EIIEHJIF- THESIS i "I {Mam in. r-z LIBRARY . Michigan Sam 3.“? University my This is to certify that the thesis entitled BIOCHEMICAL CHANGES IN THE ONCOM FERMENTATION OF PEANUT PRESS CAKE presented by Dedi Fardiaz has been accepted towards fulfillment of the requirements for __EIL._D__degree in mm. Major professor Date Oct. 27, 1980 0-7639 4 raunhss -' A “ileum ' .l "53",,”5.» '.l_ ;- ,I‘f. H OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to ram» charge from circulation recon BIOCHEMICAL CHANGES IN THE ONCOM FERMENTATION OF PEANUT PRESS CAKE BY Dedi Fardiaz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1980 ABSTRACT BIOCHEMICAL CHANGES IN THE ONCOM FERMENTATION OF PEANUT PRESS CAKE BY Dedi Fardiaz The objective of this investigation was to study some of the biochemical changes that take place in peanut press cake during fermentation by Rhizopus oligosporus and/ or Neurospora sitophila. Peanut press cake was prepared by pressing peanut seeds under a hydraulic Carver press. The cake was soaked overnight in acidified water (pH 4.5) in a refrigerator, and then washed several times. Afterwards, 1% of tapioca flour was added to the cake mass and the mixture was auto- claved at 1210C for 30 minutes. The hot mass was cooled to room temperature, drained, and inoculated with the fol- lowing mold cultures: (1) g. sitophila ATCC 14151, (2) 3. oligosporus ATCC 22959, (3) Neurospora gp. isolated from Indonesian oncom, (4) A mixed culture of (l) and (2), and (S) A mixed culture of (2) and (3). The cake was incubated at 30°C for 72 hours. At 6 hour intervals samples were drawn, freeze dried, pulverized, and analyzed for pH, free Dedi Fardiaz fatty acids, oligosaccharides, soluble protein, electropho- retic pattern of soluble protein, phytic acid, PER, di- gestibility, and carotenoids. After 36 hours of fermentation mycelia of B. gliggf sporus completely covered the peanut press cake into a com- pact semisolid product, while it took 48 hours for E. sito- phila to produce the same product. The pH gradually in- creased from about 5.1 to 7.2 in 72 hours. Approximately 40% of the peanut oil was hydrolyzed by 5. oligosporus, while only 10% by g. sitophila after 72 hours of fermenta- tion. During fermentation with either 3. oligosporus or y. sitophila, the sucrose, raffinose, and stachyose contents of peanut press cake decreased. Soluble protein of oncom increased after fermentation, and electrophoresis showed that the protein was hydrolyzed to smaller molecular weight components. At 72 hours of fermentation, about 95% of the phytic acid of the peanut press cake was hydrolyzed by 3. oligosporus, while only about 50% by g. sitophila. Only about 60-65% of the phytic acid was hydrolyzed com- pletely to inorganic phosphorus and free inositol, while the remaining phytic acid was hydrolyzed partially to other inositol phosphate forms. Fermentation did not change the protein content, apparent digestibility, and protein quality of peanut press cake. However, incorporation of 10% of sesame protein raised the PER of peanut press cake from 1.51 to 2.11. The Dedi Fardiaz mycelia and conidia of §.'sitophila contained phytofluene, neurosporene, fi-zeacarotene, 7-carotene, and B-carotene. However, the concentration of these carotenoids in oncom was too small to increase the vitamin A value of oncom. In general, Neurospora sp. isolated from Indonesian oncom was not very different from E. sitophila ATCC 14151 in its ef- fect in the oncom fermentation. Using a mixed culture of N. sitophila and B. oligosporus resulted in oncom which was low in both oligosaccharides and phytic acid. To my wife, ANDI, and my daughter, MIRI ACKNOWLEDGMENTS The author would like to thank his major professor, Dr. P. Markakis, for his encouragement and guidance through- out the course of this study and assistance in the prepara- tion of this manuscript. Appreciations are also expressed to Dr. E. S. Beneke of the Department of Botany and Plant Pathology, Dr. K. E. Stevenson, Dr. J. N. Cash, and Dr. W. Chenoweth of the Department of Food Science and Human Nutrition for their helpful suggestions as members of the graduate com- mittee. This study would not have been possible without the financial support provided by MUCIA-AID - Indonesian Higher Agricultural Education Project. Finally, the author is deeply grateful to his parents, his wife, Andi, his daughter, Miri, and his close friends for their support, encouragement, and understanding. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . Viii INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 4 Molds in oncom preparation . . . . . . . . . . 5 Biochemical Changes During Fermentation of Foods . . . . . . . . . . . . . . . . . . . 7 Changes in Lipids . . . . . . . . . . . . 7 Changes in Carbohydrates . . . . . . . . . 8 Changes in Proteins . . . . . . . . . . . 10 Changes in Other Components . . . . . . . 11 Safety of Fermented Foods . . . . . . . . . . . 12 MATERIALS AND METHODS . . . . . . . . . . . . . . . 14 Preparation of Oncom . . . . . . . . . . . . . 14 pH Determination . . . . . . . . . . . . . . . 15 Proximate Analysis . . . . . . . . . . . . . . 15 Analysis of Free Fatty Acids . . . . . . . . . 15 Extraction . . . . . . . . . . . . . . . . 15 Esterification . . . . . . . . . . . . . . 16 Gas Chromatography Conditions . . . . . . 17 Identification of the Chromatogram Peaks . . . . . . . . . . . . . . . . . . 18 Analysis of Oligosaccharides . . . . . . . . . 19 Extraction . . . . . . . . . . . . . . . . 19 Qualitative Analysis b Circular TLC O O O O O O O O O O O O O O O O O O O 2 0 Quantitative Analysis by HPLC . . . . . . 22 iv Page Analysis of Soluble Protein . . . . . . . . . . . 23 Soluble Protein Determination . . . . . . . 23 Electrophoretic Pattern of Soluble Protein . . . . . . . . . . . . . . . . . . 24 Phytic Acid Determination . . . . . . . . . . . . 26 Separation of Inositol Phosphates . . . . . . . . 30 Column Chromatography . . . . . . . . . . . 30 Phosphorus Determination of Chroma- tographic Fractions . . . . . . . . . . . . 30 Biological Evaluation of Protein Quality . . . . 34 Protein Efficiency Ratio (PER) . . . . . . . 36 Apparent Diet Digestibility . . . . . . . . 37 Apparent Nitrogen Digestibility . . . . . . 38 Analysis of Carotenoids . . . . . . . . . . . . . 38 Extraction . . . . . . . . . . . . . . . . . 38 Separation and Identification . . . . . . . 39 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 41 Growth of Molds and Changes in pH During Oncom Fermentation . . . . . . . . . . . . . . . 41 Identification of Free Fatty Acids . . . . . . . 44 Fatty Acids Liberated During Fermentation . . . . 45 Analysis of Oligosaccharides by Circular TLC and HPLC . . . . . . . . . . . . . . . ... . 55 The Soluble Protein of Oncom . . . . . . . . . . 64 Degradation of Phytic Acid in Oncom . . . . . . . 66 Biological Evaluation of Protein Quality . . . . 75 Carotenoids of N. sitophila . . . . . . . . . . . 78 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . 84 REFERENCES . . . . . . . . . . . . . . . . . . . . . . 87 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . 94 9. LIST OF TABLES Essential amino acids in reference and peanut proteins, expressed as g per 16 g N Protein quality evaluation diet (AOAC, 1975) . . . . . . . . . . . . . . . . . Free fatty acids content of oil extract- ed from oncom during fermentation (mg/g) Sucrose, raffinose, and stachyose con- tents of uninoculated cake and oncom prepared with various mold cultures . . Free inositol and inorganic phosphorus released from phytic acid during fermen- tation of oncom by 5. oligosporus . . . Ratio of phosphorus to inositol in frac- tions obtained from Dowex 1x8 (Cl’) chromatography . . . . . . . . . . . . Composition of peanut press cake, oncom, and fermented mixtures of peanut press cake and sesame flour . . . . . . . . . Protein efficiency ratio (adjusted to casein PER = 2.50) and digestibility of casein, peanut press cake, oncom, and fermented mixtures of peanut press cake and sesame flour . . . . . . . . . . . . Essential amino acids in peanut (Arachis hypogaea) and sesame seed (Sesamum indi- cum) proteins,expressed as g per 16 g N vi Page 11 37 52 62 72 75 76 77 78 Table Page 10. Identification of carotenoids in N. sitophila . . . . . . . . . . . . . . . . . . 80 11. Concentration of carotenoids found in mycelia and conidia of N. sitophila ATCC 14151 and oncom sample . . . . . . . . . . 83 Al. Linear regression equations of the methyl ester standard curves . . . . . . . . . . 94 A2. Recovery of phytic acid added to the control sample . . . . . . . . . . . . . . . . . 95 vii Figure 12. 13. LIST OF FIGURES Genus Neurospora . . . . . . . . . . . Genus Rhizopus . . . . . . . . . . . . Diagram of circular TLC . . . . . . . Standard curve for soluble protein determination . . . . . . . . . . . . Standard curve for iron determination Apparatus used for evaporating liquid in tubes . . . . . . . . . . . . . . . Standard curve for phosphorus deter- mination O O O O I O O I O O O O O O 0 Standard curve for inositol determi- nation . . . . . . . . . . . . . . . . oncom I O O O O O O O O O O O O O O 0 Changes in pH during oncom fermenta- tion 0 O I O O O O O O O O O O O O O O GLC elution pattern of the methyl es- ters of free fatty acids extracted from oncom fermented for 72 hours by N. oligosporus . . . . . . . . . . . . GLC elution pattern of the methyl es- ters of free fatty acids of a standard mixture I O O O O O O O O O O O O O 0 Mass spectra of (A) methyl myristate, (B) methyl pentadecanoate, and (C) me- thyl palmitate . . . . . . . . . . . . viii Page 21 25 29 31 33 35 42 43 46 47 48 Figure 14. 15. l6. l7. l8. 19. 20. 21. 22. 23. 24. 25. 26. Mass spectra of (A) methyl heptadeca— noate, (B) and (C) unseparated mixture of methyl stearate, methyl oleate, and methyl linoleate . . . . . . . . . . . . Mass spectra of (A) methyl linolenate, and (B) methyl arachidate . . . . . . . Mass spectra of (A) methyl behenate, and (B) methyl lignocerate . . . . . . . Circular TLC of oligosaccharides ex- tracted from fermented and unfermented peanut press cake. (1) Glucose, Fructo- se, (2) Sucrose, (3) Melibiose, (4) Raffinose, and (5) Stachyose . . . . . . HPLC Chromatogram of a standard mixture consisting of oligosaccharides, glyce- rol, and myo-inositol . . . . . . . . . Standard curves for oligosaccharides and myo-inositol . . . . . . . . . . . . . . HPLC chromatograms of (A) uninoculated cake, (B) oncom fermented for 72 hours by N. oligosporus, and (C) oncom fermen- ted for 72 hours by N. sitophila . . . . Structure of stachyose . . . . . . . . . Soluble protein of fermented and unfer- mented peanut press cake . . . . . . . . Electrophoretic pattern of soluble pro- tein. (A) Uninoculated cake, (B) Oncom fermented for 72 hours by N. sitophila, (C) Oncom fermented for 72 hours by N. oligosporus, and (D) Molecular weight peotein standard . . . . . . . . . . . . Phytic acid content of fermented and un- fermented peanut press cake . . . . . . Inorganic phosphorus content of fermented and unfermented peanut press cake . . . Elution pattern of inositol phosphates on Dowex 1x8 (Cl') column . . . . . . . . . ix Page 49 50 51 58 59 60 61 63 65 67 69 70 73 Figure Page 27. Scheme of dephosphorylation of phytic acid by phytase (Tomlinson and Ballou, 1962) O o o o o o o o o o o o I o o o o o o o o 74 28. Absorption spectra of phytofluene, fl -zeacarotene, and B-carotene ex- tracted from N. sitophila . . . . . . . . . . . 81 29. Absorption spectra of neurosporene, and 7-carotene extracted from N. sitophila . . . . . . . . . . . . . . . . . 82 INTRODUCTION Fermentation is one of the oldest methods of prepa- ration and preservation of foods. Throughout the centuries, it has been and continues to be one of the most important methods for preparing and preserving foods. In many parts of the world, especially Southeast Asia, foods prepared by fermentation are important components of diets. In general, fermented foods have some characteris- tic flavor, aroma, appearance, or consistency which make them more attractive to the consumer than the raw ingre- dients. Whitaker (1978) mentioned that some of the advan- tages of fermented foods were: preservation, improvement of texture, color, flavor and aroma, solubilization, digesti- bility, nutritional improvement, less cooking, and removal of toxic substances. Among these advantages, flavor im- provement may be one of the most important contributions of the fermentation. This is especially true for the South- east Asian whose diet is rather flat as it mainly consists of rice and vegetables. Besides imparting flavor, in many cases fermented foods make important contributions to the diet as sources of protein, calories, and some vitamins. Like in all other countries of Southeast Asia, fer- mented foods are very important in Indonesia. Some of the fermented foods produced can be catagorized as protein-rich meat substitutes. Steinkraus (1978) classified the meat analogues of Indonesia into four general types: tempeh ke- dele, oncom, tempeh bongkrek, and tempeh gembus. These products are eaten in several forms. They may be sliced, dipped in a salt brine, and fried in vegetable oil to yield a golden brown, crisp product. They may be added to soups, and they may be eaten with soy sauce. Tempeh kedele is made by fermenting dehulled par- tially cooked soybeans with molds, chiefly Rhizopus oligo- sporus (Hesseltine and Wang, 1967; van Veen and Steinkraus, 1970). Oncom is made by fermenting partially cooked peanut press cake with either Neurospora sitophila (orange or red oncom) or N. oligosporus (white oncom) (van Veen gt a}., 1968). Tempeh bongkrek is similar to oncom, except that the raw material is coconut press cake (van Veen and Stein- kraus, 1970). Tempeh gembus is similar to tempeh kedele, except that the raw material is the soybean residue remain- ing from the manufacture of soybean milk or soybean curd (Steinkraus, 1978). Traditional food fermentations are characterized by their simplicity and rapidity. Since the fermentation pro- cess is usually labor intensive, and does not require a high and expensive technology level, it is entirely appro- priate for developing countries like Indonesia. In order to produce highly acceptable nutritious fermented foods at low prices, the process should be modified or improved using an intermediate technology. This goal can only be achieved if the biochemistry of the fermentation process is fully understood. Among the Indonesian fermented foods, oncom received little scientific attention. This is the reason why oncom was chosen in this study. LITERATURE REVIEW Oncom (pronounced ontsom) is fermented peanut press cake. Itis very popular in Western Java (van Veen gt ai., 1968) where it has been prepared and consumed for centuries. Oncom can be used in soups or fried in vegetable oil for high—protein snacks. Oncom is prepared from peanut press cake called bungkil (Hesseltine and Wang, 1967). Two types of cake may be used, the commercial press cake which contains small amount of oil, and the village product which contains con- siderable amount of oil. In oncom preparation, the peanut press cake is first broken up by hand or with a knife, soaked in water overnight, washed, and pressed to remove excess water and oil. The cake mass is steamed and pressed into the form of flat cakes. The flat cakes are placed in a bamboo tray, inoculated with dry oncom from an earlier preparation, and covered with banana leaves. After two or three days the molds have grown and the oncom is ready for consumption. The finished oncom contains approximately 70% moisture, 3-9% oil, 20-30% crude protein, about 4% carbohy- drate, 1% ash, and.2% fiber (van Veen g: ai., 1968). Molds in Oncom Preparation Two different molds are involved in oncom prepara- tion. N. sitophila is used to prepare an orange or red on- com, while B. oligosporus is used to prepare a white oncom. Traditionally, the people in Indonesian villages use small dry pieces of oncom from a previous fermentation as inocu- lum. Sometimes inoculation is not necessary, since the at- mosphere in the fermentation room has been already contami- nated by the mold spores. This traditional practice can lead to contamination by undesirable microorganisms; there- fore, pure culture fermentation should be introduced to on- com producers. N. sitophila, one of several known species of the genus Neurospora, is referred to commonly as the red bread mold as a result of its prolific formation of orange to red conidia and mycelia. It is also known as the bakery mold, because it frequently infests bakeries and causes consider— able damage (Alexopoulos, 1962). The genus Neurospora can be distinguished from other genera in the family of Moni- liaceae by the presence of budding conidia which form tree- like heads as shown in Figure 1. Furthermore, distinguish- ing characteristics of N. sitophila are: (l) septate myce- lium which later may break up into cells, (2) loose network of aerial, long stranded mycelium, (3) aerial hyphae bear- ing many ovate, pink to orange-red budding conidia, which form branched chains, and are found near the top of the BUDDING CONIDIA Figure l. Genus Neurospora. SPORANGIUM —. 55:43:. . sronmenospones 5. ._-‘.';'.'},,';- ..x“*; .3 40- 4‘ APOPHYSIS COLUMELLA SPORANGIOPHORE STOLON RHIZOID Figure 2. Genus Rhizopus. (Frazier and Westhoff, 1978). oligosporus is one species of the genus Rhizopus summarized that (1) plant 5. (Figure 2). Frazier and Westhoff (1978) the distinguishing characteristics of Rhizopus are: nonseptate, (2) stolons and rhizoids, often darkening with age, (3) sporangiospores arise at the nodes, where rhizoids also are formed, (4) sporangia are large and usu- ally black, (5) hemispherical columella and cup-shaped apo- physis (base to the sporangium), (6) abundant cottony myce- lium which may fill the container, e.g. a petri dish, and (7) no sporangioles. Biochemical Changes DuringfiFermentation of Foods In food fermentations, food acts generally as a sat- isfactory medium for the growth of a variety of microorgan- isms. The biochemical changes taking place during fermen- tation are usually due to the activity of enzymes produced by microorganisms or enzymes inherent to the food (Pederson, 1971). However, microbial enzymes are more important than the inherent enzymes especially in fermented foods that un- dergo heat treatment prior to the fermentation process. These enzymes act upon plant constituents and presumably enhance the digestibility of raw materials. It has been shown that soluble solids increase during tempeh fermenta- tion (Steinkraus 33 al., 1960, 1965; van Buren 23 31., 1972). Changes in Lipids Numerous species of molds have been reported to produce lipolytic enzymes. 5. oligosporus is one of them that produces a very strong lipase activity (Wagenknecht 33 al., 1961; Alford gt 21., 1964), while N. sitophila produces lipase of weak activity (Beuchat and Worthington, 1974). Generally speaking, peanuts are considered a rich source of oil (44-56%), while peanut press cake from the village in Western Java, Indonesia contains 6-20% oil (van Veen 23 a1., 1968). The oil is composed of mixed glycer- ides of approximately 20% saturated and 80% unsaturated fatty acids (Cobb and Johnson, 1971). The major saturated fatty acids are palmitic (8.4-14.0%), stearic (l.8-3.2%), arachidic (l.0-l.7%), behenic (1.7-3.8%), and lignoceric (0.5-2.6%); whereas, the major unsaturated fatty acids are oleic (33.3-61.3%), linoleic (18.5-47.5%), and ll—eicose- noic (0.7-2.3%). It is likely that if grown in peanut press cake, both N. oligospgrus and N. sitophila will alter lipid com- ponents of the peanut. Perhaps, the most notable changes in lipids during fermentation is accumulation of free fatty acids resulted from hydrolysis of triglycerides by lipase. It was demonstrated by Sudarmadji and Markakis (1978) that the total free fatty acid content of tempeh increased from 0.04% to 10.68% after 90 hours of fermentation at 32°C by N. oligosporus. Changes in Carbohydrates In fermentation, carbohydrate splitting enzymes are important in providing substrates for the growth of micro- organisms (Whitaker, 1978). Addition of 1% tapioca to peanut press cake was shown to be beneficial for mold growth. Without it, the mold growth was slow and flavor development was poor (van Veen gt gt., 1968). Peanut press cake contains about 14-20% carbohydrates which are com- prised largely of cellulose and simple oligosaccharides. The sugar content of peanut cultivars is highly variable. Glucose, fructose, and galactose are present in small quan- tities, whereas sucrose is the most abundant sugar and va- ries from 2.9 to 6.4% depending upon genotype (Newel gt gt., 1967). The raffinose and stachyose contents of peanuts were reported to range from less than 0.1 to 0.3% and less than 0.1 to 0.5%, respectively (Hymowitz gt gt., 1972). These two oligosaccharides are considered primarily respon- sible for flatulence, as they are not hydrolyzed in the small intestine, but they are subject to microbial decompo- sition, with production of gas, in the large intestine (Rackis gt gt., 1967; Murphy, 1969). Shallenberger gt_gt. (1967) reported a marked de- crease in stachyose content during tempeh fermentation, while Sugimoto and van Buren (1970) showed that treatment of soy milk with an enzyme preparation from Aspergillus saitoi completely decomposed all the oligosaccharides to their constitutive monosaccharides. Likewise, Mital and Steinkraus (1975) demonstrated that the lactic fermentation reduced the raffinose and stachyose contents of soy milk. It is likely that microorganisms used in food fermentation which are capable of producing invertase and mgalactosidase 10 will hydrolyze raffinose and stachyose. Therefore, the fermentation process may help to reduce the flatulence characteristics of certain foods. Changes in Proteins In most fermented high protein foods, proteolysis is a major factor in the changes in texture and flavor (Whitaker, 1978). The presence of proteolytic enzyme sys- tem in the tempeh fermentation was demonstrated by Wang and Hesseltine (1966). In general, most amino acids either de- clined slightly or were unchanged as tempeh fermentation progressed (Smith gt_gt., 1964; Stillings and Hackler, 1965; Wang gt gt., 1968). However, free amino acids increased markedly in the fermented product (Stillings and Hackler, 1965). Since in general, fermentation of oilseeds showed little change in protein content except for solubility (Beuchat, 1976), it is unlikely that fermentation would im- prove the protein quality of peanut press cake. Besides being rich in oil, peanuts are also rich in protein, and peanut press cake contains 38-51% protein (van Veen gt gt., 1968). However, the quality of peanut protein is poor because it is deficient in several essential amino acids as shown in Table 1. 11 Table 1. Essential amino acids in reference and peanut proteins, expressed as g per 16 g N Amino acids Referencea Peanutb Isoleucine 4.0 3.4 Leucine 7.0 6.4 Lysine Methionine + cysteine 3.5 2.4 Phenylalanine + tyrosine 6.0 8.9 Threonine 4.0 2.6 Tryptophan 1.0 1.0 Valine 5.0 4.2 a FAQ/WHO, 1973 b FAO, 1970 Changes in Other Components Phytic acid or inositol 1,2,3,4,5,6-hexakis (dihy- drogen phosphate) is nutritionally important because of its ability to form insoluble complexes with di- and trivalent minerals, thereby, reducing their availability for absorp— tion in the intestinal tract. Defatted peanut meal was re- ported to contain approximately 1.5% of phytic acid (Erdman, 1979). The presence of phytase, an enzyme capable of hy- drolyzing phytate, has been reported in germinating seeds (Chang, 1967; Mandal gt gt., 1972; Lolas and Markakis, 1977). Reinhold (1971) suggested that yeast might contrib- ute in decreasing the phytic acid content of leavened 12 bread. Recently, it was reported that N. oligosporus pro- duced phytase which reduced the phytic acid content of soy— bean during tempeh fermentation (Sudarmadji and Markakis, 1977; Wang gt gt., 1980). No reports have been found regarding phytase production by N. sitophila. Vitamins, espeCially some B-vitamins, may change during fermentation. It was reported that the thiamine con- tent of soybean decreased during tempeh fermentation (Roe- lofsen and Talens, 1964; van Veen and Steinkraus, 1970). However, Quinn gt gt. (1975) showed that thiamine increased significantly in peanut flour fermented with N. sitophila, N. oligosporus, A. oryzae, and A. elegans. Riboflavin and niacin were shown to increase, while pantothenate was un- changed or decreased slightly, in both tempeh and oncom fermentation (Roelofsen and Talens, 1964; van Veen and Steinkraus, 1970; Quinn gt gt., 1975). Safety of Fermented Foods N. oligosporus and N. sitophila do not produce my- cotoxins (van Veen gt gt., 1968). The presence of aflatox— in in oncom, if any, may originate from peanut press cake which had been contaminated with aflatoxin. Van Veen gt gt, (1968) found that N.sitophila could destroy approximately 50% of the aflatoxin Bl present in the Indonesian peanut press cake, while R. oligosporus reduced it by about 70%. However, it is not known whether the metabolic products from degraded aflatoxin are harmless. 13 Some fermented foods may be contaminated by toxin- producing microorganisms. A good example is tempeh bong- krek (fermented coconut press cake) which is susceptible to development of bongkrek poison. Bongkrek poisoning is caused by the growth of Pseudomonas cocovenenans under con- ditions unfavorable to the molds (Nugteren and Berends, 1957). Recently, the Indonesian government has banned the production of tempeh bongkrek because of the danger of bongkrek poisoning. MATERIALS AND METHODS Preparation of Oncom Peanut press cake was prepared by pressing certain amount of shelled peanut (MSU Foodstore) under the Carver press. The peanut was first broken down into small pieces and pressed at 12,000 psi for 30 minutes. The resulting cake was soaked overnight in water which had been acidified with citric acid to pH 4.5 at refrigeration temperatures. The peanut mass was then washed several times with acidi- fied water to remove the remaining oil that rose to the surface. Afterwards, 1% of tapioca flour was added to the mass and autoclaved at 1210C for 30 minutes. The hot mass was cooled to room temperature, drained, and inoculated with the mold culture. Mold cultures used in this experiment were: (1) N. sitophila ATCC 14151, (2) N. oligosporus ATCC 22959, (3) Neurospora gp. isolated from Indonesian oncom, (4) mixed cultures of (1) and (2), and (5) mixed cultures of (2) and (3). Mold cultures were grown and stored on potato dex- trose agar slants. After one week, the mycelia and spores of each slant were harvested with 4 ml of sterilized dis- tilled water. Ten m1 of spore suspension containing 14 15 2 x 108 spores per ml were used to inoculate 1500 g of pea- nut press cake. The well mixed inoculated cake was packed tightly into disposable petri dishes (100 x 15 mm), and in- cubated at 30°C for 72 hours. At 6 hour intervals samples were drawn, freeze dried, pulverized, and stored in a freezer. pH Determination A 20 g sample of oncom was homogenized with 80 m1 of distilled water in a small Waring blender for 5 minutes. The pH determination was made directly on the homogenate using a Corning pH meter, Model 10 (E.H. Sargent and Co., Chicago, IL.). Proximate Analysis Samples used for protein efficiency ratio (PER) evaluation were subjected to proximate analysis. Moisture, crude protein, crude fat, crude fiber, and ash were deter— mined according to the methods of AOAC (1975). Analysis of Free Fatty Acids Extraction The method of Mattick and Lee (1959) was used to extract free fatty acids from the sample. Oil was first extracted from the sample with diethyl ether using a Gold- fisch extractor. One 9 of oil and 8 mg of n-heptadecanoic 16 acid (internal standard) were transferred into a 60 ml sep- aratory funnel. Thirty five ml of a mixture of diethyl ether and petroleum ether (1:1) were added to dissolve the oil, then, 6.5 ml of 95% ethanol and 12.5 ml of 1% Na2CO3 were added. The mixture was shaked several times and the aqueous layer containing the sodium salts of free fatty acids was separated into another 60 ml separatoryfunnel. Extraction of free fatty acids from the ether layer was repeated three times; first, with 1.5 m1 of 95% ethanol and 7.5 m1 of 1% Na2C03; second, with 1.5 m1 of 95% ethanol and 5.0 ml of 1% Na2C03, and finally with 6.5 m1 of distil- led water. All the aqueous phases were collected and com- bined, whereas, the ether layer containing the glycerides was discarded. To a separatory funnel containing the aqueous layer 1.5 ml of 10% H2804 was added in order to free the fatty acids. The free fatty acids were then extracted with 12.5 ml of the solvent mixture mentioned above. The ether layer was separated and transferred through Whatman # 1 filter paper containing several 9 of anhydrous NaZSO4 into a 5 m1 screw-cap vial. The solvent was evaporated to dryness by passing a stream of nitrogen gas through the vial. The ex— traction was repeated three times with fresh solvent. Esterification Free fatty acids were converted to their methyl es- ters prior to GLC analysis because these derivatives are 17 more volatile than the acids. Boron trifluoride in metha- nol (14%,w/v) was used as esterifying reagent according to the method of Supelco, Inc. (1975). Into the vial containing dry free fatty acids, 2 ml of benzene was added to dissolve the acids. Two m1 of BF3- methanol was further added into the vial and mixed well. The vial was placed in a small beaker with water and boiled for 3 minutes on a steam bath. To stop the reaction, 1 m1 of distilled water was added to the reaction mixture, which was then separated into two layers. The top layer con- tained the methyl esters dissolved in benzene, while the bottom layer was a mixture of methanol, water, and acid catalyst. To separate the two layers, the vial was cen- trifuged, and the benzene layer was transferred with a syr- inge into another vial. Two‘pl of the benzene containing methyl esters was injected into the gas chromatograph using a 10 p1 Hamilton syringe # 701 (Hamilton Co., Reno. NEV.). Gas Chromatography Conditions All gas chromatographic separations were carried out using a Perkin-Elmer 900 Gas Chromatograph equipped with a Servo/Riter II Flushmount Recorder and a Flame Ion- ization Detector (Perkin-Elmer Corp., Norwalk, CONN.). A 3 ft. x 0.125 in. o.d. stainless steel column was packed with 10% DEGS-PS on 80/100 mesh supelcoport (Supelco, Inc., Bellefonte, PA.). 18 The chromatographic conditions for methyl-ester de- rivative separations were accomplished with helium as the carrier gas at an inlet pressure of 40 psi and the flow rate of 18 ml per minute. The flame ionization detector was operated at 265°C with hydrogen pressure of 20 psi and air pressure of 40 psi. The injection port temperature was 235°C, attenuation was X64 with attenuation range X100, and chart speed was 15 in. per hour. Temperature of the column was programmed for 130 to 190°C at 10°C per minute and the column was held at 190°C for 22 minutes. Identification of the Chromatogram Peaks The Chromatogram peaks were identified with two methods: (1) by running the sample directly into a Gas Chro- matograph/Mass Spectrometer (GC/MS), and (2) by using a standard mixture RM-3 (Supelco, Inc., Bellefonte, PA.) con- taining methyl esters of myristate (C14:0), palmitate (C16:0)' stearate (C18:0)' oleate (C18:1)' linoleate (C18:2)' linolenate (C18:3)' arachidate (C20:0), behenate (C22:0)' erucate (C22:1)' and lignocerate (C24:0)' The GC/MS used was a Hewlett Packard 5840A Gas chro- matograph/HP 5985 Mass Spectrometer (Hewlett Packard Corp., Avondale, PA.). The column was a 6 ft. x 0.250 in. o. d. glass column packed with 3% DEGS on 80/100 mesh chromosorb. The helium flow rate was 25 ml per minute, and the tempera- ture was programmed from 130 to 190°C at 10°C per minute. The ion source and analyzer temperatures of the mass l9 spectrometer were maintained at 200°C. The accelerating voltage was 2000 V, ionizing potential 70 eV, repetitive scan 266.7 a.m.v. per second, and scan time 1.4 seconds. Analysis of Oligosaccharides Extraction The oligosaccharides were extracted from the defat- ted sample with 80% ethanol (Conrad and Palmer, 1976). Two g of the defatted sample was diluted in 100 m1 of 80% etha- nol and a small amount of CaCO3 was added to it to neutral- ize the acidity. The mixture was then refluxed at 70°C for 4 hours. The extract was centrifuged for 30 minutes, and the supernatant was collected. The precipitate was ex- tracted with 50 ml of 80% ethanol, and the two supernatants were combined. The supernatant was decolorized by filtering through activated charcoal Darco G-6 (Matheson, Coleman & Bell, Co.). The charcoal was washed with 20 ml of water to insure that all the sugars had passed through it. One half 9 of Ba(OH)2 was added to the filtrate and stirred using a magnetic stirrer, followed by the addition of 0.5 g of ZnSO4. This step was done to remove contaminating proteins (Delente and Ladenburg, 1972; Bau gt gt., 1978). The mix- ture was centrifuged for 30 minutes, the supernatant was desalted by passing it through Dowex 50W x 8 (H+ form) and Dowex 2 x 8 (Cl' form which had been converted to OH‘ form). 20 The desalted solution containing mainly sugars was concen- trated using a flash evaporator (Buchler Instruments, Fort Lee, N.J.) at 38°C. The concentrated solution was passed through a Sep-pak (Waters Associates Inc., Milford, MA.) to remove lipid materials, and freeze dried. Just before analysis, the freeze dried sample was dissolved in 0.5 ml of deionized water and filtered through a 0.22 pm pore-diameter membrane filter (Gelman Instrument Co., Ann Arbor, MI.) utilizing a Swinney syringe filter (Millipore Corp.). The sample was subjected to analysis by circular thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). Qualitative Analysis by Circular TLC Precoated 20 x 20 cm silica gel G-1500 plates (Schleicher & Schuell, Keene, NH.) were soaked in 0.3 M KH2P04 solution for 2 minutes, and then dried at room tem- perature for several hours. The plates were dried at 60°C for 1 hour prior to the application of the samples. In the center of a TLC plate, a circle 2 cm in di- ameter was drawn carefully with a compass. Seven samples and 1 standard mixture consisting of glucose, fructose, su— crose, melibiose, raffinose, and stachyose were applied around the perimeter of the circle, each containing approx- imately lOO‘pg sugars. The sample spots were thoroughly dried using a hair drier, then the plate was placed in the "SelectaSol" (Schleicher & Schuell, Keene, NH.) holder 21 .UAB Hoasonwo mo Emummfio .m wusmflm mmo new com: moumummmc .o musmflm 15km mmk ¢_< 32 Perchloric acid: a 70% solution Amidol reagent prepared by dissolving 2.5 g 2,4-diaminophe- nol dihydrochloride and 5079 sodium bisulfite in distilled water and diluted to 250 ml. The solution was kept in a brown bottle and discarded after 1 week. Ammonium molybdate solution: an 8.3% solution. Standard phosphorus solution: a 50 pg P per ml solution was prepared by dissolving 0.2197 g KH2p04 (dried at 1050c) in distilled water and diluted to l L. After hydrolysis with perchloric acid, 1 m1 of ami- dol reagent and 1 m1 of ammonium molybdate solution were added to each fraction, and the volume was adjusted to 10 ml with water. The solution was mixed using a Vortex mixer, and after 5-30 minutes the absorbance was measured at 675 nm with Beckman DU Model 2400 Spectrophotometer (Beckman Instruments, Inc., Fullerton, CA.). A standard curve was previously prepared using a series of standard solution containing 0 to 70 pg P per 10 m1 (Figure 7). The following linear relationship was ob- tained: A675 = 0.0125 C + 0.0021 (r = 1.000) where C was the concentration of P in pg per 10 ml. The amount of phosphorus per tube was plotted against tube number to illustrate the separation of inosi- tol phosphate. Identification of the Chromatogram was done by combining new fractions of each separated peak. These ABSORBANCE AT 675 nm 33 o 1o 20 so 40 50 60 70 P CONCENTRATION, jig/10 ml Figure 7. Standard curve for phosphorus deter- mination. 34 solutions were dried, and hydrolyzed with 6 N HCl in am- pules at 110°C for 48 hours. This step was done to liber- ate phosphorus from inositol phosphate without destroying the inositol moiety. The solution containing free inositol and inorganic phosphorus was subjected to inositol determi- nation (Agranoff gt gt., 1958) as described by Saio (1964) and to phosphorus determination (Allen, 1940). In the inositol determination, 2 ml sample contain- ing 0.01 to 0.5 pmole of inositol, 2 m1 of l M acetate buf- fer (pH 4.7), and 0.4 m1 of 0.01 M sodium periodate were mixed, and the absorbance at 260 nm was immediately read. The absorbance was read again after 30 minutes at room tem- perature and after 16 hours in water bath at 45°C. The difference in the absorbance before and after heating at 45°C was due to the oxidation of inositol. A standard curve was preViously prepared using a series of standard solution of 0 to 250 pM (Figure 8). The following relationship was obtained: 0.0024 C + 0.0066 ‘AA260 (r = 1.000) where C was the concentration of inositol in pM. Biological Evaluation of Protein Quality Five diets were prepared based on the following sources of protein: (1) casein, (2) uninoculated peanut press cake, (3) fermented peanut press cake, (4) fermented mixture of peanut press cake and sesame flour (9:1), and 35 'A Azoo Li 1 I | 0 50 100 150 200 250 INOSI‘I’OL. pM Figure 8. Standard curve for inositol deter- mination. 36 (5) fermented mixture of peanut press cake and sesame flour (8:2). These ratios refer to protein rather than the en- tire commodity. A mixed culture of N. oligosporus ATCC 22959 and N. sitophila ATCC 14151 was used to prepare the fermented diets (diet 3, 4, and 5). The fermentation was carried out at 30°C for 48 hours. Each protein source to be evaluated was incorporated into a basal diet to provide a 10% level of protein (Table 2). On the basis of proxi- mate analysis, the diets were equalized with respect to moisture, fat, ash, and crude fiber according to AOAC (1975). Fifty 21-day old male weanling rats of the Sprague- Dawley strain were used in the evaluation of protein quali- ty. The animals were housed individually in cages with a metal-screen bottom. After 3 days of acclimatization dur- ing which the animals were fed a commercial rat diet, the animals were divided into five groups, corresponding to the five experimental diets. Water and diet were offered gg libitum for 28 days. Animal weights and diet intakes were measured weekly for each animal. Protein Efficiency Ratio (PER) The PER value was calculated as the ratio of the weight gained by the animals, to the weight of protein con- sumed over the 28-day period. 37 Table 2. Protein quality evaluation diet (AOAC, 1975) g/100 g diet Caseina or sample S to provide 10 g of protein Corn oil 8 _ S x % ether extract 100 Salt mixtureb 5 - S X % ash 100 Vitamin mixtureC 1 Cellulose l _ S x % crude fiber 100 Water 5 _ S X % m01sture 100 Sucrose and corn starch (1:1) to make 100 g Teklad Test Diets, Madison, WIS. (87% protein). USP XVIII (ICN Nutritional Biochemicals, Cleveland, OH.). Composition (%): sodium chloride (NaCl), 13.93; potassium iodide (KI), 0.079; potassium phosphate monobasic (KH2P04), 38.90; magnesium sulfate (MgSO4), 5.73; calsium carbonate (CaCO3), 38.14; ferrous sulfate (FeSO4.7H20), 2.7; manganese sulfate (MnSO4.H20), 0.548; cupric sulfate (CuSO4.5H20), 0.0477; cobalt chloride (COC12.6H20), 0.0023. ICN Nutritional Biochemicals, Cleveland, OH. Composition (mg/100 g diet): vitamin A, 2000 (IU); vitamin D, 200 (IU); vitamin E, 10 (IU); menadione, 0.5; choline, 200; p-aminobenzoic acid, 10; inositol, 10; niacin, 4; Ca-D- pantothenate, 4; riboflavin, 0.8; thiamine.HC1, 0.5; PY' ridoxine.HCl, 0.5; folic acid, 0.2; biotin, 0.04; vitamin 312, 0.003. Apparent Diet Digestibility After 14 days of experimental feeding, carmine red was added to the diet as a dye marker. The red faeces were 38 collected over a 7-day period. At the end of the seventh day, the marked diet was replaced by the dye-free diet, and the PER experiment was continued. The apparent diet di- gestibility was calculated as the ratio of diet consumed, to the weight of the faecal output (dry basis) times 100. Apparent Nitrogen Digestibility The apparent nitrogen digestibility was calculated according to the following formula: nitrogen faecal Apparent nitrogen _ intake (g) - nitrogen (g) digestibility x 100 nitrogen intake (9) Analysis of Carotenoids Extraction The carotenoids of N. sitophila and oncom were ex- tracted according to the method of Liu and Luh (1977). N. sitophila ATCC 14151 was grown on potato dextrose agar in ten Roux culture bottles. The culture was incubated at 30°C for 7 days. At the end of the incubation period, the mycelia and conidia were harvested with a small amount of water and blended with a hexane/acetone mixture (1:2, v/v) for 5 minutes. The slurry was filtered through glass wool into a 500 m1 separatory funnel. The remaining mycelial residue was washed with the solvent mixture until colorless. The separatory funnel was shaken and the aqueous layer was discarded, while the organic (hexane) layer was washed with 39 25 m1 of 20% KOH in 85% methanol to saponify fatty materi- als, and the aqueous layer was discarded. The hexane layer was washed first with 25 ml of 85% methanol and then with distilled water several times. The final extract was passed through anhydrous sodium sulfate in a funnel con- taining glass wool. The filtrate was concentrated to 10 ml in a flash evaporator (Buchler Instruments, Fort Lee, N.J.), and transferred onto the chromatographic column. In determining the carotenoids of oncom fermented by N. sitophila ATCC 14151 for 72 hours, a 50 9 sample was blended with 100 m1 of water, a small amount of CaCO3, and 200 ml of the solvent used in the mold extraction. The ad- dition of CaCO3 was done to neutralize the acidity. The extraction was completed as described in the case of the mold. Separation and Identification The adsorbent used for the separation of the caro- tenoids was prepared by mixing MgO and Hyflo supercel (1:1). The mixture was suspended in hexane and wet-packed in a glass column-plugged with glass wool. A l-cm layer of an— hydrous sodium sulfate was placed on the top of 20 x 1.1 cm column of packed adsorbent. Carotenoids were separated by changing the polarity of the elution solvent according to the following order: hexane; 2%, 3%, 5%, 7%, and 10% ace- tone in hexane; and 8%, and 15% ethanol in hexane. Visible carotenoids were separated easily by watching the color 40 bands moving through the column, while the movement of phy- tofluene was traced using a UV light. The separated carotenoids fractions were dried in a flash evaporator (Buchler Instruments, Fort Lee, N.J.), and redissolved in hexane. The absorbance was scanned and re- corded with a Beckman DB-24 recording spectrophotometer (Beckman Instruments Inc., Fullerton, CA.). Carotenoid concentration was calculated based on El% at a maximum 1cm absorption peak of each component (Foppen, 1971). RESULTS AND DISCUSSION Growth of Molds and Changes in pH During Oncom Fermentation During the first 12 hours, the growth of both N. sitophila and N. oligosporus progressed slowly. After 18 hours, a rapid growth of N. oligosporus became obvious where the mycelial growth penetrated deeply into the peanut press cake mass, forming a compact semisolid product. After 36 hours, the cake was completely covered with white mycelia with small amount of black spores produced at the side of petri dish, indicating that the white oncom was ready for consumption. Unlike N. oligosporus, the growth of N. sitophila was slow. It took 24 hours for this mold to develop obvi- ous mycelial growth, and 40-48 hours to cover completely the peanut press cake mass with mycelia (Figure 9). It was noticed by touch that during fermentation by either mold, the temperature of the fermenting cake mass gradually increased above that of the incubator. Previous investigators (Steinkraus gt gt., 1960, 1965; Sudarmadji and Markakis, 1978) showed that during the tempeh fermenta- tion by N. oligosporus the temperature rose to a peak of 40-45°C. 41 42 Figure 9. Oncom. 43 O UNINOCULATED CAKE - o N. sitophila ATCC 14151 (A) 8 r x N. oligosporus ATCC 22959 (B) A Neurospora gp. (Indonesia) (C) ’ 3 Mixed culture (A) & (B) 7 . A Mixed culture (B) 5. (C) O 12 24 36 48 so 72 FERMENTATION TIME HOURS Figure 10. Changes in pH during oncom fermentation. The development of mycelial growth during fermenta- tion was accompanied by a gradual increase in pH. At the time of inoculation, the pH of peanut press cake was about 4.9-5.2. In oncom fermented by N. sitqphila the pH re- mained rather constant for the first 24 hours and then grad- ually increased to 7.2 in about 72 hours as shown in Figure 10, In oncom fermented by N. oligosporus, the gradual in- crease in pH started as early as 12 hours, and it took only 42 hours for this mold to reach pH of 7.2. It was sug- gested that an increase in pH during fermentation was due to ammonia resulted from deamination of amino acids (van Buren gt gt., 1972). 44 Identification of Free Fatty Acids Figure 11 illustrates the GLC elution pattern ob- tained for the methyl esters of free fatty acids extracted from oncom fermented by N. oligosporus after 72 hours of fermentation. Other oncom samples showed similar GLC elu- tion patterns except for their peak heights. Through com- parison with the GLC elution pattern of an RM-3 standard mixture (Figure 12), nine peaks of the oncom Chromatogram were identified as methyl esters of myristate, palmitate, stearate, oleate, linoleate, linolenate, arachidate, behen- ate, and lignocerate. Neither the uninoculated cake nor the oncom contained erucic acid. Figures 13 to 16 show the GC/MS spectra obtained for the methyl esters of free fatty acids extracted from the same oncom sample. All the esters had a base peak at m/e 74 and a large peak at m/e 87 which are usually found in the spectra of methyl esters (Kuksis gt gt,, 1976), with the exception in Figures 143 and 14C. These last two spec- tra show the mixture of M+ peaks at m/e 294, 296, 297, and 298, indicating the presence of an unseparated mixture pro— bably of methyl esters of stearate, oleate, and linoleate. Instead of 0.125 in. o.d. column used for separation shown in Figures 11 and 12, a larger diameter column (0.250 in. o.d.) was used in GC/MS separation, resulting a decrease in resolution. This is probably the reason why methyl esters of stearate, oleate, and linoleate were not resolved in GC/MS 45 system. Traces of methyl pentadecanoate (Figure 133) were detected in oncom fermented by N. oligosporus only after 72 hours of fermentation. Again, the GC/MS spectra show that neither the uninoculated cake nor oncom contained erucic acid. For quantitative analysis, calibration curves were prepared by injecting known amounts of pure methyl esters of fatty acids and plotting the amount injected versus the calculated peak areas for each compound. Linear regression equations of the methyl ester calibration curves used for calculation of free fatty acid contents are shown in Appen- dix 1. The efficiency of free fatty acid extraction was evaluated by spiking internal standard (n-heptadecanoic acid) to peanut oil prior to extraction. The recovery of added internal standard in each extraction ranged from 91 to 96%. Fatty Acids Liberated During Fermentation The peanut press cake had been autoclaved at 121°C for 30 minutes which inactivated the intrinsic enzymes; therefore, any liberation of fatty acids during fermenta- tion should have been caused by the action of lipase of the molds. Table 3 shows the free fatty acids content of oil extracted from oncom during fermentation. Myristic, lino- lenic, arachidic, behenic, and lignoceric acids were not de- tected in oncom samples at 0 hour of fermentation, while, 46 .msuommomflao .m >9 meson mm uOm OOUCOEHOM Eooco Eoum cmuomuuxm mcflom >uumm mouw mo mnmumo axnume mnu mo cuouuom coflusam Uqu .HH Onsmflm .5: .22: zoizmhmm o e a a. o. 8 on 3 q d d u 1 4 q d .1 q 4 q u - 9.. 3 /\| m we . .0. OuON N w H . o 920 a .A :— m.mpo . H dW W n1 I la a W I S . W . a 9 m0.? Wm a m cu m m 03% Numpom 3 mm w W OW N H 3 W Au V. W O .1 N . I e n. G V L M Na e m m ...... a T m I v. 3 w . N I IBI. Pmpo O 3 L.§:. T v.3 w 3 S LU] a V. O Envm. . I 1 m.{ m 3 M .Pm an N iiu w. L C I .I 3 u u . AH» A, (V A! . 875m." 56;: m xep "ta iv“ . . . . 47 hIN3A1OS O‘V‘O ‘alusnam-ew 0‘9‘0 ‘31V1lW'lVd-9W O38l ‘ - “BIO O alvuvais 9“ ‘alva‘lO-OW D Z ‘ 3‘0 ‘31V310Nl'l'9W -== €=8lo ‘31VN3‘IONI'I-9W 0:030 ‘alvaIHOVHV-ow 0‘ no ‘aIVNaHaa-ew 1:223 ‘alvonua-Ow O‘VZO ‘alvuaOONon-ON l 8 24 RETENTION TIME, GLC elution pattern of the methyl esters of free fatty acids of a standard mixture. Figure 12. 48 100 74 80‘ _ 37 (A) Methyl myristate, 604 F.W. 242 % 404 + 20‘ 242 1 04 l'llltl ' L211 N] U i O 100 "Va 200 300 100 74 80« (B) Methyl pentadeca- 87 noate, F.W. 256 60; °/o 40- 20. M+ 256 0- * J. .1. - L. 4 O 100 NW. 200 300 00 1 74 30. 87 (C) Methyl palmitate, F.W. 270.5 60.“ % 40~ M+ 20‘ 270 239 l 0* .4. O 100 NW. 200 300 Figure 13. Mass spectra of (A) methyl myristate, (B) methyl pentadecanoate, and (C) methyl palmitate. 49 100 74 80- 87 (A) Methyl heptadecanoate (internal standard), 604 F.W. 284.5 %;0‘ M+ 284 20~ 253 0‘ T 0 100 m/. 200 300 100 96 (B) Unseparated mixture of 80‘ methyl stearate (F.W. 298.5), methyl oleate (F.W. 296.5), and 264 o 60‘ methyl linoleate /o (F.W. 294.5) 40: M+ 20' 297 296 298 0‘ l 94 0 100 ml. 200 300 100 81 (C) Unseparated mixture of 80- methyl stearate (F.W. 298.5), methyl oleate 60‘ (F.W. 296.5), and methyl %» linoleate (F.W. 294.5) 40- M+ . 295 20 i 294 296 04 298 0 100 ml. 200 300 Figure 14. Mass spectra of (A) methyl heptadecanoate, (B) and (C) unseparated mixture of methyl stearate, methyl oleate, and methyl lino- leate. 50 .mumcficomum H>£DOE Amv cam .Oumcmaocfla axcuoe Amy mo muuowmm mmmz .mH onsmflm 03” com cow SE 09 o P b4 bl b P p P d- u p Io 1 d ; own ION +2 .O¢ 8 row m.mmm .3.m .oumennomnm segue: Ame .om 52. Dow 03.” 00m r ooN . .38 09 o _ _ _ 1 , .0 Now .ON +2 .ov & m.mom .3.a .oo .OumcoLocHH _>cuoz A£uos A’ _| 88 ‘03 O... X— mm :0 z: m_. J I 080 am)- No: 0: 23mm 0 (D 9:2 90 u: ... .12 (I) g) “I: C ILI in >' 2 < 5 _ a: < .— (D I I l A l 9 0 2 4 6 8 1O RETENTION TIME, Min. Figure 18. HPLC Chromatogram of a standard mixture consisting of oligosaccha- rides, glycerol, and myo—inositol. 170' 160 1 140- 120- 100- 80- 60- PEAK HEIGHT, mm T 4O 20' Figure 60 o SUCROSE v =1.234x +0.738 , r =o.999 o RAFFINOSE Y = 0.522x + 0.270 , r = 0.998 A STACHYOSE Y = 0.339x +0.119, r = 0.999 A MELIBIOSE Y = 0.794x + 1.400. r: 0.999 x MYO INOSITOL Y=L5HX +2A29. r = 0.998 x x O 20 40 60 80 100 pg SUGARS 19. Standard curves for oligosaccharides and myo-inositol. 61 .maflanDHm .m sh muse: Nb MOM cmucmfinmw Eooco ADV pom .mouommomflao .m >3 muoo: Nb How pmucwEumw Eooco Amv .mxmo omumaooocflco Adv mo mEmumoumEouno 04mm .om madman .5: .22: 29:35:. m e o m ... o o. m 4 o mmozimfi. A s % $02.92: x o 3:32“. “a mmoiofim s mmmOxm: _0_ _m_ mmOcozmu _<_ x. ..Ommoio \i \ 35050 .6532. 1 32.03 L 62 Table 4. Sucrose, raffinose, and stachyose contents of un- inoculated cake and oncom prepared with various mold cultures Content (%)a Ferm. Sample time, hours Sucrose Raffinose Stachyose UNINOCULATED CAKE 0 3.39 0.08 0.16 36 3.25 0.06 0.14 72 3.30 0.09 0.15 ONCOM FERMENTED BY: g. sitophila 18 0.14 TRb 0.08 ATCC 14151 (A) 36 TR NDC 0.04 72 TR ND TR 3. oligosporus 18 3.10 0.10 0.10 ATCC 22959 (B) 36 1.56 0.09 TR 72 0.89 0.06 ND Neurospora sp. 18 0.09 0.03 0.07 (Indonesia) (C) 36 TR TR 0.05 72 TR TR TR Mixed culture 18 0.12 TR 0.05 (A) & (B) 36 TR ND TR 72 TR ND ND Mixed culture 18 0.13 TR 0.07 (B) & (C) 36 TR ND TR 72 TR ND ND a Average of three determinations. b Trace, less than 0.01%. C Not detected. monosaccharides (Sugimoto and van Buren, 1970). The reduc- tion of sucrose and stachyose (Table 4) accompanied with the appearance of melibiose strongly indicates that the molds used in oncom fermentation possess an invertase which 63 hydrolyzes the glucose-fructose bond of the oligosaccha- ride and an a—galactosidase which hydrolyzes the other bonds of the molecule (Figure 21). memAcrosmA‘s mvgnuse “\ . E \ CH2 \7/b\CH2 : 0H oH‘\0H oH ~H 'HOCHO H OH H O OH H 0 OH E H OH H H . OH :0 CH20H H OH H OH H OH OH H GAL. GAL. GLU. rauc. MELIBIoss—l GALAC'I’OBIOSE ' L sucnose ——-—' MANNINOTRIOSE I L L RAFFINOSE . STACHYOSE #1 Figure 21. Structure of stachyose. The activity of invertase produced by N. sitophila seemed to be very strong. It eliminated sucrose and raffi- nose almost completely in 36 hours of fermentation. While, it took 72 hours for N. oligosporus to hydrolyze 70% of the existing sucrose. Stachyose content decreased during the fermentation; however, the degradation rate was much slower than that in the other oligosaccharides. The raffinose content of oncom fermented by B. oligosporus did not decrease as much as that of sucrose .539. 1; , $1."? .4'. A V “'2‘ ".1 64 and stachyose. Perhaps, this is due to raffinose formed from the partial hydrolysis of stachyose. This finding is in agreement with the report by Shallenberger £3 a1. (1967) in which the stachyose and sucrose contents decreased with- out apparent change in the raffinose content in the tempeh fermented by a Rhizopus mold over a 72-hour period. On the other hand, Worthington and Beuchat (1974) found that raf- finose and sucrose were not utilized by N. oligosporus, while small amounts of stachyose were utilized only after 68 hours of fermentation. When both N. sitophila and N. oligosporus were used as a mixed culture, all oligosac- charides were eliminated almost completely after 36 hours of fermentation. Raffinose and stachyose are thought to be involved in flatulence and their reduction in oncom is a desirable effect. The Soluble Protein of Oncom During 72 hours of fermentation, the soluble pro- tein content of uninoculated cake, as measured by the meth- od of Lowry 3E ai. (1951), remained unchanged and in the range of 70-78 mg per 9 sample. During the first 18 hours, the soluble protein content of oncom fermented by R. gliggf sporus increased to 86.4 mg per g, remained unchanged dur— ing the second 18 hours, and then increased to 125.9 mg per g at 72 hours of fermentation (Figure 22). Likewise, the soluble protein of oncom fermented by N. sitophila changed during fermentation according to the same trend as in oncom 65 300' 0 UN INOCULATED CAKE L O N. sitophila ATCC 14151 (A) X»N. oligosporus ATCC 22959 (B) b ‘ Neurospora sp. (Indonesia) (C) I Mixed culture (A) & (B) - A Mixed culture (B) & (C) 220- / FERMENTATION TIME, HOURS SOLUBLE PROTEIN. mg/g Figure 22. Soluble protein of fermented and un- fermented peanut press cake. 66 fermented by N. oligosporus except that the soluble protein content of oncom fermented by N. sitophila was much higher. Apparently the proteolytic activity in oncom fermented by N. sitophila was stronger than that in oncom fermented by N. oligosporus. Cherry and Beuchat (1976) observed that the free amino acid content was greater in peanuts inocu- lated with N. sitophila than in those inoculated with N. oligosporus during the infection period. Van Buren gt 33. (1972) reported that at the end of a 72-hour tempeh fermentation, about one half of the crude protein had become water soluble. Electrophoretic patterns of the soluble protein of uninoculated cake, and oncoms fermented for 72 hours by N. sitophila and N. oligosporus on 10% SDS gel are shown in Figure 23. These patterns are indicative of the protein hydrolysis that might have occurred during fermentation. Degradation of Phytic Acid in Oncom Recovery of added phytate ranged from 94—96% (Ap- pendix 2). The phytic acid content of uninoculated cake was 13.60 i 0.18 mg per g or 1.36% (3.83 i 0.05 mg phytate P per g). It was reported that the phytic acid content of whole oilseeds including peanut was about 1.5% (Erdman, 1979). It was assumed that there was no destruction of phytic acid during soaking overnight at refrigeration tem- perature, because pH (4.5) and temperature were low enough 67 Figure 23. Electrophoretic pattern of soluble protein. (A) Uninoculated cake, (B) Oncom fermented for 72 hours by N. sitophila, (C) Oncom fermented for 72 hours by R. oligos orus, (D) Molecular weight—protein standard. 68 to prevent any phytase activity. Lolas and Markakis (1977) reported that the pH optimum for the Navy bean phytase was 5.3 with an optimum temperature of about 50°C. Others re- ported that pH optimum of different sources of phytase ranged from 5.0 to 7.5 (Peers, 1953; Chang, 1967; Mandal gt gt., 1972). Prior to inoculation, peanut press cake was autoclaved at 121°C for 30 minutes. This step might de- crease the phytic acid content of the cake. Reddy gt gt. (1978) reported that autoclaving Black gram beans in excess of water at 116°C for 5 minutes resulted in losses of phy- tic acid and total P content due to leaching. Likewise, Tabekhia and Luh (1980) showed that cooking dry beans at 100°C for 3 hours had little effect on phytate retention, while canning the dry beans at 115.50C for 3 hours resulted in a reduction of phytate. During 72 hours of fermentation, the phytic acid content of uninoculated cake remained constant (Figure 24), while the phytic acid content of oncom decreased cosidera- bly. The decrease in phytic acid content was accompanied by an increase in inorganic phosphorus content as shown in Figure 25. The decrease in phytic acid and an increase in inorganic phosphorus content were probably due to phytase released by the molds. Autoclaving prior to inoculation should have inactivated the phytase which might be inherent to the peanuts. Therefore, any phytase activity found in oncom must have originated from the molds. 69 .mxmo mmmuq uscmwm bmucmEummco pom pmucmEumw mo ucmucoo oflom oflu>£m .wm muomfim 250: .22... 20:35.25“. Nu om we on em NP 0 l u s 'I . d d 1 1 a q - 10 IX X .. N md< mum—3.530 aux; I 3: 800.3 532.00.... :8: 0326£ .mm 22.3502 d 3. mmmwu 00.: «Banana mfladflmm x o o X can mmmahJ—JO Omx=¢ d. o / :3 5:: co: allzfiqfi amiss; 9:8 35.50022: ‘0 5/5w ‘alov OILAHd N— D!) (4‘ d1 + (T ¢p 70 .mxmo mmmum pocmwm bmucmEummc: can omucmEuwm wo ucwucoo monogamonm vacmmuocH .mm muoofim manor .mEZ. 2033;52:9— Nh Do we on vw NP 0 q‘ q u q 7 q 7 d T q u o u\\\\J‘\\\ ht ”\ X 0% .- . ... d . / o . . . x .D O)! o . mé ... a x can «255.5 09:: q . . ua< awash—:0 0522 I A0. 8023 caiocouc. ON I :3: 0332: ..mm 2232...: I x E. 93% co: ....oaaloamm 3632: x :2 ES: 095‘ 2:33.» 2032.52 0 3:0 095430052: 0 mN 71 Judging from the disappearance of phytic acid, it may be deduced that the phytase activity of N. oligosporus is greater than that of N. sitophila. Direct evidence for the presence of phytase in N. oligosporus has been recently reported by Sudarmadji and Markakis (1977) and Wang gt gt. (1980). As shown in Figure 20, free inositol was detected in oncom samples, indicating that phytic acid had been hydro- lyzed completely by phytase. However, calculations on the free inositol and inorganic phosphorus contents of oncom fer- mented by N. oligosporus (Table 5) showed that only 60-65% of phytic acid was hydrolyzed completely to free inositol and inorganic phosphorus after 72 hours of fermentation. Some phytic acid was hydrolyzed partially to different forms of inositol phosphates which could be separated on anion ex- change resins (Saio, 1964; Cosgrove, 1969; Asada gt gt., 1969). Inositol phosphates produced in oncom fermented by N. oligosporus were separated on a Dowex l x 8 (Cl‘ form) with 0 to 1.0 N HCl linear gradient elution. The peaks appearing in the chromatogram were identified by calculat- ing the molecular ratio of phosphorus to inositol (Table 6). As shown in Figure 26, phytic acid (IP6) appeared as a major peak, while inositol tri- (IP3) and tetraphos- phates (1P4) were detected as minor peaks. As the fermen- tation progressed, phytic acid was dephosphorylated to other inositol phosphate forms (inositol mono-, di—, tri-, tetran, and pentaphosphate) and inorganic phosphorus. 72 Table 5. Free inositol and inorganic phosphorus released from phytic acid during fermentation of oncom by N. oliggsporus Inositol Ferm. Initial Phytic time, phytic acid hours acid lost Released (mg/g) (mg/9) theoret- Found Released ically (mg/g) (%) (mg/g) 0 13.60 0 0 0 0 36 0.96 12.64 3.45 1.08 31.30 72 0.41 13.19 3.60 2.32 64.44 Inorganic phosphorus Ferm. Initial Phytate P time, phytate P lost hours (mg/g) (mg/9) Found Released (mg/g) (%) 0 3.83 0 O 0 36 0.27 3.56 1.72 48.31 72 0.12 3.71 2.25 60.65 A scheme of dephosphorylation of phytic acid by phytase was suggested by Tomlinson and Ballou (1962)(Figure 27). These results demonstrate that fermentation by molds, especially N. oligosporus, improves the nutritive value of peanuts by lowering their phytic acid level. However, it is not known whether these inositol phosphate forms also reduce bio- availability of minerals. 73 .GESHoo AIHUV mxa x0300 :0 mmumnmmonm Houflmocfl mo cumuumm seapoam .mm muooflm :Em. cmnaaz 20:043.“. 0: on 3 . 00/ 669/ 49/ A.” 69’ II I I I I I I I I - I ...-o. I I... I. o... no. ’ son... I coo-no.0... o... o. I I I o o I I I I I I I I I I I I I I I I I I o I I I o a o ..I- o no no... I ...... I I o o .... I. o I o ’ I I o... I I I 0 II I I I . II I II 53 . 9 Av . . ow 0 Av IVA: .1 A LO? ORE .. L. L1 I oo . I .1 _.._ am now > .Amooa .soHHmm b :flHEoev mmmu no >3 bflom ofluxcm mo coflumfi>uonmmozmwo mo memnom .nm whomam no ac do no no no mo 40....moz. K ..o no ‘ 39... 6336 no no u ‘ 1 K ..o no no ..o no no no mo ~ 0 no no no K ..o ..o 1 mo 75 Table 6. Ratio of phosphorus to inositol in fractions obtained from Dowex 1x8 (C1 ) chromatographya Phos— Ino- pmole P/ Inositol Fraction # phorus sitol pmole inositol phosphatesb (pg) (#9) identified Observed Theory 4- 10 125.1 NDC - - Inorganic phosphorus 13- 18 38.7 225.0 1.00 l IPl 23- 31 101.2 303.4 1.94 2 1P2 34- 48 201.4 423.3 2.76 3 1P3 52- 59 150.6 224.8 3.89 4 1P4 63- 79 210.1 257.6 4.73 5 IP5 82-107 418.1 416.2 5.83 6 IP6 a Sample was oncom fermented by R. oligosporus for 12 hours. b Inositol mono- (1P1), di- (1P2), tri- (IP ), tetra - (1P4), penta- (1P5), and hexaphosphate (I56) C Not detected. Biological Evaluation of Protein Quality The composition of peanut press cake, oncom, and fermented mixtures of peanut press cake and sesame flour is shown in Table 7. On a dry weight basis, the protein con- tents of peanut press cake and oncom are practically the same, 46.6 and 46.5%, respectively. This indicates that fermentation did not affect the total protein content of the cake. The PER values and apparent digestibility of the 76 Table 7. Composition of peanut press cake, oncom, and fer- mented mixtures of peanut press cake and sesame flour Carbo- Mois- Fat Pro- Fiber Ash hydrate ture tein (%, by (%) (%) (%) (%) (%) differ- ence) Peanut press cake (P) 8.8 15.3 42.5 2.7 3.2 27.5 Oncom 9.3 15.4 42.2 2.8 3.4 26.9 Fermented mixture: P + sesame , 8.5 13.8 43.2 3.0 3.3 28.2 (9.1) P fgfgfame 8.3 12.6 42.8 2.8 3.5 30.0 experimental diets are shown in Table 8. The PER and ap— parent digestibility of oncom did not differ significantly (P‘<0.05) from those of peanut press cake. This indicates that fermentation did not improve protein quality of raw materials. These results were in agreement with those re- ported by van Veen gt gt. (1968). However, when peanut protein was supplemented by 10% sesame protein, the PER of fermented product was im- proved significantly by 50%. There was no further im- provement when the level of added sesame protein was in- creased to 20%. The improvement is probably due to the high methionine content of sesame, since this amino acid is 77 Table 8. Protein efficiency ratio (adjusted to casein PER = 2.50) and digestibility of casein, peanut press cake, oncom, and fermented mixtures of peanut press cake and sesame flour Apparent digestibility _ PER (x : SEM) _ Diet Nitrogen (x 1 SEM) (x : SEM) Casein 2.50:0.05 93.1:0.8 89.8:0.8 peanut press cake 1.51:0.07a 86.7:1.3a 79.8105a Oncom (P) 1.41:0.08a 85.3:1.2a 80.0:0.6a Fermented mixture: P + sesame (9:1) 2.11:0.06b 84.9:0.6a 79.4:0.4a p + sesame (8:2) 2.13:0.06b 84.6:0.8a 79.1:0.5a SEM = standard error of means; Means with the same letter are not significantly different at P = 5% (Tukey's test). limiting nutritional value of peanut protein (Table 9). On the other hand, sesame is low in lysine and this amino acid may become limiting when the proportion of sesame protein reaches a certain level in a mixture of sesame and peanut. The supplementary effect of sesame protein on legume pro- tein has been shown by Boloorforooshan and Markakis (1979) and Akpapunam and Markakis (1980). There were no changes in the digestibility of ei- ther the diet or the protein as a result of the supplemen- tation. Therefore, poor protein quality of oncom and pea— nut press cake was mainly due to a poor over-all amino acid 78 Table 9. Essential amino acids in peanut (Arachis 'hy o aea) and sesame seed (Sesamum indi- cum) proteins.expressed as g per 16‘g Na Amino acids Peanut Sesame seed Isoleucine 3.4 3.6 Leucine 6.4 6.7 Lysine 3.5 2.7 Methionine + Cysteine 2.4 4.6 Phenylalanine + tyrosine 8.9 7.6 Threonine 2.6 3.6 Tryptophan 1.0 1.3 Valine 4.2 4.6 a FAQ, 1970 balance. These results suggest that the protein quality of oncom can only be improved by supplementation with other protein sources. Carotenoids of N. sitophila Numerous species of molds are reported to produce carotenoids. Neurospora crassa which produces a complex mixture of carotenoids was often used in studies on carot- enoid biosynthesis (Goodwin, 1954). On the other hand, N. sitophila which is also believed to produce carotenoids is rarely mentioned in a literature. This experiment was 79 done to investigate whether the carotenoids produced by N. sitophila have vitamin A value. N. sitophila ATCC 14151 was grown on potato dex- trose agar in ten Roux culture bottles at 30°C for 7 days. The yield of mycelia and conidia harvested from all culture bottles was 58.2 g wet weight. As shown in Table 10, five carotenoids extracted from mycelia and conidia of N. sito- Etttg were identified as phytofluene, neurosporene, fi-zea- carotene, 7-carotene, and fi-carotene. The identification of these carotenoids was based on their UV and visible ab- sorption spectra in hexane. Their absorption maxima were the same as those reported by Foppen (1977). The absorp- tion spectra of the N. sitophila carotenoids are presented in Figure 28 and 29. Neurosporene, fi-zeacarotene, 7-car- otene, and fi-carotene as illustrated, absorbed in the vis- ible region, indicating that the red or orange color ap- peared in conidia of the mold might be due to the presence of these carotenoids. Phytofluene, neurosporene, B-zeacarotene, and 7- carotene were found as intermediate products in the biosyn- thesis of B-carotene from mevalonic acid (Davies, 1973). Phytoene and {-carotene which were also reported as inter- mediate products in the biosynthesis of fi-carotene were not detected. The concentration of carotenoids identified above is shown in Table 11. Among the five carotenoids found in 80 Table 10. Identification of carotenoids in N. sitophila Carotenoids Absorption maxima identified in hexane (nm) Phytofluene reporteda 331 347 366 observed 330 347 367 Neurosporene reported 416 440 470 observed 416 439 469 fi—zeacarotene reported 400 425 450 observed 400 425 450 7-carotene reported 431 462 494 observed 430 460 492 B-carotene reported 425 450 476 observed 425 448 475 a Foppen (1971). N. sitophila, only’ 1- and B-carotenes were detected in oncom sample. Both of these carotenoids have vitamin A value. Based on calculations, 100 g of oncom only contains 1.92 retinol equivalents, where 0.42 and 1.50 retinol equi— valents are contributed by ‘7- and B-carotenes, respec- tively. Since the recommended daily allowance for the adult man is 1000 retinol equivalents, the oncom carot- enoids have little nutritional significance as provitamin A. 81 0.15 B-ZEACAROTENE 448 425 /\ a, I \ 330 347 11“ {456‘ .— .I ‘\ I.“ 400 I J \\ 3 67 ,'\. i 4 2 5’] \\ j I’. \‘\‘ 475 / \V" I \ \ (310 P I 1 \ ' PHYTOFLUENE / \ \ / \ ‘ I.“ ‘\ ‘ L) . 1 z x. \ < B-CAROTENE \“ m \ I: " \\ <3 ~t "’ \ D “ “. 1‘. 0.05 - ‘3 \ l ‘J b o l 1 l 1 j_ 1 J 1 250 300 350 400 450 500 Figure 28. WAVELENGTH, nm Absorption spectra of phytofluene, fl-zeaca- rotene, and p-carotene extracted from N. sitophila. 82 0.6 460 Y-CAROTENE 0.4 '- m 492 (J I: " m c n 8 2 NEUROSPORENE .‘ I’ 'u ,' i : 0‘2 L ‘. 469 3 1". i (1 ‘. u' '. \; 1 L .. x, ‘\ \\\ o J l l l l l l ‘I‘~\...j~ ‘ 300 350 400 450 500 550 WAVELENGTH, nm Figure 29. Absorption spectra of neurosporene and T-carotene extracted from N. sitophila. 83 Table 11. Concentration of carotenoids found in mycelia and conidia of N. sitophila ATCC 14151 and oncom sample EATgétigggia Oncom sample . 1%a Caroten01ds Elcm Conc. Conc. ‘ b (pg/ (ug/ Abs. Vol 1009 Abs. Vol 1009 (m1) wet (m1) wet wt.) wt.) Phytofluene 1100 0.32 25 125 NDC - - Neurosporene 2768 0.16 . 25 25 ND - - p-zeacarotene 1940 0.48 25 106 ND - - 7-carotene 3100 1.90 25 263 0.14 5 5 B-carotene 2592 1.10 50 365 0.25 5 9 a Foppen (1971). Measured at absorption maxima. C Not detected. CONCLUSIONS The pH of the fermenting oncom increased gradually from about 5.1 to 7.2 in 72 hours. Oncom seemed to be ready for consumption when pH was about 6.5-6.8, or after 36 hours fermentation by N. oligosporus or 48 hours fermen- tation by N. sitophila. During fermentation, free fatty acids were liber- ated from triglycerides as a result of lipase released by the molds. Approximately 40% of the peanut oil was hydro- lyzed by N. oligosporus, while only 10% by N. sitophila af- ter 72 hours of fermentation. The order of fatty acid lib- eration was oleic, linoleic, palmitic, and stearic acids followed by minor fatty acids. During fermentation with either 5. oligosporus or N. sitophila the sucrose, raffinose, and stachyose contents of peanut press cake decreased. Raffinose and stachyose are thought to be involved in flatulence and their reduc- tion in oncom is a desirable effect. The soluble protein content of oncom fermented by either 3. oligosporus or N. sitophila gradually increased. Electrophoresis of the soluble protein fraction of oncom showed protein hydrolysis. 84 85 During fermentation, about 95% of the phytic acid was hydrolyzed by N. oligosporus, while only about 50% by ' N; sitophila. Apparently the phytase activity of N. oligo- sporus is much stronger than that of N. sitophila. About 60-65% of the phytic acid was hydrolyzed completely to in- organic phosphorus and free inositol, while the remaining phytic acid was hydrolyzed partially to other inositol phosphate forms. Fermentation did not change the protein content, apparent digestibility and protein quality of raw materials. Therefore, the protein quality of oncom can only be improved by supplementation with other protein sources. Incorporation of 10% of sesame protein raised the PER of peanut press cake from 1.51 to 2.11. The mycelia and conidia of N. sitophila contained phytofluene, neurosporene, 6-zeacarotene, 7-carotene, and B-carotene. However, only ‘7- and 3-carotenes were de- tected in oncom. Both of these carotenoids have vitamin A value, but the concentration of these carotenoids was too small to have nutritional significant as provitamin A. In general, Neurospora gp. isolated from Indonesian oncom was not very different from N. sitophila ATCC 14151 in its effect on the oncom fermentation. Using a mixed culture of N. sitophila and N. oligosporus in oncom prepa- ration resulted in considerable decrease in both oligo- saccharides and phytic acid content. 86 For future study, it is recommended to include dif- ferent protein sources as supplements to improve the pro- tein quality of oncom. Typical oncom flavor produced dur- ing fermentation has not been analyzed; therefore, the study on flavor of oncom and its relation to sensory evalu- ation would be interesting. To encourage local oncom pro- ducers in the village area, a study on a simple, rapid and hygienic method of oncom preparation would be helpful. Preparation of oncom using a mixed culture of N. oligospo- rus and N. sitophila should be introduced to produce oncom which is low in both oligosaccharides and phytic acid. REFERENCES REFERENCES Agranoff, B.W., Bradley, R.M., and Brady, R.O. 1958. The enzymatic synthesis of inositol phosphatide. J. Biol. Chem. 233: 1077. Akpapunam, M.A., and Markakis, P. 1980. 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Linear regre351on Methyl esters range equations (#9) Me-myristate 0- 1.5 Y = 5.3805 X - 0.4448 (r = 0.9870) Me-palmitate 0- 6.0 Y = 6.9830 X - 0.6514 (r = 0.9925) Me-stearate 0- 4.5 Y = 5.5158 X + 0.0661 (r = 0.9948) Me-oleate O-22.5 Y = 4.7111 X + 5.2933 (r = 0.9854) Me-linoleate 0-22.5 Y = 3.7101 X + 0.4019 (r = 0.9977) Me-arachidate 0- 4.5 Y = 5.0738 X - 0.5069 (r = 0.9925) Me-linolenate 0- 4.5 Y = 1.9667 X - 0.3000 (r = 0.9923) Me-behenate 0- 4.5 Y = 5.1583 X - 0.6375 (r = 0.9922) Me-lignocerate 0- 4.5 Y = 3.6190 X - 0.2143 (r = 0.9977) Me-heptadecanoate 0- 8.5 Y = 8.5715 X - 0.9027 (internal standard) (r = 0.9991) 95 Appendix 2 Table A2. Recovery of phytic acid added to the control samplea PA in Addgd Total PA % Recov- Sam le control PA PA found ery p sample (mg) (mg) (mg) (mg) Control 12.05 — — - _ Control + 10 mg NaPhy 12.05 5.24 17.57 16.57 95.84 Control + 20 mg NaPhy 12.05 10.47 22.52 21.37 94.89 a b PA = phytic acid = 52.37% of standard NaPhy. Average of three determinations. (I'wmmiflllif I) W” Nm U." Em All ( (NIWINHII 56 3328 293 030 31