Chemical and Aerobic Bacterial Composition of "Terasi", a Traditional Fermented Product from Indonesia (Received October 18, 1993)

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1 Chemical and Aerobic Bacterial Composition of "Terasi", a Traditional Fermented Product from Indonesia (Received October 18, 1993) Ingrid S. SURONO*1 and Akiyoshi HOSONO*2 (*1The United Graduate School of Agricultural Science, Gifu University, Gifu, , Japan; *2Faculty of Agriculture, Shinshu University, Minamiminowa-mura, Nagano-ken, , Japan) The chemical and aerobic bacterial characterization of "Terasi" (Indonesian traditional fermented shrimp and/or fish paste) was carried out. The composition of "Terasi" samples on average was protein g/100 g, sodium chloride g/100 g, fat g/100 g, carbohydrate g/100 g, and ash (including salt) g/100 g, and the moisture content was g/100 g with ph The major amino acid in "Terasi" was glutamic acid. Aerobic bacteriological analyses showed that "Terasi" had total viable bacterial counts of 4.OX105 CFU/g and a halophilic count of 1.1X105 CFU/g. Out of 70 isolates, the predominant microbial flora in "Terasi" was Bacillus (65.7%), followed by Pseudomonas (21.4%), Micrococcus (7.2%), Kurthia (4.3%), and Sporolactobacillus (1.4%). The dominant strain was high-salttolerant Bacillus. Key words: Terasi, fermented shrimp paste, aerobic bacterial composition Introduction "Terasi" is an indigenous fermented food in Indonesia, made from fish and/or shrimp paste obtained by natural fermentation in the presence of a high salt concentration. It has a flavor reminiscent of ripe cheese. Closely related products are "Ngapi", the Philippino "Bagoong", Burmese Malaysian "Belachan" and Thai "Kappi". "Terasi" and soy pastes serve similar roles as condiments in the diet. "Terasi" in general is prepared as follows: The shrimp or fish are mixed with salt at the 10% level on the fishing boats, then spread out on the floor. Further salt is added at 5%, and the *1 Permanent address: Faculty of Agricultural Technology, Indonesia Institute of Technology, Jl. Raya Puspiptek, Serpong-Tangerang 15320, Indonesia. product is dried in the sun for about 1 to 3 days, and occasionally turned over, to decrease the moisture content from about 80 to 50% and also to minimize off-flavor. The resulting mass is minced, pressed tightly into wooden tubs to exelude all air and allowed to ferment for 1-4 weeks, then again sun dried and packed for sale. The fermentation time of "Terasi" may vary from 1 to 4 weeks, and during fermentation the ph of "Terasi" rises from 6. 0 to Later, due to lactic acid production, the ph declines to Further fermentation results in an increase of ph to 7. 8 due to ammonia production1). "Terasi" is normally produced traditionally on a small scale. The most important center for its manufacture is Bagansiapiapi, North Sumatra. Regarding the aerobic bacteria of this fish or shrimp paste, the finished product of Malaysian "Belachan", had total viable counts of bacteria of OX104 CFU/g, and a halophilic count of X 104 CFU/g. During different stages of fermentation, Bacillus, Pediococcus, Lactobacillus, Micrococcus, Sarcina, Staphylococcus, Clostridium, Brevibacterium-like, Flavobacterium-like, and Corynebacterium-like bacteria were detectedl2). The protein content of Malaysian "Belachan" was g/100 g, and the salt content was 1325 g/100 g with a ph of '. A survey on Indonesian "Terasi" from 65 samples

2 Table 1. Chemical Composition of "Terasi" 1 Mean (X) and standard deviation (SD) of six determinations Table 2. Amino Acid Composition of "Terasi" reported that its moisture content was in the range of g/100 g, ash (including salt) g/100 g, salt 4.1-4O.9g/100 g, and protein g/100 g3). However, further information about the species of microorganisms present in "Terasi" is lacking. The purpose of our study was to examine the chemical and aerobic bacterial composition of "Terasi". The results should be useful for improving "Terasi" processing and flavor. Materials and Methods 1. "Terasi" sample The present study was limited to one type of "Terasi" (n=6) produced commercially in Indonesia, which was obtained from a small producer in Indramayu, West Java, Indonesia in October, and kept in a refrigerator for 1 week until used for experiments. The "Terasi" from Bagansiapiapi was used as a starter, mixed with dried shrimp, rice bran, cassava powder, and dried fish, and sun-dried for 4-6 hours. The resulting mass was minced, placed in wooden tubs, allowed to ferment for 1 week, packed with banana leaf and over-wrapped with polyethylene film. 2. Chemical analyses Protein (total NX6. 25), fat, sodium chloride, moisture content and ash were determined according to AOAC procedures4). Analyses were carried out with 6 samples taken randomly. The ph value was measured with a ph meter (Iuchi, model CP-1, Japan). Carbohydrate was obtained by calculation. The amino acid composition was analyzed with a JEOL JLC-300 high performance liquid chromatograph (HPLC). A set of amino acid standards was included in the analysis. Acid hydrolysis was carried out with 6 N HCI. 3. Bacteriological analyses 3. 1 Media for bacterial count: Nutrient Agar (Oxoid) was used for the total bacterial count, and Nutrient Agar with 10 g/100 ml NaCl was used for the total halophilic count. Dilutions of samples were prepared with sterile 1/15M phosphate buffer (PB), ph Enumeration and isolation of bacteria: Five grams of "Terasi" sample was suspended in PB and made up to 50 ml, then homogenized by shaking vigorously for 5 minutes. Suitable decimal dilutions were prepared with PB, plated on Nutrient Agar and on Nutrient Agar with 10 g/ 100 ml NaCI, and incubated at 37C for 2 days. Colonies formed were counted5). Representative colonies of the bacteria were picked up randomly from the enumeration plate, placed in a liquid medium of the same composition without agar, incubated at 37C for growth, and purified by streaking on agar plates. Representative isolates were purified, i. e., 37 colonies from Nutrient Agar with 10 g/100 ml NaCI, and 33 colonies from Nutrient Agar plates.

3 Table 3. Characteristics of Micrococcus, Pseudomonas, Kurthia and Sporolactobacillus Isolated from "Terasi" A: lylae; B: roseus; C: kristinae; D: aeruginosa; E: cepacia; F: alcaligenes; G: fluorescens; H: gibsonir, I: inulinus; +: Positive; -: Negative; Y: Yellow; R: Red; CW: Cream white; W: White; *: Yellow, cream white, red, white. ND: Not determined

4 Table 4. Characteristics of Bacillus Isolated from "Terasi" A: brevis; B: subtilis; C: megaterium; D: circulars; E: pantothenticus; F: licheniformis; G: coagulans; H: polymyxa; I: mycoides; J: pumilus; K: sphaericus +: Positive; -: Negative; =: Small gas bubbles 3. 3 Identification: Taxonomic properties of the purified isolates were examined according to the methods of Harrigan and McCance6), and Bergey's Manual7)8). Cultures were evaluated for Gram staining, motility, catalase and oxidase tests, aerobic and anaerobic tests, spore staining, gas production from glucose, and acid production from various carbohydrates, growth at different temperatures and salt concentrations, various hydrolysis tests, citrate utilization, reduction of nitrate, ortho-nitrophenyl-b-d-galactosidase (ONPG) test, and urease test. For grampositive rods, the Voges Proskauer test was included. Results 1. Chemical analyses The chemical composition of "Terasi" is shown in Table 1. The protein content of "Terasi" was g/100 g. Protein contained glutamic acid ( g/100 g) as the major amino acid (Table 2). Data on tryptophan were not included in this work since it is completely destroyed during acid hydrolysis. The ph of "Terasi" was rather high (7. 53), with salt g/100 g, moisture g/100 g, ash (including salt) g/100g, fat g/ 100 g, and carbohydrate (by calculation) g/100 g. 2. Bacteriological Analyses Enumeration of the bacterial load of "Terasi" showed that bacteria levels of 4. 0 x 105 CFU/g and a halophilic count of 1. 1 x 105 CFU/g were present in the finished product of "Terasi". The characteristics of Micrococcus, Pseudomonas, Kurthia and 5porolactobacillus isolated are pre-

5 sented in Table 3. Three isolates were identified as M lylae, one isolate was identified as M. roseus and one isolate was identified as M krisinae. The determination of the Pseudomonas group was based on the taxonomic pro perties described in Bergey's Manual8). Three isolates were identified as P. aeruginosa, nine isolates were identified as P. cepacia, two isolates were identified as P. alcaligenes and one isolate was identified as P. fluorescens. It was also found that 3 isolates belonged to Kurthia gibsonii and one isolate was identified as Sporolactobacillus inulinus (Table 3). Of 70 isolates, it was found that 46 isolates belonged to aerobic spore-forming bacilli, and the dominant strains were identified as B, brevis, B. sphaericus, B. subtilis and B. circulans. Some of the properties of the isolates belonging to Bacillus showed slight deviations from the properties assigned in the schedule (Table 4). The isolates identified as B. brevis, B. coagulans and B. polymyxa were salt-tolerant, contrary to Bergey's Manual7). Discussion The predominant amino acid in "Terasi" protein is glutamic acid, which may be responsible for the taste, but the amount of free glutamic acid was not analyzed. Glutarnic acid is produced in fermented fish and shrimp paste as a relatively stable amino acid without secondary decomposition during fermentation9). Bacteria, which are more competitive under these circumstances, dominate the microflora. Among the bacteria present in "Terasi", the acid-producing Bacillus seems to be the most versatile group which can utilize a wide variety of energy sources such as protein, sugar and even inorganic nitrogen (ammonium compound) (Table 4). In addition, Pseudomonas also has a great ability to utilize such a wide range of energy sources. On the other hand, the non acid-producing Bacillus seems to be very poor in utilizing starch, sugar and protein, but can utilize organic acid such as citric acid (Table 4). Micrococcus is even able to utilize the protein but is very poor in utilizing carbohydrate sources such as starch and sugar (Table 3). Furthermore, Kurthia gibsonii and Sporolactobacillus inulinus (Table 3) were relatively fastidious, and hence these bacteria represented a minority of the population of aerobic bacteria in "Terasi". "Terasi" is mostly comp osed of protein, as well as nitrogenous substances formed by protein decomposition, such as ammonium compounds, and it contains a high percentage of sodium chloride (16. 75%), whereas the carbohydrate content is low. "Terasi" is fermented at temperatures between 3040C, and during the fermentation, the ph ranged between ). Bacillus was the most versatile bacteria in terms of utilizing the nutritional sources in "Terasi", as well as from the ph ysicochemical point of view (ph, salt content of "Terasi" and the temperature of its processing), and hence Bacillus dominated the bacterial population of "Terasi". During the early stage of "Terasi" fermentation, when the ph drops to 4. 5, most Pseudomonas fail to grow. Moreover, the processing of "Terasi" is not hygienic, under actual manufacturing conditions. Therefore, the Pseudomonas found in "Terasi" may represent contamination during "Terasi" processing such as mixing, sundrying, and packing. Pseudomonas grow rapidly on proteinaceous substances and are considered as active participants in microbial spoilage of seafoodsloa P. eepacia and P. aeruginosa are important potential pathogens. Strains of these species have been isolated from many clinical materials and from infected tissues in humans8j. The presence of these isolates needs confirmation by examining the microflora present in "Terasi" starter (mother culture) and "Terasi" processed under controlled laboratory conditions. In this research, Bacillus was found as the dominant flora. This finding is in agreement with the predominant presence of Bacillus in fermented fish paste and Philippino "Bagoong"11), 12). On the other hand, lactic acid bacteria are predominant in Malaysian "Belachan" and Indonesian "Terasi"131. The differences among the microflora in "Terasi" and related products may due to differences in the traditional manufacturing processes. Microbiological changes during "Terasi" fermentation are still unknown, and need to be examined. From this study, it was found that glutamic

6 acid is the dominant amino acid in the finished product of "Terasi". Mutagenic compounds, i. e., heterocyclic amines such as 2-amino-6- methyldipyrido(1, 2-a: 3', 2'-d)imidazole (GIu-P- 1) and 2-aminodipyrido(1, 2-a: 3', 2'-d)imidazole (GIu-P-2) are produced in products containing glutamic acid during heat treatment14). Since "Terasi" is consumed after heat treatment, it is of interest to study the mutagenicity of "Terasi" and to identify the mutagenic compounds present (if any). Acknowledgement Ingrid S. SURONO thanks Monbusho (Ministry of Education, Science and Culture, Japan) for a scholarship. References 1) Jennie, B. S. L., Muchtadi, D.: "Agricultural Product Microbiologyl", p (1978), Department of Education and Culture, Jakarta, Indonesia. 2) Steinkraus, K. H.: "Handbook of Indigenous Fermented Foods". p (1983), Marcel Dekker, Inc., New York. 3) Anonymous. Report on the survey of quality of dried salted fish (Peda), dried small fish (Ten) and Terasi (1981), Balai Besar Industri Hasil Pertanian (BBIHP) Institute for Research and Development of Agro-based Industry-Bogor, Indonesia. 4) AOAC. Official Methods of Analysis, 15th ed (1990), Association of Official Analytical Chemists, Washington, DC. 5) Saisithi, P., Kasemsarn, B., Liston, J., Dollar, A. M.: J. Food Sci. 31, (1966). 6) Harrigan, W. F., McCance, M. E.: "Laboratory Methods in Food and Dairy Microbiology" (1976), Academic Press, New York. 7) Sneath, P. H. A., Mair, N. S., Sharpe, M. E., Holt, J. G.: Bergey's Manual of Systematic Bacteriology., Vol. 2 (1986), The Williams and Wilkins Co., Baltimore. 8) Krieg, N. R., Holt, J. G.: Bergey's Manual of Systematic Bacteriology, Vol. 1 (1984), The Williams and Wilkins Co., Baltimore. 9) Mizutani, T., Kimizuka, A., Ruddle, K., Ishige, N.: J. Food Composition and Analysis 5, (1992). 10) Roberts, T. A., Skinner, F. A.: "Food Microbiology: Advances and Prospects", p. 217 (1983), Academic Press, London, England. 11) Cha, Y. J., Lee, E. H.: Bull. Korean Fisheries Soc. 22, (1989). 12) Fujii, T., Basuki, S. B., Tozawa, H.: Bull. Jpn. Soc. Sci. Fisheries 46, 1, 2351, 240 (1980). 13) Ohhira, I., Jeong, C. M., Miyatomo, T., Kataoka, K.: Jpn. J. Dairy and Food Sci. 39, 175-l82 (1990). 14) Sugimura, T.: Science 233, (1986).