A Substance Inhibiting the Growth of Lactic Acid Bacteria in Duhat (Sizygium Cumini Skeels) Bark

Biocontrol Science, 2000, Vol.5, No.1, 33-38

Original

A Substance Inhibiting the Growth of Lactic Acid Bacteria in Duhat (Sizygium cumini Skeels) Bark

KIYOSHI MURA*, HIDEAKI SHIRAMATSU, AND WAHACHIRO TANIMURA

Faculty of Applied Bioscience, Tokyo University of Agriculture , 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

Received 2 April 1999/Accepted 8 September 1999

We isolated a substance inhibiting the growth of lactic acid bacteria from duhat bark, which

is added to "basi" , a sugar cane wine in the Philippines, and investigated its structure . By adding gelatin to the aqueous extract of duhat bark (AEDB), 63.2% of the polyphenol components in AEDB were precipitated, and the resulting supernatant lost its inhibitory activity on

the growth of lactic acid bacteria. This result indicates that the inhibitory substance was the

polyphenol component combining to protein. In addition, we fractionated AEDB into two fractions by ultrafiltration and investigated their inhibitory activities on the growth of lactic acid bacteria. A strong inhibitory activity was found in the fraction having molecular weight (MW)

above 1•~104 containing about 53% of the polyphenol components in AEDB, indicating that the main inhibitory substance is a polyphenol component with high MW. We then separated the

polyphenol components with MW above 1•~104 by ion-exclusion chromatography using CM- Sepharose CL-6B, and obtained a polyphenol component with inhibitory activity on the growth

of lactic acid bacteria. The polyphenol component produced gallic acid, anthocyanidins of delphinidin and cyanidin, and glucose by hydrolysis with HCl, and was assumed to be condensed tannin comprised of gallic acid and leucoanthocyanin.

Key words : Duhat/Basi/Lactic acid bacteria/Polyphenol/Condensed tannin.

INTRODUCTION other materials used in "basi" production on the growth of lactic acid bacteria, acetic acid bacteria and In the northwestern Luzon Island in the Philippines yeasts. We found that many of these materials there is a sugar cane wine called "basi" (Tanimura strongly inhibited the growth of lactic acid bacteria et al., 1977). Among liquors made from sugar cane, and acetic acid bacteria, which cause deterioration rum, which is well-known throughout the world, is a during fermentation, and identified that the addition of spirituous liquor, while "basi" is non-spirituous. The barks and other materials is indispensable to the fer- characteristic of "basi" production is the addition of mentation of "basi" in the Philippines, a tropical re- barks and the likes to sugar cane juice before fermen- gion with high air temperatures. Moreover, we tation. Those materials usually used in "basi" prod- investigated the substances inhibiting the growth of uction include samac (Macharanga grandifolia Linn.) lactic acid bacteria in samac bark and fruit and identi- bark, fruit and leaves, and duhat (Sizygium cumini fied that, the main inhibitory substance was con- Skeels) bark. densed tannin comprised of catechin and leuco- In previous studies (Mura and Tanimura, 1986; anthocyanin in samac bark, and was gallotannin Mura et al., 1986, 1987 and 1996) , we investigated combining glucose or xylose with multiple galloyl radi- the impact of aqueous extracts from some barks and cals in samac fruit. In this study, we report the investigation results on *Corresponding author . Tel : +81-3-5477-2459, Fax : +81- the substance inhibiting the growth of lactic acid bac- 3-5477-2626. teria in duhat bark used in "basi" production. 34 K.MURA ET AL.

MATERIALS AND METHODS detected by the blue color using the spray of potas- sium ferricyanide-ferric chloride reagent (Nakagawa Preparation of AEDB and Torii, 1964). The polyphenol compounds sepa- Dry duhat bark, used in "basi" production in the rated by TLC were individually determined by a TLC Philippines, was used as the material in this study. scanner (CS-920; Shimadzu Co., Ltd., Kyoto) at a The bark was crushed and extracted with water of 50- wavelength of 630 nm. fold volume in boiling water for 30 min, and the extrac- tion was performed three times. All extracts were TLC and absorption spectrum measurement for filtered, concentrated under reduced pressure, and anthocyanidins what was used in this study was an aqueous extract Anthocyanidins were separated by cellulose-TLC of duhat bark (AEDB). using a HPTLC cellulose plate. Three kinds of development solvents, acetic acid-concentrated HCl-water Fractionation by ultrafiltration (30:3:10 in volume ratio), n-butanol-acetic acid-water Ultrafilter of UK-10 (MW cutoff: 1•~104) from (4:1:5 in volume ratio, upper layer) and n-butanol-2 N Advantec Toyo Co., Ltd. (Tokyo) was used and filtra- HCI (1:1 in volume ratio, upper layer) were used. The tion was carried out under a nitrogen gas pressure of anthocyanidins separated by TLC developed in acetic 4kg/cm2. acid-concentrated HCl-water were scratched up from the plate, eluted in 0.01% (w/v) HCI-methanol and Fractionation by CM-Sepharose CL-6B 0.01% (w/v) HCl-ethanol respectively, and the ab- A column made by connecting two columns of 2.5 sorption spectra of the eluates were measured. In ad- ×90 cm filled with CM-Sepharose CL-6B was used dition, each 4 ml eluate of 0.01% (w/v) HCI-ethanol and elution was performed using 0.05M borate buffer was mixed with 3 drops of 5% (w/v) aluminium (pH 7.8). chloride-ethanol and its absorption spectrum was measured again to calculate the shift of spectral Growth measurement of lactic acid bacteria maxima caused by the addition of aluminium chloride. The lactic acid bacteria, Pediococcus acidilactici ATCC 8042, was used in this study. The synthetic TLC for sugars medium of Tamura et al. (1952) was used as basal Sugars were separated by cellulose-TLC using medium (pH 6.8). Two ml of double strength basal HPTLC cellulose plate. A mixture of n-butanol- medium was mixed with 2 ml of AEDB adjusted to the pyridine-water (6:4:3 in volume ratio) was used as the same pH and sterilized at 120•Ž for 10 min. To this 4 development solvent. After development, the TLC ml medium, a drop of 100 times dilution (number of plate was immersed in silver nitrate-acetone solution, cells: about 1•~108/ml) of the preculture medium of then dried, and immersed in another solution of 0.5 N P. acidilactici, which had been cultured in 10 ml basal NaOH-ethanol. The sugars on the TLC plate were medium at 37°C for 24 h, was inoculated by using a black-brown in color (Smith, 1954; Trevelyan et al., capillary pipet and incubated at 28°C for 72 h. After 1950). To fix the color, the TLC plate was immersed incubation, the acid produced in the 4 ml culture me- in a fixer for photographic film (Fujifix; Fuji Photo Film dium was neutralized with 0.1 N NaOH, and the Co., Ltd., Tokyo). After being dried, the sugars sepa- growth of P. acidilactici was measured by the acidity rated by TLC were individually determined by TLC of the medium. scanner at a wavelength of 550 nm.

Determination of total polyphenol compound GLC and GLC-MS for gallic acid The total polyphenol compound was determined us- Gallic acid was converted to trimethylsilyl ether/ ing the method of Folin and Denis (1915) and calcu- methyl ester, and analyzed by gas-liquid chromatog- lated as D-(+) -catechin. raphy (GLC) and GLC-mass spectrometry (GLC-MS) (Horvat and Senter, 1980). The GLC device (GC- TLC for polyphenol compounds 4CM; Shimadzu Co., Ltd., Kyoto) was made with a Polyphenol compounds were separated by two- flame ionization detector and a glass column of 3 mm dimensional cellulose thin-layer chromatography ×4m packed with 3% SE-30 absorbed on Chro- (TLC) using a HPTLC cellulose plate of 10•~10 cm mosorb W of 100-120 mesh. The oven temperature from MERK (Darmstadt, Germany). As development was programmed to increase from 120 to 225•Ž at a solvents, n-butanol-acetic acid-water (4:1:2.2 in vol- speed of 4°C/min, and the nitrogen gas flow was set ume ratio) and 2% (w/w) acetic acid were used. to 30 ml/ min. The GLC-MS device (GCMS-6020; After development, polyphenol compounds were Shimadzu Co., Ltd., Kyoto) was set so that the ion INHIBITORY SUBSTANCE IN DUHAT BARK 35

source temperature was 270•Ž and the electron en- removed. In contrast with AEDB which completely in- ergy was 20 eV. hibits the growth of P. acidilactici, the supernatant showed no inhibitory activity after the precipitate had RESULTS been removed. This result suggests that the substance in AEDB inhibiting the growth of lactic acid Growth inhibition of AEDB after removal of the bacteria is the polyphenol component which binds to precipitate caused by gelatin protein, since about 63% of the polyphenol compo- As duhat bark contains 4.8% polyphenol com- nents in AEDB formed precipitate with gelatin and the pounds in its dry weight (Mura and Tanimua, 1986) , inhibitory activity on the growth of lactic acid bacteria the substance inhibiting the growth of lactic acid bac- disappeared after the removal of the precipitate. teria in duhat bark was expected to be the polyphenol compound, as in samac bark and fruit (Mura et al., Fractionation of AEDB by ultrafitration 1986, 1987 and 1996). Since some polyphenol com- AEDB was fractionated into two fractions, the high

pounds can bind to protein and form precipitate MW fraction and the low MW fraction by ultrafiltration (Oshima, 1958), we removed the precipitate in AEDB using an ultrafilter with a MW cutoff of 1•~104. The after adding gelatin and investigated the inhibitory ac- two fractions were then measured for polyphenol con- tivity of the remaining solution on the growth of lactic tent and inhibitory activity on the growth of P. acid bacteria. Fifty ml AEDB of 40 mg/ml in concen- acidilactici (Table 2). In the polyphenol compounds

tration was adjusted to pH 3 with 1 N HCI and mixed of AEDB, 53.1% was in the fraction with MW above with 20 ml of 1% (w/w) gelatin, and the resulting pre- 1•~104 and 39.2% in the fraction with MW below 1 cipitate was removed by centrifugation (Horwitz, ×104, that is, more polyphenol components having 1970). Then the polyphenol content and the inhibi- high MW above 1•~104. Both fractions inhibited the

tory activity of the supernatant on the growth of P. growth of P. acidilactici, but the fraction with high MW acidilactici were measured (Table 1). After adding above 1•~104 containing more polyphenol compo- gelatin to AEDB and removing the precipitate, 36.8% nents completely inhibited the growth of P. acidilactici, of the polyphenol compounds remained in the super- showing strong inhibitory activity. Hence, it is consid- natant, indicating that 63.2% of the polyphenol com- ered that the substance in AEDB inhibiting the growth

pounds in AEDB had been precipitated by gelatin and of lactic acid bacteria is mainly the polyphenol com-

TABLE 1. Polyphenol content of and growth inhibition of P. acidilactici by the aqueous extract of duhat bark after removal of the precipitate formed by the addition of gelatin.

TABLE 2. Polyphenol contents of and growth inhibition of P . acidilactici by the fractions prepared by ultrafiltration of the aqueous extract of duhat bark. 36 K.MURA ET AL.

ponent having high MW above 1•~104. fraction showed no inhibitory activity while the C-2 fraction completely inhibited the growth of P. Separation of polyphenol components by CM- acidilactici. Therefore, it was confirmed that the C-2 Sepharose CL-6B fraction is a high MW polyphenol component which in- The polyphenol components in the fraction having hibits the growth of lactic acid bacteria. MW above 1•~104 with strong inhibitory activity on the growth of lactic acid bacteria, were fractionated by Hydrolysis products of C-2 fraction ion-exclusion chromatography using CM-Sepharose The C-2 fraction inhibiting the growth of lactic acid CL-6B, and the fractionated polyphenol components bacteria was hydrolyzed with HCI (Ito and Oshima, were investigated for inhibitory activity on the growth 1962), and the hydrolysis products were investigated. of lactic acid bacteria. The fraction having MW above The C-2 fraction obtained by ion-exclusion chroma- 1•~104 obtained from AEDB by ultrafiltration was tography with CM-Sepharose CL-6B was desalted by eluted with 0.05 M borate buffer (pH 7.8), and the ultrafiltration using an ultrafilter with a MW cutoff of

polyphenol components were detected by spectrome- 1•~104 and then freeze-dried. Then 2 mg of the try at 280 nm. Figure 1 shows the chromatogram. freeze-dried C-2 fraction was hydrolyzed with 1ml of The polyphenol components with MW above 1 x 104 2 N HCl in boiling water for 20 min. The hydrolyzate were fractionated into two fractions of C-1 and C-2 and after the pH was adjusted to 6.0 with 1 N HCl, these fractions were desalted by ultrafiltration using an ultrafilter with a MW cutoff of 1•~104. Their inhibitory activities were then measured on the growth of P. acidilactici (Table 3). The C-1 fraction contained 3.7% and C-2 fraction 59.6% of the polyphenol com-

ponents with MW above 1•~104. As results, the C-1

FIG. 2. Two-dimensional cellulose thin-layer chromatogram of polyphenol compounds released by the hydrolysis of C-2 FIG. 1. lon-exclusion chromatogram of fraction with MW fraction. Hydrolysis of C-2 fraction was performed with 2N above 1•~104 on CM-Sepharose CL-6 B . The chroma- HCI in boiling water for 20 min. The chromatographic condi- tographic condition is described in Materials and Methods. tion is described in Materials and Methods.

TABLE 3. Polyphenol contents of and growth inhibition of P. acidilactici by the fractions separated by ion-exclusion chromatography of the fraction with MW above 1•~104 on CM-Sepharose CL-6B. INHIBITORY SUBSTANCE IN DUHAT BARK 37 was red-purple in color and some red-brown precipi- the hydrolyzate by TLC. The HCI hydrolyzate was tates were observed. neutralized with 10% (w/w) sodium carbonate and The polyphenol compounds in the hydrolyzate were filtered, and the filtrate was dried under reduced pres- then separated by two-dimensional TLC. As shown in sure. Then, 0.5ml anhydrous pyridine was added and Fig. 2, three polyphenol compounds, P-1, P-2 and P-3 the sugars were extracted. Figure 3 shows the result were detected in the hydrolyzate. The P-1, with a Rf of TLC for the extracted sugars. Almost all the sugars value confirmed to be consistent with that of gallic in the hydrolyzate were glucose. Furthermore, the acid, was defined to be gallic acid by GLC and GLC- polyphenol compounds and sugars in the HCl MS (Mura et al., 1996). As the P-2 was a red pigment hydrolylzate were individually determined by TLC and P-3 a purple one, they were investigated as scanner. Table 4 shows the yields of polyphenol anthocyanidins according to the methods of Harborne compounds and sugars produced from 1 mg of C-2 (1958 and 1967) and Bate-Smith (1950). Their Rf fraction. The main products of HCI hydrolysis were values on cellulose TLC, spectral maxima of HCl- gallic acid, delphinidin and glucose, and 1 mg of C-2 methanol and HCl-ethanol solutions, and shift of spec- fraction produced 43.2 ƒÊg of gallic acid, 12.3 ƒÊg of tral maxima due to aluminium chloride addition were delphinidin and 115 ƒÊg of glucose. measured. Based on these results, P-2 was identified to be cyanidin and P-3 to be delphinidin (Mura et al., DISCUSSION 1987). In addition, since anthocyanidins can exist in the form of glycoside, we investigated the sugars in We fractionated AEDB into two fractions by

ultrafiltration using an ultrafilter with a MW cutoff of 1•~104, and investigated their inhibitory activities on the growth of lactic acid bacteria. The fraction with MW above 1•~104, which contained about 53% of the

polyphenol components in AEDB, was found to have a strong inhibitory activity, suggesting that the

polyphenol component inhibiting the growth of lactic acid bacteria in AEDB was mainly that having high MW above 1•~104. The polyphenol components with MW above 1•~104 obtained by ultrafiltration were fur-

ther separated by ion-exclusion chromatography using CM-Sepharose CL-6B, and the polyphenol component with high MW, which inhibited the growth of lactic acid bacteria, was obtained. The high MW polyphenol component, which was

isolated by ion-exclusion chromatography using CM- Sepharose CL-6B and inhibited the growth of lactic acid bacteria, produced gallic acid, anthocyanidins and sugars upon HCI hydrolysis. The anthocyanidins were delphinidin and cyanidin. The delphinidin was thought to be derived from leucodelphinidin and FIG. 3. Cellulose thin-layer chromatogram of sugars released by the hydrolysis of C-2 fraction (A) and glucose (B). cyanidin from leucocyanidin, and these leucoantho Hydrolysis of C-2 fraction was performed with 2N HCI in cyanidins were considered to be in the form of leuco- boiling water for 20 min. The chromatographic condition is anthocyanins, glycosides with the sugars released by described in Materials and Methods. HCI hydrolysis (Bate-Smith, 1954; Ito, 1965). Hence, the high MW polyphenol component in duhat bark which inhibits the growth of lactic acid bacteria is pre- TABLE 4. Yields of hydrolysis products from C-2 fraction. sumed to be condensed tannin comprised of gallic acid and leucoanthocyanins (Nishioka, 1986). Be- sides, as most of the anthocyanidin produced by HCI hydrolysis was delphinidin, the leucoanthocyanin in this condensed tannin was thought mainly to be

leucodelphinin; and as most of the sugar produced by Hydrolysis of C-2 fraction was performed with 2N HCI in HCI hydrolysis was glucose, the leucoanthocyanin boiling water for 20 min. was considered mainly to be glucoside. 38 K. MURA ET AL.

The substance inhibiting the growth of lactic acid Ito, S., and Oshima, Y. (1962) Studies on the tannin of bacteria in samac bark had previously been identified Japanese persimmon (Diospyros Kaki L.) . Agric. Biol. to be a typical condensed tannin comprised of ca- Chem., 26, 156-161. techin and leucoanthocyanin (Mura et al., 1987) . In Mura, K., and Tanimura, W. (1986) Influence of samac bark, the present study, the inhibitory substance in duhat samac fruit, samac leaf, and duhat bark on growth of mi- croorganisms (in Japanese) . Hakkokogaku Kaishi, 64, 1- bark was reported to mainly be an unusual condensed 7. tannin comprised of gallic acid and leucoanthocyanin. Mura, K., litoi, Y., and Tanimura, W. (1986) Growth inhibition of lactic acid bacteria by samac (Macharanga grandifolia Linn.) bark (in Japanese) . Hakkokogaku ACKNOWLEDGMENT Kaishi, 64, 417-423. We thank Professor Priscilla C. Sanchez of the University Mura, K., litoi, Y., and Tanimura, W. (1987) Inhibitory sub- of the Philippinesat Los Banos for providingthe duhat bark stance for the growth of lactic acid bacteria in samac used in this study. (Macharanga grandifolia Linn.) bark (in Japanese) . Hakkokogaku Kaishi, 65, 399-404. Mura, K., Shiramatsu, H., and Tanimura, W. (1996) REFERENCES Inhibitory substance for the growth of lactic acid bacteria in samac (Macharanga grandifolia Linn.) fruit. Nippon Bate-Smith,E. C. (1950) Anthocyanins,flavones and other Shokuhin Kagaku Kogaku Kaishi, 43, 866-872. phenolic compounds. Biochem. Soc. Symp., No.3, 62-73. Nakagawa, M., and Torii, H. (1964) Studies on the flavanols Bate-Smith, E. C. (1954) Leuco-anthocyanins.Biochem. J., in tea. Agri. Biol. Chem., 28, 160-166. 58, 122-125. Nishioka, I. (1986) Chemistry of tannin (in Japanese) . Folin, 0., and Denis, W. (1915) A colorimetric method for KASEAA, 24, 428-439. the determination of phenols (and phenol derivatives) in Oshima, Y. (1958) Chemistry of plant tannin (in Japanese) . urine. J. Biol. Chem., 22, 305-308. Nippon Nogeikagaku Kaishi, 32, A81-88. Harborne, J. B. (1958) Spectral methods of characterizing Smith, R. H. (1954) The phosphatides of the latex of Hevea anthocyanins. Biochem. J., 70, 22-28. brasiliensis. Biochem. J., 57, 140-144. Harborne, J. B. (1967) The anthocyanidins. Comparative Tamura, G., Tsunoda, T., Kirimura, J., and Miyazawa, S. Biochemistryof the Flavonoids, pp.3-46, Academic Press, (1952) The microbiological determination of amino acids London and New York. by lactic acid bacteria (in Japanese) . Nippon Horvat, R. J., and Senter, S. D. (1980) A gas-liquid chroma- Nogeikagaku Kaishi, 26, 464-470. tographic method for analysis of phenolic acids in plants. Tanimura, W., Sanchez, P. C., and Kozaki, M. (1977) The J. Agric. Food Chem., 28, 1292-1295. fermented food in the Philippines. J. Agric. Sci., Tokyo Horwitz,W. (1970) Tannin. In OfficialMethods of Analysis Nogyo Daigaku, 22, 135-141. of the Association of OfficialAnalytical Chemists, 11th ed., Trevelyan, W. E., Procter, D. P., and Harrison, J. S. (1950) pp. 240-241, A.O.A.C.,Washington, D.C. Detection of sugars on paper chromatograms. Nature, Ito, S. (1965) Chemistry of leucoanthocyan (in Japanese) . 166, 444-445. Nippon Shokuhin Kogyo Gakkaishi, 12, 295-302.