang koro hasil olahan dalam ransum meningkatkan produksi bobot potong dan karkas yang lebih baik dibandingkan dengan pemberian tepung kacang koro tanpa diolah dan pada tingkat 21,5% pemberian tepung kacang koro hasil fermentasi dalam ransum menyebabkan peningkatan produksi bobot potong dan karkas paling baik.

Kata kunci: kacang koro, Mucuna pruriens, ransum, kelinci, New Zealand white.

INTRODUCTION

Rabbit (Oryctolagus cuniculus) is known as healthy meat-producing cattle with high protein content and low cholesterol and triglyceride. As an added value it also produces skin and fur, feces (droppings) and urine as organic fertilizer. New Zealand White rabbits (NZW) quickly grow large; this type of rabbit can be used as meat rabbit. Adult weight can reach 4.5 to 5 kg; and the baby can reach 10-12 rabbits (Verhoef-Verhallen 1998). Developing rabbits is a good prospect in taking over the problem of meat shortage as the protein source in order to ensure continuous availability of food at the community level (Farrell and Raharjo 1984).

Rabbit farming in Indonesia has been quite popular in the community for its easy maintenance, relatively small capital, simple cage that can be made not extensively. To feed rabbits we can use agricultural waste, kitchen waste, market waste, or other forages. In addition, by looking after rabbits family can take utilize its spare time for more productive activities in an effort to obtain value-added (Rismunandar 1981; Sitorus et al. 1982; Sarwono 1991).

Based on the observation in the field it is found that the low productivity is caused mainly by poor of food management. Foods that are given to the rabbits are generally only green forage without concentrate and any other feedstuffs. Thus, the growth rate of body weight of the rabbit is not optimal and results in low slaughter weight

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and low carcass quality. Conditions like these can be improved by using feed ingredients that can improve the quality of foods so that they can meet the needs for basic living and production. However, the problem in the implementation of maintenance is the limited availability of raw materials, particularly soybean meal. Up to now, soybean meal is still imported because the domestic production has not been able to meet the needs. Every year our country imports as much as 1.2 million tons of soybeans for feeding material only.

In relation to the problem above, it is necessary to find alternative feed ingredients, by maximizing the utilization of local raw materials to reduce the dependence on imported feed ingredients. One of the local foods ingredients that are known to have the potential to be a source of protein food is M. pruriens (Mucuna pruriens var. utilis). This plant is known for its ease and quickness to grow naturally. M. pruriens are legumes that grow in the soil surface by creeping or climbing. Like other legumes, M. pruriens can help to increase soil nitrogen levels through its symbiosis with rhizobium, which is known as a source of organic material. In addition, M. pruriens are resistant to disease, crop residues, weeds and any other weeds (Friday et al. 1999).

In Indonesia, M. pruriens are not used yet optimally as any other leguminose crops (soy beans) both as food crop and as animal food ingredients. M. pruriens have many kinds of species with different seed colors and are good source of protein because they have abundant of essential amino acids, especially leucine. Although there are differences in species, the amino acids which are contained in them have the same composition (Lubis 1972). M. pruriens seeds contain high crude protein namely as much as 28.94%. If we take a look at the nutritional content, M. pruriens can be used as animal feed for its protein, vegetable, replacing some soybean, especially for rabbits.

Koro seeds are known to contain toxic compounds that may affect the use of nutrients in the body of non-ruminant livestock, such as the acid compound cyanide/HCN (Purwo 1974). Raw koro seeds contain HCN 42.5 mg/kg. The use of raw koro seeds in the food of pigs which is more than 15% is found to decrease food intake, weight, and feed conversion (Enemalom et al. 2004). So is

the use of raw koro seeds in broiler chicken rations which is more than 10% can reduce food intake and weight (Carmen et al. 1999).

Mucuna pruriens nutritional value as animal food ingredients can be increased, if the anti-nutritional compounds they contain can be reduced or even eliminated altogether, so the potential of this food material provides a good prospect in the diversification of animal food. Treatments with water or solution through a process of soaking, dyeing, boiling, roasting, and fermentation or by a combination of these ways are a treatment that can reduce the levels of cyanide (Aisyah 1995). The treatment process through the provision of heat with method of roasting or boiling can reduce by approximately 68% cyanide acid levels in M. pruriens (Siddhuraju 1996). Fermentation of M. pruriens can remove cyanide to get the results that are safe for use as a raw material feed (Handajani 2001).

In connection with it this research examines the effects of the use of M. pruriens that is processed by heating, boiling and fermentation toward the weight of New Zealand White male rabbits.


Place and time of the study The experiment was conducted in District Pacet,

Cianjur, West Java for 8 weeks from 5 December 2008-11

Figure 1. New Zealand White rabbits

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January 2009. The altitude of the location of the study is 1050 meter above sea level, the minimum temperature was 21.3oC, and the maximum temperature was 27.2oC, with 88% humidity.

Material This study used New Zealand White male rabbits

(Figure 1) as many as 25 wean off of about 6 weeks old, with body weight ranged from 450-1270 g. The basic rations used in this study consisted of field grass in the vicinity of the study and concentrate from KUD Cipanas, Cianjur regency. The level of M. pruriens contains the results of processing a food mixture material that would be studied. The species of the field grass used consisted of bitter grass (Paspalum conjugatum), galinggang (Galinsoga parviflova), sintrong (Crassocephalum crepidioides), babadotan (Ageratum conyzoides), domdoman (Andropoghon aciculatus), and carrot leaves. The species of the field grass were obtained in the vicinity of the study. Concentrate, the species of grass used in this study, the nutrients are shown in Table 1. Five kinds of rations used in this study were (R0, R1, R2, R3 and R4) with the composition of food materials of every kind of ration experiment that can be seen in Table 2. Table 2. The composition of food ingredient in each rations experiment (based on fresh ingredients and dry ingredients.)

Ration Ration material (g) Field grass Concentrate M. pruriens

R0 (control) R1 (raw) R2 (heating) R3 (boiling) R4 (fermentation)

250 250 250 250 250

50 50 50 50 50

0.000 10.75 10.75 10.75 10.75

Note: R0 = diets without the addition of M. pruriens. R1 = rations with addition of 21.5% raw M. pruriens. R2 = rations by the addition of 21.5% heated M. pruriens. R3 = rations with addition of 21.5% boiled M. pruriens. R4 = rations with addition of 21.5% fermented M. pruriens.

Prior to rationing the treatment was given, each rabbit was measured for its maximum feed intake limit per day, then the ration was given by ad libitum twice. First the food was given in the morning at 08.00 in a form of M. pruriens from processed mixed with a concentrate that was mixed evenly mixed with boiling water and stir until soft. At 14.00 fields grass then was given. Drinking water was given once a day (early morning) by ad libitum.

M. pruriens that were given had to be previously cleaned first, and then they were sliced into small pieces and dried in the sun for two to three days by autoclave (heating). Subsequently dried M. pruriens ground to a flour, M. pruriens flour were mixed into the concentrate with homogeny according to the level of the addition. Rabbits were randomly placed in individual cages with 0.6 m long, 0.6 m wide and 0.45 m high, pedestal high was 0.30 m. Each cage was surrounded by concentrate place of 20 cm diameter

3 cm high and made of soil, and drinking water place was 20 cm and 3 cm height made of soil, the cage made of wood, bamboo, wire and woven wire. Scales used for measuring the body weight, slaughter and diet weight 5 kg, with a precision of 1 g, while to measure the weight of meat, bone and fat using an electric scale with a capacity of 400 g with a precision of 0.1 g.

Other tools used were places for food and drink, plastic buckets, plastic bags, plastic gloves, meat cleaver, scissors, cameras, plastic ropes, plastic tray, raffia rope, grinder, thermometer to measure room temperature (° C), and hygrometer to measure humidity room (%). The efforts to prevent disease is done through cage sanitation and some equipments, parasitic worm medicine Albenol-100 at a dose of 0.005% was also used for rabbit with body weight of 450-1270 g (2.25 to 6.35 mL) given orally at the time of preliminary research.

Design of research The experiment was conducted by using experiment

methods with randomized block design (RBD). Treatment that were given were five kinds of diets, namely processed and raw M. pruriens, so there were 5 kinds of rations treated respectively as follows:

R0 = control diet, containing 0% M. pruriens. R1 = rations containing 21.5% raw M. pruriens. R2 = rations containing 21.5% heated M. pruriens. R3 = rations containing 21.5% boiled M. pruriens. R4 = rations containing 21.5% fermented M. pruriens. This study used white male New Zealand wean off as

many as 25 heads of about 6 weeks old, body weight ranging from 450-1270 g. Those rabbits were grouped into five blocks of replications based on body weight, so that each group consisted of 5 rabbits. Rabbit body weight in group I ranged from 450-600 g, 601-750 g in group II, group III 751-900 g, 901-1050 g group IV, group V 1051-1270 g. The randomization results in the layout of the experiment as follows:

Table 1. Nutrient of the feed (based on dry ingredients).

Nutritional value Field grass Concentrate M. pruriens

Protein (%) 1)

14,10 - - -

14,07 - - -

28,94 (raw) 26,84 (heating) 26,89 (boiling) 32,42 (fermentation)

Fibers (%) 1) Lipids (%) 1) Ashes (%) 1) BETN (%) 1) Gross energy (kkaL/kg)1)

31,65 2,17 12,12 39,96 3741,00

11,98 1,12 19,66 53,17 1302,00

- - - - -

HCN (mg/kg) 2) - - - -

- - - -

42,5 (raw) 39,5 (heating) 24,4 (boiling) 0,93 (fermentation)

Note: 1) The result from Quality Test Laboratory PPPPTK Cianjur Farming Board 2008. 2) The result from Livestock Nutrient and Chemical Food, Pajajaran University 2006.

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Table 3. The layout of the experiment Block/repetition Treatment I II III IV V

R0 R3 R1 R4 R2

R4 R1 R4 R2 R1

R2 R4 R3 R0 R4

R1 R0 R2 R1 R3

R3 R2 R0 R3 R0

Note: I, II, III, IV, V = repetition block; R0, R1, R2, R3, R4 = Blocks per replicate R4

Data analysis Statistical model experiments are as follows: Yij = μ + αj + ri + Єij Yij = observed response of the experiment that

measured in to-i, j-th repetition i = 1, 2, 3, 4, 5 (treatment) j = 1, 2, 3, 4, (replications) μ = mean of population ri = effect of i-th treatment αj = effect of j-th group Єij = effect of experimental error/effect error

component in the i-th treatment, j-th repetition Assumptions: Єij value spread normally and independent of each other Єij expected value = 0 or E (Єij) = 0 Variety of Єij = δ2 So Єij ~ NID (0, δ2) Effect of treatment is permanent. The hypothesis was tested: H0: R0 = R1 = R2 = R3 = R4 H1: There are at least a pair of treatment (R1) which is

not the same The data gained then were analyzed by method of

variance and multiple range test followed by DMRT. Data were analyzed with ANOVA according to the experimental design (Table 4). Antarper lakuan Differences tested with Duncan multiple range test (Steel and Tori 1991).

Table 4. List of variance analysis

Source diversity Db JK KT Fhit F.0,05Group Treatment Error Total

t-1 = 4 t-1 = 4 t (r-1) = 6 rt-1 =24

JKK JKP JKG

KTB KTP

KTB/KTGKTP/KTG

Note: If F ≤ F hit 0.05, then thank HO (ns), meaning that the treatment had no significant effect. When F ≥ F hit 0.05, then thank HI (s), meaning that the treatment significantly.


Body weight The mean slaughter weight gain New Zealand White

male rabbits for each treatment during the study are presented in (Table 4). The highest mean weight gain is in the treatment of R4 which is 316 g/head, while the lowest average weight gain the lowest in the treatment of R1, that of 164 g/head.

The result of the calculation of variance showed overall weight gain New Zealand White male rabbits was significantly affected (P <0.05) by treatment. Furthermore, to know the difference between the treatment of the addition of body weight/piece of New Zealand White male rabbits that were fed with processed M. pruriens performed using the Duncan Multiple Range Test results that can be seen in Table 5.

Table 5. DMRT results of treatment effect on body weight gain, carcass weight, meat weight and bone weight in New Zealand White male rabbits (g/head).

Treatment Mean weight

Body (g) Carcas (g) Meat (g) Bone (g) R0 (control) R1 (raw) R2 (heating) R3 (boiling) R4 (fermentation)

238 ab 164 a 288 b 286 b 316 c

326.62 b 294.77 a 341.14 bc 341.15 bc 350.60 c

178.78 b 158.18 a 185.23 bc 185.50 bc 194.87 c

147.83 a 136.60 a 155.90 ab 155.65 ab 155.73 ab

Note: number followed by different letters in same column significantly different at level 0.05.

The mean weight gain of R1 is the lowest (P <0.05)

when compared with weight gain of the other treatments (Table 5). Lower body weight gain by rations in the presence of treatment R1 is because there is toxic cyanide that becomes a disturbing factor on both raw M. pruriens for human and livestock consumption(Purwo 1974). Based on the results, raw M. pruriens seeds that was used as animal feed have negative effect on the growth of livestock which can reduce feed intake, weight gain, and feed conversion (Enemalom et al. 2004). This condition is a result of cyanide compounds contained in raw M. pruriens (Carmen et al. 1999). According to Widodo (2005) the presence of cyanide in the feed caused the negative effect of losing the use of proteins, especially amino acids containing sulfur, such as: methionin, cysteine, cystine, vitamin B-12, minerals iron, copper, iodine, and the production of thyroxin. HCN compounds used in the diet causes the amino acids that contain sulfur compounds to neutralize the HCN. It causes reduction of the amino acids mainly methionin that is available to form muscle in growth period. If the HCN levels consumed too much, it will cause difficulty for cells in the body to breathe because of the disruption of cytocrom oxidase enzyme which usually ends with the death of the livestock. According to the results of this study that the diet containing high HCN causes a low growth. According to Bahri and Tarmuji (1990) cyanide enters the animal's body through breathing,

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skin, and at mostly through the digestive tract. So tolerance of cattle against cyanide depends on the ability of cattle in detoxification. These capabilities can be seen from the level of rhodanase enzyme in its liver. R0 treatment mean body weight/cut 238 g/head with ration treatment without M. pruriens contain no significant differences due to growth in one animal is influenced by the nutritional adequacy, body size, and amount of rations consumed. In the R$ treatment the fermented M. pruriens is most favored over the treatment diet compared to other M. pruriens.

Treatment with fermentation is the best way because there is no effect on body weight reduction. Allegedly R4 treatment rations shows there a big loss of HCN due to the fermentation process is always preceded by boiling and/or steaming. Treatment R2 mean body weight/pieces of 288 g/head and R3 mean body weight/pieces of 286 g/head have no difference in its effect because M. pruriens diet containing the results of heating and boiling HCN levels are still high because only soluble in during heating and boiling, and evaporate during drying. In the R2 treatment andR3 treatment the food are not so favored, and this cause nutrition deficiency resulting in low weight gain in cattle.

Carcass weights Rao et al. (1978) states that what is meant by rabbit

carcass is part of the animal's body without the blood, head, skin, feet, tail, digestive tract and its contents and the content of the chest cavity, except kidney. Average carcass weight of New Zealand White male rabbits for each treatment during the study are presented in (Table 5). The highest mean carcass weight is in treatment of R4 which is 350.60 g/rabbit, while the lowest average carcass weight is in treatment of R1, that is 294.77 g /rabbit. The result of the calculation of variance shows overall carcass weight of New Zealand White male rabbit was significantly affected (P <0.05) by treatment. Furthermore, to know the difference between the treatment of additional carcass weight of New Zealand White males who were given diets containing M. pruriens processing results performed using Duncan's Multiple Range Test of the results can be seen in Table 5.

Average carcass weight real R1 is the lowest (P <0.05) when compared with other treatments carcass weight (Table 5). Average carcass weights resulting from this research are lower (27.40%) than that recommended by Templeton (1968) that the percentage of young rabbit carcass (fryer) as much as 50-59%. The difference is possible because in addition to the nation and the environment it is also caused by rabbit breeding patterns that are generally much different.

Different carcass weight was not in line with the slaughter weight. It is presumed as a result of differences in the proportion of muscle in the meat. A good carcass composition has a high proportion of meat, low bone, and optimum fat (Berg and Butterfield 1976). The highest average carcass weight is in treatment R4 and the lowest is in R1 treatment. This is due to HCN in the diet was consumed not only affected the growth of meat but the

effect on overall growth. Usually a percentage and carcass weight are more influenced by genetic trait.

According to Forrest et al. (1975) raising livestock across nation diversity has effect on the speed of growth and body composition. He adds that if the slaughter id high, it will produce a higher carcass weight. Shafie et al. (1961) declared that young male rabbit carcass weight higher than that of female, female rabbit carcass weight then becomes higher as time goes on because more fatty carcass. Templeton (1968) and De Blass et al. (1977) added that a rabbit carcass is affected by nation, gender, age, thickness of the skin, gastrointestinal tract, fatty, quality, and quantity of ration consumed.

Meat weight The mean body weight of New Zealand White rabbit

males for each treatment during the study are presented in (Table 5). The highest mean weight gain was found on treatment R4 namely 194.87 g rabbit, while the lowest average body weight gain in treatment R1 namely 158.18 g /rabbit. The results of calculation of variance show in overall body weight gain in New Zealand White rabbit males of each rabbit was significantly affected (P <0.05) by treatment. .Furthermore, to know the difference between the treatment of meat weight encroachment New Zealand White males who were given diets containing M. pruriens from processed using Duncan multiple range test whose results can be seen in Table 5. The mean weight gain of R1 is the lowest (P <0.05) when compared with other treatments meat weight. The highest mean weight (Table 5) produced by the rabbit who ate R4 treatment (194.87 g), and then followed by R3 (185.50 g), R2 (185.23 g), R0 (178.78 g), and R1 (158.18 g). It appears that rabbits treated with R4 produce meat weight which is significantly higher (P <0.05) than rabbits treated rations consumed R0, R1, R2, and R3.

On the other hand, the weight of meat produced by the rabbit who ate rations R0, R1, R2, and R3 showed no significant differences (P> 0.05). The high weight of rabbit that consume ration treatment with level of M. pruriens (R4 = 21.5%/10.75 g/head/hr) was in line with the carcass weight. This is in accordance with the opinion by Dwiyanto et al. (1984) that with increasing carcass weight, the percentage of muscle meat tends to rise. These results provide evidence that fermented M. pruriens in a relatively small amount of 10.75 g per cow per day is thought to be able to increase the metabolism and absorption of protein. Proteins are absorbed and subsequently deposited in the form of meat.

Bone weight The main function of bone is as the framework

supporting the soft tissues of the body. Bone tissue was formed in the phase before birth (prenatal) and after birth (postnatal) with changes in the connecting tissue (Forrest et al. 1975). Sandford (1979) argues that each tissue has a growth rate that is different. The organs that develop earlier are the brain, liver, lungs, gastrointestinal tract, and bone. The development will be followed by the development of bone tissue and the last is the development of fat.

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The mean weights of bone in New Zealand White male rabbits for each treatment during the study are presented in (Table 5). The highest mean bone weight gained is in treatment R2 namely 155.90 g /rabbit, followed by treatment R4 (155.73 g /rabbit), R3 (155.65 g /rabbit) R0 (147.83 g /rabbit), while the lowest average weight gain is in treatment R1 that is 136.60 g /rabbit. The result of the calculation of variance shows that the overall weight of bone was not significantly affected (P> 0.05) by treatment. Furthermore, to know the difference of the treatment of bone weight given diets containing processed M. pruriens results performed using the Duncan Multiple Range Test results we can see Table 5.

Table 5 shows that the average bone weight of each treatment (R0, R1, R2, R3, R4) was not significantly different. From the analysis it was known that the treatment had no effect (P> 0.05) against the weight of the bone. Similarly, the results obtained after the analysis of Duncan. This means that the diversity of the weight of the bone caused by the treatment means nothing. According to Forrest et al. (1975), bone is the body component formed in the phase before birth (prenatal) and after birth (postnatal), and is developed earlier than tissue and fat (Sandford 1979). In addition, carcass bone weight negatively correlated with carcass weight, because the increase of carcass weight means the increase in fat and meat but the percentage of bone tends to decrease (Forrest et al. 1975).By considering the diversity of a relatively small weight of bone, it is presumed that it is an indication showing that the development of rabbit bone was near optimum. Thus, the provision of treatment produces no significant effect.

CONCLUSION

The provision of M. pruriens in the rabbit food increased the body weight and the carcass weight of New Zealand White male rabbits. This is supported by the results as follows: (i) Rabbit R1 treatments that use the raw M. pruriens flour in the ration were not able to neutralize the HCN content. (ii) The M. pruriens fermentation process results in a ration of 21.5% (10.75 g) as a whole is able to increase production of body weight/carcass weight of white New Zealand White male rabbits.

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protein by the fungus Rhizopus sp and its influence on the growth of broilers. [Dissertation]. Padjadjaran University. Bandung. [Indonesia]

Bahri S, Tarmuji. 1990. Cyanide poisoning in livestock and how to overcome them. Research Center for Animal Diseases. Bogor. [Indonesia]

Carmen DJ, Gernat AG, Myhrman R, Carew LB. 1999. Evaluation of raw and heated velvet beans (Mucuna pruriens) as feed ingredient for broilers. Poult Sci 78: 866-872.

De Blass YC, Tornes A, Fraga MJ, Perez E, Galves JF. 1977. Influence of weight and age on the body composition of young soe rabbit. J Animal Sci 45 (1): PP. 48-53.

Dwiyanto K, Sitorus P, Moerfiah. 1984. The role of rabbit livestock in supporting the provision of protein. Research and Development Center for Livestock. Ciawi-Bogor. [Indonesia]

Emenalom OO, Udedibie ABI, Esonu BO, Etuk EB, Emenike HI. 2004. Evaluation of unprocessed and cracked, soaked and cooked velvet beans (Mucuna pruriens) as feed ingredients for pigs. Livestock Res Rural Dev 16 (5): 33. www.lrrd.org/lrrd16/5/enem16033.htm

Farrell DJ, Raharjo YC. 1984. Potential rabbits livestock as producers of meat. Research and Development Center for Livestock. Ciawi-Bogor. [Indonesia]

Forrest YC, Aberle ED, Hendrick HB, Judge MD, Markel RA. 1975. Principle of meat science. WH Freeman. San Francisco.

Handajani S. 2001. Indigenous Mucuna tempe as functional food. Asia Pasific J Clin Nutr 10 (3): 222-225.

Friday KS, Drilling ME and Garrity DP. 1999. Imperata grassland rehabilitation using agroforestry and assisted natural regeneration. ICRAF-S.E. Asia Program. Bogor.

Lubis DA. 1972. Fodder science. PT. Pembangunan. Jakarta. [Indonesia] Purwo A. 1974. Identification and elimination of toxic compounds in

Mucuna pruriens dc and study of Mucuna pruriens dc seeds as a source of protein. [Research Report]. Bandung Instutute of Technology. Bandung. [Indonesia]

Rao DR, Sunki GR, Johnson WH, Chen CP. 1978. Effect of weaning and slaughter age on rabbit meat productions II. carcass, quality and composition. J Animal Sci 5: 578-582.

Rismunandar. 1981. Breeding rabbits. Penerbit Masa Baru. Jakarta. [Indonesia]

Sandford JC. 1979. The domestic rabbit. 2nd ed. Granada. London. Sarwono B. 1991. Superior rabbit breeding. Penebar Swadaya. Jakarta.

[Indonesia]. Shafie MM, Badreldin AL, Ghany MA, Hanafi M. 1961. Differential

growth and carcass characteristic in the Giza rabbits. Egyptian J Anim Prod 1: 135-148.

Siddhuraju P, Vijayakumari K, Janardhanan K. 1996. Chemical composition and protein quality of the little known legume velvet bean (Mucuna pruriens). J Agric Food Chem 44 (9): 2636-2641.

Sitorus PS, Partadihardjo, Raharjo YC, Putu IG, Santoso, Sudaryanto B, Nurhadi A. 1982. Report on rabbit farm in Java. Research and Development Center for Livestock. Ciawi-Bogor. [Indonesia]

Templeton GS. 1968. Meet domestic rabbit production. 4th ed. Interstate Printer and Publishers. Danville-IL.

Verhoef-Verhallen E. 1998. Encyclopaedia of rabbits and rodents. Rebo Productions. Lisse.

Widodo W. 2005. Poisonous plants in the life of cattle. UMM Press. Malang. [Indonesia]


Alternative supplementary biochemic food for growing up the fresh water lobster (Cherax quadricarinatus)

EDI PRIYONO1,♥, OKID PARAMA ASTIRIN², PRABANG SETYONO² ¹ MTs Negeri Sukoharjo. Jl. KH Agus Salim No. 48 Sukoharjo, Central Java, Indonesia

² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Manuscript received: 22 June 2009. Revision accepted: 15 October 2009.

Abstract. Priyono E, Astirin OP, Setyono P. 2009. Alternative supplementary biochemic food for growing up the fresh water lobster (Cherax quadricarinatus). Nusantara Bioscience 1: 123-130. This research denotes to know the influence of biochemic composition to the rapid grow of fresh water lobster on the stadium of post larva (PL) of 60 within three months. This research used the complete random planning dealing with 4 treatments and each treatment would get 3 times cycle. The treatments cover, group K tested animal was treted with 100% mill food containing 30% of protein. Group A is given with food and biochemic food containing 13,34% of protein. Group B is the treated with mill food which is mixed with biochemic food containing 10,7% of protein. While group C was tested by treating them with mill food and biochemic food containing 13,58%. After all the above mentioned would be set up within 3:1 comparation. The variable of this research were the length of the abdomen, cephalothorax, total length, and the wet weight. The data analysis is using ANOVA system on 95% power test completed by of SPSS version 13. The result of the research shows that mentioned treatments give us the same influence toward the growth of fresh water lobster. The composition of biochemic food with the containing protein around 10,7%,13.34% and 13,38% has given the same effect to the lobster growth on post larva 60 level. There is strong correlation between abdomen and cephalothorax and between the total length and the lobsters weight.

Key words: Cherax quadricarinatus, suplementary food, water quality.

Abstrak. Priyono E, Astirin OP, Setyono P. 2009. Alternatif penambahan suplemen hayati untuk meningkatkan pertumbuhan udang lobster air tawar (Cherax quadricarinatus). Nusantara Bioscience 1: 123-130. Penelitian ini bertujuan untuk mengetahui pengaruh pemberian suplemen hayati sehingga dapat meningkatkan pertumbuhan lobster air tawar pada stadia post larva (PL) 60 pada masa pertumbuhan 3 bulan. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL) dengan 4 macam perlakuan, masing-masing dengan 3 kali ulangan. Perlakuan yang diberikan meliputi, kelompok K hewan uji diberikan pakan pabrik 100% dengan kadar protein 30%, kelompok A hewan uji diberikan pakan pabrik ditambah suplemen hayati dengan kadar protein 13,34%, kelompok B hewan uji diberikan pakan pabrik dicampur dengan suplemen hayati kadar protein 10,7%, sedangkan kelompok C hewan uji diberikan pakan pabrik ditambah suplemen hayati dengan kadar protein 13,58% masing-masing dengan perbandingan 3:1. Variabel yang diamati adalah panjang cephalothorax, abdomen, panjang total, dan bobot basah. Analisis data dengan menggunakan ANAVA taraf uji 95% dengan bantuan SPSS versi 13. Hasil penelitian menunjukkan bahwa perlakuan yang diberikan berpengaruh sama terhadap petumbuhan lobster air tawar. Komposisi suplemen hayati dengan kadar protein antara 10,7%, 13,34%, dan 13,58% memberikan pengaruh yang sama terhadap pertumbuhan lobster stadia post larva 60. Terdapat korelasi yang sangat erat antara cephalothorax dengan abdomen, dan antara panjang total dan berat lobster .

Kata kunci: Cherax quadricarinatus, suplemen hayati, kualitas air.

INTRODUCTION

The presence of freshwater crayfish in Indonesia has not been much known to the public, even there are some people who think that lobster species can only be obtained from the catch at sea and can not be cultivated. Freshwater crayfish has long been grown in Queensland, Australia and the United States, while in Indonesia only started in 1991, and still is limited done by some breeders due to the limited number of parent who must be imported from Australia. Freshwater crayfish consists of 500 species from families Astacidae, Cambaride and Paraticidae whose habitat are in lakes, rivers, wetlands and irrigation channels (Sukmajaya and Suharjo 2003). Currently, the dominant species cultivated is Cherax spp., while the most popular species is

Cherax quadricarinatus (Red Claw). C. quacarinatus is a freshwater crayfish from Australia, found in rivers, swamps and lakes in north coast of Australia, and northeastern Queensland. In Indonesia, this population is found in Papua (Bahtiar 2006). This species is much cultivated in Indonesia because it has high resistance to parasites, high adaptability and rapid growth compared with most other lobster species, which can grow to 50 cm with a weight between 300-600 g, meat content of 30% on average abdominal and the claws of 5-10% (Wiyanto and Hartono, 2007; Showalter 2006). Shrimp life consists of several stages including; stage nauplii from 0.32 to 0.58 mm size, stage zoea 1.05 to 3.30 mm, 3.50 to 4.80 mm are respectively Mysis stage and postlarva stage (PL). In this stage it is shaped like an adult shrimp lobster so that the

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body can be distinguished between chephalothorax and abdominal (Haliman and Adijaya 2005).

Freshwater crayfish has similar characteristics with the sea water lobster, but the difference is in the maintaining. Freshwater crayfish also has an export opportunity in various countries (Petasik 2005). Lobster market share is not limited in the country but also found abroad. Export lobster cultivation tends to increase every year. In 1990, lobster exports to the Netherlands, reached 745.132 tons or 89.59% of total exports of lobster in Indonesia (826 tones) In 1995, Indonesian exports reached 182 065 tones of lobster per year, 2% of total exports (3641.3 tones). Total exports of cultivated lobster reached 94,511 tons/year (Alexander 2006a, b).

Development of the freshwater crayfish is not separated from the high market demand, especially export markets either alive or frozen. Basically the main purpose is for the consumption of lobster, but lately begun to be exploited as ornamental lobsters. Cultivation of freshwater crayfish has become the best solution for the current business or for side business, given the current economic condition is uncertain, and the lobster market is always waiting for the supply of fresh water from the farmers every day both local consumption and export. In addition to the conditions that are very supportive, biological food source for the lobster is quite abundant. Some examples are the vegetables that are categorized as biological feed, such as: kale, spinach, bean sprouts, green beans, and cabbage. These types of feed that are categorized as tubers are cassava, sweet potatoes, and white yams. The type of feed that are categorized as meat are snail, snail carp, fish, chicken. With biological feed the lobster can grow rapidly (Bahtiar 2006).

Freshwater crayfish maintenance is easy. It is very easy to obtain their food and the price is cheap. Starting from 1 month ranchers have gotten the sale of seeds. The average age of harvested of 1, 2, 3, 4 and 5 months depending on which program the farmers want. Everything is profitable only difference in profit and the initial capital only. Freshwater lobster eats everything. All the kind of food is eaten by lobsters. Feed is one of the most important parts of cultivation. Availability of food in sufficient quantities needed to support the success in the cultivation of freshwater grows out. Types of artificial feed or feed supplement that farmers use in freshwater aquaculture grows out are usually various kinds. The feed mill is quite expensive, for it is necessary to minimize the use of feed mills so as to reduce the operational cost of cultivation. Hence the need for additional research on the use of feed (biological) which have economic value and are available in the hope for improving the cultivation of freshwater crayfish to its maximum. To lobster, food is a very important, because food occupies 40-50% of the total production cost that must be replaced (Lim 2006).

Some studies on artificial food ingredient uses 4 ingredients in terms of fiber, protein and fat. Bran contains 12.5 g/100 g protein, 4.9 g/100 g fat, and 18.3 g/100 g fiber. Fish meal contains 55 g/100 g protein, 6 g fat, and 2.4 g/100 g fiber. Bean cake contains 37.7 g/100 g protein 11.5 g/100 g fat, and 13.2 g/100 g fiber. Wheat flour contains 12.2 g/100 g protein 1.5 g/100 g fat, 2.7 g/100 g

fiber (Mujiman 2004). According to Priscilla (2007) lobster feed contain 87.79% dry matter, fat 5.72%, 1.81% crude fiber, ash 2.79%, which effects on lobster growth rate.

Biological feed is more easily obtained in sufficient quantities, sustained and more durable. At least for one season of maintenance the nutritional content can be adjusted according to the needs, shapes and sizes that can be tailored to the needs of lobster. Durability in water can be adjusted with lobster eating habits, smell and taste can be set so it will look attractive and desirable. This kind of food is more available and cheaper than the food from manufacturers that are very often and sometimes difficult to obtain.

The purposes of this study are: (i) to know the difference of crayfish growth stages post larvae (PL) 60 plants fed only with feed supplement mixed with various kinds of plant. (ii) To determine the optimum composition of biological supplement to the growth of freshwater post larva stage crayfish (PL) 60.


Material Test animals. This study used test animals from species

of freshwater crayfish Cherax quadricarinatus, PL60, size; long 2 inches (5.08 cm).

Biological food. Big pink earthworms Allobophora caliginosa species are from the local yard (in the vicinity of the bin), while carrots and green bean sprouts purchased at market Sukoharjo. Feed were given taste, about one-third of weight (ad libitum) was given 1 time a day i.e. at 15.00 pm. The food is given in the afternoon because the lobster belongs to nocturnal animals and actively foraging substrate waters at night.

Experimental design The experimental design used was Completely

Randomized Design (CRD) which is an experiment that gives restriction to the allocation of treatment of the material or test area with basic assumptions. The size of the test animals are considered homogeneous, as well as tools, materials, media and environment maintenance. The method is based on experiment conducted research methods to investigate the possibility of a causal relationship with how to use one or more condition treatment to one or more experimental groups (Srigandono 1989). The study was conducted using 4 treatments with repetition 3 times. The most notable treatment is the provision of supplementary food. (i) K: control group with 100% of the feed mill, with 30%

protein content. (ii) A: group A with providing supplemental biological

protein content 13.3%, mixed with the feed mill. (iii) B: group B with biological supplementation 10.7%

protein content mixed with the feed mill. (iv) C: group C with biological supplementation 13.5%

protein content mixed with the feed mill. This treatment is given to know the composition of

biological supplements best to influence the growth of

PRIYONO et al. – Biological supplements on the growth of Cherax quadricarinatus

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lobsters PL 60.

Procedures The procedure includes the preparation of this research

study, the implementation of the core research and measurement of water quality include pH, temperature and dissolved oxygen.

Preparation. Activities of preparatory phase of this study are as follows: (i) Preparation of equipment and materials to be used. (ii) Preparing test animals which 60 PL of lobster seed length of 2 inches (5.08 cm) with the same age. (iii) Preparation of test media that is used to prepare water originating in the bin into a bath of aerated settling maintenance and 24 hours later measured the temperature, pH and Dissolved Oxygen.

Implementation. Tested animals were put in the maintenance container that had been filled with water taps which had been accommodated for 24 hours, chlorine content was decreased and the temperature was set to room temperature, as high as 15 cm and aerated to increase the supply of natural oxygen container cultivation. Lobsters fed for 8 weeks with record growth (cephalothorax length, abdominal length, total length and weight of crayfish) were performed every 2 weeks, which it is expected that within 2 weeks the lobsters have reached measurable growth. The daily activities were carried out according to treatment each feeding at 15.00 pm. Penyifonan done as much as 30% of the volume of water every day and replaced with water from reservoir. The weekly events container cleaning and maintenance was done as well as measuring its growth every 2 weeks. Eligibility and allocation of water quality referred to PP. 82 year 2001 concerning the management of water quality and water pollution control.

Observation / data collection. The data observed were growth cephalothorax length, abdominal length, total length and weight of lobster. Growth cephalothorax average length, abdominal length and total length was measured using the formula length growth Effendi (1997):

L = Lt-Lo L = length of individual average growth (cm) Lo = The mean length of individual baseline (cm) Lt = The mean length of individual end of the study (cm)

Calculation of average individual weight was calculated

using the formula Stickney (1979): W = Wt-Wo W = average weight of biomass growth (g) Wo = average weight of test animals at the beginning of

the study (g) Wt = Average weight of test animals at the end of the

study (g)

Data analysis Analysis of data is with data normality test which is

used to find out that the data obtained from a normal distribution, then the next stage is to test the homogeneity.

The objective is to find out that the data will be tested come from a homogeneous population. After the data otherwise acting normal and homogeneous test next stage of analysis of variance (ANOVA) followed by Duncan multiple range test (DMRT) at 5% significance level to find out the real differences among the treatments.


Maintenance of environmental factors Media environment maintenance is very influential on

the test animals. Water quality plays a role for lobster, remembering that organisms have a certain tolerance limits to environmental factors in which the organisms are located (Ward 1987; Handy 1992). Water quality effects on lobster growth. Water quality parameters that must be controlled during the process of research are the degree of acidity (pH), Dissolved oxygen (DO) and temperature. The measurement results of water quality during the maintenance process are shown in Table 1. This table shows that there is no significant difference between the time of observation (January, February and March). It is expected that with this evidence, then the things that affect the growth of the lobster is really from the treatment given. These water quality parameters included in Class I under Government Regulations (Peraturan Pemerintah) No. 82/2001 (Table 2), whose allocation can be used for drinking water, and others that require the designation of water quality in common with the use of these. Table 1. Water quality data on maintenance period.

Parameter UnitsObservation time

January February March Average ± SD Average ± SD Average ± SD

pH - 7,579 ± 0,083 7,410 ± 0,051 7,060 ± 0,379Suhu (t) 0C 25,425 ± 0,154 25,292 ± 0,211 25,342 ± 0,090 DO mg/L 8,089 ± 0,068 8,124 ± 0,062 8,021 ± 0,168

Table 2. Quality parameters under Government Regulations (Peraturan Pemerintah) No. 82/2001

Parameter Units Class I II III IV

Physics Temperature 0C - - - - Organic chemistry pH *) 6-9 6-9 6-9 5-9 DO **) mg/L 6 4 3 0 Note: * pH (acidity), when the naturally outside that range will be determined based on the state of nature. ** DO (dissolved oxygen), a minimum number

The degree of acidity (pH) Average pH value for maintenance is stable, the

average ranged from 7.06 (March 2008) to 7.57 (January 2008) the situation is quite qualified in the maintenance of C. quadricarinatus. Optimum pH between 7.5 to 8.5 in the maintenance of lobster (Alfrianto 1990), 7.2 to 8.5 (Bahtiar

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2006), 7-8 (Wiyanto and Hartono 2007). The observation of pH during maintenance can be seen in Figure 1, where the pH did not change significantly. Rapid change in pH will result in lobsters becoming stressed, the condition of the body will be weak, it can even result in death. The pH 4-5 is the deadly levels of acidity, no reproduction, whether for reproductive the pH is 7-9, while pH 11 is deadly levels of alkalinity (Cholik et al. 1986). PH value becomes the parameter directly related to CO2 and ammonia. Changes in pH occur if there is accumulation of CO2 respiration, so the pH will be low (Ghufron 1997). The higher the density of lobsters, the higher the concentration of CO2 in water will be, but lobsters can still tolerate the levels of CO2. Possible increased levels of CO2 in the water can be reduced by the replacement of water by 30% of overall volume. The existence of other toxic gases that allow the disruption of lobster growth is ammonia gas. Ideally the ammonia content is less than 0.01 ppm (Boyd and Lithkopper 1982).

Based on statistical analysis as listed in Table 1, it is known that the pH did not significantly affect lobsters growth because during the measurement the pH value is tolerable for lobster.

Dissolved oxygen (DO) DO levels greatly affect the metabolism of lobster,

respiration always needs oxygen necessary to the survival of lobster. Another thing to note is the accumulation of food residue and dirt from the lobster that will also reduce oxygen levels, because in order to digest the food scraps and manure needs oxygen. This results in decreased levels of dissolved oxygen in maintenance media. Oxygen dissolved in water is needed for respiration of the lobsters ranged from 4-8 mg/L, if the oxygen requirement is fulfilled, it will be better for lobster growth, so that its activities would be good also because the remaining feed and feces are decomposed by microbes.

Average value of DO during maintenance shows digit range between 8.02 to 8.12 mg / L. This indicates that the

value is stable (does not show a high fluctuation). DO optimal for maintenance of lobsters is between 4-8 mg / L (Alfrianto and Eviliawaty 1990), at least 3 mg / L (Bahtiar 2006), 3-7 mg / L (Singh 2003). DO observations during maintenance can be seen in Figure 1.

From the statistical tests as listed in Table 2, between DO with lobster growth there was a low correlation proved the value of "r" (0.2 ≤ r ≤ 0.4) that during the maintenance in dissolved oxygen it did not affect the growth of lobsters. Results of measurements carried out showed that DO in the range of 8.12 mg/L, so the situation was quite ideal for the life of lobster and did not affect its life.

Most organisms use oxygen in process of decay. This process can take place due to the activity of bacteria that describes organic materials such as food remains and lobsters feces (Alfrianto 2006). Problems of this kind if not treated quickly will occur resulting in decrease of accumulation of dissolved oxygen (DO).

Temperature Water temperature is an environmental factor affecting

the rate of metabolism. Changes in water temperature will be followed by changes in body temperature, so if there is a decrease in environmental temperature there will lower the lobster's body temperature that will decrease the rate of metabolism. The situation is even worse if the lower ambient temperature conditions can cause lobster to die.

If the ambient temperature increases, the lobster temperature will increase as well, so the metabolic rate also increases. If the increase in temperature continues to rise it will reach critical temperature where lobster will experience death from hypoxia (Afrianto and Eviliawaty 2005). An increase in water temperature, generally will result in increasing the biological activity and will result in increased need for oxygen in waters, in other words the increase in temperature water will decrease the level of solubility of oxygen, and it will reduce the ability of aquatic organisms in utilizing the available oxygen for the survival of biological processes in water (Asdak 2004)

7,5797,41

7,06y = -0,2595x + 7,8687

R2 = 0,961

6,66,8

77,27,47,67,8

Jan Peb Mar

pH

8,0898,124

8,021

y = -0,034x + 8,146R2 = 0,4214

7,95

8

8,05

8,1

8,15

Jan Peb Mar

DO

(ppm

)

25,425

25,292

25,342

y = -0,0415x + 25,436R2 = 0,3816

25,2

25,25

25,3

25,35

25,4

25,45

Jan Peb Mar

Suhu

(o C)

A B C Figure 1. Relations with the observation time: A. pH, B. DO, C. temperature. Note: pH: y shows regression equations, determinant coefficient R2 expressed in the figure showing the large R2: 0.961 means that if the pH factor for maintenance can contribute to the influence of 96.1%. This happens because during the measurement between the months of January and February the average pH did not show a high deviation (0.083 and 0.051), but in March the pH has decreased resulting in a higher deviation (0.379) so that this will result in R2 will be higher. Similarly, although the decline was still within the optimum value. DO: y indicates the regression equation, the determinant coefficient R2 states shown in the figure of R2: 0.4214 means for maintenance and DO factors can contribute only at 42.14%. This is supported by the data for DO measurement that showed no difference in the high deviation that would result in R2 which did not show high numbers. Temperature: y indicates the regression equation, the determinant coefficient R2 expressed in the figure showing the large R2: 0.3816 mean temperature factor contributing effects only as much as 38.16%. During temperature measurements it showed no difference in the height deviation so that R2 did not show high numbers.


127

Value of average temperature during maintenance ranged from 25.29 to 25.490 C, and this is a stable condition that it is qualified enough for the maintenance of lobster as proposed by Alexander (2006a, b) that the requirements temperature in the maintenance of lobster is 24-260C with a maximum fluctuation day and night 2-30C. Observations of water temperature during maintenance was shown in Figure 1. The analysis of statistical tests is listed in Table 2. Then during the maintenance, temperature was not correlated with growth, this proved the value of "r" (r ≤ 0.2). Temperature does not affect the growth of lobsters. According to Iskandar (2006a, b) optimum temperature for lobsters maintenance is 24-260 C, in accordance with temperature measurements made are at the value of tolerance so that this situation does not affect the life of lobsters.

The three elements of water quality (temperature, pH and DO) does not affect the life of lobsters because the values of the three factors are at tolerant level for lobster life, if the element of water quality is outside the tolerance it will affect the lives of lobsters.

The correlation between the length of lobsters and the water quality

Results of correlation test to determine the relationship between the growth of water-quality lobster (DO, temperature, and pH) are shown in Table 3. Based on statistical analysis as shown in Table 3, the acquisition of test results of correlation between the quality of water and the growth of lobsters are as follows: between the length of lobster with DO showed a low correlation. It can be proven all values of "r" is 0.2 ≤ r ≤ 0.4, whereas temperature and pH on the growth of the lobster is very low or not correlated, as shown by the value of "r" is r ≤ 0.2. On the whole it can be stated that the fluctuation changes in the value of DO, temperature and pH did not affect the length of lobsters. Table 3. The value of correlation between length and weight of lobster with water quality

Parameter Value r (correlation) DO Temperature pH

Length of cephalothorax (cm)

0.226 -0.033 -0.275

Length of abdomen (cm) 0.239 -0.102 -0.101 Length of total (cm) 0.244 -0.069 -0.203 Weight (g) 0.109 0.016 -0.095

Growth of lobster Lobsters cephalothorax length

To determine that the treatment given could provide a different and real effect not then ANOVA was performed. The ANOVA results can be seen in Table 5. The observation of the control group showed a mean length in initial cephalothorax maintenance is 3.075 cm, and at the end of the maintenance is 3.794 cm. The difference is 0.715 cm. For treatment group A (13.34% protein), the mean length of 3.018 cm cephalothorax initial maintenance, maintenance end 0.887 cm 3.905 cm

difference , for group B (10.7% protein), the mean length of 3.020 cm cephalothorax initial maintenance, maintenance end 3.738 cm, 0.718 cm difference, for group C (13.58% protein), the mean length of initial maintenance cephalothorax 2.909 cm 3.827 cm end maintenance, difference 0.918 cm. The mean total length cephalothorax comparisons are shown in Table 6, where the fourth-highest treatment group difference in length cephalothorax is the group C with the tabulated results from the difference between 0.918 cm. However, this difference has not shown significant differences (P <0.05) when compared with other groups. Table 5. Analysis of the results of ANOVA test toward lobster length and weight.

Parameter Average Values significance Description

Length of cephalothorax(cm)

3.005 0,252 -

Length of abdomen (cm) 2.512 0,503 - Length of total (cm) 5.524 0,335 - Weight (g) 5.822 0,361 - Note: Count with significance level p <0.05, which was not significantly different between treatments. Table 6. The mean ratio of cephalothorax total length lobster during the breeding period.

Group Initial length ±SD

Final length ±SD Accuracy Growth

length Control 3.075 ± 0.061 3.794 ± 0.037 85.197% 0.719 A 3.018 ± 0.046 3.905 ± 0.045 81.881% 0.887 B 3.020 ± 0.084 3.738 ± 0.141 84.975% 0.718 C 3.062 ± 0.111 3.748 ± 0.089 85.754% 0.686 Note: the highest abdominal growth length is A group

Growth is assumed as the network that meant the

addition of structural proteins in the body tissues as proposed by Buwono (2004). Of course, the biological supplements with low protein content will produce poor growth (group B), but otherwise if biological supplement with optimal levels of protein will produce more optimal growth as well. From the tabulated results from the long accretion cephalothorax the highest are in group C who were given supplements as much as 13.5% of biological protein. Supplement composition of group C was: 50% green bean sprouts, 30% carrot, and 14% earthworms. From the different tests carried out then, longer cephalothorax in each treatment group were not significantly different (p <0.05). This condition indicates that the feed mill (control group) was not significantly different from biological additional supplements. There is potential to reduce pure composition of feed mill with an additional biological supplement.

The length of lobster abdominal The calculation of growth of cephalothorax abdominal

steps is the entire length of the tested animals that were measured starting from the end karapace abdomen up to the

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sixth vertebra using shove (calipers) with a precision of 0.02 cm. The results are as follows: Control group mean abdominal length is 2.562 cm at the beginning of the breeding, and at the end of the breeding is 3.200 cm. The difference in average length is 0.638 cm. Group A (protein 13.34%), average length in initial breeding is 2.514 cm, and 3.32 cm at the end of breeding. The difference in average length is 0.806 cm. Group B (protein 10.7%) the mean length is 2.528 cm abdominal at the beginning of breeding, and at the end of breeding is of 3.238cm. The difference in mean length 0.71 cm. While group C (protein13, 58%), the mean length of the abdomen at the beginning of the breeding is 2.471 cm, and 3.261 cm at the end of breeding. The difference in average length is 0.79 cm. The mean length of the abdomen of lobster during the breeding period are shown in Table 7.

Table 7. Comparison of mean abdomen length of lobster during breeding



length Control 2.562 ± 0.013 3.200 ± 0.028 84.341% 0.638

A 2.514 ± 0.073 3.320 ± 0.072 80.462% 0.806 B 2.528 ± 0.079 3.238 ± 0.132 82.586% 0.710 C 2.471 ± 0.106 3.261 ± 0.064 80.509% 0.790

Note: the highest abdominal growth length is A group.

From the four groups, the highest treatment group

difference is the biological treatment using the supplement with protein content of 13:34% (A) with the numbers 0.806 cm difference. This can happen because the protein content in food is essential and should be available for the lobster. The optimal protein content will result in maximum growth of lobsters (Mudjiman 2004). Proteins in food are used for long-bone growth, including growth in the abdomen. The addition of biological supplements with high levels as much as 13:34% (A) proved to have been able to increase growth in lobster abdominal length parameters as proposed by Balazs and Ross (1976). Artificial diets with protein content of 25-35% proved to have been able to increase the growth rate of lobsters. Greatest length progress is the group A with additional supplements of protein content of 13.34%. From the statistical tests that were conducted, the power difference, the length of the abdomen of each treatment group were not significantly different (P <0.05). This condition indicates that the feed mill (control group) was not significantly different to supplement existing biological potential to reduce the pure composition of feed mill with an additional supplement biological.

Lobsters total length From the data tabulated in Table 8 above, it can explain

the growth of the total length of lobster during the breeding. In the control group which used 100% feed mill the total length in the beginning of the breeding is 5.637 cm, and at the end of the breeding it is 6.994 cm. The difference is 1.35 cm. In group A (protein 13.34%), the length average is 5.533 cm in the beginning of the breeding, and at the end of the breeding is 7.226 cm. The

difference is 1.693cm. For group B (10.7% protein) the length is 5.548 cm at the beginning of the breeding, and at the breeding end it is 6.977cm and therefore the difference is 1.429cm. For group C (13.5% protein), the average length at the beginning of the breeding is 5.380 cm, and at the breeding end its is 7.088 cm, and it has 1.708 cm difference. The mean total length of the lobster are shown in Table 8. For the entire length of the lobster we found that protein supplement that is best for the growth of lobsters is the protein levels of 13:58% (C) with the excess length of 1.708 cm. It is because the protein contained in feed is still in the optimum level (between 25-35%). According to Balazs and Ross (1976), and Buwono (2004), rapid growth is determined whether the protein is absorbed. In general, lobsters need food that between 20-60% protein content, while the optimum level is between 30-36%. When the protein content in feed is less than 6% (wet weight), then lobsters will not go grow well (Mudjiman 2006). Protein is one of the main factors affecting lobster growth. When we look at the data on the levels of protein with content of 13:58%, it means proteins that is absorbed by the lobster is also the most high-absorbed, and protein is used for the formation of body parts in addition to activities. The additional supplements with high levels of feed composition of 13:58% in group C is composed of 50% mung bean sprouts, 30% carrot and 14% earthworms, the highest proven growth difference, if compared with additional supplements with a lower protein content. Table 8. The mean ratio of total length of lobster during the breeding.

Group Initial length

±SD Final length

±SD Accuracy Growth length

Kontrol 5.637± 0.053 6.994± 0.043 84.007% 1.357 A 5.533± 0.110 7.226± 0.109 81.235% 1.693 B 5.548± 0.155 6.977± 0.275 83.865% 1.429 C 5.380± 0.216 7.088± 0.148 80.627% 1.708

Note: the highest growth of total length is C group. From the analysis test that was done, the total length of

each group was not significantly different (P <0.05). This condition indicates that the feed mill group (K) was not significantly different from biological supplements. There is potential to reduce the pure composition of feed mill with biological supplements.

Lobster weight Results tabulated in Table 9 on lobster weight shows

that the average lobster weight control treatment at the beginning of the breeding is 5.905g, and it became 11.793g at the end of the breeding, it means that the difference is 5.89 g. For group A (protein 13.34%) the weight is 5.679 g at the beginning of the breeding, and at the end of the breeding it was 13.2g, it means that the difference is 7.56 g. Group B (10.7%), average initial breeding weight is 5.850 g, average final breeding weight is 12.513g, thus the difference is 6.613 g. Group C (13.58% protein), the weight is 5.859g at initial breeding, and the weight at the end of the breeding is 12.615 g, as much as 6.756 g different.


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Food major function is to provide energy for the activity of body cells. Proteins are part of the nutrients in food working for growth and energy sources, and another function of protein is in regulating metabolism. The energy generated in the metabolism are needed by the body in the formation of components such as muscle tissue which affects the weight of lobsters. The quality of protein in food lobster not only defined by the content in food sources alone but is determined also by the balance of amino acids they contain (Murtidjo 2007). Lobsters require proteins as much as 20-40% (Lim 2006). Said lower feed quality when essential amino acid levels are also low in protein, essential amino acid balance will determine the quality of feed (Buwono 2004).

Supplements type A (protein13, 34%) proved capable of producing excess growth which is highest when compared with type B and C. Supplements produce the highest weight difference supplement with protein content of 13.34%. Protein required Lobster is a balanced protein is not the most phones, even low feed levels will show its protein a higher weight when compared with a supplement containing a higher protein (Afrianto Eviliawaty 2008). Supplements types B and C have a lower weight difference because supplements containing protein that is less obvious weight will inhibit the growth of lobsters, otherwise if it is high protein supplements will result in lobster become lazy and consequently difficult molting and weight growth is inhibited. Mean lobster weight ratio shown in Table 9. Table 9. The mean weight ratio of lobster during the breeding.



length Control 5.905± 0.368 11.793±0.327 52.910% 5.888

A 5.679± 0.090 13.242±0.690 43.472% 7.563 B 5.859± 0.501 12.615±1.795 48.282% 6.756 C 5.850± 0.322 12.513±0.768 48.685% 6.663

Note: the highest weight lobster is the group A

In the calculation on accuracy for the control group, A,

B, and C we can see the low accuracy rate for the calculation of data which was taken preliminary and final only, so that statistically it will cause high deviation, and in turn the high deviation will cause a low accuracy rate.

From the above tabulation, it can be concluded for the weight parameter, then the excess weight that most of the lobster group treated with the protein content of biological supplements is 13:34% (group A), with the result of excess weight 7.561 g. This can be explained that the proteins is organic substance which contains levels of hydrogen, nitrogen, sulfur and phosphorus. Substance is the main food that contain nitrogen. Protein is essential for life of lobster because it is an active protoplasm in all living cells, not only the protoplasm of living cells are composed of protein but also the nuclei that control the cell activity. Protein is the largest part of muscle meat, organs and bones (Murtidjo 2007).

From the tabulation, the highest weight is group A with supplementation of biological protein content 13.34%. From the analysis conducted, the test of lobsters weight did

not show significantly different results (P <0.05). This condition indicates that the feed mill group (K), not significantly different from biological supplements, there is potential to reduce the composition of feed mill with a supplement purely biological.

The composition of the optimal biological supplements. Optimal composition of supplements can be known

from the ANOVA test conducted. The test results was the growth of the three treatment given did not produce significant difference, because the levels of protein contained in supplements do not have biological difference. But it still provided the protein content in optimum boundary according to Iskandar (2006a, b) lobsters protein needs ranged from 35-40%, so the supplements produce the same growth.

Correlation between cephalothorax length with lobster abdominal

Correlation between cephalothorax length with lobster abdominal is a very close relationship (r: 0.804). If cephalothorax experience addition in length then it will be followed by the addition in abdomen. According to Showalter (2006) cephalothorax growth and abdomen are comparable or equal.

Correlation between total length with and weight of lobster Correlation between total length with weight of lobsters

is a close relationship (r: 0.777), meaning that this can be explained that the main function of the protein is to repair tissue and to encourage growth, growth that covers the length and weight of lobster (Murtidjo 2007).

CONCLUSION

The use of three kinds of biological and feed supplement plant in the ratio 1: 3 has the same effect on the growth of freshwater crayfish (Cherax quadricarinatus). In the breeding period for 3 months starting from PL 60, the biological supplementation tended to give better results compared to the feed from factory only. The composition of biological supplement with protein content 13.34%, 10.7%, 13.58% gave the same effect on the growth of C. quadricarinatus (PL 60). There is a very close correlation between cephalothorax with abdominal, and between total length with the weight of lobsters.

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Wildlife and Fisheries Science, A&M University. Texas. Sukmajaya Y, Suharjo I. 2003. Freshwater lobster fishery commodities

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Variation of morphology, isozymic and vitamin C content of dragon fruit varieties

BANATI RAHMAWATI1,♥, EDWI MAHAJOENO² ¹ SMP Negeri 5 Surakarta, Jl. Diponegoro No. 45, Surakarta 57131, Jawa Tengah, Indonesia; Tel./Fax.: +92-271-634930, email: [email protected]

² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Manuscript received: 17 July 2009. Revision accepted: 28 Augustus 2009.

Abstract. Rahmawati B, Mahajoeno E. 2009. Variation of morphology, isozymic and vitamin C content of dragon fruit varieties. Nusantara Bioscience 1: 131-137. The aims of the research was to study the variation of morphology, the band pattern of isozyme, and vitamin C content of dragon fruit (Hylocereus spp.) varieties such as super red, red and white from Pasuruan (East Java), Sukoharjo and Klaten (Central Java), and Bantul districts (Yogyakarta). Morphological character were carried include fruit, stem, and flowers of each variety of dragon fruit. The isozymic pattern was analyzed using NTSYS 2.02i. The data matrix was counted based on the DICE coefficient. The clustering was done by applying UPGMA which counted through SHAN. Vitamin C content measured by titration method then analyzed descriptively. The results showed that the higher vitamin C content was found from super red of Pasuruan (6.00) and then followed by red color (5.376) and super red (5.113) both from Bantul. The morphological variation on the stem and petal colors, and fruits were also shown by the isozymic data of three varieties of dragon fruits collected from four separated locations. Esterase (EST) showed 18 bands and forming four (4) groups based on 75% genetic similarity index. The specific band occurred on Rf 0.633 of red varieties of dragon fruit from Bantul and on Rf 0.755 from Pasuruan. The specific band also occurs on Rf 0.347 of white variety from Bantul and on Rf 0.510 and on Rf 0.633 from Klaten. Glutamic oxaloacetic transaminase (GOT) enzyme shows 12 bands and also forming four groups with a little difference for member in the fourth group. The specific band occurs on Rf 0.321 of red color fruit from Pasuruan. The specific band also occurs on the white from Pasuruan on Rf 0.446 and on Rf 0.482. The variation of dragon fruits were also supported by isozymic data indicated that the morphological character were in accordance with the genetics data.

Key words: dragon fruit, Hylocereus, morphology, isozyme, vitamin C.

Abstrak. Rahmawati B, Mahajoeno E. 2009. Variasi morfologi, isozim dan kandungan vitamin C pada varietas buah naga. Nusantara Bioscience 1: 131-137. Penelitian ini bertujuan untuk menguji keragaman variasi morfologi, pola pita isozim dan kandungan vitamin C pada buah naga (Hylocereus spp.) berdaging merah super, merah, dan putih dari Kabupaten Pasuruan (Jawa Timur), Sukoharjo dan Klaten (Jawa Tengah), serta Bantul (Yogyakarta). Data morfologi diuraikan secara deskriptif meliputi buah, batang, dan bunga dari setiap varietas buah naga. Data pola pita isozim dianalisis menggunakan program NTSYS 2.02i. Data matrik dihitung berdasarkan koefisien DICE. Pengelompokan dilakukan dengan UPGMA yang dihitung melalui SHAN. Kandungan vitamin C diketahui dengan metode titrasi dan dianalisis secara deskriptif. Hasil penelitian menunjukkan bahwa kandungan vitamin C tertinggi terdapat pada merah super Pasuruan (6,00), diikuti merah Bantul (5,376) dan merah super Bantul (5,113). Variasi morfologi terjadi pada warna batang, kelopak bunga dan rasa daging buah yang ditunjukkan juga pada pola pita isozim ketiga varietas dari empat lokasi pengamatan. Enzim esterase (EST) mengekspresikan 18 pita yang membentuk empat kelompok berdasarkan jarak kemiripan 75%. Pita spesifik muncul pada buah naga berdaging merah pada Rf 0,633 dari Bantul dan pada Rf 0,755 dari Pasuruan. Pita spesifik juga dimiliki untuk buah naga putih pada Rf 0,347 dari Bantul serta pada Rf 0,510 dan Rf 0,633 dari Klaten. Enzim glutamat oksaloasetat transaminase (GOT) mengekspresikan 12 pita dan memperlihatkan empat kelompok dengan keanggotaan sedikit berbeda di kelompok keempat. Pita spesifik muncul pada varietas buah naga berdaging merah pada Rf 0,321 dari Pasuruan. Pita spesifik juga muncul pada buah naga berdaging putih dari Pasuruan pada Rf 0,446 dan Rf 0,482. Terjadinya variasi pada buah naga yang di uji dan didukung oleh data isozim menunjukkan bahwa data genetik mendukung karakter morfologi.

Kata kunci: buah naga, Hylocereus, morfologi, isozim, vitamin C.

INTRODUCTION

Dragon fruit has high economic value and is useful for treating various types of diseases (Suryono 2006). Dragon fruit is believed to able to lower cholesterol concentration, to balance blood sugar concentration, to prevent colon cancer, to strengthen kidney function and bone, to strengthen the brain workings, increasing the sharpness of the eyes as well as cosmetic ingredients (Suryono 2006).

Dragon fruit is rich in potassium (K), ferum (Fe), sodium (Na), calcium (Ca), and fiber which are good for health compared to other fruits (Sari 2009).

Recent studies of biological diversity get much attention both nationally and internationally. The assessment of biological diversity includes diversity between or within species or populations (Murphy et al. 1993; Karcicio et al. 2003; Yunus 2007a, b; Julisaniah et al. 2008). The studies of relationship of species have been

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conducted up to the organizational structure and evolution of a genome (Purwanto et al. 2002). In order to obtain results that can strengthen taxon boundaries, examine relationship relationships, define and classify taxon particularly categories of species and taxon level below the species, it requires an accurate marker. The commonly used markers are morphological characteristics of plants. The weakness of morphological marker is that it is based on the nature of phenotypes, while the obtained genetic diversity is still a conjecture and is still influenced by environmental factors (Cahyari et al. 2004). A more accurate marker is molecular marker such as isozyme and DNA analysis.

The use of isozyme markers have many advantages because the isozymes are regulated by a single gene and have codominant character and inheritance, normally segregated according to Mendel ratio collinear with gene and is a direct product of genes. This markers are stable because they are not influenced by environmental factors, more quickly and more accurate because they do not need to wait for the plants to reproduce (Cahyarini et al. 2004). According Rahayu et al. (2006) the advantage of isozyme are, among others, producing more accurate data because the isozyme is the last gene expression, relatively simple, requiring relatively low cost compared to other molecular markers. Isozyme has several characteristics and advantages (Hadiati et al. 2002), among others: (i) the product of different alleles move at different positions in the gel, (ii) different alleles are usually inherited in an codominant way, free of epistasis, so that homozygous individuals can be distinguished from heterozygous ones, (iii) the position of the tape is often the product of a locus, making it possible to detect the number of genes encoding an enzyme by analyzing the banding pattern of the enzyme, (iv) the necessary equipment and materials are relatively inexpensive and the experiments can be done easily in the laboratory, (v) the number of samples can be analyzed in a short time, and (vi) it can be done on seed phase, so it saves time, place and money.

Vitamin C has a very important role in strengthening the immune system to fight infection. Animals and humans cannot synthesize Vitamin C, allegedly due to lack an enzyme needed to convert L-gulonic acid into ascorbic acid in foods, so that the intake of Vitamin C should be included in the diet (Airey 2005).

This study aims to determine variations in morphology, banding pattern isozyme and the content of Vitamin C in dragon fruit plant varieties with super red pulp, red pulp and white pulp taken from Pasuruan, Sukoharjo, Klaten and Bantul


Materials A morphology test is carried out on stems, fruit, and

flower of dragon fruit plants with super red pulp (Hylocereus costaricensis), red pulp (Hylocereus polyrhizus), and white pulp (Hylocereus undatus). Isozyme banding pattern test uses shoots of the dragon fruit plant stems taken from Pasuruan (East Java), Sukoharjo and

Klaten (Central Java), and Bantul districts (Yogyakarta). The material test of vitamin C is a ripe dragon fruit. Morphological observation of dragon fruit plant

The observed variables of dragon fruit plants morphology are stems, flowers, and fruits. Observation of morphology refers to Kristanto (2008). Diversity of isozyme markers

Sample subtraction. Young stems of each sample plant are weighed with an analytical balance until it reaches the weight of 100 mg and are placed in the mortar to be extracted.

Extraction of samples. Young stems are pulverized by a mortar, and then are given with a solution of 1 mL of buffer extract and are pulverized again until smooth and then are put into the eppendoRf tube. The examiner then prepares the centrifuge to cool condition (temperature is ± 0 ° C), and it is played with the speed of 700-1500 rpm for ± 20 minutes. Clear supernatant can be immediately used for electrophoresis or cooled at a temperature of 20° C for later use. The use of fresh ingredients give the best results (Arulsekar and Parfit 1986).

Preparation of polyacrylamide gel. Polyacrylamide gel consists of 2 parts, namely running a gel that lies at the bottom with a concentration of 7.5% and spacer gel located on top of running gel with a concentration of 3.75%. Polyacrylamide materials is more profitable than the core gel due to their transparent characters so it can be scanned on visible light or on ultraviolet region. In general, acrylamide gel has no charge while the starch gel contains carboxyl in small proportion which at neutral pH will be negatively charged (Nur and Adijuwana 1989).

Preparation of running gel. All the ingredients are mixed, after a homogeneous mixture is inserted into the electrophoresis glass. On the edge of the left, right and bottom are mounted with bulkhead (shield tube). Furthermore, to create a flat surface of the gel, alcohol and water are added, then alcohol and the water sucked up by aspirator, so that the top of the running gel can be poured with a spacer gel.

Preparation of spacer gel. After the solution is mixed and homogeneous, this mixture is inserted in the glass just above the running electrophoresis gels, and then a comb is mounted on the spacer gel and electrophoresis glass is heated with neon lights for ± 0.5 to 1 hour until it is condensed. Once solidified, the comb is removed so that there are holes that will be filled with supernatant.

Electrophoresis. The process of electrophoresis uses a vertical electrophoresis type, completed with its power supply. The first step, the cover is opened and the electrophoresis bath tub is filled with a solution of buffer tanks electrode for as high as ± 2 cm. This solution serves as conductor of electric current during electrophoresis in a face to face condition, then is added with solution of running buffer tank to the inside of the plate that has been mounted against it, then the supernatant solution is filled into the sample hole for as much as 5 µl with a stepper. The remaining buffer tank is loaded up again till it reaches the electrophoresis bath tub and the cover is replaced. The

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process of electrophoresis is performed with electric currents of ± 100 mA for 180-200 minutes.

Coloring process. Staining process is performed after the process of electrophoresis, the esterase enzyme dye (EST), and glutamic oxaloacetic transaminase (GOT).

Observations gel. After the coloring process and the image banding pattern on the gel is visible, then the fixation process is performed (gel is placed in a solution of ethanol 60% + distilled water and covered with the glass, then put into a refrigerator). The goal is to preserve the gel by stopping the chemical reactions that occur in the gel.

Making dendrogram. Banding pattern isozyme results are interpreted in zimogram electrophoresis, and then are converted into binary data, and are drawn their dendrogram. The measurement of migration distances (RF) is measured by measuring the visible distance of ribbon and then is divided by the longest migration distance.

Vitamin C of dragon fruit For the analysis of Vitamin C, the iodine titration

method referring to the Sudarmadji et al. (1984) is used. A total of 14.88 g of ripe dragon fruit is crushed till it becomes mush. It is added with 100 mL of aquadest. Then, the mush is refined to obtain filtrate. A total of 10 mL of filtrate is titrated with 0.01 of N standard iodine until the blue color is emerged within 15 seconds. Iodine titration volume is converted to ascorbic acid, where 1 mL of 0.01 standard iodine is equivalent with 0.88 mg of vitamin C.

Data analysis The morphological observation

of dragon fruit is described descriptively. Banding pattern isozyme is analyzed qualitatively according to whether or not the banding appears on the gel and quantitatively according to the thickness of the ribbon formed. The diversity of banding pattern is determined based on the value of RF. The resulting binary data is created in the matrix equation. The data matrix is calculated based on the DICE coefficient. The grouping is done by UPGMA (unweighted pair group with arithmetic mean) which is calculated by SHAN on NTSYS program (Rolf 1993). The results of quantitative levels of Vitamin C are analyzed descriptively.


Morphology Morphological data as shown in

Table 1 is then converted into binary data. The grouping is done by UPGMA is calculated by SHAN on NTSYS program (Rolf 1993).

Morphological dendrogram is shown in Figure 1.

Figure 1. Dragon fruit plants dendrogram based on morphological characteristics. Description: PK: white Klaten, PB: white Bantul, PS: white Sukoharjo, PP: white Pasuruan, MB: red Bantul, MP: red Pasuruan, MK: red Klaten, MS: red Sukoharjo, SB: red super Bantul, SK: red super Klaten, SS: super red Sukoharjo, SP: Super red Pasuruan.

The Similarity distance in morphology at 60% of Pasuruan super red varieties and Bantul red varieties always forms their own group. This is because the location of plantations is located near the beach with a sandy soil texture. Location of beach makes it capable of providing a more powerful influence than genetic influences. Coastal

Table 1. Morphology test results fleshy white dragon fruit plants, Red and Super red on four observation sites.

Fruit origin Super red Red White P S K B P S K B P S K B

Stem shape Triangle √ √ √ √ √ √ √ √ √ √ √Quadrangle √

Fruit shape Round √ √ √ √ √ √ √ √ √Oval √ √ √

Hollow shaft Shallow (5-15 mm) √ √ √ √ √ √Medium (16-21 mm) √ Deep (22-28 mm) √ √ √ √ √

Stem color Whitish green √ √ √ √Light green √ √Dark green √ √ √ √ √ √

Flower color Pure white √ √ √ √ √ √ √ √ √ √White √ √

Calyx color Light green √ √ √ √Reddish green √ √ √ √ √ √ √ √

Fruit color Vermilion √ √ √Red √ √Dark red √ √ √ √ √ √ √

Color of fruit flesh White √ √ √ √Red √ √ √Dark red √ √ √ √ Blackish red √

Taste of fruit flesh Sweet √ √ √ √Sweet sour √ √ √ √ √ √ √ √

Color tassels Red √ √ √Red with green tip √ √ √ √ √ √Red with yellow tip √ √ √

Thorn Dense √ √ √ √ √ √ √ √Sparse √ √ √ √

Tassels Dense √ √ √ √ √ √ √ √Sparse √ √ √ √

Note: P: Pasuruan, S: Sukoharjo, K: Klaten, B: Bantul.

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regions have a different climate, temperature, soil conditions, altitude and soil moisture. According to Suranto (2001), the emergence of variation can be caused by two factors: environmental and genetic factors. If genetic factors have a stronger influence than environmental factors, then if the plants live in an environment that is different with their native, it will show no morphological variation similar to their original place and if environmental factors are stronger than the influence of genetic factors, then the plant grew in different place will have a morphological variety.

Plant genetic traits are influenced by environmental factors. Phenotype in an individual is an interaction between genotype and its environment (Sitompul and Guritno 1995).

The gene properties are able to interact with their environment. In this case, environmental factors can influence the emergence of traits or characteristics of an individual. For example, two individuals have the same genes, but living a different environment then the second individual can only bring different characteristics and traits.

Isozymic banding pattern

Electrophoresis results show that the tested esterase isozyme can be visualized well, makes it possible to do genetic interpretation. Zimogaram of esterase banding pattern isozyme of dragon fruit plants is shown in Figure 2. This data is made to dendrogram as in Figure 3.

The style of zimogram isoenzymes electrophoresis results can be considered as phenotypic traits, the genetic testing determines the pattern of its zimogram which is encoded by genes on the same loci and genes at different loci (Sudaryono 1989). In the gel, isoenzymes can be separated by using electrophoresis and the result is zimogram banding pattern. Zimogram patterned electrophoresis typical results that can be used as distinguishing features to reflect the genetic phenotype (Sriyono 2005).

Isozymes migration on the electrophoretic process moves from the negative pole to the positive pole. Esterase enzymes express 18 bandings. Bandings at Rf of 0.591 and 0.816 are owned by all the varieties of the four sampling sites. Red dragon fruit varieties of Bantul, express the specific band at Rf 0.633 and at Rf 0.755 for Red dragon fruit varieties of Pasuruan which is not owned by the red dragon fruit plants from 3 other locations. Specific band is expressed in the phenotype morphology of the oval shape fruit whereas fruit from other regions is round, while stem color is green while other plants are dark green on their stems.

Bantul white dragon fruit express specific band of ribbon at Rf 0.347 which does not appear in other locations. Specific band is expressed at the phenotypic morphology, namely the color green of its stem, while the other is light green and round shape of fruit. Klaten also express a unique band at Rf 0.510 and 0.633 that is not owned by any other location. Specific band is expressed at the morphological phenotype, namely the fruit flavor is sweeter than fruit from other locations and the tassels color is red with green tip while the other is red with yellow tip. Nandariyah et al. (2004) says that the cultivars that have

specific properties have a difference in the taste of fruit pulp, fruit pulp texture and leaf stalks that are not owned by any other cultivar.

Based on the research conducted by Nandariyah (2007) concerning the identification of diversity in cultivars of salak manggala, it is concluded that this cultivar has a specific band that is not owned by the other cultivars and it can be connected with a prominent characteristic of this cultivar, namely the nature of the leaf with curved tip and striated skin of fruit that is not found on other cultivars.

Quantitative traits are usually controlled by many genes and strongly influenced by the environment, on the other hand, the qualitative traits has a relation with the presence or absence of bands on certain migration distance that reflects the presence or absence of amino acids forming enzyme which is the product of the gene itself (Bailey, 1983 in Setianto 2001). The difference of ribbon thickness is because of the difference of migrated molecular weight, the greater the molecular weight, the harder it is separated properly, and thus it forms a thicker ribbon. Molecules that have a large ionic strength will migrate further than the one with lower ionic strength (Cahyarini 2004).

The groups which are separated at a similarity distance above 0.75 or 75% actually still have a close resemblance. Since the similarity distance is said to be far if the distance is less than 0.60 or 60% (Cahyarini 2004). The analysis shows that at the similarity distance which is less than 75% of the 12 groups of dragon fruit being studied, there are 12 divisions of groups (Figure 3).

The results show that the grouping varieties of Sukoharjo super red is separated from the other super red. This indicates that there is genetic variation in a dragon fruit with super red pulp from four locations. Banding pattern isozymes of Sukoharjo super red dragon fruit is slightly difference with Pasuruan dragon fruit which is the first local place for planting dragon fruit. It is possible that the varieties of dragon fruit from Sukoharjo have plasticity in response to the environment. According to Fitter (1998), the different responses to environment (nutrient) is related to heredity, so that plant breeders can create the response of crop fertilization. Claudia et al. (2002) explains that the environment which is too dominant affects the enzyme activity, such as heat, temperature, and pH. This is caused by damage to the function of enzymes due to environmental conditions. A research on the insect has shown that esterase is influenced by a particular dominant environment (Hadiati 2002).

The lower level of crop genetic similarity between accessions, the higher genetic diversity among accessions is. On the contrary, the higher the genetic similarity between accessions, the lower level of genetic diversity generated (Sulistyowati 2008). The occurrence of genetic variability in populations of living species in nature can be caused by hybridization (sexual and somatic), natural mutations, and the transfer of genes from living species of the same or of different (transgenic). The Opportunities of the three factors are very much different (Baihaki 2002; Mansyah 2003).

According to the research of Pasquet et al. (1999) on wild Bambara peanut species and its cultivation, the low


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diversity of genes may be due to a strong autogamy pollination system in these species. Differences of environmental factors may emerge the isozyme expression (Supriyadi 2006). Sitompul and Guritno (1995) say that the appearance of crop plant genetic is controlled by nature under the influence of environmental factors. Dragon fruit breeding is mostly done by vegetative propagation rather than generative multiplication (Kris 2008).

Vegetative propagation produces the less varied offspring, so it is said that the genetic similarity of dragon fruit varieties is high. The high level of genetic similarity shows low levels of genetic diversity in dragon fruit. Maideliza and Masyurdin (2007) say that high gene flow is

usually found on plants that are interbreed or reproduced by seeds. Dragon fruit plants are reproduced by stem cuttings and thus have a high level of genetic similarity. Dragon fruit plants are found scattered in some areas, there is possibility that they come from a single genetic source with different genetic types and then dispersed to various places with the help of humans.

Enzyme Glutamate Oxaloacetic Transaminase (GOT)

Banding pattern isozyme of Glutamate oxaloacetic transaminase (GOT) in the super red dragon fruit can be shown in zimogram in Figure 4, and is subsequently made to dendrogram as in Figure 5

Figure 2. Esterase isozyme zimogram dragon fruit taken from four sampling sites. Note: Rf: The distance migration. Acronym stands for the same case with Figure 1.

Figure 4. GOT isozyme zimogram of dragon fruit from four sampling sites. Note: Rf: The distance migration. Acronym stands for the same case with Figure 1.

Figure 3. Dendrogram of dragon fruit plants esterase from four sampling sites. Note: Acronym stands for the same case with Figure 1.

Figure 5. GOT enzyme dendrogram dragon fruit plants from four sampling sites. Note: Acronym stands for the same case with Figure 1.

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GOT isozyme in dragon fruit plants expresses 12 ribbons. Rf 0.607 is at all dragon fruit plants from the 4 locations. Red dragon fruit varieties from Pasuruan express specific band that is at Rf 0.321 which is not owned by the plants from 3 other locations. This specific band is expressed in the morphologic phenotype, namely the taste is sweeter and the stem has a deep groove, whereas on other varieties the stem groove is shallow. White dragon fruit varieties from Pasuruan also express specific band namely at Rf 0.446 and at 0.48. This specific band is expressed in the morphologic phenotype i.e. the fruit pulp is dark red. At a similarity distance of 0.75 or 75% of similarity, the studied dragon fruit is divided into four groups (Figure 5).

This relationship analysis shows the existence of genetic variation that is high enough. Varieties that have genetic proximity, are probably derived from the elders who are closely related, on the contrary varieties which have relatively high genetic distances, are probably derived from an elder which has a distant relationships with elders of other varieties. The above results can be used as a reference in determining the parent for seed production. The more distant relationship between samples, the less successful the crossing is, but the possibility to obtain superior genotypes is greater if the crossing goes successfully. The more diverse the genetic is the higher the possibility to obtain superior genotypes. Marriage between individuals with close genetic similarity or equal relationship relationship has the effect of increased homozygosis, whereas marriage between individuals having large similarity distance or having far relationship relationship has the effect of increased heterozygosis.

Isozyme is a variation found in the same enzymes that have similar functions and are in the same individual. The enzyme is an amino acid chain in which the genetic information in it is the translation of RNA, whereas RNA is a direct transcription of DNA. Therefore, the variation in enzyme levels shows the variation in the level of DNA (genes) (Na'im 2000). Variations on banding pattern formed by GOT enzyme are less than those by esterase. Sriyono (2006) says that the difference in isoenzymes will produce different velocity when the condition of the electric field and the medium gel is semiporous, so every different enzymes will cause different banding pattern.

The classification of GOT enzyme shows that Pasuruan white varieties belong to the same group with the Bantul red. This is an interesting phenomenon. This seems to be possible because the pioneers of dragon fruit from Pasuruan, East Java, Sapta Surya also develops dragon fruit plantation in Kulonprogo and other Yogyakarta regions (Wijaya 2005). Dragon fruit seedlings introduced to Yogyakarta (Bantul) from Pasuruan is the result of the crossing between varieties of dragon fruit. To prove this phenomenon needs to be done further research using other enzymes or other more modern methods such as using DNA data, whether this occurs due to mutation or due to other factors. This phenomenon also occurs in red pineapple and green pineapple that have a different pigment but form similar banding pattern (Hadiati 2002), it also occurs in dark marie cactus and marie cactus which

have different pigment but form the same banding pattern (O'Leary and Boyle 2000). In the science of plant breeding, plant introductions have an important role to increase genetic diversity in a region. There is a need for high yielding varieties by bringing in varieties from other areas to assist in the provision of high yielding varieties for farmers and as a germplasm collection (Allard 1998).

Novarianto (2008) explains that the breeding is very dependent on the source of genetic diversity. Genetic diversity is not just about physical germplasm collection, but also assessment of the extent of genetic diversity is necessary in genetic manipulation activities towards assembling the desired varieties. Germplasm should be evaluated its genetic diversity as the basis for selection in the crossing process or the assembly of the desired variety of consumers.

Carvalho et al. (2004) explains that the polymorphism produced can be used as a basis for selecting the parent elders that can be used for plant breeding programs. To assemble the improved varieties, the thing need to be considered is the determination of crossing elders, so there is a requirement for information on the genetic distance and relationship relation. According to Hadiati (2002), in hybridization process, the farther the genetic distance between the elders is, the greater opportunity for new cultivars to be produced. In contrast, crossing process between closely related elders resulted in a narrow genetic variability.

Vitamin C contents of dragon fruit The test results of Vitamin C content of dragon fruit

from the 4 locations show differences in Vitamin C as shown in Figure 6. The highest level of Vitamin C content is Pasuruan super red dragon fruit (6:00 mg/100g), followed by Bantul red super (5113 mg/100g) and then Bantul red (5376 mg/100g). While the white pulpy dragon fruit contains Vitamin C which at least compared with other varieties.

Figure 6. Dragon fruit Vitamin C content of the super red, red and white of the four observation sites. Description: P: Pasuruan, S: Sukoharjo, K: Klaten, B: Bantul.

Super red dragon fruit from Pasuruan district contains

the highest Vitamin C compared to the three other locations. This means that the dragon fruit grown in the district of Sukoharjo, Klaten, Bantul undergo a decrease of


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its Vitamin C content. The cause is probably that a super red dragon fruit is more suitable to be planted in the area where the plant is first grown in Indonesia. Pasuruan is low-lying areas which are ideal for growing super red dragon fruit. Dragon fruit Vitamin C content of Pasuruan is not much different from one of Bantul because Bantul has similar rainfall with Pasuruan and it is also low-lying areas.

White pulp dragon fruit is Pasuruan also has the highest Vitamin C content when compared with the three other regions. This means that white pulp dragon fruit is also most suitable to be planted in the area of Pasuruan which has high light intensity due to its coastal region. and its low-lying area. According to Fitter (1998), the productivity of a community is a reflection of net photosynthesis of its component species, and strongly influenced by many factors other than light intensity. Nevertheless its total irradiation during one growing season at times when physiologically important is the important determinants for the production of maximum photosynthesis.

Kristanto (2008) suggests that the content of Vitamin C in dragon fruit ranging from 8-9 mg/100 g. The result of this study shows that Vitamin C content is lower. This is possible because the fruit is too ripe, resulting in lower number of Vitamin C. According to Winarno (1995) and de Man (1999), the content of Vitamin C in raw fruit is higher than in a mature one, and the more mature the fruit is the lesser the Vitamin C content.

CONCLUSION

There is a morphological diversity of stem color, flower petal color, fruit pulp color, and taste of fruit on dragon fruit plants of super red, red, and white from the four sampling sites, Pasuruan, Sukoharjo, Klaten and Bantul. There is a variation of banding pattern isozyme on the varieties of dragon fruit. Based on the isozyme esterase, 18 bands are appeared and are classified into four groups. Based on GOT isozyme, 12 bands appear and are classified into four groups. There are differences in Vitamin C content of dragon fruit from the four sampling sites. Dragon fruit which has the highest content of Vitamin C are Pasuruan super red, Bantul super red, and Bantul red.

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Kimpul (Xanthosoma spp.) characterization based on morphological characteristic and isozymic analysis

NURMIYATI1,♥, SUGIYARTO², SAJIDAN1,² ¹Biology Education Program, Department of Mathematics and Natural Sciences Education, Faculty of Teacher Training and Education Science, Sebelas

Maret University Surakarta 57 126, Central Java, Indonesia ²Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Manuscript received: 27 July 2009. Revision accepted: 28 September 2009.

Abstract. Nurmiyati, Sugiyarto, Sajidan. 2009. Kimpul (Xanthosoma spp.) characterization based on morphological characteristic and isozymic analysis. Nusantara Bioscience 1: 138-145. This research is aimed: (i) to know the variety of kimpul (Xanthosoma spp.) based on morphological characteristics and isozymes analysis; (ii) to know the correlation between its genetic space based on morphological characteristics and its genetic resemblance based on isozymes-banding pattern. This research results were analyzed and described by descriptive qualitative methods. Morphological observation was carried out in sub-District of Galur, Lendah and Girimulyo, Kulonprogo District, Yogyakarta. Morphological data of the kimpul plant was explored descriptively and then made dendogram. Data of isozymic banding pattern were analyzed quantitatively based on the appearance of the band on the gel, and qualitatively based on the thickness of the band formed, and then made dendogram. The correlation, between its genetic distance based on morphological characteristics and its genetic resemblance based on isozymes-banding pattern, were then analyzed grounded on coefficient correlation between product-moment and goodness of it criteria based on correlation. The results pointed out that morphologically, on eight observed samples which were consist of four different types (species), each Xanthosoma from different locations did not indicate obvious differences. Esterase was formed four different banding-patterns, Glutamate Oxaloacetate Transaminase indicated eight different banding-patterns, and Peroxidase indicated seven different banding-patterns. Correlation between morphological data and data from EST and GOT isozymic banding pattern were very good (0.967918 and 0.937113), While, the correlations between morphological data and POD isozymes were good (0.892721).

Key words: kimpul, Xanthosoma, morphological characteristic, isozyme.

Abstrak. Nurmiyati, Sugiyarto, Sajidan. 2009. Kimpul (Xanthosoma spp.) characterization based on morphological characteristic and isozymic analysis. Nusantara Bioscience 1: 138-145. Penelitian ini bertujuan untuk mengetahui keragaman tanaman kimpul (Xanthosoma spp.) berdasarkan karakter morfologi dan analisis isozim serta korelasi antara jarak genetik berdasarkan karakter morfologi dan kemiripan genetik berdasarkan pola pita isozim. Penelitian morfologi dilakukan di Kecamatan Galur, Lendah dan Girimulyo, Kabupaten Kulonprogo, Yogyakarta. Data morfologi diuraikan secara deskriptif dan dibuat dendogram hubungan kekerabatan. Data pola pita isozim dianalisis secara kuantitatif berdasarkan muncul tidaknya pita pada gel kemudian dibuat dendogram. Korelasi antara jarak genetik berdasarkan karakter morfologi dan kemiripan genetik berdasarkan pola pita isozim dianalisis berdasarkan koefisien korelasi product-moment dengan kriteria goodness of fit. Hasil penelitian menunjukkan bahwa secara morfologi pada delapan sampel dari empat macam kimpul yang ditemukan di lokasi yang berbeda tidak menunjukkan perbedaan yang nyata. Pola pita isozim Esterase yang terbentuk menunjukkan empat pola pita yang berbeda, isozim Glutamat Oksaloasetat Transaminase menunjukkan delapan pola pita yang berbeda dan isozim Peroksidase menunjukkan tujuh pola pita yang berbeda. Korelasi data morfologi dengan pola pita isozim EST dan GOT sangat baik (0,967918 dan 0,937113), sedangkan dengan isozim POD berkorelasi baik (0,892721).

Kata kunci: kimpul, Xanthosoma, karakter morfologi, isozim.

PENDAHULUAN

Indonesia is a country with abundant natural wealth. Currently, Indonesia is ranked the third in the world in terms of biodiversity. Ironically, with abundant natural wealth, Indonesia is still threatened by food crisis. The natural resource wealth that is owned by Indonesia can not guarantee the welfare of its people. Efforts to diversify the pattern of the creation of food self-sufficiency must be done to reduce the problems of rice. Increasing the food production can be done through the development and utilization of biodiversity that has not been used optimally.

Crop diversity exists and has the potential to be developed, including the classes of cereals and tubers as a source of carbohydrates and nuts for protein. Types of tubers that can be utilized more optimally include cassava, sweet potato, taro, kimpul, arrowroot and cannas which can substitute the Indonesian staple food: rice. Biological resource of tubers, such as kimpul has not been used optimally to meet food needs. Kimpul is only used as alternative food sources in certain areas in case of famine or natural disaster, though kimpul is a source of easily digestible carbohydrates and has a carbohydrate content of about 70-80% (Kusumo et al. 2002).

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The potential of these commodities is not supported by good data. According Kusumo et al. (2002) the number genotype kimpul (Xanthosoma spp.) in Indonesia has not been recorded. Therefore, to explore the potential plant data collection of kimpul a study needs to be done with the properties of importance to characterize. Characterization was conducted to determine the plant diversity in the field, either in the form diversity of morphological characters, agronomy, physiology, molecular markers or isozyme markers.


Place and time The collection and characterization of the plants were

done in Kulonprogo District, Yogyakarta, which was in Subdistricts of Galur (0-25 m asl.), Lendah (700-100 m asl.) and Girimulyo (25-500 m asl.). Isozyme compare comparison pattern analysis was conducted in the Laboratory of Plant Breeding, Faculty of Forestry, University of Gajah Mada University, Yogyakarta.

Material Material that was used for morphological characteri-

zation is a specimen kimpul (Xanthosoma spp.) from Kulonprogo. Isozyme analysis used three enzyme systems, namely: esterase (EST), glutamate oxaloacetic transaminase (GOT) and peroxidase (PER, POD) in polyacrylamide gel.

Morphological observation. The morphological characteristics that were observed

were the characters of vegetative and kormel/the tuber based guidebook by Kusumo et al. (2002) and Tjitrosoepomo (2003).

Isozyme diversity (Suranto 1991, 2000, 2001) Sampling. Young leaves of each sample plant were

taken, and then weighed with an analytical balance until it reached 100 mg and placed in the mortar to be extracted.

Samples extraction. Young stems were destroyed by mortar, and then given a solution of 1 mL buffer extract and crushed again until smooth and then it was put into the ependorf tube. Prepare centrifuge to cool (± 0°C), and played with the speed of 700-1500 rpm for ± 20 minutes. Clear supernatant can be immediately used for electrophoresis or cooled at 20°C for later use.

Preparation of polyacrylamide gel. Polyacrylamide gel consists of 2 parts, namely running a gel that lies at the bottom with a concentration of 7.5% and spacer gel located on top of running gel with a concentration of 3.75%.

Making running gel. All the ingredients were mixed in a solution; after it became homogeneous mixture then it was put inside of the electrophoresis glass, where on its right and left edge, and bottom there is a mounted bulkhead (shiled tube). Furthermore, to create a flat surface of the gel it can be added alcohol and water. Then the alcohol and the water sucked out with the aspirator running. The top of the gel can be poured with a gel spacer.

Making gel spacer. After the solution was mixed and homogeneous, this mixture was inserted in the glass just above the running electrophoresis gels. Then the sample comb mounted on the spacer gel electrophoresis and glass heated with neon lights ± 0.5 to 1 hour to condense. After the spacer gel solidified, the sample comb was removed so that there were holes that would condition with supernatant.

Electrophoresis process. Electrophoresis was carried out using a vertical electrophoresis type, complete with its power supply. The first step of the cover was opened and the electrophoresis bath tub was filled with a solution of electrode buffer tanks as high as ± 2 cm. This solution serves as the conductor of electric current during electrophoresis in a face-to-facing. Then it was added a solution of running buffer tank to the inside front plate that had been installed, but not fully. Then the supernatant solution was poured into the hole as much as 5 mL samples with a stepper. The remaining buffer tank was loaded again up to meet the electrophoresis bath tub cover and replacing it. The power supply was turned on to run the electrophoresis process with electricity currents of ± 100 mA for 180-200 min.

Staining process. Staining or coloring process was done after the process of electrophoresis, the dye were EST, GOT, and POD.

Observations gel. After the coloring process was done and the banding pattern image was visible on the gel, and then the process of fixation was made (the gel was placed in a solution of 60% ethanol plus distilled water and closed the glass and then put into refrigerator). The purpose of this fixation process was to help to preserve the gel with a way to stop the chemical reactions that occurred in the gel. Making dendogram. Isozyme banding pattern results were interpreted in zimogram electrophoresis, and then it was converted into binary data, and its dendogram was drawn. Measurement of migration distances (Rf) were measured from a distance of band that looked divided by the longest migration distance.

Data analysis The data are described in descriptive morphology.

Isozyme banding pattern data were analyzed quantitatively, which is based on whether the tape appeared on the gel and qualitatively based on the thickness of thin band formed. The correlation between genetic distance based on morphological characteristics and genetic similarity based on isozyme banding pattern was analyzed based on product-moment correlation coefficient with the criteria of goodness of fit based on the correlation according to Rohlf (1993). Genetic similarity between samples was tested using cluster analysis (group average analysis), which results in the form of dendogram or tree diagram. Banding pattern that was obtained from electrophoresis then was translated into qualitative properties (based on the presence or absence of certain bands on the migration distance). At a certain distance migration if there are no distinguishing band thick and thin bands that form the given number 1 and if there is no tape then given a code number 0. The resulting binary data created in the equation matrix (distance matrix) is

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calculated based on the DICE coefficient. Clustering/grouping is done by UPGMA (Unweigthed Pair Group with Arithmetic Mean) is calculated via tail SHAN on NTSYS program (Felsenstein 1985; Rohlf 1993). Dendogram formed as shown in Figure 1 and 3.


Characterization of morphology Results are morphologically characterization of

Xanthosoma plants in the region Kulonprogo, from eight samples taken from three different locations, were found four types of Xanthosoma with characteristics as in Table 1. Based on the results of the morphology characterization of the three study sites, there are four kinds of Xanthosoma spp. which shows the diversity of characteristics.

Kimpul Gendruk Gendruk has grown type of acalauscent. Hastate-shaped leaves are with drooping position at the

end of the petiole. Full leaf edge smooth, green and have no indentation. Not glaucous leaf surface, its upper surface is shiny and dark green, while the bottom surface is green. Leaves are not diverse and do not have hair. Sliced crosswise of the mother leaves bone and the lateral bone appears on the upper surface and does not appear on the bottom surface.

Each leaf stalk attached to the midrib of its part, where the 2/3 part is bright green top and third bottom of the green. 1/3-2/3 of the length of the leaf midrib petiole, midrib pink edge.

Bulbs are light brown or medium brown and have a smooth surface on the outside, while on the inside is white. Pink root tip with the tip position is under the ground. Ellipse-shaped tuber is ovate and cylindrical with size varied depending on the location of plant.

Kimpul Ireng Ireng has grown type acalauscent. Hastate-shaped leaves with bowl-shaped leaf are on the

tip of the leaf stalk. Full leaf edge is smooth, purple and has no indentation. Upper leaf surface is glaucous, not glossy and purplish-green, whereas the lower leaf surface color is green. Leaves are not diverse and do not have hair. Sliced crosswise of mother leaves the bone and the bone lateral leaf appear on the upper surface and does not appear on the bottom surface.

Each leaf stalk attached to the part of the stem, where the whole stalks is purple. 1/3-2/3 of the length of the leaf is midrib petiole, midrib purple edge is like stalks and the stem.

Tuber is dark brown haired and rough on the outside, while on the inside it is white to purple. Pink root tip with the tip position is under the ground. Ellipse-shaped tuber or cylindrical with a wide variety of sizes depending on the location of planting.

Kimpul Puteh Puteh has grown type of acalauscent.

Sagitate shaped leaves is with lobes <1/8 part of the long leaf-shaped bowl with leaf position at the end of the petiole. Full leaf edge is wavy, pale yellow/beige and have no indentation. Upper leaf surface and bottom is not shiny and bright green. Leaves are not diverse and do not have hair. Sliced crosswise mother leaves the bone and the bone lateral leaf appears on the upper surface and does not appear on the bottom surface.

Petiole is slightly attached to the part of the stem, 2/3 the top of the stem is bright green and 1/3 part bottom is green. 1/3-2/3 of the length of the leaf midrib petiole, midrib green edges such as stalks and the stem.

Kimpul Puteh does not produce tubers.

Kimpul Mothe This kimpul is very similar to Puteh but petiole color

(2/3 top) is green, a third of the remaining is red/purple and yellow in the inside cormel.

Dendrogram morphological characters The similarity of the observed character of the eight

samples consisting of four types of Xanthosoma in this study may indicate the closeness of relationship owned. Therefore testing the closeness of relationship that is owned by eight samples consisting of four types of Xanthosoma using the dendogram, as shown in Figure 1.

The dendogram of the eight samples tested, the similarity coefficient of 65% to form three major groups are: the first group consisted of Gendruk (Galur, Lendah and Girimulyo) and the mothe of Girimulyo, the second group consisted of Ireng derived from Galur and Lendah and the third group is Puteh of Galur and Lendah. According to Cahyarini (2004) distance is much similarity can be said when less than 0.60 or 60%. Thus, separate groups at a distance of 0.65 is actually still have a close resemblance. In this dendogram analysis, the number 1 or 100% in the dendogram indicate that group members had a perfect likeness, while getting closer to the number 0 means the similarity distance is going further.

Figure 1. Relationship of eigh sample of Xanthosoma spp. from three different locations based on morphological characters. Note: 1. Gendruk, 2. Ireng, 3. Puteh (Galur); 4. Gendruk, 5. Ireng, 6. Puteh (Lendah); 7. Gendruk, 8. Mothe (Girimulyo).


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Table 1. Morphological characteristics of Xanthosoma spp. in sub-districts of Galur, Lendah and Girimulyo, Kulonprogo District, Yogyakarta.

Morphological characteristics Galur Lendah Giri-mulyo

1 2 3 4 5 6 7 8 Growth type Acalauscent √ √ √ √ √ √ √ √ Upright on the ground - - - - - - - - Fell on the ground - - - - - - - - Petiole attachment Mutual bound √ √ - √ √ - √ √ Few bound - - √ - - √ - - No bound - - - - - - - - Position of leaf blade Upright - - - - - - - - Droop √ - - √ - - √ √ Bowl shape - √ √ - √ √ - - Leaf edge Full smooth √ √ - √ √ - √ √ Full wavy - - √ - - √ - - With lobus divided in part - - - - - - - - Divided nearly to the basal - - - - - - - - Leaf shape No basal lobe - - - - - - - - Hastate √ √ √ √ √ √ Sagitate (lobe <1/8 Long) - - √ - - √ - - Sagitate (lobe length 1/8-1/4) - - - - - - - - Sagitate (lobe> 1/4 long-leaf) - - - - - - - - Comparison of length/width of leaf blade Length 53.2551.5 41.3639.2 46.5 28.4 62.2 72.5Width 36.2533.6726.3827.9 27.9 22.6 47.7 53Color of the leaf edge Green along the leaf edge √ - - √ - - √ √ Colorless along the leaf edge - - - - - - - - Purple/red √ √ Pale yellow/beige - - √ - - √ - - Leaf sinus denuding No √ √ √ √ √ √ √ √ Few (<5mm) - - - - - - - - Splitting (5 mm-several cm) - - - - - - - - Mixed (some split. others not) - - - - - - - - Shiny leaf surface None (0) - √ √ - √ √ √ √ Upper surface √ - - √ - - - - Below surface - - - - - - - - Glaucous leaf surface None (0) √ - √ √ - √ √ √ Upper surface - √ - - √ - - - Below surface - - - - - - - - Color of the upper leaf surface Green light - - √ - - √ - - Green medium - - - - - - - - Dark green √ - - √ - - √ √ Reddish/purplish green - √ - - √ - - - Others - - - - - - - - Color of the lower leaf surface Green light - - √ - - √ - - Green medium √ √ - √ √ - √ √ Dark green - - - - - - - - Reddish/purplish green - - - - - - - - Others - - - - - - - - Diversity of leaf None (0) √ √ √ √ √ √ √ √ Present (+) - - - - - - - - Pubescence

None (0) √ √ √ √ √ √ √ √ Hair (5) - - - - - - - - Bushy-haired (9) - - - - - - - - Cross-section of main and lateral leaf veins Only on abaxial of leaf √ √ √ √ √ √ √ √ Only on adaxial of leaf - - - - - - - - On both sides of the leaves - - - - - - - - Prominent on abaxial of leaf - - - - - - - - Petiole length (cm) 58.8 99.4 51.7558.8 90.2 54 112.6133.3Petiole color (2/3 of the top) Bright green √ - √ √ - √ √ - Green - - - - - - - √ Red/purple - √ - - √ - - - Green striped red/purple - - - - - - - - Petiole color (1/3 part bottom) Bright green - - - - - - - - Green √ - √ √ - √ √ - Red/purple - √ - - √ - - √ Green striped red/purple - - - - - - - - The upper surface of petiole glaucous None (0) √ - √ √ - √ √ √ Present (+) - √ - - √ - - - Petiole length with stem Midrib < 1/3 length of stalk - - - - - - - - Midrib 1/3-2/3 length of stalk √ √ √ √ √ √ √ √ Midrib > 2/3 length of stalk - - - - - - - - Color edge of stem with petiole Just as the stalk and midrib √ √ √ √ - √ More light - - - - - - - - More dark - - - - - - - - Pink/red/purple √ - - √ - - √ - Harvesting tubers/cormel 4-6 months or less - - - - - - - - 7-12 months √ √ √ √ √ √ √ √ 13-17 months - - - - - - - - > 18 months - - - - - - - - Cormel forms Rounded - - - - - - - - Ovate - - - √ - - - - Cylindris - √ - - - - √ - Elliptics √ - - - √ - - - Mixture - - - - - - - - Cormel size Small - √ - - √ - - - Medium √ - - √ - - - - Large - - - - - - √ √ Color the exterior cormel Medium brown or light brown √ - - √ - - √ √ Dark brown - √ - - √ - - - Color the inside cormel White √ - - √ - - √ - Yellow - - - - - - - √ Orange - - - - - - - - Pink or pale - - - - - - - - White purplish - √ - - √ - - - Outer surface cormel Subtle √ - - √ - - √ - Coarse-haired - √ - - √ - - - Color of cormel tip - - - - - - - - White - - - - - - - - Pink/red √ √ - √ √ - √ - Position of cormel tip Above ground - - - - - - - - Underground √ √ - √ √ - √ - Note: 1. Gendruk, 2. Ireng, 3. Puteh (Galur); 4. Gendruk, 5. Ireng, 6. Puteh (Lendah); 7. Gendruk, 8. Mothe (Girimulyo).

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Based on morphological characters observed, Puteh from Galur and Lendah show similar properties. As shown in the dendogram, both exist at the similar coefficient of 100% which means the exactly the same or makes no difference. When compared with other types of Xanthosoma, this type forms its own group and makes little resemblance to other types. This species is clustered with other species at 46% similarity level. Ireng has a high similarity coefficient with Gendruk at 53%, while mothe has a high similarity coefficient with Gendruk at 78.6%.

Xanthosoma type that were from different locations did not show morphological characteristics are that different; in other words differences in location did not affect plant morphology. Morphological differences that appear only on the length of petiole, leaf blade length and width, and size kormel. These traits are related to the growth of each crop. Xanthosoma that usually is found in Girimulyo has a larger plant size, has the petiole and leaf blade size is larger than the two other locations. Cormel generally also has larger size, though with the same age with a second harvest in other areas. These properties appear related to physical factors/environment where Xanthosoma lives. Xanthosoma from Girimulyo area planted by the community as a plant in a garden under the trees, so Xanthosoma get shade from the plants that are above it. In this region Xanthosoma deliberately cultivated and cared quite well in the garden-courtyard. Plants constantly adapt to each face of environmental stress. Plants deal with stress, shade will make a strategy for adjustment. Adjustments to such as changes in morphological characters and physiology of plants (Djukri 2006). This character-specific changes in conditions such as shade increased leaf width but much thinner (Taiz and Zeiger 1991). Agronomic characters associated with high production potential is high habitus plants, broad leaves and roots of both (Sulistyono et al. 2002).

In the circumstances of shaded light spectrum that are active in the process of photosynthesis (wavelength 400-

700 nm) decreased. Plants will make adjustments to streamline to capture the light energy by increasing the leaf area to meet the needs of light that are active in the process of photosynthesis. Another form of adjustment is the increased plant height and chlorophyll a and b (Lambers et al. 1998). Based on the research by Anggarwulan et al. (2008) that treatment of shade variations have a significant influence on plant height that is 75% shade treatment combination gave the best plant height, while the treatment without shade produces a low plant height. Light plays an important role in plant physiological processes, especially photosynthesis, respiration and transpiration.

Characterization based on isozyme markers Analysis of the enzyme EST, GOT, and POD of the

eight samples Xanthosoma plants shown in Figure 2.

Esterase (EST) Based on isozyme analysis with dye EST on eight

samples tested Xantosoma formed four distinct banding pattern (Figure 2). Four kinds of banding pattern were different in shape and migration distance. Migration distance of each band calculated by the formula Rf (relative Ferguson) by comparing the distance of bands from each of the wells that are formed by the migration of the farthest distance (distance Loding dye) (Hames and Rickwood 1990; Julisaniah et al. 2008). Banding pattern I with the same migration distance but has a different shape, the migration distance 0.129, 0.185 and 0.222 are owned by the sample 1, 3 and 7 (quantitative and qualitative). Banding pattern II with migration distance 0.129, 0.185, 0.222, and 0.389 is owned by the sample 2 and five (quantitatively and qualitatively). Banding pattern III with migration distance 0.129, 0.185 and 0.333 are owned by samples 3 and 6 (quantitative and qualitative). IV banding pattern unique to the sample 8 has a migration distance 0.129, 0.185, 0.241, 0.259, 0.296 and 0.315.

A B C Figure 2. Zimogram of eighth sample of Xanthosoma spp. from three different locations based on the dye: A. EST, B. GOT, C. POD. Note: 1. Gendruk, 2. Ireng, 3. Puteh (Galur); 4. Gendruk, 5. Ireng, 6. Puteh (Lendah); 7. Gendruk, 8. Mothe (Girimulyo).


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Quantitative traits are usually controlled by many genes and strongly influenced by environmental factors, whereas for the qualitative nature is almost not influenced by environmental factors, so that qualitative factors are preferred because they relate to the presence or absence of certain bands on migration distance that reflects the presence or absence of amino acid enzyme which is the gene product itself (Setianto 2001). While the difference in thickness is formed thin bands due to differences in the number of molecules that is migrated, a thick band fixation of several bands. Molecules that have a large ionic strength will migrate further than the low ionic strength (Cahyarini 2004).

Glutamic oxaloacetic transaminase (GOT) Based on isozyme analysis with dye GOT, eight

samples of Xantosoma were tested to form eight distinct banding pattern (Figure 2). Banding pattern I on migration distances 0.32, 0.42 and 0.44 are owned by the sample 1. Banding pattern II on migration distances 0.32, 0.4, 0.44 and 0.46 are owned by the sample 2. Banding pattern III on migration distances 0.38, 0.42, 0.56 and 0.58 are owned by sample 3. Banding pattern IV on migration distance 0.32, 0.42, 0,440,46, 0.66 and 0.68 are owned by the sample 4. Banding pattern V of migration at a distance of 0.32 V, 0.42, 0.44 and 0.46 are owned by sample V. Banding pattern VI on migration distance 0.38, 0.42 and 0.58 are owned by the sample VI. Banding pattern VII on migration distance 0.3, 0.42, 0.44, 0.46, 0.64 and 0.66 are owned by the sample VII. Banding pattern VIII on migration distance 0.32, 0.34, 0.36, 0.4, 0.44, 0.46, 0.48, 0.62 and 0.64 are owned by the sample VIII.

Peroxidase (POD) Based on the analysis of POD isozyme with the dye on

the eight samples of Xantosoma that were tested form seven different banding pattern (Figure 2). Banding pattern I on migration distances 0.654, 0.673 and 0.75 are owned by the sample 1 and 4. The pattern of bands II at a distance of 0.75 migration is owned by sample 2. Banding pattern III on migration distances 0.096, 0.712, 0.769, 0.846 and 0.885 are owned by sample 3. IV banding pattern on migration distance 0.673 and 0.679 are owned by sample 5. Banding pattern of migration at a distance of 0.096 V, 0.712, 0.769 and 0.904 are owned by the sample No. 6. VI banding pattern on migration distance 0.673, 0.75 and 0.846 are owned by the sample 7. VII banding pattern on migration distance 0.673, 0.769, 0.865 and 0.966 are owned by the sample 8.

Genetic similarity based on isozyme markers Dendogram based on EST

Based on a dendogram in Figure 3 obtained from cluster analysis (group) that is used to determine the similarity of the samples tested, at a distance of 0.65 or 65% showed three major groups. In this dendogram analysis number one on the dendogram showed group members had perfect resemblance, while getting closer to zero means the similarity distance farther. At 65% similarity distance is divided into three major groups i.e.

group I consists of samples 1, 4, 7, 2 and 5. Group II consists of samples 3 and 6, and group III consists of the sample 8. At 86% similarity distance separation occurred in group I. Group I again split into two groups: the group It consists of samples 1, 4 and 7 and group Ib consisted of samples 2 and 5. Thus based on the diversity patterns EST band formed at a distance of 65% similarity can be separated between group I (Gendruk and Ireng) with group II and group III which is respectively Puteh and mothe. At this distance we can not distinguish between Gendruk and Ireng, both can be distinguished at a distance of 86% similarity. Based on the results of such grouping, we can not distinguish types of the same sample that derived from different locations. Gendruk from three different locations in one group, Ireng from two different locations to form a single group, as well Puteh from two different locations has also formed a group.

Dendogram based on GOT Based on the results obtained dendogram from cluster

analysis (group) that is used to determine the similarity of the samples tested, at a distance of 0.65 or 65% showed five groups. Group I consists of samples 1, 5 and 4, group II consists of sample 2, group III consists of sample 7, group IV consists of samples 8 and group V consisted of samples 3 and 6. Dendogram based on this pattern of GOT bands can separate Puteh derived from Galur and Lendah from other groups on a relatively small distance similarity as much as 17%. Between Mothe of Girimulyo Gendruk and Ireng are apart at a distance of 42.2%. At a distance of 73.4% similarity of Lendah Gendruk split from the group I, and Gendruk of Galur and Ireng of Lendah separated at a distance of 86% similarity.

Dendogram based on POD Based on the results obtained dendogram from cluster

analysis (group) that is used to determine the similarity of the samples tested, at a distance of 0.65 or 65% showed five groups. Group I consists of samples 1, 4 and 7, group II consists of sample 2, group III consists of sample 3, group IV consists of samples 6 and group V consisted of samples 5 and 8. Dendogram based on banding pattern Gendruk POD can separate from the Galur, Lendah and Girimulyo from other groups at a distance of 65% similarity. In this distance similarity of Ireng from Galur, Galur and Lendah Puteh from separating groups each forming his own group: the group II, III and IV with a single member. But at a distance of 57% similarity, Galur, Lendah, and Puteh joined in a group. At a distance of 65% similarity Lendah Ireng of parting with Ireng of Galur and joined the mothe of Girimulyo.

Relations morphology characterization and isozyme markers

The correlation between genetic distance based on morphological characteristics and genetic similarity based on isozyme banding pattern was analyzed based on product-moment correlation coefficient with the criteria of goodness of fit based on the correlation according to Rohlf (1993). The correlation between genetic distance based on

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morphological characteristics and genetic similarity based on isozyme banding pattern as in Table 2.

Correlation between morphological data and data isozyme banding pattern EST, GOT and POD in a row at the level of 0.967918, 0.937113 and 0.892721. Thus means that the results of characterization based on morphological characteristics and results of characterization based on EST and GOT isozyme markers have a very good correlation. While the results of characterization based on morphological characteristics and results of characterization based on POD isozyme markers have a good correlation. Characterization of Xanthosoma based on morphological characteristics consistent with the characterization based on isozyme markers. Table 2. The correlation between genetic distance based on morphological characters and isozyme banding pattern

Karakter yang dikorelasikan Level Kriteria

Morphology and EST Morphology and GOT Morphology and POD

0.967918 0.937113 0.892721

Very good Very good Good

Dendogram relationship of Xanthosoma spp. from

three different locations based on morphological characters, coloring EST, GOT and POD (Figure 3), indicating that Xanthosoma with the same type but originating from different areas showed no differences in the character pattern of band. At 65% similarity distance is divided into three major groups i.e. group I consists of samples 1, 4, 7 and 8. Group II consists of sample 2 and 5, and group III, i.e. samples 3 and 6. At the distance of separation of similarity of 80% occurred in group I. Group I again split into two groups: the group It consists of samples 1, 4 and 7 and Ib group consisted of samples 8. Thus it is clear that Gendruk of the three locations clustered in one group separate and distinct from other groups. Likewise mothe from Girimulyo, it is clustered in one distinct group and tend to have a closer relationship to

Gendruk. Galur and Lendah Puteh forming their own groups and has distant relationship with other groups. Ireng of Galur and Lendah formed his own group.

Based on characterization, the results indicate that each type of Xanthosoma is the same even if planted in different locations still express the same trait. This can be understood that the three places chosen as sampling sites are still in a region that is in the area of Kulonprogo, so it is possible that each of the Xanthosoma species that is in those three locations is an elder and there is no difference genetically. Stronger genetic factor influencing phenotypic expression when compared with environmental factors, so that even if planted in different locations still express the same trait. This is supported by the results of characterization based on morphologic characteristics which indicate that the same Xanthosoma species are found in different locations still showed the same morphological features.

The diversity of plant species is a manifestation of the genetic capability to respond to the potential of the existing environment. This response can be viewed from two aspects of how the environment needed to realize its potential and how plants respond to environmental values that exist. In this case each plant species Xanthosoma able to respond to any environmental value to survive. In other words each type of Xanthosoma has the capability of adaptation to the environment that is wide enough, can survive in several different environments. Species plant is said to have wide adaptation (adjustment) when completing one life cycle in different environments (Odum and Barrett 2005).

EST enzyme that has a general nature and applies to all types of plants that EST in plants is a hydrolytic enzyme that functions to withhold simple esters in organic acids, inorganic acids and phenols and alcohols have low molecular weight and soluble (Cahyarini 2004). GOT isozyme banding pattern formed on the testing of eight samples showed eight distinct banding pattern. Each sample has a banding pattern that is different from other samples. POD isozyme banding pattern formed on the

0.65

0.8

A B C D

Figure 3. Relationship of eighth sample of Xanthosoma spp. from three different locations based on the character of isozyme banding pattern. A. EST, B. GOT, C. POD, D. Combined the three. Note: 1. Gendruk, 2. Ireng, 3. Puteh (Galur); 4. Gendruk, 5. Ireng, 6. Puteh (Lendah); 7. Gendruk, 8. Mothe (Girimulyo).


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testing of eight samples shows seven different band patterns. Each sample has a different banding pattern with the other samples except sample 1 and 4 have the same band pattern. Peroxides oxidoreductase is an enzyme that contributes to the oxidation of a substrate while reducing H2O2 to H2O. Rothe (1994) says that the POD isozyme was widespread, especially in plants and present in significant amounts. With the presence of hydrogen peroxide (H2O2) catalyze the oxidation of phenol (AH2) and aromatic amines (AH2) in accordance with the following Enzim-H2O2 + AH2 enzim + A + 2 H2O.

Peroxidase has a broad spectrum and has a very important role in the process of plant physiology. This enzyme can be isolated and scattered in the cell or plant tissue, especially in plant tissues that had been developed (Butt 1980; Hartati 2001). POD in plants is a protein that catalyzes the oxidation of H2O2 with a variety of substrates. Some functions of POD in plants include the formation of wood, auxin metabolism (Gaspar et al. 1991; Groppa 1999), in response to environmental stress (Castillo and Reppin 1986; Esaki et al. 1996) and as a defense against pathogens (Lagrimi and Rothstein 1987; Mohan and Kolattukudy 1990).

CONCLUSION

There is no diversity of morphological characteristics of each type of Xanthosoma that is found at different locations. There is no diversity of EST isozyme banding pattern on each type of Xanthosoma that are found in different locations. There is a diversity of GOT isozyme banding pattern on each type of Xanthosoma found in different locations. Each sample has a banding pattern that is different from other samples. There is a diversity of POD isozyme banding pattern on each type of Xanthosoma found in different locations. Each sample has a different banding pattern with the other samples except sample 1 and 4 have the same banding pattern. There is a diversity of relevant between morphological characters and isozyme analysis. Characterization of Xanthosoma based on morphological characteristics consistent with the character-based markers isozyme terisasi EST, GOT and POD.

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University Press. Yogyakarta. [Indonesia]


Traditionally utilization of Selaginella; field research and literature review

AHMAD DWI SETYAWAN♥ Department of Biology, Faculty of Mathematic and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36a Surakarta 57126, Central Java,

Indonesia. Tel./Fax.: +92-271-663375. ♥email: [email protected]

Manuscript received: 11 September 2008. Revision accepted: 10 November 2008.

Abstract. Setyawan AD. 2009. Traditionally utilization of Selaginella; field research and literature review. Nusantara Bioscience 1: 146-158. The aims of this research were to find out traditional usage of Selaginella in medication and its other usages, especially in Java and other Indonesian Archipelago. About 200 of 700-750 world species of Selaginella was found in Indonesian Archipelago. Field research and literature review indicated that Selaginella is used traditionally to heal wound, bloody stools, internal hemorrhoid bleeding, menstrual and uterine disorder, blood expediting, enhancing body endurance and longevity of live, headache, etc. Besides that some of Selaginella are also used as raw dishes vegetable, ornamental pants, and crafts materials. The utilization of Selaginella is very limited against the amount of species and medicinal potency, it is needed an advance study on ethnobotany and phytochemistry to improve their uses.

Key word: traditional medicines, herbal, ethnobotany, Selaginella, Java.

Abstrak. Setyawan AD. 2009. Pemanfaatan Selaginella secara tradisional; penelitian lapangan dan telaah pustaka. Nusantara Bioscience 1: 146-158. Penelitian ini bertujuan untuk mengetahui manfaat Selaginella dalam pengobatan tradisional dan pemanfaatan lainnya, melalui penelitian lapangan dan telaah pustaka, khususnya di Jawa dan Kepulauan Nusantara. Sebanyak 200 dari 700-750 spesies Selaginella hadir di Kepulauan Nusantara. Secara tradisional Selaginella digunakan untuk mengobati luka, pendarahan, gangguan menstruasi dan kandungan, memperlancar peredaran darah, meningkatkan daya tahan tubuh, memperpanjang usia, mengobati sakit kepala dan lain-lain. Di samping itu beberapa jenis Selaginella juga digunakan sebagai sayuran (lalapan), tanaman hias, dan bahan baku kerajinan tangan. Pemanfaatan Selaginella sangat terbatas dibanding jumlah jenis dan potensi manfaat obatnya, sehingga diperlukan kajian etnobotani dan fitokimia lebih mendalam untuk meningkatkan pemanfaatannya.

Kata kunci: obat tradisional, tanaman obat, etnobotani, Selaginella, Jawa.

INTRODUCTION

Medicinal plants are plants that contain ingredients that can be used for treatment or becoming drug synthesis precursor (Sofowora 1982). Medicinal plants have become the leading contributor to health to mankind since time immemorial. In Indonesia, there are various systems of traditional medicine, as a result of high biological and cultural diversity in this country (Erdelen et al. 1999). The oldest and the most widespread system of traditional medication in Indonesia and the Malay Archipelago (Nusantara or Malesia) is a native herb from Java (jamu). Herbal medicine or jamu contains more than 30 species of plants. The existence of the process of making herbal relief in Borobudur temple shows that the herb has been widely known since the early 9th century (Jansen 1993). This system has been recorded since the last centuries in various serat (letters) and primbon (prophecy) (Soedibjo 1989 1990; Sutarjadi 1990). Jamu is an original vocabulary of the Java language, which means traditional medicine, in addition it has been absorbed into the Indonesian language (Riswan and Sangat-Roemantyo 2002), and the word jamu has also been used by other Malay speakers. This system is

widespread through trade and migration, since the kingdoms of Hindu Mataram (Sanjaya), Sriwijaya (Sailendra), and Majapahit. At this time, jamu plays an important role in health and economic development of Indonesia and around countries (Sidik 1994). In Indonesia, more than 75% disease was treated with jamu or traditional medicine (Al-Janabi 2001). Even according to the WHO, 80% of the developing countries depend entirely on traditional medicine to maintain the people’s health (Farnsworth et al. 1985; Bodeker et al. 2005).

Tropical forests are the habitat and the main source of medicinal plants (Stepp and Moerman 2001; Stepp 2004), due to high levels of biodiversity and endemism (Gentry 1993; Macilwain 1998). Some 40,000 species (Rifai MA, 2008, personal communication) or 15% of flowering plants can be found in Indonesia (MOSPP 1993). 10% of the plants are potential for medicinal plants (Schumacher 1996). Heyne (1927) noted the existence of 996 species of flowering plants used in traditional medicine in Indonesia, when added with algae, fungi, ferns, and gymnosperms and the number can reach 1040 species. Kazahara (1986) noted 7500 plant species in Indonesia, where 3689 species of which are medicinal plants. Zuhud et al (1994) mentions

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the 1260 species of trees in the Indonesian rain forests are used as medicinal plants.

Selaginella (cakar ayam or rane) is a medicinal plant that has not been widely used, either traditional or modern. Small amounts of the species are also used as ornamental plants and vegetables. Family Selaginellaceae Reichb has only one genus, Selaginella Pal. Beauv, consisting of 700-750 species and widespread in a cosmopolitan way (Tryon and Tryon 1982; Jermy 1990). In Nusantara or Malay Archipelago (Malesia), there are more than 200 species of Selaginella, with the highest diversity and endemism in Papuasia, Borneo, and the Philippines. In Java there are 24 with 5 endemic species. Some species are still waiting to be discovered, but a number of other species waiting to be extinct (Setyawan 2008). All species of Nusantara have small leaves resembling scales, with two different sizes: the smaller median leaves in the inner row and the larger lateral leave in the outer rows (Jermy 1990; Camus 1997).

Selaginella contains a variety of secondary metabolites such as alkaloids, phenol (flavonoids, tannins, saponins), and terpenoids (triterpene, steroid) (Chikmawati and Miftahudin 2008; Chikmawati et al. 2008). The main secondary metabolite of this plant is biflavonoid, whose type is various depending on the species. Biflavonoid that has been identified from Selaginella, among others amentoflavone, 2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone, isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, and taiwaniaflavone. These compounds act as antioxidants, anti-inflammatory, anti-cancer, anti-allergic, antimicrobial, antifungal, antibacterial, antiviral, protective against UV irradiation, vasorelaxant, heart boosters, anti-hypertensive, anti-clotting, and affect the metabolism

enzymes (Setyawan and Darusman 2008). Biflavonoid is a typical of secondary metabolites which are found only in Selaginellales, Psilotales, gymnosperms (Seigler 1998), and several species of Bryophytes and Angiosperms (DNP 1992).

This study consists of two main activities, namely the field research and literature review. The field research was conducted in several areas in Indonesia, especially Java, in order to know the traditional use of medicinal plants Selaginella, while the literature review were collected from studies around the world, especially from the Nusantara regions. The literature review is intended to strengthen and expand the knowledge about the traditional use of medicinal plants, Selaginella.


Field research The field research was conducted to know current state

of traditional use of Selaginella. The field research was conducted at 100s locations in Java and 10 locations in other islands, namely: (i) Cycloops Mountain Nature Reserve, Jayapura, Papua, (ii) Mount Meja Protected Forest, Manokwari, West Papua, (iii) Mount Gamalama, Ternate, North Maluku, (iv) Atakejawe-Lolobata National Park, Halmahera, North Maluku, (v) Mount Soputan, Minahasa, North Sulawesi, (vi) Nantu Game Reserve, Boalemo, Gorontalo, (vii) Mount Rinjani, Lombok, West Nusa Tenggara, (viii) Batu Kahu Nature Reserve, Tabanan, Bali, (ix) Mount Penumbing, Bangka-Belitung, and (x) Mount Leuser National Park, Aceh (Figure 1). The field research was conducted in the mid of 2007 to the late of 2008.

Figure 1. Selaginellas research sites in Nusantara. 1. Cycloops Mountain Nature Reserve, Jayapura, Papua, 2. Mount Meja Protected Forest, Manokwari, West Papua, 3. Mount Gamalama, Ternate, North Maluku, 4. Atakejawe-Lolobata National Park, Halmahera, North Maluku, 5. Mount Soputan, Minahasa, North Sulawesi, 6. Nantu Game Reserve, Boalemo, Gorontalo, 7. Mount Rinjani, Lombok, West Nusa Tenggara, 8. Batu Kahu Nature Reserve, Tabanan, Bali, 9. Mount Penumbing, Bangka-Belitung, and 10. Mount Leuser National Park, Aceh, 11. Java (100s sites).

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7 8

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Note: Study sites in Java included: West Java, Banten and Jakarta: Bogor Lowland (UI campus at Depok-Jakarta, Cifor educational forest, Bogor Botanic Garden, Mekarsari Park, BAU campus at Darmaga, Ciampea limestone hill); Mount Halimun Salak (Cikaniki research station, Nirmala tea plantation, Gunung Bunder, Gunung Malang, Cijeruk, Gunung Wiru, Kabandungan, Cibedug); Mount Gede-Pangrango (Cibodas Botanic Garden, Cibodas trekking); Pembarisan Mountain (Darma, Kudugede), Mount Ciremai (Linggarjati, Cilimus, Jalaksana). Central Java and Yogyakarta: Nusakambangan Island (Lempong Pucung, Ujung Alang); Mount Slamet (Baturraden, Serang); Dieng Plateau (Telaga Warna, Sikidang crater, Tuk Bimo Lukar or Serayu water spring, Batur’s ex PT. Dieng Jaya factory, Kejajar riverside, Mladi, Sikarim waterfall); Wonosobo Township, 500-1000 m.asl. (Kejiwan, Kalianget, Sambek, Tawangsari); Wonosobo’s Lowland Community Forest: 250-500 m. asl. (Kepil, Burat pine forest, Bejen water spring, Dempes pine forest, Lamuk pine forest, Ngalian pine forest, Wadaslintang dam, Wadaslintang outlet); Dieng alternatif route (Sigaluh salak plantation, Madukoro salak plantation, Talunombo agathis and pine plantation, Pagentan, Pejawaran, Batur highland); Mount Sindoro (Jumprit water spring, Candimulyo tea plantation, Kledung tobacco plantation, Damarkasian, Sojopuro, Gedekan, Ngelo, Andongsili, Blederan, Sigedang, Tambi tea plantation); Mount Sumbing (Margoyoso, Kwaderan, Rejosari, Kaliangkrik, Butuh, Batursari); Mount Telomoyo (Gedong Songo temples, Bandungan); Mount Merapi and Merbabu (Deles, Kalikuning, Plawangan hill, Kaliurang, Kalitengah Lor, Bale Rante, Ampel trekking route, Selo, Musuk, Sawangan, Kopeng, Getasan); Menoreh Mountain (Loano, Kaligesing, Girimulyo); Mount Lawu (Grojogan Sewu waterfall, Jobolarangan hill, Cemoro Sewu, Ngargoyoso forest park, Kemuning tea plantation, Jenawi rubber plantation); Sewu Mountain (Batuseribu, Nguntoronadi, Wuryantoro, Gajah Mungkur dam, Wanagama I forest, Dadapan’s Pacitan bay, Pancuran Tegalombo); East Java: Mount Wilis (Telaga Ngebel), Mount Kelud (Kelud crater area), Mount Kawi-Butak (Cuban Rondo waterfall), Mounts Bromo, Tengger, Semeru (Wonokitri village, Bromo crater, Tutur’s Kutukan river, Cuban Pelangi waterfall), Mount Argopuro (Cuban Dalungan waterfall, Bremi recreational area), Iyang Mountain (Gumitir, Boto-Mrawan, Ijen Mountain).

The data of the traditional use of Selaginella were obtained through interviews (in-depth interviews) with local residents (key person) about 2-3 people at each location. Selected respondents were adults (aged > 18 years), and were born and raised in that region. Respondents did not necessarily have high education or work as traditional healers. To maintain the spontaneity of respondents, inter-views were conducted in an informal, unstructured, using a general interview guide; by showing a number of Selaginella specimens that have been collected previously from these locations, to ensure the local name of each taxon and other data. In some locations, field studies were conducted more than one visit to deepen the research, so there were 5-10 local residents per location that were interviewed.

The data recorded include the location, scientific name, local name, the efficacy in the treatment, the used parts, single or ingredients (if ingredients, then mentioned the other ingredients that are added), the preparation procedure, the charged dose, the duration of treatment, the abstinence during treatment and the best time of collection. In addition, other non-medical use was also noted (Table 2).

All species of Selaginella found at each study site were sampled and made into herbarium, and also identified further to confirm their identity. Identification was done by referring to the Alderwereld van Rosenburgh (1915a, b; 1916 1917 1918 1920 1922) and Alston (1934 1935a, b; 1937 1940); and herbarium sheets of the Herbarium Bogoriense collection (BO) especially which has been determined by Alston in the past. Herbarium specimens are stored mainly in the Herbarium Soloense (SO), Biology Department, Sebelas Maret University of Surakarta; with the collection numbers of ADS et al. The duplicate will be sent to the Herbarium Bogoriense (BO), Research Centre for Biology, Indonesian Institute of Sciences, Cibinong Bogor and the Leiden Herbarium (L), the Netherlands.

Literature review Literature review data were collected until the end of

2008, primarily through the collection of abstracts from: Medline (www.pubmed.gov), JSTOR (www.jstor.org), HighWirePress (http://highwire.stanford.edu), BioInfoBank (http://lib.bioinfo.pl), Elsevier (www.sciencedirect.com), and Springer (www.springerlink.com). Literature review data in the form of books, journals, abstracts, articles, patents, and bibliographies were also collected from Google (www.google.com) and Yahoo (www.yahoo.com). Data collection is not restricted to language or time of publication. The data collection used the keyword

'Selaginella' and/or 'medicinal plant' and/or 'biflavonoid', including the 12 compounds of biflavonoid, namely: amentoflavone, 2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone, isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, and taiwaniaflavone. Selection was then performed manually by reading the manuscript one by one to separate the valuable data (from a trusted author and publisher) from the invaluable ones and to avoid duplication.

All the data whose strength had been proved were compiled in the tables. For traditional use, as well as field research, data collected includes location (country, place), scientific name, local name, the efficacy in the treatment, the used parts, single or ingredients (if ingredients, then mentioned the other ingredients that were added), governance way of preparation, the dose, duration of treatment, abstinence during treatment and the best time of collection. In addition, non-medical use and a list of libraries were also noted (Table 2).

Data analysis The ethnobotany data was explained descriptively and

compared with utilization in the whole world.


Species diversity Selaginella species found in the 100s sites in Java and

10 sites outside Java is listed in Table 1; photo collection of some samples are presented in Figure 2. In this study, 40 species of Selaginella has been found, where 18 species can not be identified, although by matching them with Herbarium Bogoriense collection. Some species are thought to be a new species, new records or species introductions to Nusantara. Selaginella identification of the archipelago is difficult because most libraries for the identification of old age and require revision. Selaginella species mostly not been used as medicinal plants or other purposes of economic potential, but there are at least 10 species that have been used with varying intensity (Table 2). S. involvens, S. ornata, S. willdenowii, and S. plana used as medicinal ingredients. S. ciliaris, S. singalanensis, and Selaginella sp.1 used as an ornamental plant. S. opaca, S. plana and S. wildenowii used as a vegetable. S. caudata and Selaginella sp.4 used as a wrapping of fruits and vegetables from the garden.


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Table 1. Diversity of Selaginella in the research sites. Location Scientific name Java*) Selaginella aristata Spring Selaginella ciliaris (Retz.) Spring Selaginella frondosa Warb. Selaginella intermedia (BI.) Spring Selaginella involvens (Sw.) Spring Selaginella longiaristata Hieron Selaginella opaca Warb. Selaginella ornata (Hook & Grev.) Spring Selaginella plana (Desv. ex Poir.) Hieron Selaginella remotifolia Spring Selaginella repanda (Desv. & Poir.) Spring Selaginella rothertii Alderw. Selaginella singalanensis Hieron Selaginella subalpina Alderw. Selaginella willdenowii (Desv.) Baker Selaginella zollingeriana Spring Selaginella sp.1 “hortus-mekarsari” Selaginella sp.2 “halimunensis” Selaginella sp.3 “kaliwiroensis” Cycloops Mountain Nature Reserve Selaginella angustiramea Muell. Selaginella caudata (Desv.) Spring Selaginella velutina Cesati Selaginella sp.4 Selaginella sp.5 Mount Meja Protected Forest Selaginella caudata Selaginella sp.4 Mount Gamalama Selaginella cupressina (Willd.) Spring Selaginella sp.6 Atakejawe-Lolobata National Park Selaginella angustiramea Selaginella velutina Selaginella sp.7 Selaginella sp.8 Selaginella sp.9 Mount Soputan Selaginella cupressina Selaginella sp.10 Selaginella sp.11 Nantu Game Reserve Selaginella caudata Selaginella cupressina Selaginella vonroemeri Alderw. Mount Rinjani Selaginella sp.12 “rinjaniensis” Selaginella plana Batu Kahu Nature Reserve Selaginella opaca Selaginella remotifolia Selaginella sp.13 “pseudoinvolvens” Selaginella sp.14 “pseudoplana” Mount Penumbing Selaginella ketra-ayam Alderw. Selaginella sp.15 Mount Leuser National Park Selaginella mayeri Hieron. Selaginella sp.16 Selaginella sp.17 Selaginella sp.18 Note: *) Javan selaginellas of BO collection that cannot find in this research are: S. alutacia Spring, S. stipulata (Blume) Spring, and S. subspinulosa Spring.

Medicinal use of Selaginella The traditional utilization of Selaginella based on field

studies and literature review are presented respectively in Table 2. From Table 2, it appears that the traditional use Selaginella in Java and other islands in Nusantara is still relatively rare, compared to the number of species that grow in this region. At least the local name given shows the little popularity of this plant in the community, this is certainly due to least utilization of the plants. The dominance of Javanese herbal medicine systems (jamu) in traditional medicine in Indonesia and Malaysia, which is generally made from raw materials of about 30 species of cultivated plants, especially rhizomes and spices seem to have put aside the potential use of Selaginella, whose availability in nature is affected by seasons. From the field studies, it is known that the Selaginella is useful to treat wounds, menstrual disorders and for treatments before, during, and after giving birth, and to improve fitness and endurance of the body (tonic).

It is also known that the utilization of Selaginella was not only found in Nusantara, but relatively and evenly distributed throughout the world, although the number of species that have been used relatively limited. The result of literature study also shows that the herb is commonly used to treat wounds and bleeding, either external wounds or internal injuries such as menstrual disorders and postpartum hemorrhage, and also used as a tonic to improve fitness and stamina (Table 2). The more number of utilization of Selaginella plana (Desv. ex Poir.) Hieron. in Indonesia and Malaysia compared to other species may be linked to the size distribution of these plants on the islands of Nusantara, western Nusantara region considering the ancestral home of this species (Setyawan 2008). The utilization of S. tamariscina (Beauv.) Spring and S. doederleinii Hieron indicates the presence of the influence of traditional Chinese medicine in an area, considering that both are widely used in traditional Chinese medicine recipes that are relatively advanced.

Field research In the Indonesian language, especially new libraries

generally name Selaginella as cakar ayam, referring to the leaf shape that resembles the scales on either side of the stem, like scales on a chicken leg (Dalimartha 1999); or rane uptake of Sundanese, the most common ethnic utilize this plant (Sastrapradja and Afriastini 1985). Selaginella has many local names, such as: rumput Solo, cemara kipas gunung, cakar ayam (Java), paku rane (Sunda), menter (Betawi), tai lantuan (Madura), usia (Ambon), sikili batu, lingonai (Minangkabau) (Heyne 1927; Winter and Jansen 2003), and shi shang bai or juan bai (Chinese) (Bensky et al. 2004). But the field research shows local names are now beginning not to be recognized by people; even most of the respondents did mention the local names of Selaginella shown to them, except in West Java where the Sundanese people uniformly used the word rane to name a few species from genus Selaginella, particularly S. plana. On the Dieng plateau and surrounding the word pulalata was used to name Selaginella opaca Warb. In the vicinity of Mount Argopuro, East Java where there was pretty much of the

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A B C D

E F G H

I J K L

M N O P

Q R S T

U V W X

Y Z AA BB


151

CC DD EE FF

GG HH Figure 2. Most Selaginella found in this research in Nusantara. A. S. aristata, B. S. ciliaris, C. S. frondosa, D. S. intermedia, E. S. involvens, F. S. longiaristata, G. S. opaca, H. S. ornata, I. S. plana, J. S. remotifolia , K. S. repanda, L. S. rothertii, M. S. singalanensis, N. S. subalpina, O. S. willdenowii, P. S. zollingeriana, Q. Selaginella sp.1, R. Selaginella sp.2, S. Selaginella sp.3, T. S. angustiramea, U. S. caudata, V. S. velutina, W. Selaginella sp.4, X. Selaginella sp.5, Y. S. cupressina, Z. Selaginella sp.7, AA. Selaginella sp.8, BB. Selaginella sp.9, CC. S. vonroemeri, DD. Selaginella sp.12, EE. Selaginella sp.13,FF. Selaginella sp.14, GG. S. ketra-ayam, HH. S. mayeri (source: photos Y until CC contributed by J. Kinho).

Madurese population, the word tai lantuan was still used to name a few species of Selaginella such as S. plana, S. involvens (Sw.) Spring, and S. remotifolia Spring.

In this study, most respondents did not know the local names or the benefits of Selaginella, either as raw drugs, food, ornamental plants or other benefits. Most of them were also unable to show the difference between one species of Selaginella from other species. However, many of whom were familiar with this plant, proved by the ability to indicate where the habitats of Selaginella grew when shown with the examples of the specimens and when they can be found abundantly. Generally, they identified the habitat of Selaginella as cliffs near springs, waterfalls or small water channel that was humid, wet and somewhat open; and tend to grow more abundant during the rainy season. They generally think its name was pakis or a kind of this plant. In fact, the word pakis is more properly applied to the tree ferns, such as Cyatheaceae, that have a similar appearance with pakisaji or cycads.

The field research shows the use of Selaginella in Indonesia is relatively limited, although there is a relatively large number of the species. Type of utilization is generally in the form of utilization as a medicinal plant, besides it was also noted the utilization as raw vegetables or lalapan and food wrappers from the field. Types of diseases and health problems that can be healed with this plants among others are injury, menstrual disorders and pregnancy, postpartum (puerperal), and to improve physical fitness. In the field research, the use of Selaginella as ingredients was only found in Java, while it was not found in ten other locations (islands) outside Java.

On the island of Java, the traditional use of Selaginella is generally limited in Western Java (West Java and Banten provinces). This may be related to the abundance and

higher diversity than in other parts of this island. This is supported by the habitat conditions which are more humid with higher rainfall and the level of a relatively higher slope as well, thus supporting the life of Selaginella. In East Java, the utilization of Selaginella was not found in the tribe of Javanese, Madurese, as well as the Tengger.

In Central Java, precisely in the Dieng plateau, S. opaca locally known as pulalata is used to cure wounds, menstrual disorders and to increase stamina. The name is specifically imposed only on S. opaca, whereas other species of Selaginella that grow on one plant site are not named pulalata, such as S. remotifolia and S. ciliaris (Retz.) Spring. As a drug for injury, pulalata freshly cleaned is to be chewed, and then placed on the wound as a poultice. Up to now the utilization is relatively limited and only used on small wounds, it is still used in case of accidents in the field, as first aid until a doctor or a drug is found in the nearest warung (small shops). Pulalata is also used as medicine for menstrual disorders and for increasing endurance, by boiling and eating them as vegetables. Besides, one respondent in the Kaliwiro subdistrict, Wonosobo stated that S. plana and S. ornata (Hook & Grev.) Spring shown to him was useful to strengthen the heart, although he did not know the local names of the plants, and did not know the procedure of how to use it. One respondent in Wonosobo informed that a private hospital in Yogyakarta once prescribed herbal remedies, 'jamu godog', in which one form of simples was S. plana, to treat stroke. In Banjarnegara, S. plana was used to strengthen the immunity of patients against malaria. On the slopes of Mount Lawu, between Central and East Java the respondents knew that S. opaca is needed by a company for herbal medicine ingredients, but they themselves did not know the benefit and did not use them traditionally.

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Table 2. Utilization of Selaginella medicinal plants through field research and literature review.

Scientific name

Local name Location Medical uses Single/

potionPrepa-ration Dosage

Non-medical benefits

References

Field research

S. caudata, - Manokwari - - - - Packaging Field research S. ciliaris - Wonosobo - - - - Ornamental

land cover Field research

S. involvens

Rane MHSNP Wounds, menstruation, fitness, liniment herb

Single/ potion

Raw, cooked, baked

As needed - Field research

S. opaca Pulalata Dieng, Central Java

Wounds, menstruation, body fitness Single Poultice/ cooked

At sufficiently

- Field research

Rane Bogor Wounds, menstruation, post-childbirth Single Raw/ cooked

At sufficiently

Vegetable Field research

- Mount Lawu Purchased by pharmaceutical manufacturers

- - - - Field research

S. ornata - Wonosobo Heart disease - - - - Field research Rane MHSNP Wounds, menstruation, fitness, liniment

herb Single/ potion

Raw, cooked, baked

As needed - Field research

S. plana - Wonosobo Heart disease - - - - Field research - Wonosobo Stroke Potion Cooked 5-6

handfuls - Field research

- Banjarnegara Tonic for malaria patients - Cooked ½ glass, three times a day

- Field research

Rane Bogor Injury, menstrual disorders, uterine bleeding, and post-labor tonic

- Raw/ cooked

- Vegetable, Ornamental

Field research

S. rothertii - Bogor - - - - Ornamental land cover

Field research

S. singalanensis - Wonosobo - - - - Ornamental land cover

Field research

S. willdenowii Rane MHSNP Wounds, menstruation, fitness, liniment herb

Single/ potion

Raw, cooked, baked

As needed Vegetable, Ornamental

Field research

Selaginella sp.1 - Bogor - - - - Ornamental Field research

Literature review

S. argentea - Malaysia, Sabah

Headache and high fever - - - - Ahmad and Raji 1992

S. asperula - Columbia Wound - - - - Winter and Jansen 2003

S. articulate - Columbia Treated to snake bite - - - - Winter and Jansen 2003

S. bryopteris - India Anti-inflammatory and cures veneral disease

- - - - Agarwal and Singh 1999

S. ciliaris Semerak-merak

Malaysia, Selangor

Itchy on skin - Fresh for lotion

- - Hanum and Hamza 1999

S. convoluta - Brazil Uterus illness - - - - - S. delicatula - India Gastric illness - - - - Dixit and Bhatt 1974;

Mathew et al. 1999 Laos Sedative - - - - ARCBC 2004 S. doederleinii - Cina Anti-cancer - - - - Lee et al. 1992; Lin et

al. 1994 - Korea Anti-cancer - - - - Lee et al. 1992; Lin et

al. 1994 - South East

Asia - - - - Food

supplements ARCBC 2004

S. epirrhizos - Guyana Headaches treatment - - - - DeFilipps et al. 2004 S. exaltata - Columbia Decoction or spleen disease and stomach-

aches for a prolonged period - - - - Winter and Jansen

2003 S. firmuloides - Vanuatu Post-childbirth - - - - Bourdy and Walter

1992 S. fissidentodes - Madagascar Cough - - - - Winter and Jansen

2003 S. imbricata - Zambia Not specific - - - - Cunningham 1993 - Zimbabwe Not specific - - - - Cunningham 1993 S. intermedia - South East

Asia Decoction for stomach-ache and applied as poultice over the whole body for asthma.

- - - - Winter and Jansen 2003


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S. involvens - India Life extending - - - - Dixit and Bhatt 1974; Sequiera 1998, Khare 2007

S. lepidophyll - Mexico Decoction or infusion to treat kidney stone, gastric ulcers, diarrhea, rheumatism, dyspepsia, cystisis of liver, expulsion of the placenta, blood purify

- - - - Martinez 1961; Winter and Jansen 2003

S. magnifica, Selaginella spp.

- Indonesia, BBBR NP

Headache and fever, as well as for skin care - - - - Caniago and Siebert 1998

S. moellendorffii - China Gonorrhea, bleeding, jaundice, acute hepatitis

- - - - Shi et al. 2009

S. myosurus - - Asthma, fever and fatigue - - - - Bouquet et al. 1971 - Gabon - - - - Cultural

rituals Sassen and Wan 2006

S. ornata Rane Indonesia - - - - Ornamental Sastrapradja and Afriastini 1985

- Indonesia, Java

Young leaves are eaten as vegetable and as depurative or stomachic.


S. padangensis - Sumatra, Malaya, Kalimantan,

Smoked like tobacco and used as poultice for vertigo and treated to toothache


S. parkeri - Guyana Treat headaches - - - - DeFilipps et al. 2004 - Guyana - - - Burned and

lubricate to the baby hee

- van Andel 2000

S. plana - Indonesia Not specified - - - Dish of raw vegetables

Heyne 1927

- Indonesia, KM NP

Bleeding - - - - Uluk et al. 2001

- Indonesia, MHSNP

Post-childbirth - - - - Harada et al. 2002

- Malaysia, Sabah

Headache and high fever - - - - Ahmad and Raji 1992

S. pallescens - Columbia Snake bite Winter and Jansen 2003

- Mexico Gastro-intestinal disorder Winter and Jansen 2003

- Venezuela Decoction as an emmenagogue and diuretic Winter and Jansen 2003

S. rupestris - India Tonic, puerperal tonic, sedative - - - Ornamental Khare 2007 - Sumatra and

Malaysia Decoction as protective medicines after childbirth

Winter and Jansen 2003

S. tamariscina Juan bai Cina Anti cancer, wounds, bleeding, hemorrhoids

PAM 2008; Lee et al. 1992; Lin et al. 1994

Keoun back

South Korea Anti-cancer, menstrual pain, bruises, and asthma

- - - - Lee et al. 1992; Lin et al. 1994

- Anti cancer , inhibit gastric cancer Kim 2007 - Far East

Rusia Delay the aging process Mamedov 2005

Pakong tulog

Philippine Wounds, bleeding, hemorrhoids - - - Vegetables PAM 2008

- East Asia Advanced cancer - - - - Lee et al. 2009 - - Blood purify, hematuria, prolapse of the

anus and stanching - - - - Carlo et al. 1999

S. tamariscina var. pulvinata

- - Tonic to prolong life, prevent amenorrhoea, hemorrhoid

- Boiled - - Khare 2007

S. uncinata - South China Anti-bacterial, hepatitis, tumors - - - - Ma et al. 2002 S. wallichii - - Post-childbirth - Boiled - - Khare 2007 Paku

merak Malaysia, Selangor

Cleaning sputum/cough - Boiled, for taking bath

- - Hanum and Hamza 1999

S. wallichii - Sumatra, Malaya, southern Thailand

Decoction as protective medicines after childbirth

Winter and Jansen 2003

S. wightii - India Urinary tract infections - - - - Dixit and Bhatt 1974; Mathew et al. 1999

S. willdenowii - - High fever, ashes to rub back pain - Infusion, and burned

- - Khare 2007

- Indonesia, Java

Young leaves are eaten as vegetable and as depurative or stomachic.


- Java Decoction as a protective after childbirth and tonic, treating skin disease such as itches and ringworm


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- Malaya Given internally as an infusion to treat fever and the ashes is used in liniment for backache


Selaginella spp. - Indonesia, MHSNP

Wounds - - - - Nasution 1993

Selaginella spp. Rane Indonesia, MHSNP

Post-childbirth and menstruation - - - - Setyawan and Darusman 2008

Selaginella spp. Cakar ayam

Indonesia Cancer, respiratory infection, injury, heart disorders, urinary infections, broken bones and rheumatism

Single/herb

Fresh, dry, raw/cooked

- - Dalimartha 1999; Wijayakusuma 2004

Selaginella spp. - Malaysia Endurance - - - - Batugal et al. 2004 Selaginella spp. - Sumatra,

Java Counter poison, drug fever, washing blood, menstrual blood purifier, eczema and for drug after childbirth

- - - - Warintek 2002

Note: The plant used as samples are all, especially the leaves. KMNP: Kayan Mentarang National Park, MHSNP: Mount Halimun-Salak National Park. BBBR NP: Bukit Baka Bukit Raya National Park.

In West Java, the respondents generally knew the traditional benefits of rane to treat wounds, menstrual disorders, and to improve fitness. In West Java, particularly in the lowlands of Bogor, sub-districts such as West Bogor, Darmaga, and Ciampea, S. plana. known as rane was used as drugs for injury, menstrual disorders and uterine bleeding, and post-labor tonic. As a drug for wounds, fresh herbs that have been chewed is put on the wound, while for other treatment purposes it is used by cooking or eating it straight as a vegetable. Meanwhile in the highlands of Bogor, for example in the sub-district of Pamijahan, or at sub-district Kalapanunggal, Sukabumi, the term rane is also used for some other species such as S. willdenowii (Desv.) Baker, S. involvens, and S. ornata also used for the purpose of treatment as above, besides the ash produced from burning dried herbs are used as a liniment to relieve stiffness and warm the back.

Literature review Selaginella has been prescribed in traditional medicine

of China and India, which has been thousands of years old. The utilization of these medicinal plants was also done by various other cultures, although generally limited to specific species. Selaginella can be found in the pharmacopoeia in Asia, Africa and Latin America, but not found in Europe and North America (Duke et al. 2002). The high diversity of species of Selaginella in the first three locations is likely to be the cause of this difference in the rates of utilization. The intensity of the highest utilization was carried out in China, especially for S. tamariscina (include var. pulvinata Spring), S. doederleinii, Selaginella moellendorffii Hieron, S. uncinata, and S. involvens (Chang et al. 2000; Lin et al. 1991; Wang and Wang 2001). In India, there were several species of Selaginella used as ingredients, such as S. involvens, Selaginella rupestris Spring, S. tamariscina var. pulvinata, S. wallichii Spring, S. willdenowii and others (Dixit and Bhatt 1974; Mathew et al. 1999; Khare 2007).

Selaginella traditionally used to treat several diseases such as: injury, treatment of post-childbirth, cancer, skin diseases, headaches, fever, respiratory infections, urinary tract infections, menstrual disorders, liver disorders, fractures and arthritis. The parts used are all parts of the plants, although sometimes they are called only a leaf

(herb) (Setyawan and Darusman 2008). Its use can be done singularly or in combination, fresh or dried, eaten immediately or cooked before (Dalimartha 1999; Wijayakusuma 2004). These plants are sweet and have warm effect (Bensky et al. 2004).

Nusantara (Malesia). In Nusantara, the utilization of Selaginella is still relatively limited. The Javanese traditional herbal medicine or jamu, as a traditional medicine’s most advanced systems in the region, tends to use rhizomes and spices, while the use of herbs and wild grasses is more limited.

In Kalimantan, the Dayaks in the vicinity of Kayan Mentarang NP, East Kalimantan using S. plana to treat bleeding (Uluk et al. 2001), whereas in the surrounding of Bukit Baka-Bukit Raya NP, West Kalimantan, Selaginella magnifica Warb and several other species of Selaginella are used to treat headaches and fever, as well as for skin care (Caniago and Siebert 1998). In Sabah, Malaysia, Selaginella argentea (Wall. ex Hook. & Grev.) Spring and S. plana are used to treat headaches and high fever (Ahmad and Raji 1992). In northern Borneo, the dry leaves of S. padangensis Hieron is smoked like tobacco and also used as poultice for vertigo and treated to toothache (Winter and Jansen 2003).

In Java, Sundanese people in the surrounding of Mount Halimun-Salak NP, West Java, use some species of Selaginella to treat wounds, post-childbirth, menstrual disorders, and as a tonic (Nasution 1993; Harada et al. 2002; Setyawan and Darusman 2008). S. plana leaves is drunk in decoction as tonic for treatment after childbirth (Harada et al. 2002). Selaginella intermedia (BI.) Spring is given in decoction for stomach-ache and is applied as poultice over the whole body for asthma. In Java, young leaves of S. ornata and S. willdenowii are eaten as vegetable and also as depurative or stomachic. S. willdenowii is also used in decoction as a protective medicine after childbirth and as an ingredient of tonic as well as to treat skin disease such as itches and ringworm (Winter and Jansen 2003).

In Sumatra and Java, some species of Selaginella are used to counter poison, drug fever, washing blood, menstrual blood purifier, eczema and for drug after childbirth (Warintek 2002). In Sumatra, Kalimantan, and Malaya, S. padangensis is used as poultice for vertigo and


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treated to toothache. In Sumatra and Malaysia, S. stipulate is used in decoction as protective medicines after childbirth. In Sumatra, Malaya, and southern Thailand, S. wallichii is used in decoction as protective medicines after childbirth. In Malaya, S. willdenowii is also given internally as an infusion to treat fever and the ashes is used in liniment for backache (Winter and Jansen 2003). In Kedah, Malaysia, Selaginella is used to increase body resistance (Abu-Shamah et al. 2000; Batugal et al. 2004).

In Papua New Guinea, Selaginella flabellate Spring is used to treat headaches and fever (Kambuou 1996). In the Philippines, S. tamariscina (pakung tulog) is used to treat wounds, bleeding from peptic ulcers or excessive menstruation, and hemorrhoids (PAM 2008). In the mainlands of Southeast Asia, S. doederleinii is used as drugs for various diseases and as dietary supplements, while in Laos Selaginella delicatula (Desv. ex Poir.) Alston is used to relieve tension (ARCBC 2004). In Indonesia, many species of Selaginella is offered in the form of dry powder, both local as S. plana and imports from China, in particular as S. tamariscina and S. doederleinii.

China. In China and the neighboring countries, the most widely used species is S. tamariscina. The area mentioned is the center of distribution of this species, with quite thorough distribution, both wild and cultivated plants. The checking of Herbarium Bogoriense (BO) collection indicates that in Nusantara, S. tamariscina only grows wild on the island of Flores, Sulawesi, and the Philippines (personal observation), while it may be naturalized from cultivated crop in West Kalimantan, which significantly has Chinese population. In China, the dry powder of S. tamariscina that has been cooked is used for blood clotting. Decoction of the dry powder used for amenorrhea orally either alone or mixed with some other herbs. For bleeding, in the hemorrhoid and in the uterine bleeding, the dry powder is mixed with some other plants then boiled for drinking. For single use of rectocele (NAS 1975). S. tamariscina which contains abundant of amentoflavone has been used for the treatment of advanced cancer in traditional oriental medicine (Lee et al. 2009), and has been used in traditional oriental medicine to blood purify, hematuria, prolapse of the anus and stanching (Carlo et al. 1999). It has also been used as an antioxidant, vasorelaxation, anti-HIV and anti-angiogenesis agent (Lee et al. 2009). S. tamariscina is the most useful plant Selaginella in traditional folk medicine practiced in China, Hong Kong, Japan and Korea (But et al. 1997). S. uncinata which usually grow in southern China is used to fight diseases caused by bacteria, hepatitis infections and tumors. S. moellendorffii has been used in traditional Chinese folk medicine for treatment of gonorrhea, bleeding, jaundice, and acute hepatitis (Shi et al. 2009). S. doederleinii has been used for the treatment of cardiovascular diseases (Chao et al. 1987; Lin et al. 1994; Lu et al. 2004) and an anti tumor herb used for lung, nasopharyngeal, and esophageal cancers (Jia 1985) as well as hysteromyoma (Huang 1982).

India. In India, Selaginella involvens, S. rupestris and S. tamariscina var. pulvinata are used as tonic to anti ageing (Dixit and Bhatt, 1974; Sequiera 1998; Khare

2007), meanwhile S. delicatula is used to cure bellyache, and S. wightii to cure infection of bladder (Dixit and Bhatt 1974; Mathew et al. 1999). S. rupestris is also used in decoction as sedative. S. tamariscina var. pulvinata is used in decoction to prevent amenorrhea, hemorrhage effect by pile or prolepses of the rectum. S. wallichii is treated in decoction to after childbirth. S. willdenowii is used in infusion to cure high fever, while its ash is used for liniment the backache (Khare, 2007). S. bryopteris is treated as anti-inflammatory and cures veneral disease (Agarwal and Singh 1999), its usage is also known by local indigenous people, such as Songhati people (Singh et al. 2002).

Oceania and Asia. In Vanuatu, Selaginella firmuloides Warb is used to assist child birth (Bourdy and Walter 1992). In the eastern part of Russia S. tamariscina is used to slow down the aging process (Mamedov 2005). In China and South Korea S. doederleinii is used as anticancer drugs (Lee et al. 1992; Lin et al. 1994). In South Korea, S. tamariscina which traditionally treated as anti cancer is significantly proved inhibit gastric cancer as showed in cell cycle analysis and other assay (Kim 2007). In Korea, S. tamariscina is also used to cure menstrual disorder, bruise, and asthma, while in Sri Lanka it is used to cure headache, paralysis, and to refuse black magic. S. myosurus is used to cure asthma, fever and fatigue (Bouquet et al. 1971).

Africa. In some African countries, like Zambia and Zimbabwe, Selaginella imbricata (Forsk.) Spring ex Decaisne is traded as medicinal ingredients that has lead to threatening its sustainability in nature (vulnerable, VU) (Cunningham 1993; Golding 2002). In Madagascar, S. fissidentodes is used for cough (Winter and Jansen 2003).

Latin America. In Brazil, Selaginella convoluta (Arn.) Spring is used to prevent and treat diseases related to female reproductive system (de Almeida-Agra and Dantas 2004). In Guyana, the ashes of Selaginella parkeri (Hook. & Grev.) Spring (= Selaginella pedata Klotzsch) is used by putting it hard onto the heel of the baby gently that can help the baby start walking (van Andel 2000). Selaginella parkeri and S. epirrhizos Spring are also used to treat headaches (DeFilipps et al. 2004). In Mexico, S. lepidophylla is used in decoction or infusion to treat kidney stone, gastric ulcers, diarrhea, rheumatism, dyspepsia, cystisis of liver, to facilitate the parting and expulsion of the placenta, to purify the blood (Martinez 1961; Winter and Jansen 2003). In Columbia, S. asperula treat to wound, while rhizome of S. exaltata is used in decoction for spleen disease and stomach-aches for a prolonged period, while S. articulate and S pallescens treat to snake bite. In Venezuela, S. pallescens is marketed and used in decoction as an emmenagogue and diuretic, while in Mexico, it treat to gastro-intestinal disorder (Winter and Jansen 2003).

Other uses Foodstuffs. In Nusantara (Malesia), the utilization of

Selaginella as food ingredients is very limited. In the field research, the use of Selaginella as food (vegetables) is found only in West Java, ranging from lowlands of Bogor to the area around the mountains of Halimun-Salak. In Bogor, species that is consumed in general is only S. plana,

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whereas in the plateau region, S. willdenowii is also consumed by people. Heyne (1927) notes that in West Java, young buds of S. plana can be eaten as vegetables and for medicinal purposes. PAM (2008) notes that in the Philippines, young leaves of S. tamariscina can be cooked as vegetable.

Ornamental plants. The utilization of Selaginella as an ornamental plant is found in West Java. For example, some sellers of ornamental plants in Bogor, including the Mekarsari Park at Cileungsi once sold Selaginella sp.1. which allegedly was an introduced species as for ornamental plants, whereas in the Sringanis Garden and Medicinal Plant Garden of Karyasari S. plana and S. willdenowii were once sold as medicinal plants. According Sastrapradja and Afriastini. 1985, S. ornata is planted in Bogor as an ornamental plant, although the stem is easily broken, so it must be treated with caution. The field research shows that in Kejiwan village, Wonosobo subdistrict, Wonosobo district, S. ciliaris was left to grow wild or they were planted in cemeteries to cover the surface of the soil to avoid scouring of rain urging the growth of other weeds, and also to beautify the cemetery. In Bogor Botanical Garden, S. rothertii is used as the land cover on the part of the collection of Selaginella species. In Wonosobo, Selaginella singalanensis Hieron was found as a new record with huge potential for land cover crops, because it can be grown in the surface of the soil quickly to cover the land, and can appear in blue metallic color on shaded conditions, such as S. willdenowii.

According to the author's observation (ADS 2008), about 10-15 species Selaginella potential as an ornamental plant, and comes from Java, Sumatra, Papua, Lombok and Bali, and can grow well in the experiment field in Java located in the highlands (Wonosobo, 700-800 m asl) and in the lowlands (Bogor, 200-250 m asl), but some species can grow well only in the highlands, such as S. opaca and S. remotifolia. Some remaining species only grow well in lowlands, such as S. rothertii whose seedlings are obtained from the Bogor Botanical Gardens and Bogor Agricultural University at Darmaga Campus. S. rothertii is an endemic plant of West Java, which is generally easily found in lowland that is moist and open, but there are also variants found in the highland. Observations of Selaginella collections of Herbarium Bogoriense indicate that this species ever found wild in Cibodas Botanical Gardens and the Puncak area in general. One species, i.e. S. willdenowii is endemic in western Java, grown naturally from Banten eastward to Mount Slamet, it grows well in Wonosobo, Central Java, but cannot beat the growth of other species, and on the contrary it grows dominant species in the experimental garden of Bogor. This plant has a bluish appearance that very attractive for ornamental plants. Selaginella is very attractive as an ornamental plant leaves, given his appearance can be quite diverse. In one species, sometimes there are various shapes and shades of leaves, for example S. ornata, so that in the past this species was divided into several species. Khare (2007) states that in India, S. rupestris is used as an ornamental plant.

Crafts materials. According to de Winter and Jansen (2003) reported several species of Selaginella can be used

as craft material. But in this field of research such a thing is not found and no support from also other libraries. One reason possibly because the habit of Selaginella tend to be brittle and easily broken. Some species are thought to have high chemical levels even curl when dried and not suitable for crafting, such as S. involvens.

Socio-cultural (traditional). In Gabon, Selaginella myosurus (Sw.) Alston is used for rituals or for other cultural aspects (Sassen and Wan 2006). There are no records for the utilization of Selaginella for the purpose of customs in Nusantara. Field observations in several batik shops and museums in Solo, and visits to the library palace of Solo kingdoms did not find any real pattern designs inspired from Selaginella, although there are some designs that are inspired from other ferns.

Other utilization. Other utilization, i.e. as food wrappers is found in Manokwari, West Papua. In these locations there are several species of leafy Selaginella that is wide enough to be used to wrap the sago, fruits, or other crops from the forest or fields.

Medicinal benefit of Selaginella gives opportunity for commercial effort. In Southeast Asia a wide variety of drug materials is exported and imported; Selaginella are bought and sold as drugs, and used either alone or sometimes, mixed. In Indonesia, several Selaginella is marketed as dry herbs or dry powder, both local species such as S. plana and S. willdenowii, and imported species, especially S. tamariscina and S. doederleinii. In Bangkok, Thailand imported S. tamariscina is widely marketed in traditional medicinal shops (Nitta et al. 1980). In Zambia and Zimbabwe, wild collection of S. imbricata threats the sustainability in nature (Cunningham 1993; Golding 2002). In Mexico, the popularity of S. lepidophylla has been resulted in legislation to regulate collection from the wild (Winter and Jansen 2003).

CONCLUSION

Selaginella has been used traditionally to treat wounds and bleeding such as menstruation, uterine disorders and other internal injuries. It is also used as a tonic to improve fitness and to expand life span. Several species of Selaginella are also used as food (raw vegetables), ornamental plants, handicrafts materials as well as socio-cultural and packaging materials. The utilization of Selaginella is very limited compared to the number of species and the potential benefits of the medicine, so it requires further ethnobotanical and phytochemical researches.

ACKNOWLEDGEMENTS

The author like to thank the colleagues who have sent herbarium materials, living materials and ethnobotanical data of Selaginella from outside Java, namely: Perdani C. Wijaya (Jayapura), Yohanes Y. Rahawarin (State University of Papua, Manokwari), Maya Papuangan (Hairun University, Ternate), Dr. Arrijani (Manado State University), Julius Kinho (Forestry Research Institute,


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Manado), Nur Indah Julisaniah and I Gede Mertha (Mataram University), Hartutiningsih M. Siregar (Bali Botanical Gardens), Nur Anis Hidayati (State University of Bangka Belitung), as well as Dr. Djufri (Syiah Kuala University, Banda Aceh). The author also gratefully thanks Ajis Sanjaya (Indonesian Institute of Sciences, Bitung), Edi Junaedi (Kuningan University), and Andik Wijayanto (Bogor Agricultural University) who help to collect materials in Java. The author also thanks the Herbarium Bogoriense which has given the opportunity to observe the collection for this research.

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Authors Index

Anggarwulan E 65 Astirin OP 23, 53, 123 Dharmawan R 1 Fathonah D 17 Fatmawati U 31 Handajani NS 1 Harini M 53 Hastuti D 78 Kuntjoro A 23 Latifa IC 65 Mahajoeno E 131 Maryati KT 72 Mujiyati 59 Mustofa A 105 Nurmiyati 138 Parjanto 84

Priyono E 123 Rahmawati B 131 Ruwaida IP 84 Sajidan 31, 138

Santoso U 117 Setyawan AD 43, 92, 146 Setyono P 78, 123 Sudaryatiningsih C 110 Sugiyarto 17, 38, 72, 138 Suharja 1 Supriyadi 59, 84 Supyani 110 Suranto 31, 78, 105 Sutarno 1, 23, 117 Wardani S 38

A-2

Subject Index

Adenium 78-83 allergy 1-3,7,8 atherosclerosis 53-58, 110 Azospirillum 59-61, 63, 64 Azotobacter 59-61, 63 biomass 9-13,15, 21, 22,35, 37,

48, 51, 65-67, 69, 105, 125

blood creatinine 1-3, 7, 8 body weight 2, 8, 23-29, 54, 117,

119-122 Capsicum annum 9 cashew nut apple extract 105 Central Java 9, 11, 17, 18, 24, 38, 39,

42, 59, 60, 72, 73, 75, 77, 85, 91, 92-103

Cherax quadricarinatus 123-125, 127, 129 chili 9-16, 59-64 chlorophyll 9-16, 18, 62, 66, 69-71,

142 cholesterol 8, 18, 53-58, 117, 131, coagulant 1, 54, 110, 113, 114,

116 Cr heavy metal 31-37 Dieng 17, 18, 22, 148, 150 diversity 29, 35, 41, 43-45, 48-

50, 73, 74, 77, 78-80, 83-96, 99-103, 121, 122, 131-137-147, 150, 153-154

DNA 12, 19, 31, 79, 81, 83-91, 93, 103, 132, 136

dragon fruit 131-137 Durio zibethinus 84, 90, 91 electrophoresis 31-33, 36-42, 73-86,

93-95, 101, 103, 132-134, 139, 145

esterase 14, 38, 39, 41, 42, 92, 94-99, 101, 131, 133-139, 142

ethanol 19, 33, 37, 74, 79, 85, 105, 105-109, 111-113, 133, 139

ethnobotany 146, 148, 152 evolution 43-52, 79, 83, 93, 96,

100, 101-103, 132 fertilizing 9, 11, 59, 67, 71 fixation 112, 14, 15, 59, 61, 69,

133, 139, 143 GA3 17-22, 71 global warming 43-51, 102 growth 9-12, 15-25, 27, 29, 32-

35, 37, 46, 48, 51, 59, 61-71, 79, 95, 99, 103,

105-109, 113, 117, 119-130, 137, 141-142, 145, 152

herbal 146, 148, 150-154 Hylocereus 131-132 hyperglycemic 1, 3, 8 IAA 17-22 isozyme banding patterns 38 isozyme 38-42, 77, 85, 88, 90,

92-103, 131-139, 142145

karyotype 78, 83 kimpul 65, 71, 138-140, 145 koro bean 117 leaf nitrogen 9-11, 14, 15, 69, 71 leucocytes differential count 1 linoleic acid 110, 111, 114-116 linolenic acid 110-116 manure fertilizer 59 microorganism 24, 29, 31-37, 63, 64,

108, 115 morphological characteristic 72-75, 77, 93, 132, 133,

138, 139, 141-145 morphology 17, 18, 39, 40, 42, 44,

48, 69, 72-75,78-81, 83, 90-91, 95, 112, 131-134, 137, 139, 140, 142-144, 153

Mount Merapi 38, 39, 41, 42, 72, 77 Mucuna pruriens 105-108, 117-122 new species 43, 48 New Zealand white 117-122 nitrate reductase 65-69, 71 nitrogen 9-15, 18, 22, 44, 59-71,

105-109, 118, 129 peroxidase 38-42, 92, 94, 95, 99,

102, 138, 139, 143, 145 Pimpinella alpina 17, 18, 20, 22, 49 protein banding pattern 72-83 protein 1, 2, 4, 5-12, 14-15, 19,

20, 22, 24, 25, 27, 28, 31-33, 35-37, 41, 53, 65-69, 71-83, 85, 93, 101, 105, 106, 110, 112, 113, 114, 117-124, 127-130, 138, 145

rabbits 26, 29, 58, 117-122 ramie hay 23-30 RAPD 84-87, 90, 91, 103, 137,

145 ration 117-122 Rhizopus oligosporus 110-116, Rhizopus oryzae 110-116 Salacca zalacca 38, 42, 72, 137

A-3

salak pondoh 38-42, 72-73, 75 saponin 17-22, 147 Selaginella 146-154 shade 65-71, 142, 145, 152 Sonneratia alba 92-101, 103 statistic vital 23 supplementary food 25, 123, 124 Texel sheep 23-29 tofu 110-116 traditional medicine 17, 53, 146, 148, 150,

151, 153 variation 2, 34-38, 40-42, 54, 65,

67-71, 73, 75-79, 81,

83, 84, 87, 90, 95, 97, 101-103, 131, 132, 134, 136, 137, 142, 145

VCO 1-8, 53-58 vitamin C 131-133, 136, 137 water quality 123, 125, 127, 130, white grub 38-42, 72, 73, 75-77 Xanthosoma sagittifolium 65, 67-71, 145 Xanthosoma 65, 71, 138-142, 144,

145 Zymomonas mobilis 105-109

A-4

List of Peer Reviewer

Ahmad Dwi Setyawan Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Edwi Mahajoeno Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Eko Handayanto Faculty of Agriculture, Brawijaya University, Malang 65145, Indonesia

Endang Anggarwulan Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Mahendra Kumar Rai Department of Biotechnology, SGB Amravati University, Amravati 444602, Maharashtra, India

Okid Parama Astirin Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Parjanto Faculty of Agriculture, Sebelas Maret University, Surakarta 57126, Indonesia

Prabang Setyono Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

R. Wasito Faculty of Animal Medicines, Gadjah Mada University, Yogyakarta 55284, Indonesia

Ruben Dharmawan Faculty of Medicines, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Rugayah Botany Division, Research Center for Biology, Indonesian Institute of Sciences, Cibinong-Bogor 16911, West Java, Indonesia

Sajidan Faculty Teacher Training and Education, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Sugiyarto Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Supriyadi Faculty of Agriculture, Sebelas Maret University, Surakarta 57126, Indonesia

Supyani Faculty of Agriculture, Sebelas Maret University, Surakarta 57126, Indonesia

Suranto Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Sutarno Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

A-5

Table of Contents

Vol. 1, No. 1, Pp. 1-52, March 2009 Effect of VCO to leucocyte differential count, glucose levels and blood creatinine Mus musculus Balb/c hyperglycemia and ovalbumin sensitized NOOR SOESANTI HANDAJANI, RUBEN DHARMAWAN

1-8

Biomass, chlorophyll and nitrogen content of leaves of two chili pepper varieties (Capsicum annum) in different fertilization treatments SUHARJA, SUTARNO

9-16

Effect of IAA and GA3 toward the growing and saponin content of purwaceng (Pimpinella alpina) DASIYEM FATHONAH, SUGIYARTO

17-22

Body weight and statistic vital of texel sheep in Wonosobo District by giving the jute waste as an additional woof AGUS KUNTJORO, SUTARNO, OKID PARAMA ASTIRIN

23-30

Protein expression on Cr resistant microorganism using electrophoresis method UMI FATMAWATI, SURANTO, SAJIDAN

31-37

Characterization of white grubs (Melolonthidae: Coleoptera) at salak pondoh agroecosystem in Mount Merapi based on isozymic banding patterns SRI WARDANI, SUGIYARTO

38-42

Review: Effect of global warming on plant evolution and diversity; lessons from the past and its potential recurrence in the future AHMAD DWI SETYAWAN

43-52

Vol. 1, No. 2, Pp. 53-103, July 2009 Blood cholesterol levels of hypercholesterolemic rat (Rattus norvegicus) after VCO treatment MARTI HARINI, OKID PARAMA ASTIRIN

53-58

Effect of manure and NPK to increase soil bacterial population of Azotobacter and Azospirillus in chili (Capsicum annum) cultivation MUJIYATI, SUPRIYADI

59-64

Nitrogen content, nitrate reductase activity, and biomass of kimpul (Xanthosoma sagittifolium) on shade and nitrogen fertilizer variation ISNAINI CHOIRUL LATIFA, ENDANG ANGGARWULAN

65-71

Characterization of white grub (Melolonthidae; Coleoptera) in salak plantation based on morphology and protein banding pattern KRISNANDARI TITIK MARYATI, SUGIYARTO

72-77

Variation of morphology, karyotype and protein band pattern of adenium (Adenium obesum) varieties DWI HASTUTI, SURANTO, PRABANG SETYONO

78-83

Variability analysis of Sukun durian plant (Durio zibethinus) based on RAPD marker ISMI PUJI RUWAIDA, SUPRIYADI, PARJANTO

84-91

Diversity of Sonneratia alba in coastal area of Central Java based on isozymic patterns of esterase and peroxidase AHMAD DWI SETYAWAN

92-103

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Vol. 1, No. 3, Pp. 105-154, November 2009 Activity of Zymomonas mobilis on ethanol products made of cashew nut apple (Anacardium occidentale) with different sources of nitrogen AKHMAD MUSTOFA, SURANTO

105-109

Linoleic and linolenic acid analysis of soybean tofu with Rhizopus oryzae and Rhizopus oligosporus as coagulant CICIK SUDARYATININGSIH, SUPIYANI

110-116

Slaughter weight and carcass of male New Zealand White rabbits after rationing with koro bean (Mucuna pruriens var. utilis) URIP SANTOSO, SUTARNO

117-122

Alternative supplementary biochemic food for growing up the fresh water lobster (Cherax quadricarinatus) EDI PRIYONO, OKID PARAMA ASTIRIN, PRABANG SETYONO

123-130

Variation of morphology, isozymic and vitamin C content of dragon fruit varieties BANATI RAHMAWATI, EDWI MAHAJOENO

131-137

Kimpul (Xanthosoma spp.) characterization based on morphological characteristic and isozymic analysis NURMIYATI, SUGIYARTO, SAJIDAN

138-145

Traditionaly utilization of Selaginella; field survey and literature review AHMAD DWI SETYAWAN

146-158

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Book: Rai MK, Carpinella C. 2006. Naturally occurring bioactive compounds.

Elsevier, Amsterdam. Chapter in book: Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization, biogeography,

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Abstract: Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th

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Proceeding: Alikodra HS. 2000. Biodiversity for development of local autonomous

government. In: Setyawan AD, Sutarno (eds) Toward mount Lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in Java island. Sebelas Maret University, Surakarta, 17-20 July 2000. [Indonesia]

Thesis, Dissertation: Sugiyarto. 2004. Soil macro-invertebrates diversity and inter-cropping plants

productivity in agroforestry system based on sengon. [Dissertation]. Brawijaya University, Malang. [Indonesia]

Information from internet: Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake

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Activity of Zymomonas mobilis on ethanol products made of cashew nut apple (Anacardium occidentale) with different sources of nitrogen AKHMAD MUSTOFA, SURANTO

105‐109

Linoleic and linolenic acid analysis of soybean tofu with Rhizopus oryzae and Rhizopus oligosporus as coagulant CICIK SUDARYATININGSIH, SUPIYANI

110‐116

Slaughter weight and carcass of male New Zealand White rabbits after rationing with koro bean (Mucuna pruriens var. utilis) URIP SANTOSO, SUTARNO

117‐122

Alternative supplementary biochemic food for growing up the fresh water lobster (Cherax quadricarinatus) EDI PRIYONO, OKID PARAMA ASTIRIN, PRABANG SETYONO

123‐130

Variation of morphology, isozymic and vitamin C content of dragon fruit varieties BANATI RAHMAWATI, EDWI MAHAJOENO

131‐137

Kimpul (Xanthosoma spp.) characterization based on morphological characteristic and isozymic analysis NURMIYATI, SUGIYARTO, SAJIDAN

138‐145

Traditionaly utilization of Selaginella; field research and literature review AHMAD DWI SETYAWAN

146‐158

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