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Vol. 28, 2010, No. 5: 440449 Czech J. Food Sci.

Occurrence of Biogenic Amines and Amines Degrading

Bacteria in Fish Sauce

M Z ZAMAN1, F A BAKAR1, J SELAMAT1

and J BAKAR2

1Department of Food Science and 2Department of Food Technology, Faculty of Food Science

and Technology, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Abstract

Z M.Z., B F.A., S J., BJ. . (2010): Occurrence of biogenic amines and aminesdegrading bacteria in fish sauce. Czech J. Food Sci., 28: 440449.

he contents of biogenic amines histamine, putrescine, and cadaverine in fish sauce were determined and the bacte-

ria isolated from the samples were evaluated for their amines degradation activity. Five fish sauce samples contained

62.5393.3 ppm of histamine, 5.6242.8 ppm of putrescine, and 187.1704 .7 ppm of cadaverine. hirty three bacterial

isolates produced all three amines, seven isolates produced one or two amines, and one isolate did not produce any

amine in differential agar media. Since the strains that produced amines were not supposed to degrade them, only

eight isolates were further identified and evaluated for their amines degrading capability. Bacillus amyloliquefaciens

FS-05 and Staphylococcus carnosusFS-19 degraded histamine up to 59.9% and 29.1% from its initial concentration,respectively. Staphylococcus intermediusFS-20 and Bacillus subtilis FS-12 degraded putrescine and cadaverine up to

30.4% and 28.9%, respectively. Most isolates tolerated the salt concentration of up to 15% and temperature of up to 45C.

he current study provided new information on biogenic amines degrading bacteria, isolated from high-salt-content

food products. he amines degradation activity of the bacteria is considered as strain rather than species specific.

Keywords: fish sauce; biogenic amines; amines degradation; Bacillus amyloliquefaciens; Staphylococcus carnosus

Biogenic amines are low molecular weight basicnitrogenous compounds occurring in many foods,

mainly due to the amino acids decarboxylationactivities of certain microbes (H et al. 1994;K & K 1998). heir presence is undesir-able because it can result in toxicological effectsto consumers such as hypertension, headache,diarrhea, rash, and localised inflammation wheningested in excessive amounts (H et al. 1994;S 1996). Biogenic amines are widely pres-ent in food products, especially fermented foodssuch as fish sauce, cheese, beer, and sauerkraut(H et al. 1994; S 1996). Fish sauce

is a popular fermented fish product used as acondiment in Southeast Asia. It is considered an

important source of dietary proteins and aminoacids. It contains about 20 g/l of nitrogen 80% of

which is in the form of amino acids (S etal .1996). Despite its nutritional value, severalauthors reported the presence in fish sauce ofhigh levels of biogenic amines predominated byhistamine, putrescine, cadaverine, and tyramine(M et al.2002; S et al.2002; C et al.2006; et al.2006). yramine was present atlower levels and was occasionally found in consider-able amounts in some samples (S et al. 2002;C et al. 2006; S et al. 2009). Other aminessuch as phenylethylamine, spermine, spermidine,and agmatine are considered as trace amines infish sauce (M et al.2002; S et al.2002;

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Czech J. Food Sci. Vol. 28, 2010, No. 5: 440449

et al.2006). Histamine is considered as themost active amine and is related to almost allfood amines poisoning incidences. Furthermore,the occurrence of putrescine and cadaverine canpotentiate and enhance the toxicity of histamine(S 1996). Unfortunately, it is very dif ficultto remove amines from food products once theyare formed, even with the heat treatment such asautoclaving (L et al.1992).

Biogenic amines are physiologically degradedthrough oxidative deamination catalysed by aminesoxidase by the following reaction: R-CH

2-NH-R' +

O2

+ H2OR-CHO + H2N-R' + H2O2 (M

et al.1979; I et al.1993; Y et al.1993). Monoamine and diamine oxidases are ubiq-uitous and play an important role in the metabo-

lism of amines in human, plant, and animal cells.Furthermore, these enzymes have also been foundin some bacterial strains. Monoamine oxidasewas found in some strains ofEnterobacter iaceaefamily such asKlebsiella,Enterobacter,Eschericia,Salmonella, Serratia, and Proteus (M etal .1979). I et al. (1993) reported that

Micrococcus rubenspossesses putrescine oxidase.Some strains of the food fermenting microorgan-isms such asMicrococcus sp. andBrevibacteriumlinensexhibit the ability to degrade histamine

and tyramine, while the strains of Lactobacil lusplantarum,Lactobacillus sake,Lactobacillus pen-tosus, and Pediococcus acidilact ici only degradehistamine (L et al.1998). M- et al.(2000) also found that some strainsof Staphylococcus xylosusisolated from artisanalfermented sausages had a remarkable ability todegrade histamine and tyramine. Since the lastfew years, the potential role of microorganismspossessing amines oxidase activity has become ofa particular interest in the prevention or reduc-tion of biogenic amines accumulation in food

products, especially fermented food. Land H (1998) reported that MicrococcusvariansLH 1540 decreased a huge amount oftyramine during ripening of fermented sausages.Reduction of histamine and tyramine accumula-tion in salted and fermented anchovy was reportedwhen Staphylococcus xylosushad been applied asthe protective culture (M & H 2009). heformation of tyramine, putrescine, and cadaverinein sauerkraut was significantly suppressed by theinoculation withLactobacillus plantarum(K

et al. 2000). However, no literature data are knownon the degradation of biogenic amines by bacteria

from fish sauce. Hence, the purpose of this researchwas to isolate bacteria from fish sauce and exam-ine their amino acid decarboxylation activity. heselected isolates were examined for their abilityto degrade amines, namely histamine, putrescine,and cadaverine. Biogenic amines content in fishsauce was also determined. he finding of thisresearch was expected to provide some informa-tion on amines degrading bacteria originated fromhigh-salt-content products such as fish sauce.

MATERIAL AND METHODS

Fish sauce sample s. Five fish sauce sampleswere obtained from different traditional factories

in umpat, Kelantan, Malaysia. he samples weretransported for analysis in sealed glass bottles atambient temperature to the Laboratory of FoodSafety and Quality, Faculty of Food Science andechnology, Universiti Putra Malaysia. All sampleswere made from anchovy and were fermented withsea salt for 6 months (2 samples) and 12 months(3 samples) at ambient temperature. he starterculture or proteolytic enzymes were not appliedduring the samples fermentation. he samples wereused for microbiological analysis immediately after

their arrival in the laboratory and the rest werestored at 4C prior to chemical analysis.Determination of pH value and salt content.

he pH value of fish sauce was determined by directmeasurement with an electronic pH meter (Mettleroledo 8603, Schwerzenbach, Switzerland). hesalt content of each sample was determined witha salt meter (Atago ES-421, okyo, Japan) afterten fold dilution.

Analysis, isolation, and identification of bacte-

ria. he samples (25 ml) were mixed with 225 ml ofpeptone water containing 0.85% of sodium chloride

and then homogenised in stomacher bag (Bagmixer400, Model L, Interscience, Paris, France) for 2 min-utes. Further, ten fold dilutions were made and then100 l of each dilution was spread onto agar plates.Aerobic plate count agar and skim milk agar, bothsupplemented with 3% of sodium chloride, wereused to determine total aerobic and proteolyticbacteria count in fish sauce, respectively. he bacte-rial colonies were counted after the plates incuba-tion at 37C for 48 hours. he bacterial numberswere then expressed as log colony forming unit

(CFU)/ml. o isolate bacteria from fish sauce, eachsample (25 ml) was separately added to 225 ml of

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trypticase soy broth (Merck, Darmstadt, Germany)supplemented with 3% of sodium chloride in 500 mlflask. he flasks were then shaken (100 rpm) inan incubator (Infors, Bottmingen, Switzerland) at37C for 24 hours. he cultures were spread ontrypticase soy agar plates supplemented with 3% ofsodium chloride and were then incubated at 37Cfor 48 hours. he bacterial colonies that developedon the plates were picked based on their coloniesappearance and subsequently streaked on freshtrypticase soy agar plates until pure isolates wereobtained. he isolates were then screened for theirdecarboxylase activity toward histidine, ornithine,and lysine (Sigma-Aldrich, St. Louis, USA) in dif-ferential medium proposed by J and N- (1989). he colour change of the medium

surrounding the bacterial colonies from yellow topurple indicated that the particular bacteria coulddecarboxylase amino acids. Based on the results,the isolates which did not possess the three aminoacid decarboxylase activities were selected for thebiogenic amines degradation studies. hese isolateswere identified using Gen III Microlog IdentificationSystem (Hayward, USA). he bacterial culture andidentification procedure were performed followingfrom the manufacturers instruction.

Bi og en ic am in es de grada tio n by ba ct er ial

isolates. Biogenic amines degradation test wascarried out according to the method developedby L et al.(1998). An overnight cultureof each isolate was harvested, washed with 0.05Mphosphate buffer (pH 7), and the cell pellet wasresuspended in 0.05M phosphate buffer supple-mented with 100 ppm of histamine, putrescine, andcadaverine (Merck, Darmstadt, Germany). he cellsuspension (20 ml) adjusted to a concentration of107cells/ml was incubated in a 100 ml f lask for 24 hat 37C, under shaking at 150 rpm. he sampleswere then taken and added to an equal amount

of 1M HCl (Merck, Darmstadt, Germany). hemixture was boiled for 10 minutes and centrifuged(Sigma 3-18K, Sartorius, Gettingen, Germany) at9000 g for 10 minutes. he supernatant was frozenat 20C prior to biogenic amines analysis.

Screening for the tolerance to various ranges

of the environmental factors. A trypticase soybroth medium was used in this series of studies.An overnight culture of each isolate was usedas an inoculum from which cells were obtainedby centrifugation and were then resuspended in

10 ml of 0.85% saline. 100 l of each cell suspen-sion was inoculated into individual bottles. he

temperatures tested were 30C, 37C, 45C, 50C,and 55C, the concentrations of NaCl were 0%,5%, 7%, 10%, and 15%, and the pH values testedwere 5, 6, 7, 8, and 9. o achieve the desired pH

value, the medium was adjusted with 1M HCl or1M NaOH. he bottles were then incubated for48 h at a specific temperature or at 37C for thetest of pH and salt concentration. At the end ofthe incubation, the turbidity in each bottle wasconsidered as the growth indication.

Determination of biogenic amines . Biogenicamines in the samples were determined using HPLCaccording to the method proposed by H etal. (1997) and modified by O et al.(2002).Briefly, the fish sauce samples were transferredto 50 ml centrifuge tubes and homogenised with

20 ml of 6% trichloroacetic acid (Merck, Darm-stadt, Germany) for 3 minutes. he homogenateswere centrifuged at 10 000 g for 10 min at 4C)and filtered through Whatman paper No. 1. hefiltrates were then placed in a volumetric flask and6% trichloroacetic acid was added to a final volumeof 50 ml. A series of mixed standard amine solutionswere also prepared to obtain the standard curve foreach amine. o 1 ml of standard amine solution andeach sample, 1 ml of 2M sodium hydroxide (Merck,Darmstadt, Germany) was added, followed by 10 l

of benzoyl chloride (Merck, Darmstadt, Germany).he solution was mixed using a vortex mixer andthen allowed to stand at 30C for 40 minutes. hebenzoylation was then stopped by adding 2 ml ofsaturated NaCl solution, and the mixture was ex-tracted with 3 ml of diethyl ether (Merck, Darmstadt,Germany). After centrifugation, the upper layer wastransferred into a tube and evaporated to drynessin a stream of nitrogen. he residue was then dis-solved in 1 ml of acetonitrile (Merck, Darmstadt,Germany). HPLC determination was performedwith a Waters 600 controller and a pump, a Waters

in-line degasser, a Waters 2996 photodiode arraydetector, and Empower 2 Software. A SunfireMC

18, 5 m, 150 4.0 mm column (Waters, Milford,

USA) was used with water and acetonitrile as themobile phase set for linear gradient at the flow rateof 1 ml/minute. he sample volume injected was20 l and it was monitored at the wavelength of254 nm. he detection limits for histamine, putres-cine, and cadaverine were 0.16 ppm, 0.09 ppm, and0.07 ppm, respectively. he quantification limitsfor histamine, putrescine, and cadaverine were

0.53 ppm, 0.31 ppm, and 0.24 ppm, respectively.

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Statistical analysis. he significance of dif-ference was determined by using the analysis of

vari ance (ANOVA). he comparison of meanswas carried out with Duncans multiple rangetests (DMR). Pearsons correlation test was ap-plied to determine the relationship between the

variables in the samples . All statistical analyseswere performed using the Statistical Package forSocial Sciences, SPSS Version 16.0 for Windows(SPSS Inc., Chicago, USA). he value of P < 0.05was used to indicate significant deviations.

RESULT AND DISCUSSION

he values of the pH, salt content, aerobic plate

count (APC), and proteolytic bacteria count infish sauces are presented in able 1. he level ofpH and salt content in all samples ranged from4.8% to 5.7%, and 15.6% to 25.7%, respectively.he results were about in the same range withseveral fish sauces from different countries oforigin as reported by P et al.(2001). he dif-ferences in the salt content between the sampleswere mainly due to the different ratios of fish andsalt applied by manufacturers at the beginning offermentation. It was proved that salt content was

not much changed in the course of fermentation(D et al.2006). L etal.(2001) stated that Malaysian fish sauce pro-ducers use the fish to salt ratio of 35:1. hisratio complies with he Malaysian Food Act 1983and Regulation 1985 which require fish sauce tocontain not less than 15% of salt (A2004). he high salt concentration can preventthe growth of spoilage microorganisms such as

Es cherichiasp., Serratiasp., Ps eudomona ssp.,

and Clostridium sp. in fish sauce (Let al.2001). In general, significant correlationswere found between the pH value, salt content,aerobic plate count, proteolytic bacteria count,and biogenic amines content (able 2). he resultshowed that the levels of biogenic amines in fishsauces depended on other related variables such asaerobic and proteolytic bacteria counts. A correla-tion between aerobic bacteria count and amines,namely putrescine, cadaverine, and tyramine,was also found in other products (V et al.2005). However, no significant correlation wasobserved between putrescine and salt (r= 0.459,

P= 0.085) and histamine and pH (r= 0.501, P=0.057), in agreement with the correlation previ-ously found in fermented fish products in aiwan

( et al.2006).Figure 1 shows the biogenic amines contents in

fish sauce samples. Histamine content ranged from62.5 ppm to 393.3 ppm. Putrescine and cadaver-ine contents ranged from 5.6 ppm to 242.8 ppmand 187.1 ppm to 704.6 ppm, respectively. It wasreported that the highest histamine, putrescine,and cadaverine contents in fish sauce were 1220,1257, and 1429 ppm, respectively (Z et al.2009). Histamine content in all samples exceeded50 ppm, the defect level imposed by the US FDA.

However, none of the samples exceeded the levelof 500 ppm, that is the amount which could behazardous for the consumer health. Fish sauce Bhad a higher content of histamine as comparedto all other samples. his could be attributed tothe higher contents of aerobic and proteolyticbacteria in the particular sample. Biogenic aminesaccumulation in fish products results mainly fromdecarboxylation activity of bacteria toward freeamino acids (B et al.1990). Proteolytic bacteria

able 1. Te value of pH, salt content, aerobic plate count and proteolytic bacteria count of fish sauce

Fish sauce pH Salt content (%)Aerobic plate count Proteolytic bacteria

(log CFU/ml)

A 5.4 0.1c 25.7 0.3c 5.02 0.1a 3.63 0.1a

B 5.7 0.1d 25.3 0.5c 5.53 0.5b 3.97 0.1b

C 5.1 0.1b 18.8 0.3b 4.97 0.1a 3.62 0.2a

D 5.3 0.1c 19.1 0.1b 5.18 0.1ab 3.76 0.1a

E 4.8 0.2a 15.6 0.8a 4.92 0.2a 3.65 0.2a

Te numbers represent mean SD of three replications. Values followed with same letters within a column are not signifi-

cantly different in Duncans multiple range test (P < 0.05)

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play an important role in releasing amino acidsfrom the protein ti ssues offering the substratefor decarboxylation activity by amines producingbacteria. Pearsons correlation analysis of the data

revealed a good positive relationship betweenthe aerobic and proteolytic bacteria counts andbiogenic amines contents. Putrescine content inall samples was considered to be in a lower rangeexcept for sample B (242.8 ppm), particularly whencompared to the previous report on Korean fishsauce (C et al.2006). his could be due to the

low presence of putrescine producing bacteriaduring the fermentation of the samples. Unlike ofputrescine, most of samples have a high contentof cadaverine, the level reaching 704.6 ppm in

sample B. Cadaverine content normally increasesduring fish sauce fermentation because more lysine(precursor of cadaverine) is released from the pro-tein complex of fish. P et al. (2001) reportedthat lysine as well as histidine, aspartate, glutamateand alanine, was dominant in most of fish saucesamples. he presence of bacteria capable to de-

able 2. Correlation between biogenic amines content, salt, pH value, aerobic plate count (APC) and proteolyticbacteria

Putrescine Cadaverine Histamine Salt APCProteolytic

bacteria pH

Putrescine 1.000

Cadaverine 0.981** (0.000) 1.000

Histamine 0.923** (0.000) 0.923** (0.000) 1.000

Salt 0.459 (0.085) 0.613* (0.015) 0.571* (0.026) 1.000

APC 0.715** (0.003) 0.764** (0.001) 0.720** (0.002) 0.640* (0.010) 1.000Proteolyticbacteria

0.774** (0.001) 0.833** (0.000) 0.742** (0.002) 0.643** (0.010) 0.610* (0.016) 1.000

pH 0.586* (0.022) 0.689** (0.004) 0.501 (0.057) 0.755** (0.001) 0.555* (0.032) 0.629* (0.012) 1.000

Pearson correlationP-value: *significant at the 0.05 level; **significant at the 0.01 level

a

d

b bc

a

c

a

b

ab

a

c

a

bab

0

100

200

300

400

500

600

700

800

A B C D E

Fishsauces

Concentration(ppm)

Putrescine

Cadaverine

Histamine

Figure 1. Biogenic amines content in fish sauce samples. Bars represent standard deviation of three replicates. Te samecolor histogram marked with same letters are not significantly different in Duncans multiple range test (P< 0.05)

c

b

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carboxylase lysine will result in the accumulationof cadaverine in the product. Putrescine and cadav-erine are potentiators of histamine toxicity as theyinhibit the histamine-metabolising enzyme in thesmall intestine (S 1996; L & O2000). Hence, the high content of both amines inthe presence of even a small amount of histamine

could possibly alter histamine toxicity.Forty one bacterial isolates were obtained fromfish sauce samples. All were tested and found to begram positive, 33 cocci and 8 rod shaped bacteria.All isolates were qualitatively tested for their amino

acid decarboxylase activity toward histidine, or-nithine, and lysine. hirty three isolates exhibitedthe ability to produce all tested amines in qualita-tive medium. wo isolates produced two amines,five isolates produced one amine, and one isolatedid not produce any amine (able 3). he abilityof bacteria to produce more than one amine hadbeen revealed by many studies. D et al.(2000) suggested that attention must be given tobiogenic amines production when selecting bacteriafor starter culture. Moreover, L et al.(1998) suggested that strains possessing amino aciddecarboxylase activity might not degrade biogenicamines. It is quite difficult to find bacteria withoutany amino acid decarboxylase activity involved infood fermentation, particularly in the products

with a high protein content such as fish sauce.herefore, the isolates which did not produce allthe three amines were further screened for theirability to degrade amines and tolerance to severalenvironmental conditions.

able 3 shows that all isolates had the abilityto degrade the amines tested, although with dif-ferent efficiencies. Histamine is the most activeamine and abundant in fish sauce and other fishproducts. Hence, the isolates that exhibited a highhistamine degradation activity were of particular

interest.Bacillus amyloliquefaciensFS-05 exhib-ited a significantly higher potential to degradehistamine than other isolates (able 4). hese rodshaped bacteria degrade histamine to about 59.9%of its initial content within 24 h, while the other

able 3. Amino acids degradation by bacteria isolated

from fish sauce

Code and identity of isolates

Amino acidsdegradation

His Orn Lys

FS-05 Bacillus amyloliquefaciens + +

FS-12 Bacillus subtilis + +

FS-13 Bacillus humi +

FS-14 Bacillus amyloliquefaciens +

FS-19 Staphylococcus carnosus

FS-20 Staphylococcus intermedius +

FS-22 Staphylococcus condiment +

FS-25 Staphylococcus carnosus +

His histidine, histamine precursor; Orn ornithine, pu-

trescine precursor; Lys lysine, cadaverine precursor

able 4. Biogenic amines degradation by bacteria isolated from fish sauce after incubation in 0.05M phosphate buffersupplemented with 100 ppm of amines for 24 h at 37C

Bacterialisolates

Histamine Putrescine Cadaverine

ppm degradation (%) ppm degradation (%) ppm degradation (%)

FS-05 40.1 6.8 59.9c 92.5 2.6 7.5ab 73.6 2.7 26.4b

FS-12 65.8 3.3 34.2b 70.3 5.2 29.7 c 71.1 9.6 28.9b

FS-13 67.2 5.7 32.8b 85.2 5.5 14.8b 76.1 8.2 23.9b

FS-14 73.4 9.3 26.6b 69.7 6.3 30.0c 77.7 13.9 22.3b

FS-19 70.9 4.3 29.1b 92.1 3.8 7.8ab 91.6 3.6 8.4 a

FS-20 89.1 1.2 10.8a 69.6 5.1 30.4c 71.9 3.8 28.0b

FS-22 72.6 9.4 27.4b 95.6 1.3 4.4a 93.2 2.4 6.8a

FS-25 95.1 3.9 4.9a 86.1 1.2 13.9b 92.6 2.6 7.4a

Te numbers represent mean SD of three replications. Values followed with same letters within a column are not signifi-cantly different in Duncans multiple range test (P < 0.05)

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cultures have a lower activity in degrading thisamine. he Bacill i isolated from fish sauce suchas Bacillu s amyloliquefaciens , Bacillu s subtilis,

Bacillus megaterium, andBacillus licheniformisarewell known for their protein degradation activity(F et al.2008). Little is known about the aminedegradation activity of the bacteria belonging tothis genus. In agreement with our results, otherstudies foundBacillus sp. LMG 21002 andBacil-lus megateriumKL-197 isolated from fish sauce

to contain histamine degrading enzymes (K &K 2006). M and H (2009) also describedhistamine degrading enzymes in some strains of

Bacillus coagulans. Histamine degradation activitywas also found in isolates identified as Staphy-lococcus. Staphylococcus condimentFS-22 andStaphylococcus carnosusFS-19 reduced histaminecontent to about 27.4% and 29.1%, respectively.he species of Staphylococcus , common bacteriaisolated from fish sauce, play an important rolein the protein degradation and flavour or aroma

development ( 1991; U & L1996; L et al. 2001; F et al.

2004; Y et al. 2007). Some speciesare known as amines producing bacteria, whileothers are known as amine degrading bacteria.M et al.(2000) observed in their studythat strains of Staphylococcus xylosushad a highability to degrade histamine in a buffer system,degrading up to 100% of histamine. In contrastwith our findings, L et al.(1998) re-ported that strains of Staphylococcus carnosusdidnot possess histamine degradation activity. his

result suggested that amines degradation activitywas not species but strains specific. In additionto histamine degradation activity, we also foundthat the isolates tested in our study had the abilityto degrade putrescine and cadaverine. Bacillusamyloliquefaciens FS-14 andBacillus subtilisFS-12reduced putrescine and cadaverine contents to30.0% and 28.9%, respectively. StaphylococcusintermediusFS-20 degraded 30.4% and 28.0% ofputrescine and cadaverine, respectively. his maybe of further interest since only little informationexists on putrescine and cadaverine degradingbacteria. he activity to degrade putrescine was

able 5. olerance of the bacterial isolates to ranges of temperature, NaCl concentration, and pH

Environmental factorsIsolates

FS-05 FS-12 FS-13 FS-14 FS-19 FS-20 FS-22 FS-25

emperature (C), SB + 5% NaCl, incubated for 48 h30 + + + + + + + +

37 + + + + + + + +

45 + + + + + +

50 + +

55

NaCl concentration (%), SB, incubated at 37C for 48 h

0 + + + + + + + +

5 + + + + + + + +

7 + + + + + + + +

10 + + + + + + + +

15 + + - + + + + -

pH, SB + 5% NaCl, incubated at 37 C for 48 h

5 + + - - + + + +

6 + + + + + + + +

7 + + + + + + + +

8 + + + + + + + +

9 + + - - + + + +

+ indicate growth; indicate no growth

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also found in other bacteria such asMicrococcusrubens (I et al. 1993).

he isolates could grow in the temperature rangebetween 30C and 50C, but none could with-stand 55C (able 5). Six isolates could grow at45C, although only Bacillus subtilisFS-12 and

Bacillus amyloliquefaciensFS-14 could grow attemperatures of up to 50C. However, most iso-lates could grow at the temperatures preferablefor fish sauce fermentation, generally between35C and 45C (L et al.2001). Mostisolates also tolerated pH range suitable for fishsauce (able 5). P et al.(2001) found in theirresearch that the average pH value of fish saucefrom various countries ranged from 4.9 to 6.1.Regarding the salt tolerance, most isolates could

grow in media with salt concentration of up to 15%except forBacillus humi FS-13 and Staphylococ-cus carnosusFS-25 (able 5). Bacteria will losetheir turgor pressure when cultivated in a highsalt concentration medium which leads to the cellphysiology and metabolism disturbance (L etal. 1998). Some bacteria overcome this effect byregulating the osmotic pressure between the insideand outside of the cell (K 1987). his traitwas found in halotolerant and halophilic bacteriawhich could grow at the salt concentration of up

to 5% and more than 12% of salt, respectively(F & W 1988). Hence, the find-ings of this research confirmed that most bacteriaisolated from fish sauce which exhibited aminesdegradation activity should belong to halotolerantor halophilic groups. However, amines degradationactivity of the bacteria in a buffer system wouldprobably be not the same in complex substances.M and H (2009) reported that Staphylo-coccus xylosusNo. 0538 showed a lower activityin complex substances as compared to the buffersystem. hey found that the bacteria reduced

16% of overall biogenic amines when applied asstarter culture in salted and fermented anchovy.herefore, the isolates should be further examinedin food systems such as those applying fish saucefermentation.

CONCLUSION

Histamine content in fish sauce samples exceededthe defect level of 50 ppm designed by US FDA.

he product was still considered as probably safesince the consumption of fish sauce mainly as

condiment might not lead to an excessive intakeof this amine. However, the presence of high levelsof amines in fish sauce showed that the productlacked necessary hygiene during fermentation. hefindings of this research indicated that, within thebacterial flora of fish sauce, most of bacteria pos-sessed the potential to produce biogenic amines,particularly histamine, putrescine, and cadaverinewhile others, however, were able to degrade thosethree amines. he strains of genera Bacillus andStaphylococcus found in this research were ableto degrade one or more of the amines. It may bealso concluded that amines degradation activitywas rather strain than species specific. he aminesoxidase activity as well as safety of these bacteriacan be considered as the criteria for the selection

of the bacteria used to reduce amine accumulationin fish sauce and other related product.

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Received for publication November 24, 2009

Accepted after corrections January 18, 2010

Corresponding author:

Assoc. Prof. Dr. F A B, Universiti Putra Malaysia, Faculty of Food Science and echnology,

Department of Food Science, 43400 Serdang, Selangor D.E., Malaysia

tel.: + 603 894 683 75, e-mail: [email protected]

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