Biocontrol Science, 2004, Vol. 9, No. 4, 117-122

Note

Antioxidant and Antibacterial Activities of Bacteria from Ngapi, a Burmese Salted and Fermented Shrimp

WIN AUNG, NAING NAYLIN, ZHIBING ZHENG, YUKA WATANABE AND FUMIO HASHINAGA*

Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan

Received 17 March 2004/ Accepted 30 August 2004

A total of 33 bacterial strains from ngapi, a Burmese salted and fermented , were screened for antioxidant and antibacterial activities. Two strains, NP1-1 and NP3-2, which showed high free radical scavenging and antibacterial activities, were selected. Radical scavenging activities of the ethyl acetate extracts were compared with those of BHA using DPPH. The antioxidant activity of extracts was determined according to the thiocyanate method. Moreover, the filtrate extracts from the two strains were found to have broad inhibi- tory activity against target bacteria. NP1-1 and NP3-2 strains were respectively identified as Bacillus subtilis and Bacillus sp. based on the 16S rDNA sequences.

Key words : Ngapi/Salted and fermented shrimp paste/Antioxidant and antibacterial activities/ 16S rDNA sequence.

Burma () is a country in Southeast Asia. does the quality of the finished product (Surono and In Southeast Asia, traditional fish products represent Hosono, 1994). a significantly large part of the total utilization of fish It has been traditionally known in Burma that the and are a major source of animal protein. As a result, fermented marine products are not only nutritionally the food safety of these products are vitally important, and dietarily valuable, but they may take some func- especially considering that most of them go to mar- tional roles in health. Montano et al. (2001) reported ginalized sectors of the population (Soon-Eong et al., that all the fish and shrimp condiments analyzed con- 2002). In those regions, salted and or tained measurable quantities of polyunsaturated fatty shrimp pastes are important flavor enhancers and acids, including docosahexaenoic acid (DHA). The dietary supplements. Closely related products are metabolites produced by microorganisms may serve called ngapi in Burma, kapi in Thailand, padec in as antioxidants (Hayashi et al., 1995), synergists Laos, ki in Cambodia, terasi in Indonesia (Soon-Eong (Ishikawa et al., 1985), scavengers (Kato et al., et al., 2002; Surono and Hosono, 1994), blacan in 1993), lipoxygenase inhibitors (Nihei et al., 1993) Malaysia, and in the Philippines (Montano and/or metal chelating agents (Kunze et al., 1992). et al., 2001; Steinkraus, 1996). Although the names Derivatives from food constituents produced by the are different, the process to make ngapi is quite simi- action of microbial enzymes have antioxidant and/or lar with that to make terasi, an indigenous Indonesian synergistic activity (Murakami et al., 1984). Further- fermented food made from fish and/or shrimp paste more, microorganisms can decompose lipid perox- obtained by natural fermentation in the presence of a ides and use them as nutrients (Rashid et al., 1992). high salt concentration. However, the processing Studies on the antibacterial compounds produced method varies depending on the processor and so by the bacterial flora in ngapi may contribute to: (1) reduce microbial contamination during the fermenta- *Corresponding author . Tel & Fax : +81-99-285-8666. tion and prolong the shelf life of ngapi and, (2) pro- vide new bacteriocin-like inhibitory substances with 118 W. AUNG ET AL.

activity in the ngapi, as well as on food pathogens or One Ioopful of freshly grown culture on nutrient agar microorganisms which cause spoilage. For this rea- slant containing 2% agar in NB was inoculated into 5 son, the development of new types of antioxidants ml of the same NB medium and incubated at 30•Ž for and bacteriocins from microorganisms may be possi- 1 d in a shaker incubator (150rpm). One-day-old cul- ble. There have been a few reports on the distribution ture was inoculated into a 500ml Erlenmeyer flask and identification of bacteria in these foods (Stein- containing 200ml NB and adjusted to get the final kraus, 1996; Surono and Hosono, 1994). Moreover, suspension of 107CFU/ml. It was then incubated in a

Surono and Hosono (1994) stated that more than reciprocal shaker incubator (130rpm) at 30•Ž for 2 d. 50% of all isolates from Indonesian terasi belonged to In the extraction of bacterial antioxidant and anti- aerobic spore-forming bacilli, and the dominant bacterial compounds, 200m1 of 2-day-incubated liquid strains were identified as B. brevis, B. sphaericus, B. culture were mixed with a 1.5 fold volume of ethyl subtilis and B. circu/ans. However, to our knowledge, acetate. The mixture was then shaken in a separatory there is no report on the antioxidant and antibacterial funnel and the ethyl acetate layer was collected. The activities of bacteria from ngapi. In this paper, there- ethyl acetate extracts were passed through Advantec fore, we isolated bacteria from ngapi, and two strains No. 2 filter paper (110mm) (Toyo Roshi Co., Ltd., of the culture extracts, which possessed high free Japan). The solvent was removed using a vacuum ro- radical scavenging and antibacterial activities, were tary evaporator below 35•Ž. The residues were dis- identified on the basis of the 16S rDNA gene se- solved with 10ml of 50% ethanol and kept at -80•Ž for quences. 4 h. The frozen extract solution was then lyophilized Three ngapi samples used in this work were col- until it reached a constant weight. The lyophilized ex- lected from local markets in Burma. After collection, tract was dissolved in 99.5% ethanol to get the con- the samples were kept at 4•Ž for bacterial isolation centration of 10 mg • ml-1. and identification. The radical scavenging activity of the ethyl acetate For bacterial isolation, three kinds of liquid media, extracts from the broth filtrate of tested bacteria was 0.1, 0.5 or 1.0M NaCI with 0.5% beef extract (Difco, compared with that of butylated hydroxyanisole

Detroit, MI, USA) and 1% peptone (Wako Pure (BHA) using 1,1-dipheny1-2-picrylhydrazyl (DPPH). Chemical Industries, Ltd., Japan) were used as en- Besides, antioxidant activities of extracts were deter- richment media for the isolation of microorganisms. mined according to the thiocyanate method using 2, To a 5 ml aliquot of this medium 1g of a source sam- 2,-azobis (2-amidinopropane) dihydrochloride (AAPH)

ple was inoculated and the mixture was incubated in as described by Terasawa et al. (2001). a shaker incubator at 30•Ž for 3 d. Thereafter, 2% Bacterial strains of Bacillus cereus IFO 15305, agar streak plates with compositions the same as the Bacillus subtilis IFO 13719, Micrococcus luteus IFO above media were made. Bacterial colonies of dis- 12708, Staphylococcus aureus IFO 14462, Esche- tinct morphologies that were visible after 3-7 d were richia coli B IFO 13168, Escherichia coli C IFO 13891

streaked onto new agar plates in order to obtain pure and Salmonella enteritidis IFO 3313 were used as tar-

colonies and subsequently grown in 5ml nutrient get bacteria in the antibacterial assay. broth (NB) medium containing 0.25% NaCI, 0.5% The paper disc method was used for the antibacte- beef extract and 1% peptone (pH 6.6). Aliquots of 1 rial activity test. Fifteen microliters of the extract solu-

ml from each bacterial culture were preserved at -80 •Ž tion (10mg • ml-1 ) were impregnated into 6mm

, and bacterial strains were grown from this stock diameter sterilized discs made from Advantec filter

when needed. paper. The discs were then left for 20min under the Bacterial strains for the antioxidant and antibacte- blower of a clean bench and two more Impregnations rial activity tests were selected based on the result of of 15 ƒÊl were made so that each disc contains 450

co-incubation of isolates and target bacterial strains. g of the compound. To a blank disc, the sameƒÊ Briefly, freshly grown cultures were stab-inoculated amount of ethanol, 45 ƒÊl (15 ,ƒÊl •~ 3) was impreg-

into NB agar plates seeded with target pathogens. In nated. A suspension containing ca. 106 CFU/ml of 1- this test, Bacillus cereus IFO 15305, Micrococcus day-old target bacteria grown in NB medium was

luteus IFO 12708, Escherichia coli B IFO 13168 and prepared with 3ml of NB agar containing 0.5% agar. Salmonella enteritidis IFO 3313 were used as target This suspension was transferred to a Petri dish con-

pathogens. The plates were incubated at 30•Ž over- taining 20ml of NB solid medium containing 2% agar night. The size of the transparent zone, which ap- and spread evenly with a glass rod. The discs, each

peared around each colony, was measured the next containing 450 ƒÊg of the test extract, and one blank morning. disc were put on each dish and incubated at an in- NB medium was used for ethyl acetate extraction. verted position at 37•Ž. The diameter of the inhibition ANTIOXIDANT ACTIVITY OF BACTERIA FROM NGAPI 119

zone around the disc was measured after 24h. Each agreed with that statement because we could isolate experiment was repeated three times. only three strains and a few yeast strains (data not

The two bacterial strains NP1-1 and NP3-2 were shown) from the NP2 sample. Besides, the NP2 identified on the basis of the 16S rDNA gene se- sample was thought to provide a difficult environment quences. DNA extraction, amplification and sequenc- for bacteria growth with its higher salt content, and ing were carried out as described in our previous added fillers and dyes. study (Aung et al., 2004). In bacterial isolation, especially from samples NP1 The three ngapi samples (1) that of superior qual- and NP3, a high number of colonies, which came up ity without additives, (2) that of inferior quality with on a NB agar plate were very similar to each other in fillers and dyes and (3) that which was at least six terms of shape, size, and color. According to the co- months old, were denoted as NP1, NP2 and NP3 re- incubation test, most of the strains showed activity of spectively. In ngapi processing, fermentation in the a different spectrum (data not shown). Among them, wooden tubs is carried out for generally up to 4-5 7strains, NP1-1, NP1-2, NP1-3, NP1-5, NP1-7, NP3-2 months (Thein et al., 1987) to get finished products. and NP3-4, which showed antibacterial activity During the fermentation period, the hydrolyzed liquid against all of the target pathogens, were selected to which comes out from the small holes at the bottom be tested for antioxidant and antimicrobial activities of the vessels is used as shrimp . The no. 3 of the ethyl acetate extracts. sample was thought to have had a longer storage To optimize the culturing time, 1 ml of NB culture time at the place of production and its value is less was drawn daily from 0 to 7 d incubation. Liquid cul- than that of superior quality ngapi. From the three ture was centrifuged at 5,000 •~g for 5 min at room ngapi samples, we picked and spread onto agar temperature, then 0.2 ml of supernatant was taken plates a total of 33 bacteria of different sizes and de- and the scavenging activity was measured using the grees of compactness, which are shown in Table 1. DPPH method. We found that the best incubation It is regarded that the long shelf life of shrimp paste time for the NB medium was 2 d (data not shown). is mainly due to its high salt content, 5-25% (w/w) Thus, we used 2 d incubation time for the next experi- (Adnan and Owens, 1984; Thein et al., 1987), but ments to produce ethyl acetate extracts from the NB there might also be osmotolerant and/or halophillic medium. microorganisms associated with these foods The DPPH radical is considered to be a model of (Chaiyanan et al., 1999). We used three NB media the lipophilic radical. A chain reaction in lipophilic containing 0.1, 0.5 and 1.0M NaCI in bacterial isola- radicals was initiated by the lipid autoxidation. The tion because of the wide range of salt content in the scavenging effects of ethyl acetate extracts and BHA samples. According to the Surono and Hosono on the DPPH radical were compared and shown in (1994), in the shrimp paste, the acid-producing Fig.1. According to the results shown in Fig.1, ethyl Bacillus seems to be the most versatile group which acetate extracts had significant scavenging effects at can utilize a wide variety of energy sources, and non- various levels on the DPPH radical and these effects acid producing Bacillus has poor ability in utilizing increased approximately doubly in all extracts of starch, sugar and protein. Our isolation results also strains when the concentration increased from 100 to

TABLE 1. Isolation of bacteria from ngapi samples. 120 W. AUNG ET AL.

ings and to study the production of antioxidants in mi-

croorganisms, the 2 strains, NP1-1 and NP3-2, which showed high free radical scavenging activities, were tested by the thiocyanate method. The antioxidant ef- fects of the ethyl acetate extracts from the filtrate of

the 2 strains on the peroxidation of linoleic acid after 15 h incubation are shown in Fig. 2. Although the ac- tivity was lower compared to that of BHA, this study showed that the thiocyanate method with (AAPH)

could be used to obtain quick results. Table 2 shows that the ethyl acetate extracts from NP1-1 and NP3-2 were effective against all target

bacteria and the effective zones of NP1-1 were wider than those of NP3-2 except against Bacillus subtilis. Although the strains NP1-2 and NP3-4 were found to have stronger bacterial activity on some target bacte- FIG. 1. Radical scavenging activities of the ethyl acetate rial strains, we selected and identified only NP1-1 and extracts at concentrations of 100 and 200 ppm. NP3-2 strains for their high free radical scavenging activity in this study. The control with ethanol (45 ƒÊl/ 200 ppm. It is suggested that the scavenging activity disc) shows no effect in the experiment. of the ethyl acetate extracts comes from the bacterial In the 16S rDNA sequences analysis, both strains strains growing in the NB medium. Among them, the NP1-1 and NP3-2 belonged to the Bacillus species. scavenging activities from NP1-1 and NP3-2 were The sequence of 714 by of strain NP1-1 was shown 61.5 and 61.7% respectively at the concentration of to have the highest 16S rDNA similarity of 99.75% to 200 ppm. These two stains were therefore selected to Bacillus subtilis (AB018487, accession No. of Gen- be tested again by the thiocyanate method for the in- Bank/EMBL/DDBJ/PDB database). However, the hibition of peroxidation. sequence of 722 by of strain NP3-2 was shown to Rashid et al. (1992) reported that microorganisms have a 99.46% identity with a wide range of Bacillus might survive in an environment that contained perox- species, which have the same scores. This result of ide oil. In order to examine the validity of these find- Bacillus being found in ngapi, therefore, is in agree-

TABLE 2. Antibacterial activity of bacteria isolated form ngapi samples.

FIG. 2. Antioxidant activity of the ethyl acetate extracts from the broth filtrate of NP1-1 and NP3-2 as measured by the thiocyanate method using AAPH after 15 h incubation. •¡

, NP1-1; •£, NP3-2; •Ÿ, BHA. a No activity. ANTIOXIDANT ACTIVITY OF BACTERIA FROM NCAPI 121

ment with the finding of the predominant presence of Chaiyanan, S., Maugel, T., Huq, A., Robb, F.R., and Colwell, Bacillus in Indonesian terasi as stated by Surono and R.R. (1999) Polyphasic taxonomy of novel Halobacillus, Hosono (1994). The nucleotide sequence data re- H. thailandenis sp. nov. isolated from . Syst. ported in this paper will appear in the DDBJ/ EMBL/ Appl. Microbiol., 22, 360-365. GenBank nucleotide sequence databases with the Corvey, C., Stein,T., DOsterhus, S, Karas, M., and Entian, K.D. (2003) Activation of subtilin precursors by Bacillus accession numbers AB168129 for NP1-1 and subtilis extracellular serine proteases subtilisin (AprE),

AB168130 for NP3-2 strain. WprA, and Vpr. Blochem. Biophys. Res. Commun., 304, According to the previous reports (Abee, 1995; 48-54. Corvey et al., 2003), Bacillus subtilis strains produce Hayashi, K., Suzuki, K., Kawaguchi, M., Nakajima, T., the peptide antibiotic (lantibiotic) subtilin. Moreover, Suzuki, T., Numata, M., and Nakamura, T. (1995) bacteriocins are generally effective against closely Isolation of an antioxidant from Penicillium roquefortil IFO related species (Baba and Schneewind, 1998; Parret 5956. Blosci. Biotechnol. Blochem., 59, 319-320. Hechard, Y., and Sahl, H.G. (2002) Mode of action of

and Mot, 2002). The bacteriocin gene is usually as- modified and unmodified bacteriocins from Gram-positive sociated with an immunity protein and bacteria can bacteria. Blochimie, 84, 545-557. be protected from their own bacteriocin (Hechard Ishikawa, Y., Morimoto, K., and Hamasaki, T. (1985) and Sahl, 2002). Thus, the extracts from NP1-1 and Metabolites of Eurotium species, their antioxidative prop- NP3-2 strains were thought to be associated with erties and synergism with tocopherol. J. Food Sc!., 50, bacteriocin-like antibacterial compounds. 1742-1744. Kato, S., Shindo, K., Yamagishi, Y., Matsuoka, M., Kawai, In conclusion, the results obtained in this study H., and Mochizuki, J. (1993) Phenazoviridin, a novel free

clearly demonstrate that the ethyl acetate extract of radical scavenger from Streptomyces sp., Taxonomy, fer- NP1-1 and NP3-2 strains exhibited a significant anti- mentation, isolation, structure elucidation and biological oxidant activity and a broad-spectrum of antibacterial properties. J. Antibiot., 46, 1485-1493. activities. Further evaluation of the safety of ethyl Kunze. B., Trowitzsch-Kienast, W., Hofle, G., and acetate extracts should be conducted, however. Reichenbach, H. (1992) Nannochelins A, B and C, new iron-chelating compounds from Nannocrystis exedens Further study is also needed on the NP3-2 strain (myxobacteria). Production, isolation, physicochemical (e.g., studies with the full length of the 16S rDNA se- and biological properties. J. Antibiot., 45, 147-150. quence, DNA-DNA hybridization) in order to resolve Montano, N.N., Gavino, G., and Gavino, V.C. (2001) this deep phylogenetic relationship among the Polyunsaturated fatty acid contents of some traditional Bacillus species. fish and shrimp paste condiments of the Philippines. Food Chem., 75, 155-158. Murakami, H., Asakawa, T., Terao, J., and Matsushita, S.

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Assistant Prof. Matsujiro Ishibashi (Dept. of Biochemical Nihei, Y., Yamamoto, H., Hasegawa, M., Hanada, M., Science and Technology, Faculty of Agriculture, Fukagawa, Y., and Oki, T. (1993) Epocarbazolins A and

Kagoshima Univ.) for their guidance and many helpful sug- B, novel 5-lipoxygenase inhibitors. Taxonomy, fermenta-

gestions. We also thank Assistant Prof. Takeshi Shimogiri tion, isolation, structures and biological activities. J. and Researcher Keiji Kinoshita (Dept. of Animal Breeding Antibiot., 46, 25-33.

and Genetics, Faculty of Agriculture, Kagoshima Univ.) for Parret, A.H.A., and Mot, R.D. (2002) Bacteria killing their their helpful discussion on the analysis of DNAsequences. own kind: novel bacteriocins of Pseudomonas and other

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