(15) Food Preservation Science VOL. 29 NO. 3 2003 [Article] 147

Influences of Barks Addition on the Growth of Microorganisms and the Ingredients of Basi in Basi Fermentation

MURA Kiyoshi', SANCHEZ Priscilla C.*2 and TANIMURA WahaChirO*1

* 1 Faculty of Applied Bioscience, Tokyo University of Agriculture 1 1 1, Sakuragaoka, Setagaya-ku, Tokyo 156 - 8502, Japan * 2 Institute of Food Science and Technology,University of the Philippines at Los Banos College, Laguna 4031, Philippines

Using a mixture of samac bark and fruit in equal quantity (SBF) , which is used in the sugarcane

wine •\ "basi" brewing in the Pangasinan of Philippines, we have prepared basi with and without the

addition of barks, and have studied the impact of barks addition on the growth of microorganisms and

the ingredients of basi. No impact of SBF addition was seen on the growth of yeasts like Saccharomyces cerevisiae, and no difference was found in the alcohol yields between basi with and

without SBF addition. The growth of lactic acid bacteria was completely inhibited by SBF, indicating

that the growth of lactic acid bacteria the cause of souring can be suppressed by the addition of

barks in basi fermentation. Besides, the basi after fermentation with the addition of SBF had a strong astringency resulted from the polyphenol components dissolved from SBF. Though basi with SBF

addition had a sugar concentration as high as 11.2%, as its sweetness had been moderated by the

astringency of polyphenol, it had a mild taste, showing an important significance of barks addition to

confer astringency in basi fermentation. (Received Aug. 31, 2001 ; Accepted Jan. 27, 2003)

There is a sugarcane wine called"basi"in the identified that, the major inhibitory substance is a northwest of Luzon in the Philippines from old high molecular weight condensed tannin consisting times1). Basi is a fermented liquor and has a strong of catechin and leucoanthocyanin in samac bark, a sweetness and astringency. The preparation method gallotannin with multiple galloyl radicals and m- of basi is handed down from generation to digalloyl radicals bound to the glucose or xylose of generation among the people and some barks are sugar in samac fruit, and a high molecular weight added to the sugarcane juice before fermentation. condensed tannin consisting of gallic acid and

Usually, the barks used in basi preparation are bark, leucoanthocyanin in duhat bark3)•`6). fruit and leaf of samac(Macharanga grandifolia In this study, together with the elucidation on

Linn.) and bark of duhat (Sizygium cumini Skeels). basi ingredients by analyzing the basi produced in

We have previously investigated the infiuence of the Philippines, we have performed the fermentation various aqueous extracts of barks added to basi on of basi with and without the addition of barks, the growths of lactic acid bacteria, acetic acid compared the transition of microorganisms and the bacteria and yeasts, and have found that most of change of ingredients during the fermentation, and the aqueous extracts intensely inhibit the growths investigated the impact of barks addition on the of lactic acid bacteria and acetic acid bacteria, growth of microorganisms and the ingredients of which are the cause of souring during basi basi. fermentation, suggesting that barks addition brings Materials and Methods about an oPPortune control of microorganisms in basi fermentation2). Moreover, we have studied 1 . Materials substances inhibiting the growth of lactic acid Basi produced in the La Union of Philippines bacteria in the barks added to basi and have were used as materials to analyze the ingredients.

* 1 E-mail : MURA Kiyoshi ; [email protected] * 2 E-mail : [email protected] 148 Food Preservation Science VOL. 29 NO. 3 2003 (16)

Fig. 1 Procedures for the fermentation of basi

The bubod made in the La Union was used as removed beforehand from the material solution for

starter in basi fermentation, and air-dry samac bark the automatic carboxylic acid analyzer and this was

and fruit used in the Pangasinan'were used as done by adding 2 ml 10% HClO4 to 8 ml of the

barks to add to basi. material solution , refrigerating overnight , and

2. Basi fermentation removing the protein precipitate by centrifugation11).

The fermentation of basi with and without the 4 . Measurement of viable cell count

addition of barks was carried out according to the Viable cell count was measured by plate culture

procedures shown in Fig. 1. To 650ml water, 350 g method12). Yeasts and molds were cultured on YM

brown sugar was added and dissolved by heating, agar media (yeast extract 0.3%, malt extract 0.3%,

then the solution was divided into 400ml each in peptone 0.5%, glucose 1.0%, agar 1.5%, pH 5.8) at two Erlenmeyer flasks of 1,000ml volume and 28•Ž for 23 days, and their viable cell counts were

sterilized at 120•Ž for 10 min. After sterilization, to calculated respectively. For yeasts, those with

one flask 4 g crushed bubod alone was added and filamentous rim in a colony were calculated as R

to the other both 4 g crushed bubod and 10 g yeast and others as S yeast. Lactic acid bacteria

mixture of samac bark and fruit in equal quantity were cultured on GYP agar media containing CaCO3

(SBF) were added, and both were fermented at 25 (glucose 1.0%, yeast extract 0.5%, peptone 0.5%,

℃ for 12 days. CaCO3 0.5%, agar 1.5%, pH 6. 8) at 37°C for 23

3 . Ingredient analysis days and the viable cell count was calculated as

Alcohol was determined by hydrometer method7), colonies with a clear zone due to the elution of

total sugar by Somogyi-Nelson method8) after CaCO3 from the media.

hydrolysis of sucrose, total acid by alkaline titration Results method9), and polyphenol by Folin-Denis method10).

The hydrolysis of sucrose was carried out at 65•Ž 1 . Basi ingredients for 15 min after adding 5 id of 25% HC1 to 50ml of The ingredients of basi produced in the

material solution. Total sugar was calculated as Philippines wine were shown in Table 1 and

glucose, total acid as succinic acid, and polyphenol compared with them of red wine13) which contains

as D- ( + ) -catechin. polyphenol of grape seeds. The alcohol content of

Organic acids were determined individually using basi was similar to that of the red wine. The total

an automatic carboxylic acid analyzer (S - 500 ; sugar content of basi was as high as that of

Seishin Pharmacy, Ltd., Tokyo) . Protein has to be Edufuaule wine having high sugar content. The

Table 1 Comparison of ingredients between basi and red wine

* These values were quoted from 'Constituents in Fermented Foods' (1999)13) (17) 〔Article〕 Influences of Barks Addition on Basi 149

Table 2 Viable cell counts in bubod

* Colonies with filamentous rim were counted as R yeast. * * Colonies with smooth surface were counted as S yeast.

total acid content of basi was lower than that of bacterium was Pediococcus14),15)

the red wine. The polyphenol content of basi was Using the bubod as a starter, we have carried

higher than that of the red wine and the sweetness out fermentation of basi with and without SBF

of sugar was moderated by the strong astringency addition, and have compared the transition of of polyphenol. microorganisms in both during the fermentation 2. Influence of SBF addition on the growth of process. The results were showed in Fig. 2. No microorganisms in basi fermentation significant difference in the growth of S yeasts with

The microorganisms included in the bubod used S. cerevisiae was seen between basi with SBF and

as a starter in basi fermentation were yeasts, molds that without SBF, regardless of a slight growth

and lactic acid bacteria, as shown in Table 2. In the inhibition in the early stage of fermentation in basi bubod, the number of yeasts with filamentous rim with SBF. Nearly no growth of R yeasts with S. in a colony (R yeasts) were 3.8 X 1O8CFU/ g and the fibuligera was seen in either basi with SBF or that number of other yeasts (S yeasts) were 6.6 •~ 107CFU without SBF, and all died out on the 4th day of /g. The main R yeast was identified fermentation when the growth of S yeasts became Sacchromycopsis fibuligera and the main S yeast was significant. In lactic acid bacteria, significant growth identified Saccharomyces cerevisiae. And the main was seen in the early stage of fermentation in basi mold was Rhizopus and the main lactic acid without SBF, while no growth at all was observed

Fig. 2 Changes of the viable cell counts of yeasts and lactic Fig. 3 Changes of the ingredients during the fermentation acid bacteria during the fermentation of basi with and of basi with and without the addition of samac bark and without the addition of samac bark and fruit fruit

The fermentation of basi was carried out according to the The fermentation of basi was carried out according to the procedures shown in Fig. 1. procedures shown in Fig. 1. 150 Food Preservation Science VOL. 29 NO. 3 2003 (18)

Table 3 Ingredients of basi with and without the addition of samac bark and fruit

The fermentation of basi was carried out according to the procedures shown in Fig. 1.

Table 4 Organic acids in basi with and without the addition of samac bark and fruit (mg/100ml)

The fermentation of basi was carried out according to the procedures shown in Fig. 1. in basi with SBF. This result indicated that the with SBF. In addition, organic acids have been growth of lactic acid bacteria was completely compared on the 12th day of fermentation and the inhibited by the addition of SBF. On the other hand, result was shown in Table 4. There was no no growth of molds was found in either basi with difference in the total acid content between the two or without SBF. types of basi, but difference was seen in individual 3. Influence of SBF addition on the basi organic acid. In basi without SBF, lactic acid and ingredients in basi fermentation acetic acid were numerous. In basi with SBF, acetic The change of ingredients during fermentation acid was numerous but lactic acid was less. process had been compared between basi with and Discussion without SBF, and the result was shown in Fig. 3. The production of alcohol was late in basi with SBF The preparation method of basi is different from in the early days and this is consistent with the area to area1). In some areas, starter is used for result that the growth of S yeasts is late in this fermentation while in other areas, fermentation is basi compared to that without SBF. Nevertheless, performed naturally without starter. In areas where no difference was seen in the alcohol yield between starter is used, bubod is usually used as a starter. two types of basi from the 6th day of fermentation. Bubod is made according to the following process : Similarly, the production of acids was also late in To some smashed rice and ginger, water is added basi with SBF in the early days, but the acid yield and the mixture is kneaded ; next some crushed was the same in two types of basi from the 8th old bubod is added and the mixture is made into a day of fermentation. disc ; then the disc is left indoors for several days Table 3 showed the result of the comparison of to let microorganisms grow and is finally air-dried ingredients in two types of basi after 12 days of completely in the open. In the Philippines, bubod is fermentation. The alcohol content was 12.3ml/ 100ml used as a starter mainly in tapuy, a rice wine. In in basi without SBF and 12. 7ml/100ml in basi with this study, using the bubod as a starter, we have SBF, being a little higher in the latter. Polyphenol carried out fermentation of basi with and without was the ingredient having significantly different the addition of SBF, and have compared the contents between basi with SBF and that without transition of microorganisms and the change of SBF ; its content was 28mg/100ml in basi without ingredients in the two basi. In the fermentation of SBF and 144mg/100ml, about 5-fold higher in basi basi with SBF, SBF was added in 2.5% to the (19) 〔Article〕 Influences of Barks Addition on Basi 151

sugar solution based on the preparation method in the sweetness is too strong to drink as an alcoholic the Pangasinan. beverage. Whereas in basi with SBF, regardless of

In both basi with SBF and that without SBF, its 11. 2 g /100ml sugar, it has a mild taste since its significant growth of S yeasts with S. cerevisiae sweetness has been moderated by the strong which performs alcohol fermentation being the astringency of polyphenol. Therefore the conferring majority is seen from the beginning of fermentation, of astringency by adding barks is an important and no influence of SBF addition is found on the significance for basi and that is similar to the growth of S yeasts. R yeasts with S. fibuligera being conferring of astringency on red wine by adding the majority die out from the 4th day of grape seeds. fermentation when the growth of S yeasts becomes significantly in both basi with and without SBF, and References this is thought to be resulted from the alcohol 1) TANIMURA,W., SANCHEZ,P. C. and KOZAKI,M. : produced by S. cerevisiae. In contrast to the The fermented food in the Philippines. (Part 2 ) significant growth in the early stage of fermentation Basi (Sugarcane wine) , J. Agric. Sci., Tokyo Nogyo in basi without SBF, the growth of lactic acid Daigaku, 22, 135 (1977) bacteria is not seen at all in basi with SBF, 2) MURA,K. and TANIMURA,W. : Influence of samac showing a complete inhibition of growth of lactic bark, samac fruit, samac leaf, and duhat bark on acid bacteria by SBF addition. In the previous growth of microorganisms, HakkokogakuKaishi, 64, paper2), we have reported that most of the aqueous 1 (1986) extracts of barks added to basi inhibit the growth 3) MURA,K., IITOI,Y. and TANIMURA,W. : Growth of lactic acid bacteria. In this study, we have inhibition of lactic acid bacteria by samac confirmed that the growth of lactic acid bacteria (Macharanga grandifolia Linn.) bark, Hakkokogaku the cause of souring •\ is inhibited by the addition of Kaishi, 64, 417 (1986) barks in basi fermentation. Besides, NISHIYAMA, et al .17) 4) MURA, K., IITOI, Y. and TANIMURA,W. : reported that souring to occur by acetic acid Inhibitory substance for the growth of lactic acid bacteria and lactic acid bacteria is prevented by the bacteria in samac (Macharanga grandifolia Linn.) addition of mangrove bark in the fermentation of bark, HakkokogakuKaishi, 65, 399 (1987) tuba, a nipa sap wine in Philippines. 5) MURA,K., SHIRAMATSU,H. and TANIMURA,W. :

In basi with SBF, the growth of S yeasts is a Inhibitory substance for the growth of lactic acid little late and as a result the production of alcohol bacteria in samac (Macharanga grandifolia Linn.) is also slightly late but on the 12th day of fruit, Nippon Shokuhin Kagaku Kogaku Kaishi, 43, 866 fermentation, no difference in the alcohol yield is (1996) found between basi with SBF and that without SBF. 6) MURA,K., SHIRAMATSU,H. and TANIMURA,W. :

On the 12th day of fermentation, the total acid A substance inhibiting the growth of lactic acid contents are the same in the two basi, but the bacteria in duhat (Sizygium cumini Skeels) bark, lactic acid contents are greatly different, being Biocontrol Science, 5 , 33 (2000)

150.8mg/100ml in basi without SBF and 57. 4mg/ 7) WILLIAMS,S. : Official Methods of Analysis of

100ml in basi with SBF. Since no growth of lactic the Association of Official Analytical Chemists acid bacteria is found in basi with SBF, the low ( Association of Official Analytical Chemists , lactic acid content in this basi is thought to be due Arlington) , p. 176, 220 (1984) to the lack of growth of lactic acid bacteria. 8) ANDO, T., TERAYAMA,H., NISHIZAWA,K. and

However, basi with SBF includes lots of acetic acid YAMAKAWA, T . : Methods of Study on and the total acid content is the same as that of Biochemistry, I (Asakura Syoten, Tokyo) , p. 255 basi without SBF. This result suggests that acid (1974) production of yeasts is encouraged by addition of 9) WILLIAMS,S. : Official Methods of Analysis of SBF. Besides, in basi with SBF, the polyphenol the Association of Official Analytical Chemists content reaches to 144mg/100ml on the 12th day of ( Association of Official Analytical Chemists , fermentation, resulting in a strong astringency in Arlington) , p. 224 (1984) the basi. In basi without SBF, the sugar content is 10) FOLIN, O.and DENIS,W. : A colorimetric method

13.5 g /100ml on the 12th day of fermentation and for the determination of phenols (and phenol 152 Food Preservation Science VOL. 29 NO. 3 2003 (20)

derivatives) in urine, J. Biol. Chem., 22, 305 (1915) バシ発酵における樹皮類添加の微生物の生育 11) YANAGIDA,F. : Handbook of Experiments in ならびにバシ成分への影響 Zymurgy and Food Science (Shokuhin Kenkyu-sha, Tokyo) , p. 198 (1984) 村 清 司*1・SANcHEzPriscillaC.*2・ 谷 村 和 八 郎*1 12) HARRIGAN,W. F. : Laboratory Methods in Food * 1 東 京 農 業 大 学 応 用 生 物 科 学 部 Microbiology (Academic Press, San Diego) , p. 53 (〒156-8502東 京 都 世 田 谷 区 桜 丘1-1-1) (1998) * 2 Institute of Food Science and Technology, 13) Brewing Society of Japan : Constituents in University of the Philippines at Los Banos Fermented Foods (Brewing Society of Japan, (College, Laguna 4031, Philippines) Tokyo) , p. 291 (1999) 14) MURA,K., Koizumi, Y., TACHI,H. and UCHIMURA, フ ィリピンではバ シ(甘 薦 酒)の 発 酵の際 に樹 皮類 が T. : Floras of yeasts and molds in Chinese 添加 され るこ とか ら,発 酵 過程 における樹皮類 添加 の微 starter, "Murcha" from Nepal, J. Brew. Soc. Japan, 生物 の生育 な らび にバ シ成分 への影響 につ いて検 討 した。 91, 895 (1996) 酵母 の生育 に対 して は樹皮類 添加 の影 響 はほ とん どみ ら 15) Koizumi, Y., MURA,K. and UCHIMURA,T. : Floras れず,発 酵後 のアル コール生成量 に影響 はみ られ なかっ of lactic acid bacteria in Chinese starter , た。 しか し,乳 酸 菌 は樹皮類 の添加 に よ り完全 に生育 を "M urcha" from Nepal, J. Brew. Soc. Japan, 91, 901 抑制 され,バ シの発 酵 では樹 皮類 の添 加 に よって酸敗 の (1996) 原 因 となる乳酸 菌の生育 が抑 え られる ことが確 認 された。 16) TANIMURA,W., SANCHEZ,P. C. and KOZAKI,M. : また,樹 皮類 を添 加す る ことによ りそれ らのポ リフェ ノ The fermented food in the Philippines. (Part 1 ) ール成分が溶 出 して渋味 を呈 し,原 料糖 の甘 味が緩和 さ Tapuy (Rice wine) , J. Agric. Sci., Tokyo Nogyo れて飲 みやす い味 となった ことか ら,バ シの製造 では渋 Daigaku, 22, 118 (1977) 味 の付 与 も樹皮類 添加 の意義の一 つであ る と考 え られた。 17) NISHIYAMA,R., SANCHEZ,P. C. and KOZAKI,M. : (平 成13年8月31日 受 付,平 成15年1月27日 受 理) Inhibitory function of mangrove bark toward cell growth of microorganisms, HakkokogakuKaishi, 56, 712 (1978)