One of the Chinese Yeasts), Biityu (Rice Wine

Proc. Jap. Assoc. Mycotoxicol. No.2,1976 1

Fermented foods and related microorganisms in Southeast Asia

Michio KOZAKI

小崎道雄:東南アジアの発酵食品と微生物

Many original fermented foods have been kept from ancient in central Asia. These foods representing sun dried original cheese, Indian butter ghee and others, were spread gradually to Europe, China and other places and developed to characteristic fermented foods in several countries. As a matter of course the peoples in Southeast Asia have a variety of fermented food using microorganisms for their production as shown in Tables 1 and 2, especially using ingeniously many molds, Aspergillus, Mucor, Rhizopus, Neurospora and Monascus etc. Large number of studies on fermented foods in Southeast Asia have been carried out since CALMETTEI) first presented Mucor rouxii which has strong saccharifying activity to fermented alcohol. Detailed studies by NAKAZAWAand TAKEDA developed chinese fermented foods, Peka (one of the chinese yeasts), Biityu (rice wine), Anka (red wine), Tubityu (glutenous rice wine) and other alcohol beverages and isolated many microorganisms from these fermented foods. They also found Rhizopus javanicus from raggi which is used for chinese yeast in Indonesia,

Table 1. Main fermented foods using molds, yeasts or bacteria in Southeast Asia

*1 Tokyo University of Agriculture, Department of Agricultural Chemistry, 1-1, Sakuragaoka, Setagaya-ku, Tokyo(東京農業大学農芸化学科) 2 Proc. Jap. Assoc. Mycotoxicol.

Table 2. Main fermented foods using molds plus bacteria, molds plus yeasts, yeasts plus bacteria and molds, yeasts plus bacteria in Southeast Asia

and this strain is now used for alcohol production in Japan. In a survey of microorganisms from Korean chuizu (one of chinese yeasts), NAGANISHI isolated sacchrifying molds and alcohol producing yeasts. In this report he describes Endomyces hordei (Endomycopsisfibrigera) having strong amylases, but he did not think this strain as dominant and as a key mold to decompose starch. Recently, HESSELTINE, ORILL, STEINKRAUS, and NAKANO reported several fermented foods in Asia, Tempeh, Idli, Fish sauce and Raggi, and the number of related reports is on the increase. Formerly the author had been visiting professor of UP*1, College of Agriculture through SEARCA, and at that time started a project "The study on fermented foods in Southeast Asia." This project has still been continued by a member of the Department of Food Science and Technology and a Japanese professor in UP. The present paper reports a part of our project "Native alcohol beverages and related microorganisms and Philippine delicious food Nata."

Palm wine

Palm wine referred to Toddy, Tuba, Tuack etc. in Southeast Asia is a naturally fermented alcohol beverage for peasants from the sap of palm inflorescence. It is made principally from palm trees, coconut palm being the representative one. Nippa, Sugar, Buni and Palmyra are also often used to produce this wine. Photo.1 showed Nippa palm wine correcting.

*1 University of the Philippines *2 Southeast Asian Regional Center for Graduate Study and Research in Agriculture No.2,1976 3

Photo.1. (left) Nippa palm wine correcting (right) Fruit of Nippa

The methods of obtaining palm wine have been described in detail by BROWNING and SYMONS, so that the author deals with it only briefly here. The young inflorescence is sliced thinly twice for one day in the morning and evening, and after that small bamboo tubes hang to the inflorescence in order to collect oozed sap. A piece of mangrove (=Tangal; Vateria indicans) bark is placed inside the bamboo tube in Philippines. It is left as it is for eight to ten hours, so that the sap is allowed to trickle into the tube. Fermentation begins immediately by yeasts and the other microorganisms remaining already in the used tube and brings about 4% alcohol concentration. Palm wine samples were collected from five places, two different firms in Los Baftos, Mayondon, College and Palo town in Leyte island, counted and isolated the microorganisms using

GYP medium for bacteria and malt extract plus sap for yeasts. Dominant yeasts in palm wine are shown in Table 3. From palm wine which is not added with Tangal bark, the author isolated two species of yeasts at proportions of 62 to 38: they were Saccharomyces chevalieri and terminal budding yeast Kloeckera apiculata. These yeasts and bacteria in bamboo tube after four hours hanging period are shown in Photo.2.

Table 3. Yeasts species and their ratio isolated from "Tuba" 4 Proc. Jap. Assoc. Mycotoxicol.

Photo.2. Microorganisms (Sacch. chevalieri, Kloeckera apiculata and bacteria) in palm wine (without Tangal bark) after 4 hours hanging

On the contrary, there was only Sacch. chevalieri in Tangal bark-added palm wine in Los Banos, as also same as Bayan sample added with Tangal bark, although angular formed yeast was isolated from this latter wine. It was identified as a new species of Candida and details will be reported later. It has also been able to isolate Sacch. chevalieri from Tangal bark-added palm wine collected in Leyte island. As a result of these investigations, it was suggested that dominant yeast in Philippine palm wine is Sacch. chevalieri.

FAPARUSI stated that palm wine at first consists of Sacch. cerevisiae as predominant, and also isolated Schizosaccharomyces pombe from palm wine. But the palm wine in Philippines could not be found these yeasts.

Palm wine becomes no sour and can be preserved with the use of Tangal bark, because of having the effective chemical composition of the bark which was identified polyphenols as tannin using paper partition chromatography and other methods. The effect of water extracts of the bark was studied with lactic acid bacteria, Acelobacter, and yeasts isolated from palm wine. The results are shown in Fig.1. Lactic acid bacteria could not be grown when 3% Tangal bark was added to the medium, except L. arabinosus. Acetobacter aceti can be grown in green tea but not in the medium with bark. While isolated yeasts Sacch. chevalieri and Sacch. baili var. baili don't prevent growth with the bark, Sacch. cerevisiae inhibited strongly and Kloeckera apiculata stops completely; for this reason it is a matter of course, this yeast could not be found in Tangal bark-added palm wine. The influence of the bark for isolating yeasts was studied in detail as Fig.2, from where the results show that total cell numbers of all isolated yeasts increase as in case of the control. Therefore, the aim of the add of Tangal bark is to prevent the growth of other bacteria and to assist yeast fermentation.

Rice wine

Asian peoples almost live on rice from old times, and therefore have their characteristic wines No.2,1976 5

Fig.1 Effect of tangal bark extract to yeasts, lactic acid bacteria and acetic acid bacteria

C•cControl, T•cTangal added, G•cGreen tea added

Fig.2. The chemical changes and viable yeast cell numbers in Tuba fermentation between normal and tangal bark-added sample (tangal 3%) in several countries like Sake in Japan, i.e. Tapuy in Philippines, Ou in Thailand, Brem in Bari island of Indonesia (Photo.3), Makkari in Republic of Korea, Chaoching-chu in China, Bakhar or Murcha in northern India and Sonti in India. Palm wine is widely made in tropical and sub- tropical areas, but on the contrary the rice wine is drunk regularly at limited local regions in Asia, for instance Tapuy is made only by Ifugao and Igorot tribes of northern Luzon island in Philippines and Ou made only in the northeast in Thailand. 6 Proc. Jap. Assoc. Mycotoxicol,

Photo.3. Prom in Inioncsia

Photo.4. Bubods

The procedure of rice wine making is similar to the artifact of Chinese wine "Bityu", and chinese yeasts used seed of rice wine are called Bubod or Binokbok in Igorot and Ifugao (Photo. 4), Bukhar in India and Raggi in Indonesia. Bubods were collected from several rice wine making places of northern Luzon, analysed their chemical composition and counted the dominant microorganisms. As the analytical results of chemical composition of bubod it resembled that of rice. However, the microorganisms in bubod were mainly yeasts and lactic acid bacteria, while only a few molds could be isolated No.2,1976 7

Table 4. Number of isolated molds, yeasts and lactic acid bacteria from Bubods

Table 5. Saccharifying activity of isolated filamentous yeasts (unit/ml)

V...violet, B...blue, BV...bluish violet, RV...reddish violet as shown in Table 4.

According to reports of NAKAZAWA et al., NAGANISHI and many other workers, saccharifying activity of these wines were mostly in Mucorales, but the saccharifying activity of molds that the author isolated was weaker in comparison with filamentous yeasts from same samples. The amylase activity of filamentous yeasts was therefore studied using YP medium (1.0% soluble starch, 0.5% peptone and 1.0% yeast ex., pH 6.8), and results are shown in Table 5. All isolated strains had saccharifying activity and especially among them the amylase activity of No.66 strain was 3.48 unit per ml. of harvested culture medium, and this activity is stronger about twice than that of the type culture of Endomycopsis fibligera IFO 0103. This strain No.66 was identified with Endomycopsis fibligera as based mainly on standard methods described in "The yeasts" edited by LODDER. However, it should be emphasized that Ifugao wine, Tapuy, brews by only yeasts from cooked rice, and that is, both starch-sacchrifying and fermentation of produced sugar take place in the same jar by Endomycopsis and Saccharomyces belonging to yeasts. This new phenomenon of wine making has not yet been published. It is supposed that Ou in Thailand, Brem, Bakhar and other rice wines produced in Southeast Asia were sacch- aryfied by Endomycopsis group and then fermented by Saccharomyces resembling Tapuy in Philippines. Because these rice wines also come into use of chinese yeasts for seed, these chinese yeasts just resemble to Bubod. 8 Proc. Jap. Assoc. Mycotoxicol.

Sugar cane wine

Racial minority called Ilocano tribe in Luzon island has a unique wine called Basi or sometimes Ilocano wine made from sugar cane. This wine tastes a little sweetish and is a good flavored alcohol beverage . In case of brewered sugar cane wine, it may generally mean rum which distilled molasses, but this wine is never distilled. Essential steps in Basi making is mentioned briefly. The sugar cane harvested is pressed with the use of primitive crushers. The squeezed juice is concentrated in a big pan till about half and next added the dried samac leaves and seeds. After filtration using cheese cloth, cooling, the concentrated juice transfers to earthen jars, and is added a piece of chinese yeasts powder as a starter and cooked rice. This is allowed to ferment for about 7-10 days and then continuously matured for one month or more. Dominant microorganisms in Basi were identified with Saccharomyces, Endomycopsis and lactic acid bacteria. However, these sugar cane wines are shown a taste very delicious like the grape wine because of its harmony of sourness and sweetness with the alcohol concentration.

Nata

Nata is a Philippine delicacy as shown in Photo.5, which is a thick clean white mucilagenous mat on the surface of coconut water or other fruits juices containing sugar . Harvested Nata is cutting to about one centimeter square and is then eaten as a dessert "Haro haro" with other pieces of fruits . Nata is also resembling Japanese "mitsu-mame" which is prepared with agar and some fruits and is a favorite among girls or younger generation . This microorganisms formed mucilagenous substance identified by LAPUZ et al . and DIMAGUILA in the same year as Acetobacter xylinum. The author collected two Nata samples at Alaminos, Laguna. After purifying bacteria from Nata, isolated strains were also identified according to Bergey's "Determinative bacteriology." As this results, isolated strains

Photo.5. Aata No.2,1976 9

were classified to Acelobacler xylinum. After seedling, coconut water is allowed to stand in a warm cultivation room for 2 weeks, and then developed a thick creamy mat of Acetobacler xylinum in the bottle. Nata is a unique food in the world as no other countries have it, but the tea fungus which is consumed all over the world has a long and interesting history and is not very different from Nata. But the former is produced by growing of only Acelobacler xylinum, while the latter is matured by fermentation of A. xylinum and yeasts such as Sacch. sp., Kloeckera apiculata, Torulopsis famata, Pichia membranae faciens, and Bretanomyces inlermedius. Finally Nata seems to be suitable as a diet food.

Beside briefly stated above, widely known, other fermented foods are found frequently in Asia, as rice cake, many kinds of pickles, carabao cheese and fish sauces etc., and some of these fermented foods have not yet been studied in detail. However, to summarize our interpretation of the results, we can explain that dominant yeast in Tuba was identified with Saccharomyces chevalieri, and a new species of Candida sp. was also found.

In addition, the results of analysis of the effective principle in Tangal bark were essentially shown as polyphenol compounds. It particularly emphasizes that, in case of Philippine rice wine Tapuy, these microorganisms concerned to saccharification of rice starch and alcohol fermentation belonged to two genera Endomycopsis and Saccharomyces; in other words, we found that yeast groups were only engaged in rice wine making, although it has been considered that rice starch in rice wine making is decomposed by molds and then producing sugar is allowed to ferment by Saccharomvices .

These new phenomenons , have not yet been published. I wish to thank Professor K. KITAHARA for his kind suggestions, and also thank Director J. D. DRIRON Jr, of SEARCA.

References

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