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"ABSTRAKBlood lipid is one of the factors which might cause atheroschlerosis especially if hyperlipidemia occurs (blood total cholesterol > 200 mg/dl and triglyceride a 150 mg/di). Consumption of foods with !ow lipid content can prevent hyperlipidemia. Soybeans and products made from them, such as soybean tempe, were reported to have a hypolipidemic effect. Those foods can decrease the human blood cholesterol and trigyceride level.Ergosterol is a sterol in cell membranes of microorganism, especially fungi and was found in the hyphae and spores of fungi. Ergosterol in Rhizopus spp. has not yet been studied, while Rhizopus moulds are used in the fermentation of soybean cotyledons into soybean tempe, a product which is widely consumed by all strata of the Indonesian community. The University of Indonesia Culture Collection (UICC) has a number of Rhizopus species, which are most isolated from Indonesian soybean tempe. Morphologically two groups of Rhizopus moulds can be distinguished according to length of hyphae, measurements of the sporangiospores, and growth temperatures. The groups are macro-Rhizopus and micro-Rhizopus.This thesis consists of two parts. Part i is on the ergosterol content of the dry biomass of each UICC Rhizopus strain and in the soybean tempe. In Part II is studies the potency of ergosterol to decrease the blood lipid level in rats The studies were carried out at the Laboratory of Microbiology and Laboratory of Plant and Animal Reproduction Departement of Biology, Faculty of Mathematics and Natural Sciences University of Indonesia; at the Laboratory of Clinical Chemistry, Medical Technologies, of The Ministry of Health; Jakarta Ill College; and also Laboratory of Chemical Analysis LIPI Bandung, on June 2006 - April 2007.The aim of this study was to analyse the ergosterol content in the dry biomass of Rhizopus and in the soybean tempe obtained. Five UICC Rhizopus strain respectively UICC 52 (Rh. arrhizus), UICC 128 (Rh. oryzae), UICC 531 (Rh. microsporus var. microsporus), UICC 550 (Rh. microsporus var. oligosporus), and UICC 551 (Rh. microporus var. oligosporus); and to know the potency of ergosterol to decrease the blood lipid level in rats which were fed with the respective biomass of the five Rhizopus spp. and compared with the rats fed with the soybean tempe using the five mentioned Rhizopus spp. as inoculum the respective fermentation processes. The rats (Rattus norvegicus strain Wistar) were obtained from the Laboratory of Animal Drug Testing, Gunung Sindur, Bogor. The rats were males and 2 - 3 months old. Analysis of the ergosterol content was carried out using HPLC. The Diagnostic Liquicolour Cholesteroloxidase 4-aminophenazone (CHOD-PAP) method was used for the measurement of blood total cholesterol level, and Gliserophosphoxidase Paraaminophenazone (GPO-PAP) for measurement of triglyceride level of the rats. Soybean tempe was prepared using an inoculum of each Rhizopus strain, and then powdered. The rats were given palm oil orally during 7 days to boost up the blood lipid level. Then they were fed with the fungal biomass or with the soybean tempe powder.The results obtained were : the ergosterol content in the dry biomass of the five UICC Rhizopus spp. studied varied between (278,8 - 673,17) mg/g, while in the soybean tempe (3,3 - 8,1) mg/g. Both were able to decrease the blood lipid level in rats. However, soybean tempe gave a higher decrease (58 %) in comparison with the results of the dry biomass of the Rhizopus spp. (51.6 %)."

"SUMMARY This research was carried out to study the diversity of Rhizopus in Irian Jaya. Tempe samples were collected from regions in Irian Jaya. The aim of this research was to Isolate and Identify Rhizopus spp. from traditionally made tempe, to make usar from the Rhizopus sp. isolates, and to produce tempe from the chosen usar. Isolation was done using direct plating method. Nineteen Rhizopus spp. isolates have been isolated from soybean tempe collected from several regions in Irian Jaya. Examination of their morphological characteristics, growth temperature, and spore ornamentation, showed that eighteen isolates belong to the Rh. microsporus group: two isolates were Rh. microsporus var. oligosporus, sixteen isolates were Rh. microsporus var. chinensis. One isolate was Rh. oryzae. The isolates were examined for their ability to produce usar. Out of the nineteen isolates that were tested, only seven produced good usar (inoculum): six isolates from Rh. microsporus var. chinensis, and one of Rh. oryzae. The amount of spores before and after inoculum production were determined by Total Plate Count (TPC) method. The average of viable spores from all isolates before the inoculum production was ((2.5-3.2)x14' cetllmly, and after the inoculum production was ((1.2-1.8)x104 celllg). The usars (Inocula) from seven Isolates were examined to produce tempe. The Rh. oryzae inoculum produced better tempe than the Rh. microsporus var. chinensis Inoculum based on colour, texture, aroma and shelf-life."

"Inoculum for making tempe is usually made from the mould Rhizopus oryzae or Rhizopus oligospons. Large scale use of an inoculum from one source only (LKN-LIPI) might threaten the biodiversity of microorganisms in traditionally made inoculum. The potential use of other Rhizopus moulds as inoculum for tempe production should be examined. Rh. microsporus var. rhizopodifonnis UICC 520 isolated from Hibiscus from Manado and Rh. microsporus var. chinensis UICC 521 isolated from tempe from Aceh were examined for their potentials to produce tempe.Rhizopus has also been known to be able to transform antioxidants such as isoflavone in tempe. The potential of Rhizopus microsporus v. Tiegh. var. chinensis (Saito) UICC 521 to transform glucoside isoflavones in soybean tempe has been evaluated.The results showed that Rh. microsporus var. rhizopodiformis UICC 520 has little potential as a tempe inoculum. Rh. microsporus var. chinensis UICC 521, however, is very potential as an inoculum to produce good tempe and produces abundant spores ((3-10)x106 cell/ml) on rice substrate.Two types of aglycon isoflavone, daidzein and genistein, were detected by TLC separation. The total isoflavone concentration during fermentation period of 24, 48, 72 hours was determined using HPLC. At 24 hours the concentration was 0.142 mg/g tempe, while at 48 and 72 hours the concentration were 0.181 and 0.135 mg/g tempe, respectively. The highest total isoflavone concentration was detected at 48 hours fermentation period. Hence, there was an increase of aglycon isoflavones up to 6000% after transformation of isoflavones in soybean by Rh. microsporus var. chinensis UICC 521 when compared to the initial concentration before transformation."

"Thesis Supervisors: Dr. Suyanto Pawiroharsono; Prof. Dr. Indrawati GandjarSUMMARYFood deterioration is often due to lipid oxidation, excluding bacterial andenzymatic spoilage. The end-products of lipid oxidation, such as aldehydes,ketones, and alcohols are responsible for unacceptable off-tiavors and off-odors in food. Lipid oxidation can be inhibited by antioxidants.Soybean tempe is the most popular indigenous fermented food inIndonesia. Soybeans are known to contain isotiavones. Four major forms areknown respectively as acetylglycosides, malonylglycosides, glycosides, andaglycones.Tempe were produced from soybean fermentation by Rhizopus oryzaeUICC 524 and Rhizopus microsporus var. chinensis UICC 521. The tempesamples were extracted with methanol and the extraction defatted with hexa»ne. The isoflavone aglycones were isolated using column chromatography,and then anaiyzed using a gradient elution reverse phase of high-pressureliquid chromatography (HPLC). After HPLC analysis, isotiavone aglyconeswere evaporated to dryness and added to soybean oil at the 100, 200, 300,iii ' and 400-ppm concentration in test tubes, then heated at 170°C for 30 minu-tes. The oxidation of soybean oil was measured using the thiobarbituric acid(TBA) test. The result, called thiobarbituric acid reactive substances(TBARS) value, was expressed as pmolll and compared to the syntheticantioxidant, buthylated hidroxytoluene (BHT), at the same concentration.The profile of isoflavone aglycones isolated contains daidzein andgenistein. No factor-2 (6,7,4'-trihidroxyisoflavone) and glycitein were detec-ted. Daidzein resulted from biotransformation of daidzin was dominant inboth tempe samples. The isoflavone biotransformation was much greater byR. microsporus var. chinensis UICC 521 than by R. oryzae UICC 524, exceptfor the 24 hours incubation. After 72 hours of incubation, the total isoflavoneaglycones in tempe using R. microsporus var. chfnensis UICC 521 was721.6 pglg and when using R. oryzae UICC 524, 268.2 p.glg.The oxidized soybean oil without any antioxidants had a TBARS value of327.32 1 20.31 pmol/1. Addition of the antioxidants showed a decreasedTBARS value following increasing concentration for both. For concentrationuntil 300 ppm, the TBARS values of oxidized soybean oil added with isofla-vone aglycones were greater than when added with BHT, respectively55.40 zl: 2.77 pmol!! and 45.20 i 2.63 pmolll. However at concentration of400 ppm, the TBARS values of oxidized soybean oil added with isoftavoneaglycones and added with BHT did not show a significant difference.ix + -51 pp; 6 append; 5 plates; 3 tables.Bilb 35 (1964-1999).iv"

"ABSTRAKProtease asam yang dihasilkan oleh kapang memiliki peranan penting dalam industri pangan dan farmasi. Rhizopus spp. merupakan salah satu jenis kapang yang memiliki aktivitas proteolitik dan tidak menghasilkan toksin. Kemampuan Rhizopus dalam menghasilkan enzim proteolitik bervariasi baik antar spesies maupun antar galur dalam spesies yang sama. Untuk memperoleh enzim dengan aktivitas proteolitik yang tinggi, perlu diperhatikan faktor lingkungan yang mempengaruhi aktivitasnya. Tingkat keasaman (pH) dan suhu merupakan faktor lingkungan yang sangat menentukan aktivitas enzim proteolitik. Setiap enzim memiliki pH optimum yang khas, yaitu pH lingkungan yang menyebabkan aktivitasnya maksimum. Enzim yang dihasilkan oleh mikroorganisme juga mempunyai suhu optimum tertentu untuk mengkatalisis suatu reaksi enzimatis. Indonesia dikenal kaya akan keanekaragaman hayati kapang. Untuk memanfaatkan keanekaragaman hayati kapang indigenous Indonesia, perlu dilakukan penelitian untuk mengetahui potensi produksi protease asam ekstra selular dari Rh. microsporus v. Tiegh. var. rhizopodiformis (Cohn) Schipper & Stalpers dan Rh. microsporus v. Tiegh. var. chinensis (Saito) Schipper & Stalpers koleksi University of Indonesia Culture Collection (UICC). Penelitian ini bertujuan mengetahui potensi biakan Rh. microsporus var. rhizopodiformis UICC 519, 520, dan Rh. microsporus var. chinensis UICC 521 dalam menghasilkan enzim proteolitik, dan penentuan waktu fermentasi, pH, dan suhu optimum aktivitas proteolitik pada kapang terpilih. Aktivitas proteolitik semi kuantitatif dari ketiga biakan dipelajari berdasarkan kemampuannya membentuk zona bening pada medium 4% (b/v) Skim Milk Agar (SMA) dengan cara pencawanan (plating) spora tunggal pada suhu ruang selama 28-29 jam. Filtrat kultur fermentasi Rh. microsporus var. rhizopodiformis UICC 520 yang diekstraksi dari medium dedak padi digunakan untuk uji aktivitas proteolitik. Uji aktivitas proteolitik kuantitatif dari filtrat kultur dilakukan dengan mengukur jumlah tirosin yang dihasilkan dari hidrolisis substrat kasein pada suhu suhu 37°C ± 2°C selama 30 menit. Setiap labu yang berisi medium fermentasi steril diinokulasi dengan 1 ml suspensi spora Rh. microsporus var. rhizopodiformis UICC 520 yang berisi 3,9-4,6 x(10 pangkat 5)koloni/ml. Medium fermentasi selanjutnya diinkubasi pada suhu ruang (26--30°C) tanpa pengocokan. Protease kasar diekstraksi dari medium fermentasi pada 0, 24, 48, 72, 96, dan 120 jam untuk mengetahui waktu fermentasi optimum. Untuk menentukan pH optimum aktivitas proteolitik, kasein sebagai substrat enzim dilarutkan dalam larutan dapar hingga diperoleh pH 2,0; 3,0; 4,0; 5,0; 6,0; 7,0; dan 8,0. Untuk menentukan suhu optimum aktivitas proteolitik, sampel direaksikan dengan substrat kasein 0,7% (b/v) dalam dapar glisin-HCI pH 3,0 dan dinkubasikan pada suhu 35, 40, 45, 50, dan 55°C. Berdasarkan diameter zona bening yang terbentuk dari pertumbuhan spora tunggal pada medium SMA dapat disimpulkan bahwa Rh. microsporus var. rhizopodiformis UICC 520 memiliki kemampuan menghasilkan protease lebih cepat dibandingkan Rh. microsporus var. rhizopodiformis UICC 519 dan Rh. microsporus var. chinensis UICC 521 dalam waktu yang sama (28-29 jam). Aktivitas proteolitik Rh. microsporus var. rhizopodiformis UICC 520 dengan medium fermentasi dedak padi mencapai maksimum, yaitu sebesar 0,0944 U/ml pada inkubasi selama 72 jam. Peningkatan aktivitas proteolitik yang cepat diikuti dengan penurunan pH filtrat kultur. Enzim proteolitik dalam filtrat kultur Rh. microsporus var. rhizopodiformis UICC 520 aktif pada kisaran pH substrat 2,0-6,0 dan memiliki dua puncak aktivitas, yaitu puncak tertinggi pada substrat kasein dengan pH 2,0-3,0 (0,0772--0,0912 U/ml) dan puncak aktivitas kedua yang lebih rendah dari puncak pertama, yaitu pada pH substrat 5,0-6,0 (0,0603-0,0649 U/ml). Enzim proteolitik dari filtrat kuitur Rh. microsporus var. rhizopodiformis UICC 520 aktif pada kisaran suhu 35--50°C. Aktivitas proteolitik tertinggi diperoleh pada suhu 45° C.

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