Ethnic Fermented Foods and Beverages of Indonesia 14

Home , Bagoong, Bagoong fried rice, Brem, Burmese fried rice, Chinese pickles, Chinese sticky rice

Ingrid Suryanti Surono

14.1 Introduction which are of Malay extraction. The remainder of the natives is Melanesian (in Papua and the east- Indonesia is the largest archipelago in the world ern islands). There are ethnic Chinese, Indians, extending some 2000 km from north to south and and Arabs concentrated mostly in urban areas more than 5000 km from east to west and con- throughout the archipelago. There are about 300 sists of 17,508 islands, about 6000 of which are ethnic groups, each with cultural identities devel- inhabited, scattered over both sides of the equator oped over centuries and infl uenced by Indian, (Fig. 14.1 ). The archipelago stretches over more Arabic, Chinese, and European sources, and 742 than one tenth of the equator between Southeast different languages and dialects. Major ethnic Asia and Australia. The largest islands are the groups are Javanese (45 %), Sundanese (14 %), Kalimantan, Sumatra, Papua, Sulawesi, and Java. Madurese (7.5 %), Coastal Malays (7.5 %), and Indonesia lies between latitudes 11°S and 6°N others (26 %) (Expat website Association 2015 ). and longitudes 95°E and 141°E and consists of The agricultural sector of Indonesia comprises islands (CIA 2015 ). large plantations (both state owned and private) The temperature ranges between 16 and 35 °C that tend to focus on commodities which are with humidity ranging from 60 % to 98 %. There are important export products (palm oil and rubber) two seasons, the rainy monsoon season which usu- and smallholder production modes that focus on ally lasts from November through May, with the rice, soybeans, corn, fruits, and vegetables. heaviest rainfall from November through March, According to FAO of the United Nations (2015), followed by the dry season which is driest between the top 11 products of Indonesia in 2012 include June and September. Rainfall varies throughout paddy rice, palm oil, rubber, chicken, cassava, Indonesia, averaging 706 mm (28 in.) yearly. maize, coconuts, banana, palm kernels, mango, Indonesia has a population of 255,993,674 and mangosteens. people (estimated per July 2015) and is the fi fth Rice is a staple in Indonesia, except in Papua most populous nation in the world after China, and Maluku where people sustain themselves India, EU, and the United States, the majority of with sago, which is a type of tapioca; sweet pota- toes; and cassava. Indonesian cuisine is as varied I. S. Surono (*) as its culture, and the food in Indonesia is as Food Technology Department, Faculty of diverse as its geography with the infl uences from Engineering , Bina Nusantara University , China, Europe, and even India, rich in fl avors; Jl. Jalur Sutera Barat Kav. 21, Alam Sutera Campus , Serpong-Tangerang 15143 , Indonesia soy-based dishes, such as variations of tofu ( tahu ) e-mail: [email protected]; [email protected] and tempe , are also very popular (Table 14.1 ).

Fig. 14.1 Map of Indonesia

Fermentation is one of the oldest and most such as cereals, legumes, tubers, fruits, vegeta- economic methods in preserving the quality and bles, animal such as meat and milk, and marine safety of foods; it not only prolongs the shelf life sources; many of them are made only on home but also reduces volume, shortens cooking times, scale in traditional methods of preparation passed provides better nutritional bioavailability, on from generation to generation using relatively enhances fl avor and aroma, and can be consid- simple equipment at very low cost with insuffi - ered as a functional food that exerts health- cient hygienic precautions (Surono and Hosono promoting benefi ts (Tamang 2015 ). A rich variety 1994a , b ). of indigenous traditional fermented foods involv- Most of the traditional food fermentations are ing yeast, mold, bacteria, and their combination, conducted by natural, spontaneous fermentation owned by each area, are an important part of the involving mixed benefi cial microbes from staple culture, identity, and heritage and have certain ingredients and environmental surrounding as distinct sensory characteristics as a result of home industry. As a consequence, pure and sin- metabolite accumulation produced by microbes gle culture will not be involved; natural contami- involved, contributing to fl avor, texture, and nation and inconsistent quality of the product aroma. Traditional fermented foods and bever- may occur due to lack of sterility and the use of ages are also considered as important part of diet natural fermentation (Nout and Sarkar 1999 ). due to its high nutritive value, digestibility, and Based on the substrate used, fermented foods and reduced antinutrient compounds. Fermentation beverages can be classifi ed into: may assist in the detoxifi cation of certain undesirable compounds such as toxin and antinutri- Fermented grain, cereals, and legume foods ents which may be present in raw foods, such as Fermented fruits and vegetable products phytates, polyphenols, and tannins (Sharma and Fermented milk products Kapoor 1996 ). The manufacture of fermented Fermented fi sh and meat products foods uses diverse raw materials as substrates, Fermented roots and tuber products 14 Ethnic Fermented Foods and Beverages of Indonesia 343

Table 14.1 Ethnic fermented foods and beverages of Indonesia Regions of Nature and consumption in Foods Substrates uses Microorganisms Indonesia References Tempe Soybeans Side dish Rz. oligosporus All regions Astuti et al. 2000 Oncom Soybean, Side dish N. sitophila , Rz. West Java Sastraatmadja et al. (2002 ), peanut oligosporus Hoo ( 1986 ), Aﬁ fah et al. 2014 , Sulchan and Nur ( 2007 ), and Sumi and Yatagai (2006 ) Gembus Soybean Side dish Rz. oligosporus Central Java Kuswanto (2004 ), Sulchan and Rukmi (2007 ), Sulchan and Nur (2007 ). and Fatimah ( 1998 ) Kecap Soybean Condiment A. oryzae , A. sojae , Rz. All regions Steinkraus (1995 ) and oryzae , Rz. oligosporus Judoamidjojo (1986 ) Acar Vegetables Condiment Lb. plantarum All regions Lennox and Eﬁ uvwere (2013 ) Sayur asin Vegetables Condiment Lb. plantarum , Leu. West Java Sulistiani et al. (2014 ) and mesenteroides , Lb. Puspito and Fleet (1985 ) confusus , Lb. curvatus , P. pentosaceus Tauco Soybean Condiment R. oligosporus , Rz. West Java Winarno et al. ( 1973 ) oryzae , A. oryzae , Lb. delbrueckii , Hansenula sp. Tempoyak Flesh of Condiment Ent. gallinarum UP-9, Sumatra Wirawati (2002 ), Pato and durian (Durio Ent. faecalis UP-11, Surono (2013 ), Widowati zibethinus ) Oenococcus , et al. (2013 ), and Yuliana Leuconostoc , and Garcia (2009 ) Enterococcus , Lactococcus , Pediococcus acidilactici , Lactobacillus. Leuconostoc sp. Mandai Inner part of Condiment P. pentosaceus , Lb. Kalimantan Emmawati (2014 ), Rahayu cempedak or plantarum , Lb. pentosus (2003 ), (2010 ) jackfruit Brem Cassava, Snack, Rz. oryzae , M. rouxii , A. Central Java, Basuki (1977 ), Saono et al. glutinous rice beverages oryzae , S. cerevisiae , Bali ( 1984 ), and Aryanta (2000 ) Acetobacter aceti Tuak Juice of plant Beverages S. cerevisiae , C. tropicalis North Sumatra, Hermansyah et al. (2015 ) Nusa Tenggara Dadih Buffalo milk Beverage Lac. lactis subsp. lactis , West Sumatra Imai et al. (1987 ) and Lb. brevis , Lb. plantarum , Surono (2003a , b ) Lb. casei , Lb. paracasei , and Leu. mesenteroides Urutan Meat, pork Side dish Lb. plantarum , Lb. Bali Antara et al. (2002 ) and farciminis , and obligate Aryanta (1998 ) heterofermentative lactobacilli Lb. fermentum and Lb. hilgardii . Besides, P. acidilactici and P. pentosaceus (continued) 344 I.S. Surono

Table 14.1 (continued) Regions of Nature and consumption in Foods Substrates uses Microorganisms Indonesia References Peda Fish Side dish Lb. plantarum , Lb. Java Rahayu (2003 ) curvatus , Lb. murinus and Strep. thermophilus Terasi Fish and Condiment Bacillus sp. and Sumatra, Java Surono and Hosono shrimp Pseudomonas sp. (1994a ) Telur asin Duck egg Side dish Lb. plantarum , Lb. casei All region Suprapti (2002) and subsp. rhamnosus , Saputra ( 2013 ) Enterococcus gallinarum , and P. acidilactici Tape Cassava, Snack Rz. oryzae , M. rouxii , A. West Java, Aryanta (1988) and glutinous rice oryzae , S. cerevisiae , E. Central Java Uchimura et al. (1998 ) burtonii , H. anomala , and P. pentosaceus. Lb. plantarum and Lb. fermentum Growol Cassava Snack Coryneform , Yogyakarta Suharni (1984 ) Streptococcus , Bacillus , Actinobacteria Lactobacillus , and yeast Gatot Cassava Staple food P. pentosaceus , Yogyakarta Ichsyani (2014 ) Saccharomyces sp. TR7 , Lb. plantarum 250 Mut7 FNCC

14.2 Traditional Fermented Foods 14.3 History, Manufacture, of Indonesia Biochemical and Nutritional Value, and Socioeconomics 14.2.1 Fermented Grains/Legumes of Tempe and Cereals 14.3.1 History of Tempe Tempe , oncom , tauco, and kecap are all Indonesian legumes and grain fermented foods. The word tempe appears to have originated in Tempe and oncom are solid fermented foods, Central Java, Indonesia. It is not derived from tauco is in the form of paste or slurry, and kecap Chinese (as other soy foods in Indonesia), and it is a liquid fermented food. Historically, most tra- does not start with the preﬁ x tau or tao (as do ditional soy protein foods originated from China tauci , tauco , taugé , taujiong , tahu , takua ) (Astuti and were introduced later to other countries in the 1999 ). The earliest known tempe reference is East and Southeast Asia (Smith 1963 ). Tempe is found in the Serat Centhini manuscript and ﬁ rst unique among major traditional soya foods, cited in History of Tempeh (Shurtleff and Aoyagi because it is the only fermented soya food prod- 1979) and then in The Book of Tempeh (Shurtleff uct that did not originate from China or Japan and Aoyagi 1985 ), so it is presumed that tempe (Shurtleff and Aoyagi 2007 ). Today, Japan leads existed in Java in the early 1600s. The Serat to industrialization, technology development, Centhini (the Centhini manuscript), a classic equipment manufacture, and worldwide soybean- work of modern Javanese literature and a kind of based food marketing. encyclopedia, was probably written around 1815. 14 Ethnic Fermented Foods and Beverages of Indonesia 345

“Serat” means manuscript or work or tale. duction, it can be preserved by drying or freezing “Centhini” (also spelled “Centini”) refers to a (after blanching to inactivate the mold and its character in the book written in verse, and the enzymes). information is given often very detailed on many There are two distinct fermentation periods. different subjects – not just religion but also vari- The fi rst occurs during soaking of the soybean ous aspects of Javanese culture and life. On one and results in acidifi cation by lactic acid bacterial page the word tempe appears, indicating that fermentation. Second is fungal fermentation and tempe was produced in the early seventeenth cen- results in mycelial growth and partial digestion of tury (Okada 1988 ). the enzymes from the mold (Steinkraus et al. Prinsen Geerligs (1896 ), a Dutchman, is the fi rst 1960 ). In the preparation, the soybeans (Glycine to spell the word tempeh (with an “h” on the end) and max) are soaked overnight in three volumes of also the fi rst to name the tempeh mold as Rhizopus water containing 10 mL of 0.85 % lactic acid per oryzae. Other authors from the Dutch use the spelling liter of water or Lb. plantarum , a lactic acid- témpé (Gericke and Roorda 1875 ; Heyne 1913 ) or producing bacterium that can be added to the tèmpé (Vorderman 1902 ; Stahel 1946 ). soak water in place of lactic acid. The soak water In 1905, Dr Kendo Saito of Tokyo Imperial is acidifi ed to about pH 5.0 to inhibit the growth University described that the main tempeh micro- of microorganisms which can cause spoilage, organism is Rhizopus oligosporus (Kendo 1905 ). boiled, drained, cooled, and spread out on a tray, Tempe was introduced to the Japanese by Dr followed by mixing with a little molded tempe Nakano Masahiro in 1958 and some published cake from a former batch or adding fermentation papers on tempeh were written by Japanese sci- starter containing the spores of Rz. oligosporus entist (Nakano 1959 ; Ohta et al. 1964 ; Ohta 1965 , (Fig. 14.4 ); Wang et al. ( 1975 ) recommended 10 6 1971; Nakano 1967, Watanabe et al. 1971 ). spores per 100 g cooked soybean for optimal fer- Indonesians pronounce the word tempe, which is mentation, then wrapped with banana leaves, and the correct spelling in Indonesian language. Van kept overnight for about for 24–36 h at room tem- Veen (1962 ) reported that the attempt to intro- perature until the mass is bound by cottony myce- duce tempeh to Indian population by missionar- lium of the mold into a solid white cake. Tempe is ies in Travancore, in Southern India in 1936, was often produced in Indonesia using Hibiscus tilia- not successful, since they did not have any inter- ceus leaves, called usar . The undersides of the est in this unknown fermentation product. leaves are covered in downy hairs known techni- cally as trichomes to which the spores of Rz. oligosporus can be found adhering. During 14.3.2 Manufacturing Tempe, fermentation, there is some biochemical reaction Biochemical and Nutritional occurred involving some enzymes; hence, tempe Values is more digestible as compared to soybean (VanVeen and Schaefer 1950 ). The fermentation Tempe is legumes’ fermentation with the aid of eliminates the beany fl avor of raw soybeans and mold, Rhizopus sp. The hydrated, cooked, dehu- gives the product a bland but attractive fl avor lled whole soybeans are fermented by Rhizopus (Hesseltine and Wang 1967 ). Martinelli and sp. molds. It is a moist solid cake with a mild, Hesseltine (1964 ) introduced the use of plastic pleasant taste. According to Steinkraus (1980 ), bags as containers for tempe fermentation, which tempe is a single cell protein grown on edible is perforated to provide the moderate aeration substrate. Temp e fermentation is similar to cheese necessary for mold growth without excessive fermentation since the hydrolysis of protein and sporulation, resulting in an attractive creamy, lipid occurs, fl avor intensifi es, and free amino white fresh tempe cake (Figs. 14.2 and 14.3 ). acid is released (Steinkraus 1983 ). The tradi- This new idea and new technology is quickly tional product is highly perishable and is usually transferred to tempe makers in Java and becomes consumed the day it is made. In industrial pro- widely used (Fig. 14.4 and 14.5 ). 346 I.S. Surono

Fig. 14.5 Rz. oligosporus ( http://www.mycology.ade- laide.edu.au )

Fig. 14.2 Tempe in plastic bag 14.3.3 Microbes Involve in Tempe Manufacture

The microbes in tempe are complex, involving mixed culture fermentation by molds, yeasts, lactic acid bacteria, and various other bacteria. The major genus of importance is the mold Rhizopus with different species such as Rz. microsporus , Rz. oligosporus , and Rz. oryzae (Nout and Kiers 2005 ). Rz. oligosporus is a species that can grow between 30 and 42 °C (optimum 25–37 °C), characterized by an inability to ferment sucrose; Fig. 14.3 Fermented soybean cake, Tempe high proteolytic and lipolytic activity; and ability to release free ammonia after 48–72 h fermentation (Wang and Hesseltine 1965 , 1979; Steinkraus 1983 ), grow on wheat or other cereal substrates without producing noticeable amount of organic acids due to minimal amylase activity (Wang and Hesseltine 1979), inhibit production of afl atoxin, and biosynthesize B vitamins (Murata et al. 1968 ). Lactic acid bacteria play a role in the acidifi - cation of the soya beans during soaking, thereby preventing the growth of spoilage microorganisms (Ashenafi and Busse 1991 ; Nout et al. 1987), improving the shelf life of tempe . During fermentation, lactic acid bacteria grow up to 109 Fig. 14.4 Adding inoculums of Rz. oligosporus to the cfu/g-1 in fi nal tempe products. dehulled cooked soybeans (Source: Rumah Tempe Indonesia) 14 Ethnic Fermented Foods and Beverages of Indonesia 347

Samson et al. ( 1987) reported that the micro- The main sugars in soybean are sucrose, bial load of 110 commercial tempe samples in the stachyose, and raffi nose, and the last two are oli- Netherlands was more than 107 cfu/g aerobic gosaccharides which are considered primarily plate counts, predominated by Enterobacteriaceae responsible for fl atulence. Soaking and boiling and lactic acid bacteria. Sixty-nine percent of the treatment can reduce stachyose, raffi nose, and samples contained yeast more than 10 5 cfu/g. sucrose at the amount of 51 %, 48 %, and 41 % of Some samples also contained Staph. aureus , B. the original content, respectively (Kasmidjo cereus, or E. coli . Ashenafi (1994 ) found high 1989/1990). Shallenberger et al. ( 1976 ) reported numbers of enterobacteria, enterococci, and further decrease of stachyose and sucrose during staphylococci, whereas Mulyowidarso et al. the fermentation of tempe which might be due to ( 1991) found high numbers of Bacillus species in the activity of bacteria, since Rz. oligosporus is tempe. The contribution of bacteria and yeasts to not able to utilize stachyose, raffi nose, and the properties of tempe is in developing fl avor sucrose. Sorenson and Hesseltine (1966 ) reported and substrate modifi cation and in the safety of the that glucose, fructose, galactose, and maltose product (Nout and Rombouts 1990 ). supported excellent growth of the mold. On the other hand, raffi nose was relatively constant. The amino acid composition of soybeans 14.4 Biochemical Changes apparently is not signifi cantly changed by fer- and Nutritional Value mentation, but free amino acids and ammonia increased (Wang et al. 1968 ). Lysine and methio- During fermentation, there are some changes in nine have been found to decrease during the the chemical composition of the soybeans. The course of long fermentation. Wang et al. (1968 ) mold, Rhizopus spp., produces a variety of carbo- also found insignifi cant increase in PER of soy- hydrases, lipases, and proteases, which degrade beans after fermentation, which might be attrib- the macronutrients into substances of lower uted to better availability of amino acids liberated molecular mass, with a higher water solubility. from the beans during fermentation and to better Also vitamins, phytochemicals, and anti- digestibility due to increase in soluble solids and oxidative constituents are formed (Astuti et al. nitrogen. 2000 ; Nout and Kiers 2005 ). Hesseltine (1965 ) stated that the total fat Soaking, washing, dehulling, and cooking (ether extractable) of tempe remained relatively cause considerable loss of solids due to solution constant throughout fermentation, although about into the water (Steinkraus et al. 1960 ). one third of soybean oil was hydrolyzed due to its Fermentation of soybeans by the tempe mold also lipolytic activity into fatty acids by the tempe causes an increase in soluble solids from 13.0 % mold into palmitic, stearic, oleic, linoleic, and in cooked soybeans to 27.5 % in tempe (Steinkraus linolenic acids, with linoleic acid predominant. et al. 1960 ) and explains the higher digestibility Linolenic acid is the only fatty acid utilized by of tempe compared to plain cooked soybeans as the mold, and about 40 % of this fatty acid is used stated by VanVeen and Schaefer (1950 ). During (Hesseltine 1965 ). The lipid content of tempe is fermentation, the pH gradually increases from lower than that of unfermented soybeans since 5.0 to 7.5 due to ammonia production in the later the lipase enzyme hydrolyses triacylglycerol into stages of fermentation (Hand 1966 ). Fresh, prop- free fatty acids around 40–50 % during soybean erly fermented temp e has been reported to have a fermentation (Pawiroharsono 1997 ). pH value of 7.25 (Ilyas et al. 1970 ). The strains Furthermore, the fatty acids are used as a source with good amylolytic activity are unsuited for of energy for the mold. During tempe fermenta- fermentation of tempe since they will break down tion the lipid contents decrease about 26 % starch to simple sugars which are then used to (Astuti 1994 ). A study by Graham et al. shows produce organic acids which will lower the pH that the mold of Rz. oligosporus and Rz. stoloni- and inhibit the growth of the mold. fer uses linoleic acid, oleic acid, and palmitic 348 I.S. Surono acid as energy sources, which rapidly decrease of soybean and wheat was comparable to that of during fermentation, and palmitic acid, stearic milk casein. Tempe fermentation does not alter acid, and linoleic acid by 63.4, 59.25, and 55.78, amino acid profi les but make them bioavailable. respectively (Astuti 1994 ). The protein content and nutritive value make Fatty acids in soybean are rich in unsaturated tempe a good substitute for meat (Steinkraus fatty acids (around 80 %), mainly oleic acid, lin- 1983 ). During the fermentation process, the lev- oleic acid, and linolenic acid. The concentration els of anti-nutritional constituents are decreased, of oleic acid and linoleic acid increases propor- and the nutritional quality and digestibility of the tionally with duration of fermentation time, but fermented product are improved due to the enzy- linolenic acid is decreased, and the optimal con- matic activity of the mold (Nout and Kiers 2005 ). centration is achieved on 24 h of fermentation The mold also contributes to the development of (Wagenknecht et al. 1961 ). a desirable texture, taste, and aroma of the prod- A 30.7 % reduction of phytic acid occurred in uct (Hachmeister and Fung 1993 ). the tempe fermentation (Egounlety and Aworh 2003), while Van der Riet et al. (1987 ) reported that phytic acid was reduced by about 65 % as a 14.5.1 Antibacterial and Enzymes result of the action of phytase enzyme produced in Tempe by Rz. oligosporus . Phytic acid is known as an antinutrient factor which is able to bind divalent The high digestibility of tempe has been observed minerals, thus lowering the mineral bioavailabil- during World War II when prisoners suffering ity. Therefore, the decrease in phytic acid has a from dysentery were able to digest tempe much benefi cial effect on mineral bioavailability (Wang better than soya beans (Steinkraus 1996 ; Tibbott et al. 1980 ; Astuti 1994 ). 2004 ). Pediatric research in Indonesia indicated Rz. oligosporus caused almost complete that in infants, the recovery after acute bacterial destruction of phytic acid, due to phytase activity diarrhea was faster when tempe was consumed as of the mold and improved bioavailability of iron an ingredient of the infant food formula (Karyadi (Sudarmadji and Markakis 1977 ; Fardiaz and and Lukito 1996 , 2000 ; Soenarto et al. 1997 ). Markakis 1981 ; Sutardi and Buckle 1985 ). Tempe showed a strong bioactivity in vitro by Phytates adversely affect nutritional status by reducing the adhesion of enterotoxigenic chelating minerals and making them unavailable diarrhea- causing E. coli to animal and human for use by humans. intestinal cells (Kiers et al. 2002 ; Roubos-van den Hil et al. 2009 ), and tempe intake was associated with better memory (Hogervorst et al. 2008 ). 14.5 Nutritional Value of Tempe Tempe has antibacterial effect against Lb. bul- garicus , Strep. thermophilus , Bacillus sp., and Tempe contains 157 calories per 100 g, proteins Listeria sp., although growth of Lb. plantarum , (12.7 %), carbohydrates and fats (4 %), and vita- isolated from tempe , was not affected. No anti- mins B1 (0.17 mg) and B12 (2.9 μg); it is low in bacterial activity of tempe against E. coli or cholesterol and saturated fat; is high in fi ber and Salmonella was observed (Kiers et al. 2002 ; most B vitamins including B12; and has good- Kobayasi et al. 1992; Wang et al. 1969 , 1972 ). quality protein (19.5 % ), comparable to protein Tempe was found to possess anti-diarrhea- content of meat products(Shurtleff and Aoyagi associated bacteria. On the one hand, tempe 1979 ; Okada 1989 ). On a 40 % dry solids basis inhibits the adhesion of ETEC to intestinal cells, (Steinkraus 1983 ), it contains all essential amino which can be of interest in the recovery and pre- acids and is rich in lysine which is lacking in vention of diarrhea in humans. On the other hand, cereal grains, but methionine is limited (Shurtleff tempe has antibacterial against B. cereus cells and Aoyagi 1979 ). Wang et al. (1968 ) found that and spores, which can be of interest in food pres- the nutritive value of tempe made from a mixture ervation and pathogen control. The anti-adhesion 14 Ethnic Fermented Foods and Beverages of Indonesia 349 activity is caused by an interaction between eggs. Over the last four decades, the attitude ETEC and tempe extracts, which results in a loss toward tempe has changed, and it is now consid- of adhesion capability of ETEC to the intestinal ered as inexpensive food with high nutritive val- cells (Roubos-van den Hil et al. 2009 ). This bio- ues (Syarief 1997 ). activity is found in tempe derived from legumi- In 1982 a company in Southern Netherlands nous seeds, but not with tempe derived from was making 6000–8000 lb/week of tempe , mak- cereals. The bioactive component(s) are released ing it the largest tempe manufacturing company or formed during fermentation by enzymatic deg- in the world. In early 1979, there were 13 com- radation of leguminous matter (Roubos-van den mercial tempe shops in the United States, one in Hil et al. 2010 ). Fermentation with several other Canada, and four in the Netherlands (Shurtleff microorganisms also resulted in the formation of and Aoyagi 1979 ). The total sales of refrigerated bioactive components, such as carbohydrate and tempe as meat alternatives in the natural food arabinose, which is an important monosaccharide channel for the year ending August 2011 were at constituent supposed to originate from arabinan least $51.6 million, and 19.3 % of this was refrig- or arabinogalactan chains of the pectic cell wall erated tempe , while in the mainstream/mass mar- polysaccharides of legumes (Roubos-van den Hil ket (including conventional supermarket chains), et al. 2010 ). Tempe contains enzymes with throm- sales of refrigerated meat alternatives for the year bolytic activity, which can digest thrombotic pro- ending August 2011 were at least $65.9 million, tein (fi brin) and which will be inactivated by and 4.47 % of this was refrigerated tempe heating above 65 °C (Sumi and Okamoto 2003 ). (Shurtleff and Aoyagi 2011 ). Aoki et al. (2003 ) revealed that tempe contains In Indonesia, tempe is consumed as a protein- γ-aminobutyric acid that suppresses the elevation rich meat substitute by all economic groups due of blood pressure; contains dietary fi ber, sapo- to its low-cost production, low price, and nutri- nins, isofl avones, and superoxide dismutase tional value (Karyadi and Lukito 1996 ). Outside which eliminates active oxygen; and has an anti- Indonesia, tempe gains interest as a major protein carcinogenic effect. source other than meat, especially nutritional and health functionality (Astuti 2000; Nout and Kiers 2005 ; Steinkraus 1996). Rumah Tempe Indonesia 14.5.2 Socioeconomy of Tempe (RTI) or Indonesia Tempe House was launched on the 6th of June 2012 in Bogor, West Java Tempe is an indigenous fermented food of Province, as a model of Tempe factory for the Indonesia and the most extensively studied effi cient, hygienic, and eco-friendly issue, worldwide. For most of Indonesian people, tempe implementing good manufacturing practice and is a meat substitute, and the price is affordable for good hygiene practice. everyone. Throughout Indonesia tempe is consumed by people of low as well as high socioeconomic level. The tempe makers produce at home 14.6 O n c o m and Gembus : using 10–150 kg of soybean daily, and the pro- Microbiology, Nutritive ducers are united in Cooperatives of Producers of Value, and Potential Health Tempeh and Tofu in Indonesia. Urban population Benefi t growth has stimulated a rise in the number of tempe processors in many cities throughout Fermentation on solid waste of soybean, peanut Indonesia, in response to the demand for rela- residue, or shredded coconut residue also con- tively inexpensive foods. The signifi cance of ducted in Indonesia produced oncom , gembus , tempe industry as part of informal sector plays an and bongkrek, respectively. Oncom is one of the important role. Temp e was formerly considered traditional fermented foods of West Java, a as an inferior food due to its low costs compared Sundanese ethnic cuisine of Indonesia, involving to other protein foods such as meats, fi sh, and several molds and closely related to tempe . 350 I.S. Surono

Fig. 14.6 Oncom Bandung

Oncom is made from the by-products of tofu Tofu oncom is made from soybean residues or peanut press cake residue after the oil has been and peanut oncom is based on peanut, well- pressed out and cassava tailings when extracting known traditional fermented foods in West Java. the starch (Fig. 14.6 ). There are two kinds of The oncom cultures N. intermedia var. oncomen- oncom : red oncom and black oncom . The solid sis had bright yellow and large macroconidia. by-product of tofu or peanut press cakes covered Generally, red oncom is made from solid tofu with massive coat of living conidia is called red waste, i.e., the soy residue after its protein has oncom because of the glistening orange color of been taken for tofu making, while the black the conidia of the microorganisms, and the oncom is generally made from the peanut dregs thicker the conidial layer, the higher the commer- mixed with cassava dregs or cassava powder, i.e., cial value of the product (Sastraatmadja et al. tapioca, in order to make a better texture and to 2002 ; Wood 1998 ). make it more tender. Although both the substrate Since oncom production uses by-products to material is a kind of waste, its nutrient is still high make food, it increases the economic effi ciency enough to be exploited by human. Tofu waste of food production . Black oncom is made by still contains high nutrient values; however, most using Rz. oligosporus and other types of Mucor of its organoleptic properties are less preferred. (Sastraatmadja et al. 2002 ; Wood 1998 ), while Fermented tofu waste, i.e., red oncom , is pre- red oncom is made by involving Neurospora , par- ferred as food product than the waste without ticularly N. crassa , N. intermedia var. oncomen- fermentation. sis, and N. sitophila (Hoo 1986). It is the only Tofu waste might contain protein similar to human food produced from Neurospora . N. inter- tofu and soy, although it has undergone many media var. oncomensis had bright yellow and changes because of certain treatments during the large macroconidia in contrast to wild N. inter- manufacturing process of tofu , such as heating. media with pink and small macroconidia (Hoo The fl avor of oncom can be described as strong, 1986 ). fruity, almond-like, and somewhat alcoholic, but In the production of oncom, sanitation and when fried, it takes mincemeat fl avor, while the hygiene are important to prevent bacterial or alcoholic fl avor which is present due to sugar mold contamination such as A. fl avus (which pro- degradation will vaporize and disappear. The best duces afl atoxin), even though afl atoxin- producing oncom in Indonesia is oncom Bandung (Fig. molds (Aspergillus spp.) are naturally present on 14.6 ); instead of using peanut press cake, raw peanut press cake when the peanut has already peanuts are used as the main ingredients. contaminated with the molds. N. intermedia var. The high nutrient content of tofu and its large oncomensis and Rz. oligosporus reduce the afl a- amounts provide a signifi cant opportunity to be toxin produced by A. fl avus (Nout 1989 ). Soybean used as a growth media for enzyme-producing is the best substrate for growing Rz. oligosporus microbes for health. Oncom has 187 kcal per 100 to produce tempe , but oncom has not been as g, protein 13 %, fat 6 %, carbohydrate 22.6 %, thoroughly studied. vitamin B1 0.09 mg, and vitamin B 12 3.1 μg 14 Ethnic Fermented Foods and Beverages of Indonesia 351

(Winarno 1989 ). In vivo study revealed that red oligosporus . Hygienic conditions should be taken oncom reduces the cholesterol levels of rats, sug- into consideration in preventing P. cocovenenans gesting potential health benefi t for humans. The contamination and outgrowth of the mold that fi brinolytic activities in oncom also show poten- produces two toxins, toxofl avin and bongkrek tial prevention toward cardiovascular diseases. acid. Bacillus licheniformis RO3 with high fi brinolytic Consumption of tempe bongkrek is associated activities was isolated from red oncom (Afi fah with a food-borne human intoxication and sig- et al. 2015 ). nifi cant numbers of deaths annually. Since 1975, Gembus is also made from solid soybean tempe bongkrek production has been banned by waste of tofu (Kuswanto 2004 ), fermented by Rz. local authorities for safety reasons. oligosporus , involving B. pumilus 2.g which has Garcia et al. (1999 ) found that 40 % and 50 % high proteolytic and fi brinolytic activities (Afi fah coconut fat concentrations in the substrate (shred- et al. 2013 ). Gembus is a variety of tempe , but ded coconut residue from coconut milk produc- whose substrate is different (solid tofu waste and tion) support production of 1.4 mg/g bongkrek soybean, respectively). Microbial fi brinolytic acid, while less than 10 % coconut fat supporting enzymes from food-grade microorganisms have growth of the P. cocovenenans yields no bong- the potential to be developed as additives for krek acid. Oleic acid was most stimulatory in functional foods and as drugs to prevent or cure production of bongkrek acid (2.62 mg/g dry sub- cardiovascular diseases (Afi fah et al. 2014 ). strate). Lauric, myristic, and palmitic acids also Like the soy tempe , gembus tempe contains stimulated production of bongkrek acid but at several substances such as fi ber, polyunsaturated lower levels. fatty acids, ergosterol, and isofl avonoids, which may have infl uences on lowering the level of blood lipids (Sumi and Yatagai 2006 ). 14.7 Tauco ( Miso -Like Product) Gembus tempe contains 65 calories, protein (3.41 %), carbohydrate (11.94%), fat (0.2%), cal- Tauco is a yellow-colored saline paste, Indonesian cium (143 mg), iron (0.4 mg), and vitamin B1 style miso , made from fermented yellow soy- (0.09 mg) (Sulchan and Nur 2007 ). Sulchan and beans and a yellow-colored saline paste with a Rukmi (2007 ) reported that gembus tempe con- meat-like fl avor and used in Chinese and tains energy (77.70 kcal), protein (4.07 g), lipid Indonesian cuisines as fl avoring agent (Fig. 14.7 ). (0.23 g), total carbohydrate (14.25 g), fi ber (4.69 The name comes from its pronunciation in the g), ash (0.84 g), calcium (159.98 mg), phospho- Hokkien dialect of the Chinese language, and it rus (59.69 mg), iron (0.48 mg), and water (6 %). originates from China. Tauco is often used as Gembus tempe , which is made of the solid condiment and fl avoring for stir-fried dishes of soybean waste of tofu (Kuswanto 2004 ), rich in Indonesian cuisine traditions such as Sundanese fi ber (4.69 %), and contains a threefold greater and Javanese cuisines. Cianjur town is the center level in fi ber compared to tempe (1.40 g %). The of tauco production. amount of essential fatty acid content in gembus To make tauco, the soybeans are soaked in tempe , mainly linoleic and linolenic acid, 21.51 % fresh water, the hulls are removed, and the seeds and 1.82 %, respectively (Fatimah 1998 ). are boiled and spread on bamboo trays to cool. Sulchan and Rukmi (2007 ) reported that gem- Rice or glutinous rice fl our is roasted until golden bus tempe did not contain cysteine, proline, and brown, then mixed with the seeds, and set aside tryptophan, whereas methionine was found at for 3–5 days to ferment between hibiscus (waroe ) 11.9 mg/100 g, in extreme contrast with tempe leaves on fl at trays, involving mold in the fermen- containing cysteine (70 mg/100 g) and methio- tation by Rz. oligosporus , Rz. oryzae , and A. ory- nine (168 mg/100 g). zae followed by brine (20 %) fermentation for Tempe bongkrek is a freshly fermented coco- 20–30 days involving Lb. delbrueckii and nut press cake or shredded coconut residue by Rz. Hansenula sp. After the second phase of fermen- 352 I.S. Surono

Fig. 14.7 Viscous liquid tauco , sweet tauco (left ), salty tauco ( right ) tation is completed, the brine is drained; palm little or no innovation in the process since ancient sugar (25 %) is added and the mixture is cooked times. Black soybeans are boiled to undergo and stored for 24 h or placed directly into bottles. spontaneous solid-state fermentation (SSF) Microorganisms present in tauco are A. oryzae , before being subjected to brine fermentation. Rz. oligosporus , Rz. oryzae , Hansenula sp., After the brine is fi ltered, the fi ltrate is boiled Zygosaccharomyces soyae, and Lb. delbrueckii together with caramel and spices, yielding the (Winarno et al. 1973 ). fi nal product, kecap (Roling et al. 1994 ). When the mass has molded, it is sun-dried for It is a liquid, brown-colored condiment, made a few days until very hard, and the soybean koji by a two-stage batch fermentation which involves for making tauco is used. Remove the leaves and the biochemical activities of mold (Rz. oryzae or put this mass of soybean koji into salt water. On Rz. oligosporus ), lactic acid bacteria the third or fourth day, add some yeast and some ( Lactobacillus sp.), and yeast (S. rouxii ). There cane sugar syrup. Continue the soaking and fer- are two types of kecap , sweetened soy sauce mentation in salt water for 3–4 weeks. Tauco is (kecap manis ) and salty soy sauce (kecap asin ). available in viscous liquid form (Fig. 14.7 ) or According to Codex Alimentarius Commission semisolid form which is obtained by sun-drying (FAO/WHO 2004a , b ), soy sauce is a clear liquid the liquid product to a fi nal moisture content of seasoning obtained by fermentation of soybean 25 %. and/or by hydrolysis of soybean or other vegetable protein sources to produce soy extract which is further processed into sweet soy sauce or salty 14.8 K e c a p (Soy Sauce) soy sauce. Naturally brewed soy sauce is the product In the nineteenth century, sinologist Samuel obtained by A. oryzae and/or A. sojae and/or Rz. Wells Williams wrote that in China, the best soy oryzae and/or Rz. oligosporus as main starter(s) sauce is “made by boiling beans soft, adding an and cultured in either soybean or soybean and equal quantity of wheat or barley, and leaving the cereal grains with or without addition of bacteria mass to ferment; a portion of salt and three times and/or mold and/or yeast and/or enzyme. Non- as much water are afterwards put in, and the brewed soy sauce is the product obtained by whole compound left for 2 or 3 months when the hydrolization of soybean and/or other vegetable liquid is pressed and strained” (Williams 1848 ). protein by using acids or enzymes in the brine or Kecap is an Indonesian soy sauce and usually salt water, namely, “Hydrolyzed Vegetable traditionally made by small-scale producers, with Protein,” and classifi ed as delicious agents (taste 14 Ethnic Fermented Foods and Beverages of Indonesia 353 enhancer agents), not included in the category of and 70 L of hot water (58 °C) and 30 kg of salt soy sauce (FAO/WHO 2004a , b ). While mixed were added to prepare kecap mash and allowed to soy sauce is the product obtained by brewed soy ferment for 2 months at room temperature with sauce and hydrolyzed vegetable protein, propor- exposure to sunlight. tion added by brewed soy sauce is not less than 50 % (FAO/WHO 2004a , b ). Kecap is made by spreading cooked soybeans 14.8.1 Preparation of Kecap Koji on a bamboo tray and leaving for a period to make molded soybeans (kecap koji ). The molded Kecap koji were prepared without inoculum by soybeans are then mixed with salt solution to the conventional method in home industry, but in carry out the second stage of fermentation under the factory, using the starter culture, hence, the 20 % brine solution for 14–120 days at room tem- kecap koji fermentation in the factory is faster perature (Steinkraus 1995 ). Then the fermented than in home industry, 3 and 9 days, respectively. mash is fi ltered. To make kecap manis , the fi ltrate Koji culturing is in a mixture of equal amount of is mixed with palm sugar and spices, boiled for boiled soybeans and roasted wheat to form a 4–5 h and fi ltered (Steinkraus 1995 ). Kecap grain mixture, then Aspergillus spores are added manis contains 26–65 % carbohydrate, 0.3 % (the cultures are called koji in Japanese) total nitrogen, and 3–9 % salt (Judoamidjojo (Judoamidjojo 1986 ). After sun-drying, the mois- 1986 ). ture contents decreased to 7–8 %. Kecap manufactured in home industry does not usually use any inoculum in kecap koji preparation; molds grow on the surface of cooked soy- 14.8.2 Kecap Mash Fermentation beans as the result of infection from the environment such as the air and the previously Kecap mashes were prepared with kecap koji and used trays (Judoamidjojo 1986 ; Nikkuni et al. allowed to ferment for 2 months; the pH value of 2002 ; Steinkraus 1995 ). Molds isolated from the mash reached 5.5 and contained about 21 % kecap koji were mostly of Aspergillus sp. salt. The contents of formol nitrogen and water- (Judoamidjojo 1986 ; Nikkuni et al. 2002 ), and soluble nitrogen increased with the fermentation afl atoxin producers were found from Indonesian time and showed higher as compared to without soybean koji samples (Nikkuni et al. 2002 ). the starter culture. The molds involve in brewing According to Sadjono et al. (1992 ), approxi- soy sauce are A. oryzae and A. sojae (Fig. 14.8 ), mately 47 % of 32 samples of traditionally fer- strains with high proteolytic capacity mented Indonesian kecap tested contained (Maheshwari et al. 2010 ). S. cerevisiae is also afl atoxin B1 at more than 5 μg/kg. Therefore, the involved, and the yeast will convert some of the possibility of afl atoxin contamination cannot be sugars to ethanol, and further biochemical ruled out in traditional koji making process, and changes contribute to fl avor development of soy it is thus necessary to use a pure culture starter for sauce. Bacillus sp. may also grow in soy sauce food safety concern. ingredients and generate odors and ammonia, Kecap mash prepared according to the tradi- while Lactobacillus species will produce lactic tional method is as follows: black soybeans (40 acid and lower the pH. kg) were soaked in water overnight, boiled for about 3 h, spread on ten bamboo trays (ca. 90 cm in diameter), inoculated with 120 g of the starter 14.8.3 Traditional Brine culture, and left for 3 days in the koji fermenta- Fermentation tion room at room temperature by inoculated starter culture. The molded soybeans (kecap koji ) The Aspergillus sp. breaks down the grain pro- were sun-dried for 2 days, winnowed to remove teins into free amino acid and protein fragments the hulls and spores, and placed in a plastic pail, and starches into simple sugars. This amino- 354 I.S. Surono

of glutamine to glutamic acid, rapidly decreased but did not completely disappear. Even after formol nitrogen production had stopped, protease and leucine aminopeptidase activities were present. Therefore, the exhaustion of digestible proteins was more likely the cause of the termination of amino acid production (Roling and van Verseveld 1996 ).

14.8.6 Fermentation by Lactic Acid Bacteria

Fig. 14.8 Molded soy and wheat by A. sojae cultures in traditional fermentation of kecap (Source https://ja.wiki- Staphylococci and enterobacteriaceae involved in pedia.org/wiki/%E3%83%95%E3%82%A1%E3%82%A SSF decreased rapidly after addition of salt. Only 4%E3%83%AB:Shoyukoji.jpg) bacilli was observed after 1 week in brine solution. Numbers of salt-tolerant bacteria were high glycosidic reaction gives soy sauce its dark brown at the start of fermentation (107–8 cfu/ml), but color. dropped rapidly within 2 days before increasing again. After 1 week of brine fermentation, only the lactic acid bacterium T. halophila was iso- 14.8.4 Brine Fermentation lated from samples and reached around 10 8 cfu/ at Industrial Manufacturer ml. The pH of the brine dropped from 5.0–5.1 to After 43 h SSF followed with brine fermentation 4.4–4.6, and concentrations of lactate and acetate for 4 months. About 8000 kg SSF material is increased up to 164 mM and 69 mM, respec- mixed with 16,000 l brine. Final salt concentra- tively. Fructose completely disappeared. Lactic tion of the brine is 15 %. Three overlapping acid fermentation took about 2.5–3 weeks, then phases occurred in brine fermentation: fi rst, no further changes in lactate and acetate concen- amino acid production (based on formol nitrogen trations occurred, and the number of T. halophila production), followed by lactic acid fermentation declined (Roling and van Verseveld 1996 ). (based on acetate and lactate production), and lastly yeast fermentation (based on ethanol and glycerol production) (Roling and van Verseveld 14.8.7 Yeast Fermentation 1996 ). Immediately after the preparation of brine, high number of yeast was observed (104–5 cfu/ml). 14.8.5 Amino Acid Production Yeast fermentation in brine fermentation started after 5 days, and in some cases, after 42 days Amino acid production started directly after the there was no yeast fermentation observed. During preparation of brine, and amino acids were pro- yeast fermentation a slight increase in yeast num- duced during the fi rst 3 weeks, and glutamic acid, ber, ethanol, and glycerol concentrations were mainly responsible for fl avor, is produced during observed. Zygosaccharomyces rouxii was the a longer period, and less than 15 % of the fi nal dominant yeast species. Glucose concentration glutamic acid is formed during the last 3 months dropped during the yeast fermentation, but galac- and continued to increase slowly after 4 months tose concentration remained unchanged. In brine (Roling and van Verseveld 1996 ). The activity of fermentation heat-dependent browning reactions glutaminase, which is responsible for conversion (Maillard reactions) took place during the entire 14 Ethnic Fermented Foods and Beverages of Indonesia 355 period of fermentation (Roling and van Verseveld duction methods and is also conducted by kecap 1996 ). manufacturer to speed up the fermentation. The The fi ltrate brine undergoes several post- addition of enzymes should be allowed for fermentation treatments, such as the addition of brewed kecap . In traditional kecap manufactur- caramelized sugar and subsequent boiling for ing, microorganisms are added for the sole pur- several hours, resulting in a thick, strong brown pose of producing enzymes that hydrolyzed soy color and evaporation of volatile compounds proteins for development of the characteristic such as ethanol. taste attributes of soy sauce. Whether produced The organic acids formed during the growth of traditionally, or added directly, enzymes carry T. halophila have a preserving effect on kecap . out the same function. The fully fermented grain Amino acids contribute to the fl avor of kecap , slurry is placed into cloth-lined containers and directly as glutamic acid or indirectly via Maillard pressed to separate the solids from the liquid reactions during boiling of the mixture of brine kecap. The isolated solids are used as fertilizer or extract and caramel (Yokotsuka 1986 ). The fed to animals while the liquid kecap is processed amino acid content and lactic acid concentration further. Finally, the raw kecap is pasteurized to do not change much after 4 weeks; hence, brine eliminate any active yeasts and molds remaining fermentation for 1 month seems to be suffi cient in the soy sauce and then fi ltered. The kecap can for industrial kecap production. be aged or directly bottled and sold (Fig. 14.9 ). Addition of food-grade enzymes in brewing In traditional practice, the liquid is extracted, kecap represents continuous innovation in pro- clarifi ed, and fi ltered before introduction of

Fig. 14.9 Kecap manis (sweet soy sauce) 356 I.S. Surono desired taste and fl avor by addition of brown 14.8.10 Food Safety Concern sugar, spices, and certain additives (enhancer, on Carcinogens preservatives and or coloring, and molasses); fi nally it is pasteurized and packaged. Sweet soy Soy sauce may contain ethyl carbamate, a Group sauce in Indonesia is mostly produced by 2A carcinogen (Matsudo et al. 1993 ). In 2001, medium-large enterprise (approx. 60 %) while the UK Food Standards Agency found that 22 % the remaining 40 % produced by small-medium of tested samples contained a chemical carcino- enterprise (FAO/WHO 2004b ). gen named 3-MCPD (3-monochloropropane- 1,2- Industrial manufacturers use defatted yellow diol) at levels considerably higher than those soybean fl akes and wheat instead of black soy- deemed safe by the EU (0.02 mg/kg) from vari- beans only. SSF is well controlled and inocu- ous soy sauces manufactured in mainland China, lated; however, brine fermentation is spontaneous Taiwan, Hong Kong, and Thailand, made from and subjected to tropical weather conditions for 4 hydrolyzed soy protein, rather than being natu- months (Wilfred et al. 1996). During brine fer- rally fermented (Hamlet et al. 2002 ; Crews et al. mentation in traditional kecap manufacture, 2003). About two thirds of these samples also amino acid production and growth of the lactic contained a second carcinogenic chemical named acid bacterium Tetragenococcus halophila (until 1,3-DCP (1,3-dichloropropan-2-ol) which should recently known as Pediococcus halophilus ) take not be present at any levels in food. Both chemi- place. Few or no obvious yeast fermentation is cals have cancer potential, and the agency recom- observed (Roling et al. 1994 ). mended to withdraw from shelves and avoided 3-MCPD and 1,3-DCP , chloropropanol carcinogens. The same carcinogens were found in soy 14.8.8 Socioeconomic Value sauces manufactured in Vietnam, causing a food scare in 2007 . Continuous lifetime exposure to In Indonesia, during 2003, total production of high levels of 3-MCPD could pose a health risk, sweet soy sauce and salty soy sauce was approxi- and Health Canada has established 1.0 part per mately 80.000 tons (90 %) and 31.200 tons million (ppm) as a guideline for importers of the (10 %), respectively, and the potential growth is sauces and considered to be a very safe level (Fu about 3.6 % per year. As condiment, consump- et al. 2007 ). tion of sweet soy sauce is about 0.9 l/capita/year. Besides being part of daily Indonesian cuisine, kecap or sweet soy sauce also served as 14.9 Fermented Fruits Indonesian typical sauces for instant noodle and and Vegetable Products other products. 14.9.1 Acar Pickles

14.8.9 Quality Criteria of Soy Sauce Acar is a type of vegetable pickle s (Fig. 14.10 ) made in Indonesia , Malaysia, and Singapore , The total nitrogen should be not less than 0.4 % usually prepared in bulk as it easily is stored in a w/w in salty soy sauce and not less than 0.15 % in well-sealed glass jar in refrigerator for a week sweet soy sauce, the soluble solid contents, exclu- and served as the condiment for any meals. It is a sive of added salt not less than 6 % (w/v), and the localized version of the Mughlai Achaar . It is sugar content for sweet soy sauce is not less than known as atjar in Dutch cuisine , derived from 30 %, and salt content for salty soy sauce is not Indonesian acar . In Indonesia , acar is commonly less than 10 % (FAO/WHO 2004a ). made from small chunks of cucumber, carrot, 14 Ethnic Fermented Foods and Beverages of Indonesia 357

while PG was effective in causing excessive soft-

ening. When CaCl2 was present in PG-containing solutions, softening by PG was inhibited (Lennox and Eﬁ uvwere 2013 ; Buescher et al. 1979 ).

14.9.1.1 Microbial Changes During Acar Fermentation The common microorganisms usually isolated from cucumber are enteropathogenic bacteria, lactic acid bacteria (LAB), Pseudomonas spp., E. Fig. 14.10 Mixture of cucumber and carrot acar carotovora, and some fungi, and the heterotro- phic plate counts in the produce range between shallot, young bamboo shoot, eggplant, chili, and 4.0 × 10 2 and 5.7 × 10 2 cfu/g-1 (Nahaisi et al. occasionally pineapple and marinated in a sweet 2005 ). and sour solution of sugar and vinegar. The changes in microfl ora of the fermenting Acar is a very popular accompaniment in cucumber in brine solution were reported by many of Indonesian dishes, such as nasi goreng Lennox and Efi uvwere ( 2013 ), with the initial (fried rice), friend noodle, sate, and almost all counts of 4.2 × 10 6 cfu/ml, 1.4 × 10 3 cfu/ml, varieties of soto . It is very easy to prepare at 5.0 × 10 6 cfu/ml, and 1.2 × 10 3 cfu/ml for lactic home; the key to a successful acar is to use the acid bacteria (LAB), fungi, E. coli , and freshest ingredients possible. Just like common Salmonella – Shigella, respectively, in the fer- pickles , the sour taste of vegetable acar may menting brine solution. There was sharp increase freshen up the meal, especially the fi shy dish in counts of LAB and indicator organisms in the such as grilled fi sh or the rich and oily dish such brine on the third day, and thereafter they started as mutton satay to neutralize the fatty taste. to decline but the indicators were fi nally inhibited Various microorganisms are usually associ- by the 12th day. Salmonella – Shigella showed ated with fresh fruits and vegetables as normal very slight increase on the third day but were fl ora, transit fl ora, spoilage, and pathogenic fi nally inhibited by the 12th day. The fungi counts organisms. Cucumber comes in contact with soil fl uctuated and reached their peak on the sixth insects and animals during its growth and harvest day, but were fi nally inhibited also by the 15th from the fi eld, and therefore, the microbial fl ora day. LAB persisted to the end of the fermentation will include soil microorganisms and those from with the total count of 2.1 × 10 cfu/ml. There was contaminated irrigation water; direct contamina- no growth of Salmonella – Shigella within the tion by wild animals, birds, and insects; and fruit during fermentation. On the third day of transportation with contaminated containers fermentation, only LAB and indicator organisms (Reina et al. 2002 ; Heaton and Jones 2007 ; grew with counts of 5 × 103 cfu/g and 3 × 103 Williamson et al. 2003). cfu/g, respectively. The indicator organisms were Cucumber (Cucumis sativus ) is one of the pri- inhibited within the cucumber by the sixth day. mary vegetables often fermented to obtain pick- The changes in the microbial fl ora in the brine les involving a mixed microbial fermentation in and cucumber could have been due to the com- which desirable and undesirable bacteria and petitive nature of the microorganisms in any envi- fungi interact and compete during the initial ronment and proved to be effective in eliminating stages of the fermentation. The harvested cucum- pathogenic organisms and other contaminants bers were naturally fermented in 10 % (w/v) and also preserved the cucumber.

NaCl solution for 30 days with and without CaCl2 There is usually succession of LAB during and/or polygalacturonase (PG). CaCl2 and PG fermentation of pickled cucumber. The epiphytic treatments did not interfere with fermentation. LAB which occur naturally on the surface of the

Separately, CaCl2 enhanced fi rmness of pickles cucumber initiate fermentation and effectively 358 I.S. Surono control the microbial ecology of the fermentation pH falls from 6.5 to 4.2 in 8 days of fermentation by consuming the glucose and fructose present, (Puspito and Fleet 1985 ). Lactic acid, acetic acid, producing lactic acid, and lowering the brine pH succinic acid, ethanol, and glycerol are produced which favors Lb. plantarum , a homofermenta- during 2–14 days of fermentation. Hydrolysis of tive, acid-tolerant LAB which takes over the fer- starch and maltose resulted in glucose, which is mentation and does not result in production of utilized by microbes for their growth during carbon dioxide from sugars (Lennox and fermentation. Efi uvwere 2013 ). The lactic acid they produce is Manufacture of fermented mustard cabbage effective in inhibiting the growth of other bacte- leaves was made by addition of the salt to vegeta- ria that may decompose or spoil the cucumber bles, allowing the growth of certain fermentable due to metabolic products such as lactic acid like microorganisms, resulting in sensory changes bacteriocins, peroxides, and peptides that can bearing acidic and unique characteristics to the inhibit other bacteria (DeVuyst and Vandamme sayur asin (Chiou 2004 ). Epiphytic lactic acid 1994 ; Sapers and Annous 2004 ). bacteria (LAB) of which initially amounted to slightly between 10 and 1000 cfu/g plant (0.001–1 % of the total population of microor- 14.9.2 Sayur Asin ganisms) became dominant within the microorganism population in the fermented mustard Sayur asin (Fig. 14.11) is an ethnic, fermented cabbage as a result of the anaerobic condition on mustard cabbage leaf ( Brassica juncea var. the vegetables (Daeschel et al. 1987 ; Azcarate rugosa ) product from Indonesia (Puspito and and Todd 2010 ). After 2 days of fermentation, the Fleet 1985 ). Mustard cabbage leaves are sorted, lactic acid was produced at 0.8–1.5 % in 2.5 % washed, withered, wilted, and rubbed or squeezed brine and the pH reached 3.4 (Sulistiani et al. with 2.5–5 % salt. Liquid from boiled rice (air 2014 ). A combination of acidic conditions and tajin) is added to provide fermentable carbohy- salt concentrations suppressed the growth of drate to assure that suffi cient acid is produced undesirable microorganisms, hence, preserving during fermentation. Fermentation is initiated by the vegetables. Leuc. mesenteroides , Lb. confusus, and Lb. cur- Pederson (1971 ) reported that 2.25–2.5 % salt vatus and later dominated by Lb. plantarum and allowed exclusive growth of lactic acid bacteria, P. pentosaceus . Starch degrading species of suppressed the growth of spoilage bacteria, and Bacillus , Staphylococcus , and Corynebacterium inhibited pectinolytic and proteolytic enzymes exhibited limited growth during the fi rst day of that can cause softening and putrefaction (Swain fermentation. The yeasts, C. sake and C. guillier- et al. 2014); the osmosis process will draw out mondii, also contributed to the fermentation. The the water and nutrients from vegetables as growth

Fig. 14.11 Sayur asin on sale in the traditional market 14 Ethnic Fermented Foods and Beverages of Indonesia 359 medium, facilitating metabolism of sugar into from sayur asin . The spontaneous fermentation lactic acid during fermentation. Low salt concen- of sayur asin involved diverse variation of lactic tration such as less than 2.25 % will support the acid bacteria. growth of proteolytic bacteria, while adding more According to Chao et al. (2009 ), the variety of than 10 % salt will enable the growth of halo- LAB population in the fermented mustard was philic bacteria and cause fermentation failure. In infl uenced by different fermentation process and general the higher the salt concentration, the treatments, especially the salt concentration. slower fermentation. For a short time fermenta- During the process, the squeezed and salt treat- tion preferably 2.5–10 % brine solution is used ment diffused water and nutrient out of the vege- (Swain et al. 2014 ). table tissues by high osmotic pressure (Chiou Sulistiani et al. ( 2014) reported identifi cation 2004 ). In addition, the varying conditions of of 246 lactic acid bacteria isolates from sayur anaerobiosis, moisture levels, and temperature asin based on 16S rDNA sequence data. The bac- resulted in changes in the population balance and teria belong to 11 species, viz., Lb. farciminis (15 selected for spontaneous fermentation by lactic isolates), Lb. fermentum (83 isolates), Lb. namu- acid bacteria (Azcarate and Todd 2010 ). The rensis (18 isolates), Lb. plantarum (107 isolates), growth of the lactic acid bacteria is also infl u- Lb. helveticus (1 isolate), Lb. brevis (1 isolate), enced by nutrient movement from plant material Lb. versmoldensis (3 isolates), Lb. casei (12 iso- into the surrounding liquid (Daeschel et al. 1984). lates), Lb. rhamnosus (2 isolates), Lb. fabifermentans (3 isolates), and Lb. satsumensis (1 isolate), and revealed that Lb. plantarum and Lb. 14.9.3 Brem fermentum are common LAB used in sayur asin production from Central Java, Indonesia, and Brem is traditional fermented food or fermented have also been reported to be found in different beverage , a non-distilled ethnic alcoholic drink fermented foods (Ludwig et al. 2009). These spe- from Indonesia prepared from glutinous rice. It is cies have been characterized into different fer- a dried, starchy, sweet–sour rice extract and is mentation types: obligately homofermentative, eaten as a snack. There are two types of brem : facultatively heterofermentative, and obligately brem cake (solid), which is yellowish-white, heterofermentative (Felis and Franco 2007), and sweet–sour snack usually eaten in Madiun , where they have been reported to be not pathogenic to it is prepared in blocks of 0.5 × 5 to 7 cm (Fig. human or animal (Azcarate and Todd 2010 ). 14.12 ), and in Wonogiri (Fig. 14.13), where it is Therefore, the sayur asin is safe for human sweet, very soluble, white, and thin circular consumption. blocks of 5 cm diameter, and brem beverage Several species which were not found by (liquid), which is made of rice wine from Bali Puspito and Fleet ( 1985 ) have been determined and Nusa Tenggara, but mostly known from Bali by molecular identifi cation. These include Lb. (Basuki 1977 ). namurensis , Lb. versmoldensis , Lb. rhamnosus Brem cake from Madiun (Fig. 14.12 ) and (previously known as Lb. casei subsp. rhamno- Wonogiri (Fig. 14.13 ) is believed by Indonesian sus ), Lb. fabifermentans , and Lb. satsumensis . The result showed that each sample consisted of various species, predominated by Lb. plantarum and Lb. fermentum, with Lb. fermentum and Lb. plantarum being more acid tolerant and often dominating the fermentation processes of vegetables and cereals (Sulistiani et al. 2014 ). In another study, Swain et al. (2014 ) reported that Leu. mesenteroides , Lb. confusus , Lb. curvatus , P. pentosaceus, and Lb. plantarum have been isolated Fig. 14.12 Brem Madiun 360 I.S. Surono

Fig. 14.13 Brem Wonogiri

consumer to be important for stimulating the a solid-state fermentation of steamed glutinous blood system. It is also reported to prevent der- rice by a traditional inoculum (ragi ), extraction matitis , probably due to the presence of signifi - of the liquor, and further liquid-state fermenta- cant amounts of B vitamins produced by the tion without additional inoculation. The quality microorganisms. This product is consumed as a of this product is inconsistent due to the inconsis- snack and is not part of the daily family diet. tency of the microorganisms in ragi as a conse- All three types of brem are made from the liq- quence of spontaneous fermentation. In the uid portions of tapé ketan (fermented glutinous Philippines, rice wine is called tapuy, while in rice). The glutinous rice is steamed and spread on Japan it is called sake (Aryanta 2000 ). the trays lined by banana leaves to cool, then The liquid portion of tapé ketan is aged for 7 0.2 % powdered ragi (inoculum), the same ragi months, during which solids precipitate, leaving for tape singkong fermentation is added to the a clarifi ed brem, known as brem Bali, and is cooled rice and mixed thoroughly, incubated at decanted and bottled (Basuki 1977 ). Alcohol room temperature (30 °C) for 3 days aerobically, content of brem Bali (Fig. 14.14 ) is 6.1 % the juicy rice called tape is pressed out and trans- (Winarno 1986). Brem with improved ragi is ferred to the fermenting jars, fermented anaerobi- produced which has more desirable fl avor than cally at room temperature for 8–10 weeks. After conventionally made brem (Saono et al. 1984 ). fermentation, the juice is siphoned carefully into Brem beverage consumed and holds important sterilized bottles and stored in a cool room for use in temple ceremonies of Hinduism called aging around 8–12 months (Aryanta 1980 ). Tetabuhan, an offering beverage for Buto Kala During brem production, the fi ltrate of tapé (lit. Kala the Giant) in order to evoke harmony. ketan is boiled down, poured onto a table, cov- Brem Bali beverage can be either white or red ered with banana leaves, and left to cool at ambi- depending on the proportions of white and black ent temperature over 8–12 h ( brem Madiun) or glutinous rice used in production; it is very sweet sun-dried for 1 day to produce brem Wonogiri to semisweet, yet acidic, and contains alcohol (Campbell-Platt 1987 ). with varying degree, usually from 5 % to 14 %. Brem beverage is a traditional rice wine of Liquid brem is made from fermented mash of Bali island. The process of brem making involves black/white glutinous rice using a dry starter 14 Ethnic Fermented Foods and Beverages of Indonesia 361

The sugar content of brem decreased during the fermentation, due to the decomposition of simple sugars into ethanol and carbon dioxide by the yeast’s enzyme activity (Pederson 1971 ) through Embden–Meyerhof–Parnas (Aurand and Woods 1973). During fermentation of brem , the pH decreased, which might be due to more production of some organic acids at longer fermentation time. After 10 weeks, the pH reach 4.0 and contained 3 % reducing sugar, 6 % ethanol, and 0.6 % total acidity (as acetic acid), due to the oxidation of ethanol to acetic acid by the activity of Acetobacter aceti .

Fig. 14.14 Brem Bali beverage 14.9.4 Tuak (Palm Wine) called ragi tape . Glutinous rice is soaked and Tuak (palm wine) is one of the indigenous alco- drained, steamed for 1 h, and then cooled down. holic beverages most widely known in North The cooled glutinous rice is then inoculated with Sumatra region of Indonesia. The Arecaceae ragi tape and amylolysis begins. A honey-like such as palm sap of aren ( Arenga pinnata ) and rice syrup settles in the bottom of the malting nipa ( Nypa fruticans) called nira , a sweet juice vessel. Following 3 days of conversion from the with pH between 5.5–6.5 % and 80–90 % mois- starch to sugar, yeast culture is added and alco- ture content, is fermented spontaneously through holic fermentation begins. Alcoholic fermenta- the application of one or more several kinds of tion typically goes on for 2 weeks. woodbark or root, called raru (Xylocarpus woodbark or a variety of forest mangosteen), into the 14.9.3.1 Microbiological sap water of sugar palm (Arenga pinnata ) with and Biochemical Changes the involvement of natural yeasts for 2–3 days. Aryanta (1980 ), Lotong ( 1985 ), and Uchimura The sweet taste of the palm sap is due to the (1998) reported the microbes in ragi . Aryanta presence of sugars (sucrose, glucose, fructose, (1980 ) reported that during the fi rst 3 days of fer- and maltose). The sugar content is 12.30– mentation, the population of molds in brem (ragi 17.40 %, and the reducing sugar is 0.5–1 %. In NKL as inoculum) was 3.5 × 105 cfu/ml and addition to sugar, the juice contains other ingredi- decreased to 5.9 × 102 cfu/ml; the yeast was at ents such as protein, fat, water, starch, and ash as 5.5 × 104 cfu/ml and increased to 4.9 × 106 cfu/ml, well as organic acids (citric, malic, succinic, and after 2 weeks of fermentation, it then lactic, fumaric) that play a role in the formation decreased until the sixth week, and no mold was of specifi c brown sugar fl avor (Judoamidjojo found. No yeast was isolated at the eighth week 1985). Hence, palm sap is a good medium for the of fermentation. On the third day of fermenta- growth of microorganisms such as bacteria, tion, bacterial count was 6.5 × 10 cfu/ml which fungi, and yeast and needs to be preserved as increased until the second week (8.0 × 106 cfu/ soon as possible. The presence of microorgan- ml) and then decreased until sixth week. On the isms may spoil the palm sap which is character- eighth week of fermentation, no bacteria were ized by the formation of mucus to become turbid, isolated (Aryanta 1980 ). murky, green, white, and frothy sour taste. 362 I.S. Surono

In North Sumatra, Tuak is produced by spontaneous fermentation of the palm sap ( Arenga pinnata) in the presence of raru wood or several kinds of woodskin or roots (like nirih – Xylocarpus woodskin or a kind of forest mangosteen) for overnight incubation. Native people in Gorontalo, North Sulawesi, called tuak as “bohito” in their native language (Latief and Latief 2014 ). Hermansyah et al. ( 2015 ) isolated and identifi ed culture independent method for Candida tropicalis from North Sumatra’s tuak among Fig. 14.15 Fermented durian, tempoyak other yeasts. The C. tropicalis isolate is able to utilize glucose for more rapid and higher production of ethanol at high temperature of 42 °C as called pakua lo bohito consumes tuak for making compared with S. cerevisiae. However, the opti- social interaction, as Japanese drink sake. mum temperature of C. tropicalis isolates is 30 °C as displayed by its ability to produce 6.55 % (v/v) and 4.58 % ethanol from 100 g/l glucose 14.9.5 Tempoyak fermentation at 30 °C and 42 °C, respectively. Rahayu dan Kuswanto ( 1988) revealed that the Tempoyak is a traditional condiment (Fig. 14.15 ) alcohol content in tuak was 3–10 %, which made from the fl esh or raw pulp (aril) of the depends on fermentation medium (aren or nipa ) durian fruit (Durio zibethinus ), a kind of tropical and fermentation time, and the most important fruit, naturally fermented at room temperature in thing is the natural indigenous microbes involved a tightly closed container and normally prepared during fermentation. Hartanto (1997 ) revealed from excess, poor-quality or overripe fruits that alcohol content of tuak is quite similar to (Ganjar 2000 ). This product is popular among wine (6–12 %). people living in Riau Province, Sumatra, Indonesia, as well as in Malaysia. Interestingly, 14.9.4.1 Social Aspect even though the fresh durian pulp is fermented Tuak contains alcohol; hence, Moslem commu- without any heat application, there is no record of nity does not drink tuak . Non-Moslem communi- food-borne illness caused by the consumption of ties in some areas in Indonesia consume tuak and tempoyak. Tempoyak has a long history of safe also used it as traditional remedy and in some consumption, in Riau, Sumatra. ritual or traditional ceremony in Sumatra and The fermentation process may involve salt or Flores islands (Ikegami 1997 ; Ola 2009 ). The without salt, and usually low amount of salt, ethnic tribe Batak Toba believe that tuak is good 1.3 %, is added to support the growth of desired for new mothers after giving birth to augment lactic acid bacteria besides yeast as saccharolytic their breast milk production and to remove the microbes. Tempoyak is manufactured by mixing impurities through sweat. In North Sumatra, durian pulp with salt and allowing it to ferment there is traditional ceremony to respect old gen- for 3–7 days, producing a distinctive durian smell eration, namely, manuan ompu - ompu dan manu- and creamy yellow color with sour and salty taste langi (Ikegami 1997 ). Likewise, traditional (the sour taste dominates). Tempoyak is con- ceremony called Lewak Tapo in Lamaholot eth- sumed with rice or added to cooking dishes as nic group of Adonara island, East Flores. Latief condiment (Ganjar 2000 ). and Latief (2014 ) reported that in Momala vil- The initial pH of tempoyak is in the range of lage, Gorontalo, North Sulawesi, a community 6.62–6.83, and after 2 days of fermentation, the 14 Ethnic Fermented Foods and Beverages of Indonesia 363 pH is in the range of 3.96–4.08 (Leisner et al. i.e., durian, and contribute to the nutritional, 2001 ; Merican 1977 ; Amin et al. 2004 ). The total organoleptic, and health properties of tempoyak . acidity of tempoyak is around 3.6 % as acetic acid Pato and Surono (2013 ) identifi ed two poten- and the fi nal pH value is 3.8–4.6 (Steinkrauss tial probiotic strains isolated from tempoyak, et al. 1996; Merican 1977 ). Suan (1996 ) reported Enterococcus sp. UP-9 and Enterococcus sp. that tempoyak has 2.0 % ash, 67 % moisture, UP-11, by PCR the 16S rRNA gene sequences 4.5 % total sugar, 2.5 % crude fi ber, and 1.4 % fat. using specifi c primers and sequencing of ampli- fi ed region by using an automated sequencer, and showed 97 % homology to Ent. gallinarum 14.9.6 Lactic Acid Bacteria and Ent. faecalis, and named Enterococcus gal- Involvement linarum UP-9 and Enterococcus faecalis sp. During Tempoyak UP-11, respectively. This fi nding is contradictory Fermentation to the previous report by Leisner et al. ( 2000 ) where Lactobacillus was the dominant lactic acid Several LAB found to be involved in the fermen- bacteria in tempoyak due to its natural fermenta- tation of tempoyak are Lb. plantarum , Leu. mes- tion. Enterococcus sp. and Lactobacillus sp. were enteroides subsp. mesenteroides , Streptococcus the predominant genus in tempoyak and rela- faecalis (Ohhira et al. 1990 ), Leu. mesenteroides , tively resistant to acid as the isolates were origi- Lb. brevis , Lb. mali , Lb. fermentum (Leisner et al. nated from tempoyak with pH 3.69. Ent. 2001), and Lb. durianis (Leisner et al. 2002 ). gallinarum UP-9 and Ent. faecalis UP-11 showed Wirawati ( 2002) isolated Lb. plantarum , Lb. potential probiotic properties and were able to coryniformis , and Lb. casei from tempoyak. reduce cholesterol level by different mechanisms, Yuliana and Dizon (2011 ) found Lb. plantarum , namely, deconjugating taurocholic acid and cho- Lactobacillus sp., W. paramesenteroides , and P. lesterol binding (Pato and Surono 2013 ). acidilactici in tempoyak . Amin et al. (2004 ) reported that addition of In another study, Widowati et al. (2013 ) 1 % salt in tempoyak fermentation for 10 days reported the involvement of Oenococcus , showed the highest lactic acid bacteria viable Leuconostoc , Enterococcus , Lactococcus , counts, and at 8–10 days fermentation, the viable Pediococcus, and Lactobacillus. Leuconostoc sp. lactic acid bacterial counts were comparable in the presence of 2 % and 4 % salt, respectively, between 1 % and 2 % salt addition. The higher the at 20 ± 2 °C during early stages of 4 weeks fer- salt concentration, the lower the lactic acid bacte- mentation, whereas heterofermentative ria viable counts; however, addition of 2 % salt Lactobacillus sp. and homofermentative produced the most preferred tempoyak by sen- Lactobacillus sp. dominated the bacterial popula- sory evaluation. tion in the middle stage. At the end of fermenta- The involvement of lactic acid bacteria in tem- tion, homofermentative Lactobacillus sp. and poyak fermentation might be due to the total Pediococcus sp. were found during tempoyak sugar content in durian fruit, 15–20 % (Ketsa and fermentation. Daengkanit 1998 ), and 17 % saccharose which While Pato and Surono ( 2013) isolated Ent. may favor the growth of lactic acid bacteria and gallinarum and Ent. faecalis , out of 12 isolates yeast (Leisner 2001). classifi ed as genus Lactobacillus sp. isolates and the other 32 Enterococcus sp. isolates from tempoyak in Riau, . Almost all LAB isolated from 14.9.7 Mandai tempoyak were relatively resistant to acid as indicated by the reduction in the number of colonies Mandai is a fermented product made from cem- between 0.76 and 2.82 log cycles at pH 3.0 after pedak (Artocarpus champeden) or jackfruit 2 h incubation. These lactic acid bacteria play an ( Artocarpus heterophyllus ) inner peel in brine essential role in preserving raw food materials, solution, a traditional fermented food of native 364 I.S. Surono people in the province of Central, South, and East growth of aerobic microorganisms and otherwise Kalimantan. Dami , inner part of peel, is the supports the growth of microorganisms that are nonedible part of the fruit. The dami is cleaned microaerophilic and anaerobic (Emmawati and soaked in a 5–15 % brine solution for 2 2014 ). Mandai fermentation in 10 % and 15 % weeks. Fermented mandai is seasoned and con- brine solution facilitates the growth of lactic acid sumed as a side dish of rice. It tastes good and is bacteria, as shown by higher viable counts of lac- savory and its texture resembles that of meat, tic acid bacteria. On the other hand, 5 % brine making these foods popular. Mandai fermenta- solution may facilitate other microbes to grow, tion process is part of an effort to preserve and to and the competition for the nutrients may sup- utilize the waste from jackfruit consumption. press the growth of lactic acid bacteria. Mandai generally can be kept for 1 year or more. Biochemical aspects such as reducing sugar Rahayu (2003 ) found nine isolates of lactic acid and N-total decreased to 0.240 % at day 14 and bacteria from mandai cempedak and identifi ed 0.159 % at 21 day, respectively. Substrates for by molecular detection as Lactobacillus planta- salinity increased in the third week to 4.941 % rum and, P. pentosaceus. Emmawati (2014 ) and relatively stable. The pH value of the sub- reported the involvement of Lb. plantarum in strate is in the range of 3.71–6.02 (Emmawati mandai fermentation. 2014 ). The dynamic changes of microbes during 14.9.7.1 Dynamic Changes of Lactic mandai fermentation was reported by Emmawati Acid Bacteria (2014 ) that on days 4–8 of fermentation, cocci and Biochemical Changes isolates predominate the microbe population. On During Mandai day 8, the viable counts of lactic acid bacteria Fermentation were decreased in mandai fermentation. The lac- The total viable count of lactic acid bacteria was tic acid bacteria isolated from day 8 at 15 % brine reported to increase during mandai fermentation solution of fermentation reveal that the isolates in 10 % and 15 % brine solution, but not in of 5 % are halotolerant or halophilic cocci. However, at brine solution. The initial viable count of lactic day 12, the cocci isolates were not found, proba- acid bacteria was approximately 6–7 log cfu/ml bly due to acidic environment as a result of more and the fi nal viable count was in the range of metabolites produced, including organic acids, 7.0–7.7 cfu/ml (Emmawati et al. 2015 ; Nur 2009 ) thus lowering the pH and making the condition to until day 14 of fermentation. Salt concentration become acidic with the fi nal pH in the range of signifi cantly infl uences the amount of lactic acid 4.16–4.8. bacteria. The higher the concentration of salt added during mandai fermentation, the higher the viability of lactic acid bacteria counts. Salt in the 14.9.8 Low Salt Concentration fermentation of mandai added environmental on Microbial and Biochemical selection factors. Changes in Mandai Effect of salinity on the growth of lactic acid Fermentation bacteria during fermentation was also reported by Ji et al. ( 2007 ) in cabbage fermentation. In general, mandai is made in high salt concen- Higher salt concentration will decrease the total tration. Nur ( 2009) reported the fermentation of viable count of lactic acid bacteria. Eight to 12 % mandai in 10 % (w/v) brine solution for 14 days. brine solution will inhibit the growth of lactic Microbial succession occurred during acid bacteria at the beginning of fermentation and fermentation. Yeast cells grew dominantly then increase at the end of fermentation. Salt (2.8 × 109 cfu/g) on day 5, but bacteria were dom- affects microbial growth by reducing the avail- inant at day 14 (1.1 × 107 cfu/g). The highest ability of water in the cell. The presence of salt decrease of reducing sugar and N-total contents also lowers the reduction potential of limiting the were 0.240 % at day 14 and 0.159 % at day 5, 14 Ethnic Fermented Foods and Beverages of Indonesia 365 respectively. The pH value was varied within the range of 3.71–6.12 for the whole period of fermentation. Biochemical parameters such as reduction sugar, N-total, pH, and salinity of substrates were changed. Reducing sugar content decreased to 0.240 % on day 14 and the levels of N-total also declined in day 5 to 0.159 %. The pH value of the substrate was in the range of 3.71–6.02 (Nur 2009 ). The addition of salt to organic substrates leads to a series of spontaneous fermentation and microbial selection that leads to a succession of microbes. Salt in high concentrations can inhibit the growth of spoilage and pathogenic microbes due to the decrease in the value of water activity

(aw ) and ionized salt into ions Cl toxic. Treatment with high salt on the one hand affects aroma pres- Fig. 14.16 Dadih product ervation and formation and on the other hand poses a concern for the health of the consumer, safety, portability, and novelty to milk nutrients especially hypertension. for the indigenous people in West Sumatra. Rahayu (2003 ) and Lindayani and Hartayanie The higher protein content in buffalo milk (2013 ) isolated Lb. pentosus from mandai of results in custard-like consistency at the end of Semarang city, which can grow at 10 °C, 45 °C, fermentation. In addition, higher fat content and 50 °C and at pH 4.4 in 6.5 % brine solution. enriches the fl avor developed in the dadih prod- Lb. pentosus is heterofermentative lactic acid ucts. A good-quality dadih is fi rm with uniform bacteria that cannot grow at pH 9.6 and in 18 % consistency and has a creamy-white color, pleas- brine solution. ant aroma, and acidic taste with smooth and glossy surface; its cut surface is trim and free from cracks and air bubbles. 14.10 Fermented Milk Products Dadih and dahi are Indonesian and Indian yogurts, respectively, which seem to share the 14.10.1 Dadih same root word. The body and texture of yogurt depend largely on the composition of milk Dadih (dadiah , in native language) is an employed in its manufacture, whereas the manu- Indonesian traditional fermented milk made out facture of dadih and dahi is simpler than Western- of buffalo or cow’s milk produced and consumed type yogurt, without any starter cultures involved by the West Sumatran Minangkabau ethnic group (Surono and Hosono 2011 ). Dadih is served at of Indonesia (Fig. 14.16 ). It is one of the very weddings and during inauguration of an honor- popular dairy products in Bukittinggi, Padang able title “Datuk” in West Sumatra during the Panjang, Solok, Lima Puluh Kota, and Tanah ethnic tradition or “adat” ceremony. Generally, Datar (Surono and Hosono 1996a ). dadih is consumed during breakfast with rice It is a signifi cant dairy product in the diet after adding sliced shallot and chili ( sambal ), or resembling yogurt and is similar to dahi of India it is mixed with palm sugar and coconut milk, with a distinctive thicker consistency, smooth being served as a topping of steamed traditional texture, and pleasant fl avor due to its higher total glutinous rice fl akes, a corn fl ake-like product, solid content, higher fat content, and casein con- called ampiang dadih . tent as compared to cow’s milk. Dadih provided 366 I.S. Surono

14.10.1.1 Manufacturing of Dadih Various indigenous lactic acid bacteria (LAB) The manufacturing method of dadih is quite sim- involved in the dadih fermentation may vary ilar to the dahi of India, except for the heat treat- from time to time, from one place to another due ment of raw milk and the starter cultures being to the natural fermentation without any starter incorporated. In dahi making, the raw cow or culture involved (Surono 2000 ; Akuzawa and buffalo milk, or a combination of both, is pasteur- Surono 2002). Interestingly, with minimum ized and then fermented using leftover dahi from hygiene practice implemented, there was no the previous lot as starter cultures (Indian product failure and no food poisoning reported Standard Institution 1980 ). In Indonesia, dadih is among people consuming dadih . Instead, the a homemade product by the traditional way, older generation believes that consuming dadih involving the milk of water buffaloes without any may provide a benefi cial effect to their health. heat application to buffalo milk while manufac- Some dadih LAB have antimutagenicity, hypo- turing. The milk is neither boiled nor inoculated cholesterolemic properties, anti-pathogenic with any starter culture. The fresh unheated buf- properties, and immunomodulatory properties falo milk is placed in bamboo tubes covered with (Surono and Hosono 1996a , b ; Pato et al. 2004 ; banana leaves, incubated at the ambient tempera- Surono et al. 2011 ). ture (28–30 °C) overnight, and allowed to fer- Hosono et al. (1989 ) reported that Leu. ment naturally until it acquires a thick consistency paramesenteroides predominates in dadih fer- (Akuzawa and Surono 2002 ). mentation, responsible for producing aromatic compounds such as diacetyl, acetic acid, and 14.10.1.2 Important Lactic Acid other volatile compounds. Surono and Nurani Bacteria ( 2001 ) found that Lactobacillus sp., Lactococcus During Fermentation sp., and Leuconostoc sp. were dominant in dadih . The buffalo milk was poured into bamboo tubes Surono (2003b ) reported that among 20 colonies and kept overnight at room temperature, stimu- of dadih LAB isolated from Bukittinggi, West lating the mesophilic indigenous LAB derived Sumatra, fi ve strains were identifi ed as Lac. lactis from the fresh raw milk to dominate and grow, subsp. lactis , three strains of Lb. brevis, and three allowing natural fermentation. Consequently, the each of Lb. plantarum , Lb. casei , Lb. paracasei , fermentation of dadih is much longer than yogurt, and Leu. mesenteroides . Fresh dadih contains 24 and 4 h, respectively, due to different types of 4.3 × 108 cfu/g, dominated by lactic acid bacteria, LAB involved in the fermentation process at the which was 4.0 × 108 cfu/g. Lc. cremoris , Lc. lac- incubation temperature, 28–30 °C and 45 °C, tis , Lb. casei subsp. casei , and Lb. casei subsp. involving mesophilic cultures and thermophilic rhamnosus were also found. Several strains cultures, respectively, besides thicker consistency belonging to Ent. faecalis subsp. liquefaciens of dadih . Bamboo tube is hygroscopic and aided were also found in dadih , indicating that the way in keeping the product from wheying off. of manufacturing dadih did not implement good The milk is fermented by indigenous LAB of hygiene practices, since microbes belong to the the buffalo milk. Its natural fermentation pro- Enterococci group (Hosono et al. 1990 ). Surono vides different strains of indigenous lactic bacte- and Nurani (2001 ) reported that the total viable ria involved in each fermentation (Akuzawa and lactic acid bacteria count was in the range of Surono 2002 ). The natural indigenous LAB 1.42 × 10 8 –3.80 × 108 cfu/g in dadih originated observed in dadih could be derived from the from Bukittinggi and Padang Panjang area of bamboo tubes, buffalo milk, or banana leaves West Sumatra. involved in milk fermentation, and buffalo milk Diverse microbes have been observed to has been observed to contribute the most, while involve in dadih fermentation due to traditional bamboo tubes, banana leaves, and personal way of dadih manufacture. Surono et al. (1983 ) hygiene practice may also contribute. reported the involvement of yeast-like fungi at 14 Ethnic Fermented Foods and Beverages of Indonesia 367

1.1 × 107 cfu/g, identifi ed as Endomyces lactis , 14.10.2 Probiotic Bacteria Isolated which is commonly found in dairy products. Imai from Dadih et al. (1987 ) reported that the major bacterial species responsible for dadih fermentation were Lb. Dadih and several dairy products have been casei subsp. casei and Lb. plantarum . Microbial reported to consist of probiotic bacteria, which isolates of dadih have also been reported to when consumed alive and in an adequate amount exhibit probiotic attributes. confer health benefi t to the host (FAO/WHO 2002). The older generation believes that con- 14.10.1.3 Biochemical Changes suming dadih may provide a benefi cial effect to During Buffalo Milk their health. This fact has inspired more explora- Fermentation tion of the powerful indigenous LAB involved A consortium of LAB, which could be homofer- during dadih fermentation, excluding the con- mentative and heterofermentative natural starter taminants and the pathogens from the milk itself cultures producing lactic acid, with the involve- as well as environmental surroundings. ment of beta-galactosidase from lactic starter cul- Collado et al. (2007a ) reported that all the fi ve tures, results in coagulation of buffalo milk strains of dadih origin showed good adhesion beginning at pH below 5.0 and completing at 4.6 property, and the most adhesive was Lb. planta- (Surono (2003a ). Texture, body, and acid fl avor rum strain IS-10506. All LAB strains isolated of dadih owe their origin to lactic acid produced from dadih -fermented milk were able to signifi - during fermentation. cantly reduce the adhesion levels of all the patho- Small quantities of fl avor compounds are gen- gens tested. Lb. plantarum IS-10506 and Ent. erated through carbohydrate catabolism, via vol- faecium IS-27526 had the highest inhibition abil- atile fatty acids, ethanol, acetoin, acetic acid, ities. The inhibitory, competitive, and displacing butanone, diacetyl, and acetaldehyde. Homolactic properties against pathogens were also observed. starter cultures in dadih such as lactobacilli, lac- Hence, the two strains are promising candidates tococci, pediococci, and streptococci yield lactic for future probiotics. acid as 95 % of the fermentation output. Furthermore, Surono et al. ( 2010) reported a Heterolactic starter cultures, such as Lb. brevis , signifi cant increase of viable fecal LAB of rats Lb. fermentum, and Leuconostoc sp., contribute after 3 days of administration with Lb. plantarum to fl avor compounds. There are two important IS-10506 and Lb. plantarum IS-20506 at roles of lactic acid in dadih manufacture, which 1.2 × 1010 –1.6 × 1010 cfu/g each, by 3.25–3.5 and helps to destabilize the casein micelles and gives 0.35–0.65 log cycles, respectively, and continued the dadih its distinctive and characteristic sharp the increment after 7 days, by 1.8–2.0 and 2.1– acidic taste. 2.3 log cycles, respectively. The abilities of dadih During fermentation, natural LAB multiply LAB isolates in detoxifying mutagens (Hosono viable counts of 10 5 –10 9 cfu/g (Judoamidjojo et al. 1990 ; Surono and Hosono 1996a , b ) and et al. 1983 ; Hosono et al. 1989 ; Surono and cyanobacterial toxins have been reported. The Nurani 2001 ) and occupy about 1 % volume of mutagen absorbed and bound to the cell wall, dadih product. These LAB cells contain cell while the cyanobacterial toxin was being metab- walls, enzymes, nucleic acids, cellular proteins, olized (Surono et al. 2008 , 2009; Nybom et al. lipids, and carbohydrates. Beta-galactosidase 2008 ). activity contributes a major conversion of lactose Many researchers reported hypocholesterol- into LAB in dadih , which is benefi cial for lactase- emic activity of dadih. Hosono and Tono-oka defi cient people. (1995 ) reported that Lc. lactis subsp. lactis biovar. diacetylactis R-43 and R-22 of dadih ori- 368 I.S. Surono gin showed high-cholesterol-binding abilities, 14.11 Fermented Fish, Meat, 33.91 % and 29.73 %, respectively. Surono and Egg Products (2003b ) reported that Lc. lactis subsp. lactis strain IS-10285 and IS-29862 possess 14.11.1 Ikan Peda (Fish Pickled taurocholate-deconjugating abilities. Pato et al. with Salt) ( 2004 ) found that rats fed with fermented milk made from Lc. Lactis subsp. lactis strain IS-10285 Lightly salted fermented fi sh are mostly pro- showed signifi cant ( p < 0.05) lower total bile duced in Southeast Asia (Ishige 1993 ). Fish acids in serum. All these attributes show dadih as salted and packed in Thailand and then shipped potential health-benefi ting product. The probiotic to Malaysia and Indonesia accidentally under- properties of several strains isolated from dadih went fermentation because the fi sh were still not may provide the evidence on how strong the completely dried. These fi sh developed a distinct indigenous LAB derived from the fresh raw buf- fl avor upon arriving in Indonesia and were named falo milk are in combating the contaminants, pedah Siam (Van Veen 1965 ). both spoilage bacteria and pathogens, during the Ikan peda (Fig. 14.17) is a wet fermented fi sh spontaneous fermentation of dadih (Collado made from mackerel (Scomber kanagurta ) or et al. 2007b ). Kembung fi sh (Rastrelliger neglectus ) mixed Surono et al. ( 2011 ) reported signifi cantly with 20–30 % salt fermented in two steps. In the increased total salivary secretory IgA (sIgA) primary fermentation, the eviscerated fi sh are fer- level and bodyweight of children (p < 0.05) com- mented with high salt concentration (25 %, w/w) pared to placebo in a pilot randomized controlled in a vessel and in sealed container for 3 days. The trial on Ent. faecium IS-27526 isolated from fi sh are then washed, drained, and piled in dadih on children supplemented with lyophilized wooden boxes, putting banana leaves between Ent. faecium IS-27526 (2.31 × 10 8 cfu/g/) in the fi sh; then they are sprinkled with 30 % salt, 125 ml ultrahigh-temperature low-fat milk for 90 covered with banana leaves, and fermented for days. Changes of total salivary sIgA levels were another 1 week or longer, and an aroma charac- signifi cantly higher in underweight children sup- teristic of ikan peda is developed. Then the ikan plemented with probiotic, while weight gain was peda is dried in the air (Putro 1993 ). observed signifi cantly in children with normal Rahayu (2003 ) isolated Lb. plantarum , Lb. bodyweight supplemented with probiotic. curvatus , Lb. murinus , and Strep. thermophilus In a 90-day randomized double-blind placebo- controlled pre-post trial , Surono et al. (2014 ) has been conducted on Indonesian children aged 12–24 months supplemented with microencapsu- lated Lb. plantarum IS-10506 of dadih origin, at 10 10 cfu/g as probiotic, and 20 mg zinc sulfate monohydrate (8 mg zinc elemental) showed signifi cant increase of fecal sIgA in probiotic group (p < 0.01), and in probiotic and zinc group (p < 0.027), as compared to placebo group. Changes of serum zinc concentration in the combination of probiotic and zinc group showed the highest elevation after supplementation. Supplementing probiotic Lb. plantarum IS-10506 and zinc for 90 days resulted in a signifi cant increase of humoral immune response as well as improved zinc status of the young children (Surono et al. 2014 ). Fig. 14.17 Ikan peda 14 Ethnic Fermented Foods and Beverages of Indonesia 369 from ikan peda . During fermentation, enzymes Bagoong , Burmese Ngapi , Malaysian Belacan , derived from the fi sh and halophilic bacteria, and Thai Kappi . The most important center of its including lactic acid bacteria, are involved which manufacture is Bagansiapiapi, North Sumatra produce lactic acid and preserved the fi sh. (Surono and Hosono 1994a ). According to Van Veen (1965 ), the best ikan peda has moisture content of 44–47 %, 7–14 % 14.11.2.1 Manufacturing Terasi fat, 21–22 % protein, and 15–17 % NaCl. Terasi in general is prepared as follows: The Ikan peda is described as being fatty, partly shrimp or fi sh are mixed with salt at10 % level on dried salty fi sh with reddish brown color, moist the fi shing boats and then spread out on the fl oor. and slightly pasty with a fl abby texture, and a Further salt is added at 5 %, and the product is specifi c fl avor, which is cheesy, tasty, salty, and dried in the sun for about 1–3 days, occasionally often mixed with mild rancid fl avor. Prolonged turned over to decrease the moisture content from storage during retailing will facilitate the devel- about 80 % to 50 % and also to minimize off fl a- opment of rancid fl avor accompanied by a change vor. The resulting mass is minced, pressed tightly in meat color to reddish brown (Van Veen 1965 ; into wooden tubs to exclude the air and allow to Hanafi ah 1987). ferment 1–4 weeks (Surono and Hosono 1994a ).

14.11.2.2 Chemical Composition 14.11.2 Terasi (Shrimp Paste) of Terasi The protein content of terasi was 25.42 g/100 g, Terasi is one of the salty fermented products that with glutamic acid as dominant amino acid, 17.73 undergo alkaline fermentation, made from fi sh g/100 g. The pH of terasi was 7.53, with a salt and/or shrimp which is in the form of paste (Fig. content of 16.75 g/100 g, a fat content of 6.11 14.18 ). Terasi and tauco serve similar roles as g/100 g, and a carbohydrate content of 1.94 g/100 condiments due to the presence of glutamic acid g. Its moisture content was 37.41 % (Surono and and its specifi c fl avor. Hosono 1994a ). Terasi is an indigenous fermented food in Indonesia, salted fi sh or shrimp, mixed with 14.11.2.3 Microbial and Biochemical 2–5 % salt, dried repeatedly, ground into a fi ne Changes of Terasi paste, and allowed to ferment naturally for a Surono and Hosono (1994a ) reported that acid- period of several weeks until the desired fl avor producing Bacillus sp. and Pseudomonas sp. has developed, followed by sun-drying for 1–3 were dominating the microbial population days. It has a fl avor reminiscence of ripe cheese. involved during terasi fermentation. On the other Closely related products are the Filipino hand, Kurthia gibsonii and Sporolactobacillus

Fig. 14.18 ( a ) Terasi fermentation, (b ) Terasi in block 370 I.S. Surono inulinus represented minority of aerobic bacteria early stage of fermentation were B. coagulans , B. population in terasi, while Micrococcus sp. had megaterium , and B. subtilis, while in the later the ability to utilize protein but poor in utilizing stage of fermentation, B. licheniformis , M. colpo- carbohydrate. genes , M. roseus , M. varians , and Staphylococcus According to Surono and Hosono ( 1994a ), sp. were found in terasi (Surono and Hosono during the early stage of terasi fermentation, the 1994a ). pH drops to 4.5 and most Pseudomonas sp. failed Surono and Hosono (1994a ) reported that ter- to grow. Since terasi are manufactured without asi starter was composed of B. brevis , B. pumilus , any concern on good hygiene practice, B. megaterium , B. coagulans , B. subtilis, and M. Pseudomonas sp. may represent contaminants kristinae in the proportion of 39.1 %, 26.1 %, during mixing, drying, and packaging of terasi. 8.7 %, 8.7 %, 8.7 %, and 8.7 %, respectively. All This kind of bacteria grows well in proteinaceous of the microfl ora found in terasi starter were salt food and actively spoils seafoods. tolerant, as shown by their capability to grow on In the presence of 10 % salt, during fermenta- agar plates in the presence of 10 % NaCl. The tion, the species Bacillus , Pediococcus , dominant fl ora were Bacillus sp., which are halo- Lactobacillus , Micrococcus , Sarcina , philic and aerobic, grow in the temperature range Staphylococcus , Clostridium , Brevibacterium , of 10–50 °C, and have esterase (C4) and esterase Flavobacterium , and Corynebacterium were lipase (C8) activities. None of the isolates had the decreased. At the beginning of terasi fermenta- ability to produce gas from glucose, even though tion, there was an increase in total bacteria domi- most of the isolates could produce acid from glu- nated by lactic acid bacteria, micrococci, and cose (Surono and Hosono 1994a ). Only B. pumi- bacilli, which decrease at the end of fermenta- lus , B. coagulans , and M. kristinae had the ability tion. The endogenous enzymes from B. subtilis to hydrolyze long-chain fatty acids. Terasi has a and B. coagulans together with the enzymes typically characteristic aroma of cheese and derived from intestines of the fi sh hydrolyzed the ammonia. The cheesy odor is produced by low- protein (Surono and Hosono 1994b ). The bacte- molecular-weight fatty acids, and the ammonia- rial enzymes were mainly responsible for the cal odor is due to the presence of amines and deamination and decarboxylation of amino acids ammonia (Dougan and Howard 1975 ). to form lower fatty acids and amides, producing T. muriaticus strain was found and reported to characteristic fl avor of terasi . The combination of produce histamine in fermented fi sh in Japan and salt and the microbial degradation products of Thailand. Viable cell lactic acid bacterial counts protein, fat, and carbohydrate may contribute to in terasi were 104 –10 6 cfu/g. All the isolates were the taste and aroma of the terasi (Aryanta 2000 ). catalase negative, were Gram-positive cocci, and The endogenous proteolytic enzymes, rather were able to grow at 15 % NaCl and classifi ed than bacteria, are responsible for the hydrolysis into two types: the Tetragenococcus halophilus of fi sh muscle, prior to the bacterial activity. group and the T. muriaticus group as revealed by Furthermore, the halophiles could possibly a restriction fragment length polymorphism hydrolyze short-chain fatty acids; hence, the (RFLP) analysis and sequencing of the 16S rRNA presence of esterase (C4) and esterase lipase (C8) gene (Kobayashi et al. 2000, 2003). activities in all of the bacteria presented in terasi starter supports the hypothesis that bacterial enzymes hydrolyze the fat, producing low- 14.11.3 Urutan (Traditional Balinese molecular- weight fatty acids that are responsible Pork Sausage) for the cheesy odor Surono and Hosono (1994b ). The total halophilic count of terasi was Urutan (Fig. 14.19 ) is usually prepared to cele- 1.1 × 10 5 cfu/g, dominated by halophilic Bacillus brate the Galungan Day, which is a special holy sp. B. pumilus is the dominant species throughout day for Hindus in Bali. A day before the feast, the fermentation. Other bacteria responsible for Balinese people slaughter pigs to prepare tradi- 14 Ethnic Fermented Foods and Beverages of Indonesia 371

responsible for the characteristic ﬂ avor development, and contributes to the preservation of the fermented product. The pork meat inside the casing became more compact and solid after fermentation. Traditionally, urutan was manufactured without any addition of nitrite and/ or nitrate, and the color became dark red at the end of fermentation. The products were dried under the sun, and the juice (mixture of oil and water) was dripped so that the casing becomes dry and wrinkled (Antara et al. 2002 ).

14.11.3.1 Microbial and Biochemical Changes During Urutan Fermentation The total viable lactic acid bacterial counts were remarkably increased from the initial count of 1.72 × 105 –1.84 × 108 cfu/g on day 1 and then decreased to 4.98 × 107 cfu/g at days 2–5 (Antara et al. 2002 ). The results also showed that lactic acid bacteria predominated the total microbial population. The sharp reduction of pH on the fi rst Fig. 14.19 Urutan , fermented pork sausage (Picture day of fermentation affected the growth of courtesy of Prof. Nyoman Semadi Antara) Enterobacteriaceae, which were not detected at day 2 of fermentation. Soluble protein decreased tional foods. Due to the excessive fresh pork at the fi rst day of fermentation. Total acidity available, it needs to be preserved to prolong the increased signifi cantly after day 1 of fermenta- shelf life of the meat which can then be consumed tion, and it remained constant until the end of the on the following days or weeks. Two common process. The carbon sources in the product pri- Balinese practices to preserve the pork meat are marily sugar and spices were utilized by homo- by drying dendeng and fermenting urutan . fermentative lactic acid bacteria and converted Urutan is a Balinese traditional fermented into lactic acid as the lactic acid bacteria grow sausage, which is made of chopped lean pork and and multiply during fermentation. There was no fat which are mixed with spices (garlic, turmeric, butyric acid detected, and only small amounts of aromatic ginger, chili, and pepper), sugar, and succinic and propionic acids were produced salt. The mixture is fi lled into cleaned pig intes- (Antara et al. 2004 ). tine and fermented spontaneously and then sun- Antara et al. ( 2002) found that in urutan , dried for 2–5 days (Aryanta 2000 ). 77.5 % of bacteria are lactobacilli, and the other Urutan has a different microbial ecology, 22.5 % are pediococci. Further molecular identi- compared with other fermented sausages, and fi cation by 16S rDNA sequence revealed that Lb. this is primarily due to spontaneous fermentation plantarum , Lb. farciminis, and obligate heterofer- which occurs during the drying process, and the mentative lactobacilli Lb. fermentum and Lb. hil- quality of the product varies from time to time, gardii . Besides, P. acidilactici and P. pentosaceus from place to place. The use of spices and high- were also detected (Antara et al. 2002 ). Microbial temperature fermentation is of special interest in succession occurred during fermentation of uru- relation to the characteristics of lactic acid bacte- tan, dominated at initial growth by Lb. planta- ria involved in the process, and their distribution rum , predominated during 2 days of fermentation, plays an important role in food fermentation, then followed by P. acidilactici , and fi nally, Lb. 372 I.S. Surono farciminis was found to be predominant at the The heterofermentative lactobacilli were only last stage of fermentation (Antara et al. 2004 ). existent at the fi nal stage of fermentation as P. acidilactici was found after 2 days and shown by the lactic acid produced, and the slightly decreased until the end of fermentation. absence of gas in urutan indicated that the role of On the other hand, Lb. farciminis was distributed the heterofermentative lactobacilli was not sig- in an increasing manner from day 2 until the end nifi cant since, as reported, the heterofermentative of fermentation process. The heterofermentative lactobacilli ( Lb. fermentum and Lb. hilgardii ) lactobacilli ( Lb. fermentum and Lb. hilgardii ) were not signifi cantly detected during the whole were not signifi cantly detected during the whole fermentation. The existence of these species was fermentation, only detected in small number on only in small number on the third and fi fth days, the third and fi fth days, respectively (Antara et al. respectively (Antara et al. 2002 ). 2004 ). Most of lactic acid bacteria present in uru- The denaturation of protein and the absence of tan during fermentation were identifi ed as the bubbles from CO2 made the texture of urutan homofermentative type, which produced mostly more compact and solid (Antara et al. 2002 ). lactic acid and a small amount of acetic acid as Inconsistent of quality and risk of fermentation well as carbon dioxide (Aryanta 1998 ). failure are the common problems on traditional The rapid growth of LAB during the fi rst day fermented products which are produced under of fermentation of urutan is in agreement with spontaneous natural fermentation indicating off- those reported for salami; the LAB in this prod- odor development. Antara et al. ( 2004 ) reported uct decreased after 2 days of fermentation and that at pH 4.3–4.5, urutan (Balinese dry fer- remained constant until the end of the process mented sausage) using mixed LAB as the starter (Antara et al. 2002 ), in contrast to the LAB culture might inhibit the growth of Clostridia. growth in salami whereby the LAB population starts to increase on the fi rst day, reaching a max- 14.11.3.2 Telur Asin (Salted egg) imum of 7 days, and remains constant until the Telur asin (salted egg) is an alkaline-fermented end of the ripening period (Coppola et al. 1998 , ethnic food in Indonesia (Fig. 14.20 ). Alkaline- 2000). The difference of LAB growth in urutan fermented foods constitute a group of less-known might be due to the use of local types of spices, food products that are widely consumed in especially aromatic ginger and turmeric, and the Southeast Asia and African countries. Traditional high concentration of garlic suppressed the processing of telur asin is carried out by coating growth of LAB in urutan (Antara et al. 2002 ). method. The fresh duck eggs are coated with a Lb. plantarum dominating the total lactic acid muddy paste containing ash or red brick powder bacterial population during fermentation makes and salt at 1:1, allowed to ferment in a jar for urutan different from salami where Lb. sake and 15–20 days, and the telur asin can be preserved Lb. curvatus were present as the dominant species for 2–3 weeks (Margono et al. 2000 ). Suprapti (Coppola et al. 2000 ). The presence of P. acidilac- (2002) reported that the use of ash as coating tici in urutan, which was also distributed signifi - agent will produce telur asin with pale yellow cantly, could be due to the high fermentation and grayish surrounding the yolk, while the use temperature. This species is widely used for rapid of red brick powder produces telur asin with red- fermentation with temperature above 30 °C dish yolk. (Ordonez et al. 1999 ). The existence of P. acidilac- In alkaline-fermented foods, the protein of the tici as bacteriocin producer plays an important role raw materials is broken down into amino acids for the succession. The bacteriocin produced in and peptides; ammonia is released during the fer- this product inhibited the growth of Lb. plantarum mentation, raising the pH of the fi nal products and P. pentosaceus, but the growth of Lb. farcimi- and giving the food a strong ammoniacal smell in nis was stimulated by the intrinsic condition of spontaneous fermentation by mixed bacteria urutan after 3 days until the end of fermentation. cultures. 14 Ethnic Fermented Foods and Beverages of Indonesia 373

Fig. 14.20 Boiled telur asin , telur asin covered with muddy ash and salt, smoked telur asin

Saputra (2013 ) reported that Lb. plantarum , deep-fried in coconut oil and baked before con- Lb. casei subsp. rhamnosus , Enterococcus gal- sumption. The alcohol content ranged from 3 % linarum, and P. acidilactici have been isolated to 8.5 % v/v (Cronk et al. 1977 ). The glutinous from telur asin and inhibited the pathogen, espe- rice lipids are hydrolyzed during tapé ketan fer- cially E. coli and Staph aureus by organic acids mentation (Cronk et al. 1979 ). produced and bacteriocin produced by P. acidi- Tape is a traditional fermented food in lactici. Telur asin may also be smoked, produc- Indonesia and also in Malaysia, the Philippines, ing rich unique fl avor of salty and smoked taste. and Vietnam, made from raw material containing starch and ragi , the microbial starter. Ragi is a mixture of rice fl our, spices, sugar cage, water, 14.12 Fermented Roots and Tuber and yeasts and used as inoculums in alcoholic Products fermentation in natural fermentation. Aryanta (1988) isolated Rz. oryzae , M. rouxii , Asp. ory- 14.12.1 Tapé , Growol , and Gatot zae , S. cerevisiae , E. burtonii , H. anomala , and P. pentosaceus from ragi (NKL brand) obtained Tapé is a sweet and sour fermented food with an from Denpasar. On the other hand, Winarni alcoholic fl avor, prepared from glutinous rice or ( 1988 ) isolated some amylolytic molds and cassava or other cereals by using ragi starter in yeasts from four types of local commercial ragi. Indonesia (Campbell-Platt 1994). It is eaten as P. pentosaceus is the acid producer commonly dessert or delicacy in Indonesia. There are vari- found in ragi and most abundantly isolated from ous starchy substrates used to prepare tapé , such newly collected ragi samples in Indonesia, fol- as cassava (tapé singkong ), glutinous rice (tapé lowed by a small number of Lb. plantarum and ketan), maize (tapé jagung), and millet (tapé can- Lb. fermentum strains (Uchimura et al. 1998 ). tel). The raw material is washed, soaked, steamed, Tape singkong (Fig. 14.21 ) is a fermented cooled to room temperature on a woven bamboo steam cassava by ragi tape starter, involving tray, sprinkled with ragi powder, packed in small Amylomyces rouxii and E. burtonii , S . cerevisiae , banana leaves, and fermented for 2–3 days at Rhizopus sp., and Hansenula sp. (Steinkraus room temperature, and a soft juicy mass of tapé is 1983 ; Hassan et al. 1986 ; Suliantari and Rahayu produced (Saono et al. 1977 ). Tapé singkong is 1990 ). 374 I.S. Surono

whereas Sm. fi buligera produces α-amylase and Rhizopus sp. produces glucoamylase (Suprianto et al. 1989 ). Growol is fermented raw cassava tubers, whereas gatot is fermented dried cassava tubers involving lactic acid bacteria. These products are popular in certain part of Java. The manufacture of fermented tubers is by soaking the peeled raw cassava for growol and dried cassava for gatot for several days until the tubers become soft. Growol Fig. 14.21 Tape singkong (fermented cassava) is a traditional fermented food made from cassava which has sour taste, only found in Yogyakarta area, especially Kulon Progo and the The fungi Rz. arrhizus var. rouxii strain TT is surrounding area. The peeled and sliced cassava able to utilize starch and convert it to alcohol, was soaked in the water for 4 days, drained, and producing soft, juicy, sweet tape which lack sour- crushed before being steamed. ness, and 1.88 % (w/v) ethanol was produced The fermentation occurs naturally; after 72 h of fermentation (Hassan et al. 1986 ). Coryneform , Streptococcus , Bacillus , and The yeast is inoculated together with the fungi to Actinobacteria grow at the beginning of the fer- be able to do the fermentation, while inoculat- mentation followed by Lactobacillus and yeast ing yeast only, the fermentation does not occur. until the end of fermentation. Suharni (1984) reported that lactic acid bacteria dominated and 14.12.1.1 Biochemical Changes found at 1.64 × 108 cfu/g. Lactobacillus casei The fermentation process of tape singkong has subsp. rhamnosus TGR2 isolated from growol two main stages: the conversion of starch into produces extracellular metabolites which remain simple sugars carried out by amylase producers stable at room temperature and has resistance to (molds and yeasts) and the conversion of sugars heating at 98 °C for 30 min, at pH 3–8 (Rahayu into alcohol and acids carried out by certain 1995). yeasts. Gatot, dried cassava, is a traditional fermented Esters are also formed due to the reaction food in Gunung Kidul, Yogyakarta. Cassava is between alcohol and acids which contribute to peeled dried to get the typical black color of gatot the sweet–sour taste with a mild alcoholic fl avor as a staple food. Ichsyani (2014 ) reported that P. of the product (Djien 1972 ; Karim and Hassan pentosaceus and Saccharomyces sp. TR7 isolated 1986). The pH of tape singkong decreased from from gatot have amylolytic activity and able to an initial value of 5.65–5.15 during the fi rst 4 reduce cyanide in cassava. Lb. plantarum 250 days of fermentation, while total acidity increased Mut7 FNCC also has been isolated from gatot from 3.5 mg/100 g to 5.2 mg/100 g. During the (Harmayani et al. 2001) and showed the ability in fi rst 4 days of fermentation, reducing sugar suppressing bacteroides (Sari 2014 ). increased from 7.9 % to 16.0 % and decreased to 12.73 % after this time (Rahayu 1980 ). Tape singkong is sweet, slightly sour, and aro- 14.13 Conclusion matic, but too much acid content in tape is undesirable. The nutrient contents of tape singkong Traditional fermented foods are mostly carried are as follows: 0.5 % protein, 0.1 % fat, 42.5 % out involving mixed cultures. 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