ANTTBACTERTAL TEST OF PANGIUM ( Pang ium edule Reinw ) EXTRACT AGAINST THE GROWTH OF FISH SPOILAGE BACTERIA

An antibacterial test was conducted on fresh and fermented Pangium edule Reinw. Seed extracts against gram positive and negative spoilage bacteria associated with fish. Seed extraction was cJrried out by rnaceration with water, water-ethanol (1;1), and n-hexane. Antibacterial activities were determined by an agar diffusion method. Water extract of fresh Fangium edule seed inhibited the growth of gram positive and negative bacteria at concentrations of 40-80 mg/mL. water-ethanol extract inhibited the growth of gram positive and negative bacteria at concentrations of 30-80 mg/mL, while nhexane extract showed no inhibition. On the other hand, extract of fermented Pangium edule seed using water, water-ethanol, and n-hexane showed no antibacterial activity against gram positive and n"gJtiu" bacteria. The minimum inhibitory concentration values of fresh Pangium edule seed water extiact were 3.49-11.73 mg/mL, mostly effective against Pseudomonas fiuorescens. Meanwhile, the minimum inhibitory concenlration of water-ethanol extract were 6.04-10.54 mg/ml, mostly effective against Enterobacter aerogenes, Alcaligenes eutrophus, and Staphylococcus aureLts' KEyWORDS: antibacterial activity, agar diffusion method, gram positive and fish spoilage bacteria, minimum inhibitory concentration, Pangium edule Reinw INTRODUCTTON choulmogric acid (2 cyclopentene 1 tridecanoic/ C,,sH32O2) are unsaturated cyclic fatty acids as the Many places in lndonesia are not properly acidifl-cationproductof oleicandlinoleicacidcontained facilitated'by basic infrastructures so that in remote in the kernel. Pangium also contains polar and areas but fish is highly produced, problems of quantity nonpolar antioxidants. The nonpolars were d-, d-, and and quality loss arise due to the lack of ice needed to ii-tocotrienol, though dominated by 6-tocotrienol preserve ine tirn. To overcome this, fishermen use (Puspitasari-Nienaber et al',1994). The antioxidant iresh pangiu m (pangium edule Reinw) seed, called content is reduced during initial phase of seed picung,to preserve fiih traditionally for many decades. germination and some of i-tocotrienol change to 6'considering its biochemical properties, pangium has and a-tocotrienol. However, when the hypocotyls a great chalenge as a natural preservative, especially synthetize the chlorophyl, the tocotrienols increased for fish and fish products. The lack in quaniity, tne agarn and at the same time, the tocoferol is high price, and the low quality of ice, are known as synthetized. On the other hand, the polar antioxidants reason in promoting the misuses of such illegal are possibly carboxylic acid and sugars, which is preservatives like formalin in fish handling in remote believe to be a glycon of phenolic conjugate. The total areas (Heruwati et at.,20a4; Sampurno, 2006). The phenolic.contents as well as the antioxidant activity benefits of pangium for fish handling are noi only of pangi.um kernei are increased during seed attributed to its anti-bacteria and antioxidani germination. The antioxidant might protect the cell properties, but pangium is also believed in producing from potential oxidation-induces deterioration iypical ftavor on fish and fish product. (Andarwulan ef a/., 1999)' Some studies revealed that pangiurn contains Experiment using chopped pangium seed mixed many substances such as flavoring agent, cyanlde with.granular salt, and overspread on the gutted acio (Heyne, .1 9S9; Bishop, 1 997), antioitoant (Anwar, mackerel fish (Rasfre//rgrer brachysoma) at ratio of 2, .1992; Panghegar, 1990; Adidjaja, 1991; Romlah, 4, and 6% kernel and 2 and 3% salt (w/w to fish 1992), tannin and antimicroniai agents including weight) revealed that pangium was significantly hidnocarpic acid, and chaulmogriclcid (Hilditch & reduced the pH and TVB content of fish, although it Williams, 1964). The cyanide acid is believed to be a had no effect on other parameter assessed' At the derivative product of gynocardine glucosides by the same time, salt also showed a significant effect on role of gynocardasJ, while hidnocarpic aciO 1Z both TVB content and the number of HrS producing cyclopentene 1 undecanoic/c,uHrror) and bacteria. However, pH,TVBcontentaswellasnumber Corresponding author: Jl. K. S. Tubun petamburan Vl, Jakarta-lndonesia'10260, Telp. 021-536501 57, e-mail: endang-heruwati@yahoo.com 65 Ind.Fislt Res.J. Vol 1S No.2 Descember-2A79: 65_73 of enteric and H"S proclucing bacteria increased during fish storage. Sensory evaluation resulted ihat from the texture. odor and flavor point of vrew treatments with 2% pangiurn (whether mixed with 2 or 3% salt) was reJected at day-6, while the one wiilr pangtum 67o iwith 2 or 3oh salt), and combination of 4% pangiunr with 3% salt was stilt accepted by the panelists at day-9 Neverthetess. treatments wlth htgh content of pangiurn were not preferred bv the panelists due io the flsh appearance whrcn were rather brownrsh in color. This undesrrable color may be Broduced by the reaction between tannin (tannic acid) with ferric conrpound from fish meat (rnyogiobine). The tannic acid contained in fish were betwee n 16.7_23.2 ppm at initial and 7 .7-11 .A ppnr at the encl of experiment; while for cyanide, the content in fish were between 20.644.4 ppm at initiat and 2.5-19.2 ppm at the end of experiment depending on the amount of pangiurn added (lieruwati et at.,2A07). Though it has been proved both by experiments and by commercial praciices that pangii.rm coirld prolonE the shetfiife of fish, yet, it stili very limited information available on what active substances contained in pangium that responsible in fish preservation, and how long they could delay the spoilage of fish. For this purpose, a study was conducted on the antibacterial acti,rity of water, water_ ethanol, and n-hexane extracts of fresh and fermented pangium against some species of bacteria responsible in fish spoilage. lt was expecied that baseo on resulted information on extract that has highest preservative activity. experiment could be followed by isolation, characterization, and purification of ihe active substances. in ihe iong run, the produciion of natural preservative for fresh fish could be realized. IVIATERIALS AN N flfl ETN.IODS Ripe pangiu m (Pangium edrle Reinw) frurts were collected from Pabuaran, Cileungsi, Bogor. The seeds then were drawn out and washed with water. Fermented seeds were made by boiling the seed for 6 hrs, followed by rvrapping in plastic bag and keeping at room temperature for about 40 days. Two species of gram positive bacteria i.e. Micrococctts luteus, and Staphytococcus aureus, six species of gram negative i.e. Alcaligenes eutrophus, Enterobacter aerogenes, Ftavobacteriunt gleum, P se u d o m o nas fhroresce n s, S a I m o n e t I a ty p h i m u ri u m, and Serraf ia marcescens were useo in this experiment. These cultures were obtained from Microb,iological Laboratory of Research Institute for Marine and Fisheries Froduct Frocessing and oo tsiotech notogy, Jakarta and Microbiolog ical Laboratory of the Banciung Institute of Technology, Bandung. Chloram phenrcoi (l ndofarma.) at concentration of 10 mg/rnL was used as a positive contrcl, while water and water-ethanot ('1:i ) and n hexane were used as negative control. llutrientAqar (NA) (Difco) was used as the gi'owth medium for the culiures, and Mueller Hinton Agar (MHA) (Difco) was used in antibacterial foci Preparation of Pangiunn Extracts Fresh and fermented pangium seeds were cleaned and washed, tlren the shells were cracked to get the kernel. The kernelwas homogenized and freeze_dried at -40"C followed by grinding them into powoer. Extraction was cjone by nracerating 200 g of fresh or fermented pangium powder in .1,000 mL of distilted water by shaking at 30 rpm for 3x24 hrs. After filtration using Whatman paper na.42, the filtrate was driecl usf ng vacuLtm evaporato r at72 mbar to obtain crude water extract. Afier filtration, the residue was collected and remacerated using 1.000 rnl of mixture of water_ ethanol ('1:1) wiih rhe same procedure as water extraction. Filtrate of the later extraction was dried by using vacuLlm evaporaior at lZSmbar to getcrude water-ethanol extract. The residue of second extraction was tlren remacerated using 1,000 mL n_ hexane by the same procedure. ln this last extracticn, evaporation of the filtrate vras done at 335 mbar to get crude n-hexane extract. All crude extracts were then freeze-dried, weighed, and kept in capped botile until used (Harborne, 1998;Andanvulan ef a/., 1g9g). Freparation of Test Culture All cultures were grown at slanted NA for 24 hrs at 37'C, an ose of each culture was then grown in 20 mL of nutrient broth, incubated in a shaking water bath at 160 rprn, f or 24 hrs 37"C. The optical density was measured using spectrophotometer at d 600 nm to get an absorbance of 0,5-0,6. To make sure that a total number of 106-'108cfu/mL had been achieved, counting of the totalbacteria using Mc Farland method (DG of Drug & Food Control, .1gg5) was also conducted.

iresh pangiu m (pangium edule Reinw) seed, called content is reduced during initial phase of seed picung,to preserve fiih traditionally for many decades.germination and some of i-tocotrienol change to 6- 'considering its biochemical properties, pangium has and a-tocotrienol.However, when the hypocotyls a great chalenge as a natural preservative, especially synthetize the chlorophyl, the tocotrienols increased for fish and fish products.The lack in quaniity, tne agarn and at the same time, the tocoferol is high price, and the low quality of ice, are known as synthetized.On the other hand, the polar antioxidants reason in promoting the misuses of such illegal are possibly carboxylic acid and sugars, which is preservatives like formalin in fish handling in remote believe to be a glycon of phenolic conjugate.The total areas (Heruwati et at.,20a4;Sampurno, 2006).The phenolic.contentsas well as the antioxidant activity benefits of pangium for fish handling are noi only of pangi.umkernei are increased during seed attributed to its anti-bacteria and antioxidani germination.The antioxidant might protect the cell properties, but pangium is also believed in producing from potential oxidation-induces deterioration iypical ftavor on fish and fish product.
(Andarwulan ef a/., 1999)' Some studies revealed that pangiurn contains Experiment using chopped pangium seed mixed many substances such as flavoring agent, cyanlde with.granularsalt, and overspread on the gutted acio (Heyne, .1 9S9; Bishop, 1 997), antioitoant (Anwar, mackerel fish (Rasfre//rgrer brachysoma) at ratio of 2, .1992;Panghegar, 1990; Adidjaja, 1991; Romlah, 4, and 6% kernel and 2 and 3% salt (w/w to fish 1992), tannin and antimicroniai agents including weight) revealed that pangium was significantly hidnocarpic acid, and chaulmogriclcid (Hilditch & reduced the pH and TVB content of fish, although it Williams, 1964).The cyanide acid is believed to be a had no effect on other parameter assessed' At the derivative product of gynocardine glucosides by the same time, salt also showed a significant effect on role of gynocardasJ, while hidnocarpic aciO 1Z both TVB content and the number of HrS producing cyclopentene 1 undecanoic/c,uHrror) and bacteria.However, pH,TVBcontentaswellasnumber Ind.Fislt Res.J. Vol 1S No.2 Descember-2A79: 65_73 of enteric and H"S proclucing bacteria increased during fish storage.Sensory evaluation resulted ihat from the texture.odor and flavor point of vrew treatments with 2% pangiurn (whether mixed with 2 or 3% salt) was reJected at day-6, while the one wiilr pangtum 67o iwith 2 or 3oh salt), and combination of 4% pangiunr with 3% salt was stilt accepted by the panelists at day-9 Neverthetess.treatments wlth htgh content of pangiurn were not preferred bv the panelists due io the flsh appearance whrcn were rather brownrsh in color.This undesrrable color may be Broduced by the reaction between tannin (tannic acid) with ferric conrpound from fish meat (rnyogiobine).The tannic acid contained in fish were betwee n 16.7_23.2ppm at initial and 7 .7-11.A ppnr at the encl of experiment; while for cyanide, the content in fish were between 20.6- 44.4 ppm at initiat and 2.5-19.2ppm at the end of experiment depending on the amount of pangiurn added (lieruwati et at.,2A07).
Though it has been proved both by experiments and by commercial praciices that pangii.rmcoirld prolonE the shetfiife of fish, yet, it stili very limited information available on what active substances contained in pangium that responsible in fish preservation, and how long they could delay the spoilage of fish.For this purpose, a study was conducted on the antibacterial acti,rity of water, water_ ethanol, and n-hexane extracts of fresh and fermented pangium against some species of bacteria responsible in fish spoilage.lt was expecied that baseo on resulted information on extract that has highest preservative activity.experiment could be followed by isolation, characterization, and purification of ihe active substances.in ihe iong run, the produciion of natural preservative for fresh fish could be realized.
Fermented seeds were made by boiling the seed for 6 hrs, followed by rvrapping in plastic bag and keeping at room temperature for about 40 days.Chloram phenrcoi (l ndofarma.)at concentration of 10 mg/rnL was used as a positive contrcl, while water and water-ethanot ('1:i ) and n hexane were used as negative control.llutrientAqar (NA) (Difco) was used as the gi'owth medium for the culiures, and Mueller Hinton Agar (MHA) (Difco) was used in antibacterial foci Preparation of Pangiunn Extracts Fresh and fermented pangium seeds were cleaned and washed, tlren the shells were cracked to get the kernel.The kernelwas homogenized and freeze_dried at -40"C followed by grinding them into powoer.
Extraction was cjone by nracerating 200 g of fresh or fermented pangium powder in .1,000mL of distilted water by shaking at 30 rpm for 3x24 hrs.After filtration using Whatman paper na.42, the filtrate was driecl usf ng vacuLtm evaporato r at72 mbar to obtain crude water extract.Afier filtration, the residue was collected and remacerated using 1.000 rnl of mixture of water_ ethanol ('1:1) wiih rhe same procedure as water extraction.Filtrate of the later extraction was dried by using vacuLlm evaporaior at lZSmbar to getcrude water-ethanol extract.The residue of second extraction was tlren remacerated using 1,000 mL n_ hexane by the same procedure.ln this last extracticn, evaporation of the filtrate vras done at 335 mbar to get crude n-hexane extract.All crude extracts were then freeze-dried, weighed, and kept in capped botile until used (Harborne, 1998;Andanvulan ef a/., 1g9g).

Freparation of Test Culture
All cultures were grown at slanted NA for 24 hrs at 37'C, an ose of each culture was then grown in 20 mL of nutrient broth, incubated in a shaking water bath at 160 rprn, f or 24 hrs 37"C.The optical density was measured using spectrophotometer at d 600 nm to get an absorbance of 0,5-0,6.To make sure that a total number of 106-'108cfu/mL had been achieved, counting of the totalbacteria using Mc Farland method (DG of Drug & Food Control, .1gg5)was also conducted.

Antibacterial Test
Each 800 g of freeze-dried crude extract was dissolved in its appropriate solvent (i.e.water, water_ ethanol, n-hexane), added with phosphate buffer at pH 7 to make a stock solution containino g0 mg extracVmL.Each stock solution was then OiLteO into test solution at concentration of 10, 20, 30, 40, 50,  60, 70, and 80 mg/ml (DG of Drug & Food Control,   1995).
Antibacterial test was conducted using agar plate diffusion method.Twenty pL of test bacteria were inoculated into test tubes containing 15 mL of MHA medium, homogenized, and poured into sterile plates.Sterile paper disks (6 mm in diameter) that had been dipped in 20 iL of pangium extract at concentration of 10, 20, 30, 40, 50, 60, 70, and B0 mg/ml were then put onto the surface of solid MHA plates which had been cultured by tested bacteria, and incubated at 37'C for 24 hrs.Six paper disks were put in each plate, two of them were positive control (chloramphenicol at concentration of 10 mg/mL) and negative control (appropriate solvent), respectively.The inhibition area were assessed by measuring the diameters of clear zone surrounding the paper disks (DG of Drug & Food Control, 1995).Experimentwas conducted using 3 replications.
Antibacterial activity of the extract was determined based on the clear zone area.Low activity is the one having clear zone of less than 5 mm, medium activity is between 5-10 mm, while strong activity is between 11-20 mm, and very strong is the one having clear zone between 21-30 mm (Morales ef a/., 2003 in lsmaini,2007).

Minimum lnhibition Concentration
Minimum inhibition concentration was calculated using the linear regression between Mo (concentration of pangium extract te;t solution) and x2 (square of inhibition area)which crossed the ordinat at MT. MIC was determined as anti-ln of YoxMt (Bloomfield, 1991   ln Nuraida et al.,2000).
Out of 200 g of raw material, extraction of fresh pangium seed using water yielded 4.93 g powder, while extraction using water-ethanol and n-hexane yielded 5.45 and 1.08 g powder respectively.On the other hand, the yield of water, water-ethanol, and nhexane extract of 200 g of fermented seed were 1 5.43, 20.53, and 1.13 g powder respectively.For both fresh and fermented seeds, extraction using water-ethanol gave the highest yield because the solveltt effectively drawn out polar and semipolar substances contained in the pangium seed such as the flavonoids, phenol, tannin, and perhaps small amount of terpenoids, saponins, alcaloids, and steroids.These substances are produced as a biodegradation of fat and protein contained in the seed (Harborne, 1998).Claeson ef a/.('1998);Andarwulan ef a/.(1999)stated thatwaterethanol is a good solvent to extract the polyphenols, phenols, glycocides, and flavonoids from plants.
According to Dicko ef a/. ( 2006), phenolic substances are easily dissolved in polar solvent, due to its aromatic ring having one or more hydroxyl group (s).
Water extraction of fresh and fermented seed yielded less of extract because as a polar solvent, water could only pulled out polar substances such as flavonoids and tannin, as well as anionic substances, such as thiocianates, nitrates, chlorides, and sulphates.Wahyono (2004) found that extraction of Scurrula atropurpurea with water could drawn out the flavonoids.On the other hand, extraction of fresh and fermented pangium seed with n-hexan produced the least yield because n-hexane is a non-polar solvent so it only could extract the non polar substances especially those containing oil and fat such as triterpenoids (kamphor, linalool) and steroids.lnhibition Rate of Each Extract Against the Growth of Gram Positive and Negative Bacteria a. Waterextractof fresh and fermented pangium seed Water extract of fresh pangium seed could inhibit the growth of gram positive bacteria (Staphylococcus aureus and Micrococcus luteus) at concentration of 40-80 mg/ml, with the diameter of inhibition zone of 5.00-8.50mm for S. aureus and 0.33-1.33mm for M. luteus (Appendix 1).This inhibition was possibly due to the antibacterial substances contained in the extract such as phenols, flavonoids, alkaloids, tannins, and anionic substances like thiocianates, nitrates, chlorides, and sulphates.The mechanism of bacterial inhibition by phenols is by bonding of hydroxyl group of phenol with bacterial protein membrane, leading to the disruption of membrane permeability, affecting the electron transport system, and the rupture of peptidoglycan layer of bacterial cell walls.The inhibition mechanism of anionic substances to bacterial growth is through the inhibition of extracellular enzyme during oxidation process of disulphide bonds (El Astal et al., 2005).However, water extract at concentration of 10-30 mg/mL could not inhibit the growth of S. aureus and M. /ufeus due to low toxicity of the extract so it couldn't be able to lnd.Fish Res.J. Vol.15 No.2 Descember-2)19: 65-73 rupture the peptidoglycan layer of bacterial cell membrane.
Water extract of fresh seed could inhibit the growth of gram negative bacteria, Alcaligenes eutrophusand Enterobacter aerogenes at concentration of 30-80 mg/ mL, with inhibition zone diameter of 3.33-2.00mm for A. eutrophus, and 0.16-1.67mm lor E. aerogenes, while for Serratia marcescens, Pseudomonas fluorescens, Flavobacterium gleum, and Salmoneila typhimurium the effective doses were 40-80 mg/mL, giving zone diameter of 0.83-2.67mm, 1.00-1.1 7 mm, 0.50-1.17mm, and 1.00-3.00mm for S. marcescens, P. fluorescens, F. gleum, and S. typhimurium respectively (Appendix 2).The mechanism of gram negative bacteria inhibition by phenols was possibly because the diffusion of phenol into the surface of the bacterialcell, especially at hydrophobic lipid bilayer, therefore the hydrolytic enzyme in lipopolysaccharide and the phospholipid layer were inhibited resulting in the loss of the protection of bacterial cytoplasmic membrane.
For the fermented seed.water extract at concentration of 10-80 mg/mL didn't show any antibacterialactivity (Appendix 1 and 2)against gram positive (5.aureusand M. luteus) and gram negative bacteria (A.eutrophus, E. aerogenes, S. marcescens, P. fluorescens, F. gleum, and S. typhimurium), This is presumably due to the decomposition of antibacterial substances or related enzymes during the boiling of the seed before fermentation process.
b. Water-ethanol extract of fresh and fermented pangium Water-ethanol (1 :1) extract of fresh pangium could inhibit the growth of gram positive S. aureus at concentration of 30-80 mg/mL with zone diameter of 7.00-4.67mm (Figure 1) and M. luteus al concentration of 40-80 mg/mL with zone diameter of 1.00-3.00mm (Appendix 1).The inhibition mechanisms were possibly similar to the inhibition of water extract because the polarity of both solvents were nearly similar, so that the same substances were also extracted.Dicko et al. (2006) reported that proantocyianidine (condensed tannin) is toxic to microorgAnism by inhibition of hydrolytic enzymes, and inactivaticjn of protein transport in envelope cell.According to Gilbert (1984) biosynthetic disturbance of peptidoglycan is also due to the diffusion of toxic phenolic into the cell membrane, causing increase in permeability, thus resulted in the increase of intracellular pressure to peptidoglycan.The diameter of clear zones of water-ethanol extract treatments on S. aureus was wider than that of M. luteus i.e. 5.00- 8.50 mm, 7.00-14.67mm, and 0.33-1.33mm, 1.00-3.00mm presumably because S. aureus is more sensitive to phenols, alkaloids and flavonoids than M. luteus.ElAstal et al. (2005) revealed that S. aureus was very sensitive to ethanol extract ol Thymus vulgaris and Salvia officinalis at concentration between 2.5-40.0mg/mL.
Water-ethanol extract of fresh pangium could inhibit the growth of gram negative E. aerogenes and A.
Water-ethanol extract of fermented pangium seed could inhibit the growth of gram positive S. aureus and gram negative S. rnarcescens at concentration of 70-80 mg/mL with zone diameter of 5.33-6.00mm, and 2.67-3.50mm respectively, but not at concentration of 10-60 mglml (Appendix 1 and 2)' The nrechanism of inhibition was presumably because the fermented seed extract contains antioxidantswhich could perform as an antibacteria Andanruulan et al'   (1999); Anwar (1992) revealed that fermented pangium contained many antioxidants such as tocopherols, tocotrienols, and antioxidants originated from primary amine groups and aromatic (benzene) groups.c. n-hexane extract of trestr and fermented pangrum It was noted from the experiment that n-hexane extract of fresh and fermented pangium at all concentration could not inhibit all bacteria tested.This probably because the extracts contain fats and fatty acid substances, that might hindered the diffusion of antibacterial agents into the peptidoglycan of the cell wall of gram positive bacteria or hydrophobic lipid bilayer of the gram negative bacteria.Moshi & Mbwambo (2005) also found that non polar extract of Terminalia sericea could not suppress the growth of S. aureus, S. typhii, and P aeruginosa.Similar to this, Priyono (200a); Indriyanto (2004) reported that n-hexan extracts of Parkia timoriana seeds, Ruta angttstifolia leaves, and Parameria barbata stem shell could not also inhibit S. aureus, M. luteus, and S' typhosa growth.we"e medium against S. aureus, low to medium against E. aerogenes, and low against M. Iuteus, A' eutrophus, P. fluorescens, S. marcescens, F. glettm, Table 1.
Minimum inhibitory concentration of water and water-ethanol extract of fresh pangium seed against various species of bacteria and S. typhimurium.Meanwhile, the antibacterial activity of water-ethanol extracts were medium to high activity against S. aureus, A. eutrophus, and E. aerogenes, and low against M. luteus, P' fluorescens, S. marcescens, F. gleum, and S. typhimurium.El Astal et a/" (2005) showed that water extract of Thymusvulgaris had high antibacterial activity against S. aureus, and Psetrdomonas aeruginosa while Digrak ef al. ( 1 999) showed thal Ph lo mis bou rgei extract had high activity against Eacitlus subf/is, S. aureus, E coti, P. aeruginosa, and Profeus vulgaris.
e. Minimum inhibitory concentration of water and water-ethanol extract of fresh pangium The nninimurn inhibitory concentration of fresh pangium water extract were 3.49 mg/mL against P fluorescens and between 8-9 mg/ml against S, aureLls, M. tutetts, and E. aerogenes' The minimum inhibitory concentration against A. eutrophtrs, F. gleum, S. rnarcescens, and S. typhimuriuln were somewhat higher, reaching more than 10 mg/mL (Table 1).Based on the minimum inhibitory concentration values, water extract of fresh pangium at concentration of about 4 mgiml was effective enough to inhibit P fluorescens, but for other species, higher extract concentration is needed.EiAstalef al. (2005)reported that water extract ol Thymus vulgaris could inhibit S. aureus at minimurn inhibitory concentration value of 2.5 mg/ml, E. coti of 20 mglmL, S. typhiof 10 mg/ mL, and P. aerugirtosa at minimum inhibitory concentration value of 20 mg/mL.
The minrmum inhibitory concentration of fresh pangium water-ethanol extract were 6-7 mg/ml against S. aureus, A. eutrophus, and E. aerogenes, and 8-10 mg/mL against E gleum and S. marcescens, and more than 10 mg/mL against P. fluorescens, M. luteus, and S. typhimurium (Table 1).Salvat ef a/. (2001)stated that water-ethanol extract of Vassobla breviflora was effectively suppress S. aureus at minimum inhibitory concentration of 0.25 mg/mL, while for F. aeruginosa and S. typhimuritrnr, the inhibition was at minimum inhibitory concentration of 1 mg/mL respectivelY.2. Water and water-ethanol extracts of fresh seed showed an effective antibacterial activity against gram positive and negative bacteria at concentration of 40-80 mg/mL, but n-hexane extract didn't show any antibacterial activity at all concentration ( 1 0-80 mg/mL).
3. Waier, water-ethanol, as well as n-hexane extract of fermented seed didn't show any antibacterial activity against gram positive or negative at all concentration (1 0-80 mg/mL).
4. Water extract of fresh seed showed high antibacterial activity against p fluorescens with the rninimum inhibitory concentration of 3.49 mg/ mL, while the water-ethanol extract showed medium to high antibacterial activity against S.
aureus and E. aerogenes with the minimum inhibitory concentration of 7.17 and 6.04 mg/mL respectively.

RECOMMENDATIONS
Though traditionally, pangir:m kernel has been widely employed as spices, yet, to be accepted in preservation of fish and fish product in areas where pangrum has not been popular is not easy.Further studies are important to provide the maximum benefit of pangium as preservative, especially by characterizing the active substances of pangium that responsible to prolong the shelf life of wet fish as well as developing method of industrialization, whether in crude or pure basis.