The influences of fish infusion broth on the biogenic amines formation by lactic acid bacteria.

The influences of fish infusion decarboxylase broth (IDB) on biogenic amines (BA) formation by lactic acid bacteria (LAB) were investigated. BA productions by single LAB strains were tested in five different fish (anchovy, mackerel, white shark, sardine and gilthead seabream) IDB. The result of the study showed that significant differences in ammonia (AMN) and BA production were observed among the LAB strains in fish IDB (p < 0.05). The highest AMN and TMA production by LAB strains were observed for white shark IDB. The all tested bacteria had decarboxylation activity in fish IDB. The uppermost accumulated amines by LAB strains were tyramine (TYM), dopamine, serotonin and spermidine. The maximum histamine production was observed in sardine (101.69 mg/L) and mackerel (100.84 mg/L) IDB by Leuconostoc mesenteroides subsp. cremoris and Pediococcus acidophilus, respectively. Lactobacillus delbrueckii subsp. lactis and Pediococcus acidophilus had a high TYM producing capability (2943 mg/L and 1157 mg/L) in sardine IDB.

Introduction

Biogenic amines (BA) production in seafood are one of the considerable public concern since histamine and possibly other biogenic amines such as cadaverine and putrescine are responsible for histamine fish poisoning (Taylor 1986; Jorgensen ) and serve as chemical indicators of fish spoilage (Alberto ). The toxicity of histamine appears to be enhanced by cadaverine (CAD) and putrescine (PUT) since they inhibit the histamine-detoxifying enzymes, which are diamine oxidase and histamine N-methyltransferase (Stratton ; Yongsawatdigul ). Biogenic amines such as PUT, CAD, spermidine (SPD), spermine (SPN), histamine (HIM), tyramine (TYM) and tryptamine (TRP) are widely distributed in proteinaceous foods (Krizek ). Most studies have been focused on BA formation in fish and meat products (Ruiz-Cappillas and Jimenez-Colmenero, 2004) due to their proteinaceous nature and propensity to form BA from free amino acids (Magwamba 2010).

Accumulation of BA in foods requires (I) the availability of precursors (i.e. amino acids), (II) the presence of microorganisms with amino acid decarboxylases enzyme, which either derived from environmental contamination or from an added starter culture and (III) favourable conditions that allow bacterial growth, decarboxylase synthesis and decarboxylase activity (Bodmer ; Karovicova and Kohajdova, 2005; Stadnik and Dolatowski, 2010). Although amino acid decarboxylases are not widely distributed among bacteria, species of many genera such as Bacillus, Citrobacter, Clostridium, Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella, Photobacterium and the lactic acid bacteria (Lactobacillus, Pediococcus, and Streptococcus) are capable of decarboxylating one or more amino acids (Halasz ; Silla-Santos 1996; Karovicova and Kohajdova, 2005; Özogul and Özogul, 2005).

BA production by bio-preservative features of lactic acid bacteria (LAB) have been reported (Connil ). Some strains of LAB synthesize histamine because of their ability to possess the histidine decarboxylase gene (Landete ; Lucas ). The presence of BA in fermented foods is due to the decarboxylase activity of the LAB used as starter culture, and the action of some spoilage bacteria (Marcobal ). High BA contents have been reported in some fish products such as fish sauce, fish paste, fish salads, cold-smoked fish (Leuschner and Hammes, 1999; Petaja ; Yongsawatdigul ; Jorgensen ; Gonzalez-Rodriguez ; Thapa ; Udomsil ; Zaman ; Zhong-Yi ). The production of BA by LAB to be selected as starter cultures is not a desirable feature for food industry (Buchenhuskes 1993). Thus some safety aspects of the LAB isolates of food products must be investigated before considering their use for the bio-preservation (Matamoros ). Before the use of the LAB in the fish product, the quantitative analysis of BA is very essential to confirm the bacterial production. A great amount of research has been focused on BA production in fish and fish products. However no study has been found on BA production by single LAB strains which is used in fish infusion broth. Therefore, the aim of the study was to investigate the function of some commercially important LAB strains on biogenic amine production in different fish infusion decarboxylase broth (IDB).

Materials and Methods

Bacterial strains

The used LAB species were Lactococcus lactis subsp. cremoris (MG 1363), Lactococcus lactis subsp. lactis (IL 1403), Lactobacillus plantarum (FI8595) and Streptococcus thermophilus (NCFB2392). They were obtained from Sutcu Imam University, Kahramanmaras, Turkey in BGML stock culture. Leuconostoc mesenteroides subsp. cremoris (DSMZ 20346), Lactobacillus acidophilus (ATCC 11975), Pediococcus acidophilus (ATCC 25741) and Lactobacillus delbrueckii subsp. lactis (ATCC 10697) were purchased from Institute of Refik Saydam Hifzisihha (Ankara, Turkey).

Fish species

In the present study, fish decarboxylase infusion broth was prepared using five different fish species which were gilthead seabream (Sparus aurata), sardine (Sardinella aurita) anchovy (Engraulis encrasicolus), white shark (Carcharodon carchairas), and mackerel (Scomber scombrus).

Biogenic amine analysis

Culture media and bacterial extraction

Fish infusion broth was prepared according to method of Okuzumi et al. (1982) with minor modifications. Two hundred fifty grams of fish flesh was homogenised with 2 volumes of water (w/v), steamed at 100 °C for 1 hour and filtered. The filtrate was enriched with 1% glucose and 0.5% NaCl. In order to decarboxylate amino acid by bacteria, 3 mg pyridoxal HCl addition was made in each infusion broth before autoclaving process.

MRS and M17 broth were used for propagation of LAB cultures. Lactic acid bacterial strains were incubated at 37 °C for 24 hour which after 0.5 mL of these bacterial cultures was removed and put into the fish IDB to decarboxylate amino acid.

For extraction of LAB cultures, 5 mL of the fish IDB containing LAB strains were removed to separate bottles and then 2 mL trichloroacetic acid was added. They were centrifuged at 3000xg for 10 min and then filtered through a Whatman filter paper (Whatman GmbH, Dassel, Germany). After that, 4 mL of bacterial supernatant was taken for derivatisation from each of LAB bacterial strains.

Chemical reagents

All BA standards were purchased from Sigma-Aldrich (Munich, Germany). The mobile phase consisted of acetonitrile and HPLC grade water for amine analyses.

Preparation of standard amine solution

HIM dihydrochloride (165.7 mg), TYM hydrochloride (126.7 mg), TRP hydrochloride (122.8 mg), PUT dihydrochloride (182.9 mg), 2-phenylethylamine (PHEN) hydrochloride (130.1 mg), CAD dihydrochloride (171.4 mg), SPD trihydrochloride (175.3 mg), SPN tetrahydrochloride (172.0 mg), 5-hydroxytryptamine (Serotonin, SER) (133.9 mg), 3-hydroxytyramine hydrochloride (Dopamine, DOP) (123.8 mg), agmatine (AGM) sulphate (175.4 mg), trimethylamine (TMA) hydrochloride (161.7 mg) and ammonium chloride (296.9 mg) were dissolved in 10 mL HPLC grade water. The final concentration of free base for each amine was 10 mg mL−1 solution.

Derivatisation of extract from bacterial broth culture

A stock solution was prepared by dissolving 2% benzoyl chloride in acetonitrile to enhance the reaction with amines. For derivatisation of standard amine solutions, 100 μL was taken (4 mL for extracted bacterial cultures) from each free base standard solution (10 mg mL−1). 1 mL of sodium hydroxide (2 M) was added, followed by 1 mL of 2% benzoyl chloride (dissolved in acetonitrile) and the solution mixed on a vortex mixer for 1 min. The reaction mixture was left at room temperature for 5 min and then centrifuged for 10 min. After that, the benzoylation was stopped by adding 2 mL of saturated sodium chloride solution and the solution extracted twice with 2 mL of diethyl ether. The upper organic layer was transferred into a clean tube after mixing. Afterwards, the organic layer was evaporated to dryness in a stream of nitrogen. The residue was dissolved in 1 mL of acetonitrile and 10 μL aliquots were injected into the HPLC.

Analytical method

BA analysis was done using the method of Özogul (2004) and measured in mg amines per litre broth. The confirmation of BA production was accomplished using a rapid HPLC method with a reversed phase column by using a gradient elution program. Same analytic method was used for ammonia and trimethylamine (TMA) separation.

HPLC apparatus and column

A Shimadzu Prominence HPLC apparatus (Shimadzu, Kyoto, Japan) equipped with a SPD-M20A diode array detector and two binary gradient pumps (Shimadzu LC-10AT), auto sampler (SIL 20AC), column oven (CTO-20AC), and a communication bus module (CBM-20A) with valve unit FCV-11AL was used. The column was a reverse-phase, ODS Hypersil 5, 250×4.6 mm (Phenomenex, Macclesfield, Cheshire, UK) for the BA analyses.

Statistical analysis

The mean value and standard deviation were calculated from the data obtained from the four samples for each treatment. One way ANOVA was used to determine the significance of differences at p < 0.05. All statistics were performed using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL. USA).

Results

Ammonia (AMN) and BA production by LAB strains in five different fish IDB were given in Tables 1 to 5. Significant differences in AMN were found among the LAB strains and fish IDB (p < 0.05). The all used bacteria produced more than 200 mg/L of AMN. Lc. lactis subsp. cremoris produced the highest amount of AMN (591.72 mg/L) in sardine IDB,(p < 0.05). The lowest AMN production was observed by Strep. thermophilus in sardine infusion broth (Table 2), whereas LAB strains had the highest ability to produce AMN in white shark infusion broth (Table 5).

Table 1

Biogenic amines production (mg/L) by lactic acid bacteria in anchovy (Engraulis encrasicolus) infusion decarboxylase broth.

	Lb.delb.subsp.lactis*	Leu.mes. subsp. crem	Lb.acidophilus	Ped.acidophilus	Lc. cremoris	Lc.lactis	Lb.plantarum	Strep.thermophilus
AMN	530.36x ± 29.71ycd	572.68 ± 7.93bc	360.68 ± 21.35e	670.95 ± 36.79a	281.96 ± 22.08f	407.76 ± 16.65e	597.09 ± 35.21b	495.79 ± 22.54d
PUT	39.61 ± 2.51cd	44.16 ± 2.15c	37.85 ± 0.37cd	139.98 ± 10.11a	14.49 ± 2.05e	32.09 ± 1.20d	38.56 ± 1.48cd	60.94 ± 2.37b
CAD	39.06 ± 1.01c	41.47 ± 2.01c	33.64 ± 2.22d	110.51 ± 3.17a	17.80 ± 1.42e	32.46 ± 2.66d	33.40 ± 1.99d	48.76 ± 2.00b
SPD	43.84 ± 3.50e	100.72 ± 10.06c	58.53 ± 2.24d	196.83 ± 4.06a	24.55 ± 2.00f	55.32 ± 1.14de	64.75 ± 3.24d	149.05 ± 10.45b
TRPT	17.76 ± 0.37b	14.26 ± 1.34dc	6.11 ± 0.38e	1.64 ± 0.12f	12.45 ± 1.10d	15.61 ± 0.52bc	6.08 ± 0.11e	32.13 ± 2.69a
PHEN	39.72 ± 2.99d	38.12 ± 2.36de	21.21 ± 2.30f	16.44 ± 0.63f	99.28 ± 10.30b	116.27 ± 7.54a	27.78 ± 0.81ef	67.37 ± 2.02c
SPN	63.02 ± 5.80b	63.96 ± 4.09b	36.74 ± 2.19cd	17.14 ± 0.20e	0.89 ± 0.01f	40.10 ± 2.82c	29.77 ± 2.81d	116.71 ± 4.47a
HIM	47.10 ± 1.42a	22.88 ± 1.40b	19.960.49c	2.69 ± 0.16e	4.77 ± 0.19e	11.77 ± 0.15d	22.39 ± 0.47b	22.25 ± 1.79b
SER	589.27 ± 25.24a	307.15 ± 6.70c	80.18 ± 4.41f	135.09 ± 6.86e	301.84 ± 15.43c	477.28 ± 17.08b	469.59 ± 2.83b	206.40 ± 4.59d
TYM	75.33 ± 4.16e	56.29 ± 4.13f	69.74 ± 3.87ef	224.42 ± 12.50b	153.64 ± 8.18d	61.02 ± 2.38ef	170.30 ± 6.79c	241.57 ± 8.21a
TMA	414.60 ± 17.91a	0.07 ± 0.00d	1.34 ± 0.11d	7.17 ± 0.13d	97.91 ± 2.82c	247.56 ± 8.21b	5.60 ± 0.35d	2.47 ± 0.17d
DOP	11.39 ± 0.60g	128.50 ± 7.88d	103.89 ± 9.77e	200.71 ± 5.97b	554.30 ± 17.18a	48.53 ± 3.14f	201.43 ± 7.29b	163.85 ± 13.05c
AGM	34.26 ± 2.99d	20.27 ± 2.01e	42.51 ± 2.92c	40.87 ± 2.19cd	55.12 ± 1.49b	47.37 ± 1.56c	47.21 ± 3.43c	100.32 ± 5.12a

Lactococcus lactis subsp. cremoris MG 1363. Lactococcus lactis subsp. lactis IL 1403. Lactobacillus plantarum FI8595. Leuconostoc mesenteroides subsp. cremoris DSMZ 20346. Lactobacillus acidophilus ATCC 11975. Pediococcus acidophilus ATCC 25741. Lactobacillus delbrueckii subsp. lactis ATCC 10697 and Streptococcus thermophilus, AMN, ammonia; PUT, putrescine; CAD, cadaverine; HIM, histamine; SPD, spermidine; TRPT, tryptamine; PHEN, 2-Phenyl-ethylamine; SPN, spermine; SER, serotonin; TYM, tyramine; TMA, trimethylamine; DOP; Dopamine, AGM, agmatine.

Mean value.

Standard deviation (n = 4).

Different lowercase letters (a–g) in a row indicate significant differences (p < 0.05) among bacteria.

Table 5

Biogenic amines production (mg/L) by lactic acid bacteria in white shark (Carcharodon carchairas) infusion decarboxylase broth.

	Lb.delb.subsp.lactis*	Leu.mes. subsp. crem	Lb.acidophilus	Ped.acidophilus	Lc. cremoris	Lc.lactis	Lb.plantarum	S.thermophilus
AMN	3424.54x ± 106.34yc	2712.24 ± 112.13e	3526.19 ± 264.71c	4348.38 ± 264.03b	2769.90 ± 173.46de	4991.38 ± 211.26a	3306.16 ± 179.91c	3193.06 ± 81.83cd
PUT	6.62 ± 0.40de	123.24 ± 9.56a	16.96 ± 0.67c	13.25 ± 0.99cd	2.37 ± 0.14e	6.13 ± 0.60de	41.86 ± 3.02b	1.85 ± 0.08e
CAD	7.51 ± 0.34de	8.29 ± 0.22d	27.82 ± 1.50a	12.50 ± 0.35c	2.59 ± 0.24f	2.36 ± 0.10f	14.24 ± 0.81b	6.74 ± 0.12e
SPD	36.88 ± 3.06b	33.79 ± 1.63b	626.58 ± 58.19a	57.16 ± 3.31b	23.59 ± 1.15b	15.12 ± 1.24b	44.42 ± 1.34b	17.28 ± 1.30b
TRPT	21.22 ± 1.12c	39.84 ± 1.34a	1.18 ± 0.10f	14.83 ± 0.71d	1.63 ± 0.06f	8.28 ± 0.70e	34.32 ± 2.86b	0.57 ± 0.02f
PHEN	90.37 ± 3.23a	87.33 ± 3.35a	16.82 ± 1.56cd	69.10 ± 2.26b	20.36 ± 0.59c	67.31 ± 2.87b	20.48 ± 1.39c	14.06 ± 0.88d
SPN	25.95 ± 2.76d	0.00 ± 0.00e	30.49 ± 2.10c	22.87 ± 0.53d	0.00 ± 0.00e	0.00 ± 0.00e	116.21 ± 3.69a	66.20 ± 1.69b
HIM	38.21 ± 1.28a	22.16 ± 1.25c	28.26 ± 0.75b	26.15 ± 1.78b	16.95 ± 1.21d	2.69 ± 0.13f	19.84 ± 0.44c	9.24 ± 0.24e
SER	183.18 ± 4.38c	124.09 ± 5.40d	223.11 ± 4.74b	98.62 ± 1.83e	239.63 ± 13.51b	100.37 ± 5.40e	428.37 ± 17.10a	222.07 ± 11.20b
TYM	40.14 ± 3.15c	42.08 ± 3.42c	75.83 ± 5.30a	28.38 ± 2.34d	15.70 ± 0.92e	8.86 ± 0.36e	29.06 ± 1.68d	53.68 ± 3.44b
TMA	4.61 ± 0.38b	2.80 ± 0.14b	8.31 ± 0.33b	2.64 ± 0.09b	1.45 ± 0.04b	4.08 ± 0.19b	784.46 ± 8.74a	786.75 ± 18.60a
DOP	365.16 ± 13.58a	125.53 ± 2.76d	356.09 ± 22.35a	205.81 ± 13.72c	100.99 ± 5.32d	223.40 ± 5.75bc	340.09 ± 22.17a	251.70 ± 8.55b
AGM	18.13 ± 0.71cd	18.14 ± 0.67cd	41.62 ± 0.32a	17.52 ± 0.33cd	14.40 ± 1.29d	18.82 ± 1.07cd	31.85 ± 0.90b	20.15 ± 1.48c

Lactococcus lactis subsp. cremoris MG 1363. Lactococcus lactis subsp. lactis IL 1403. Lactobacillus plantarum FI8595. Leuconostoc mesenteroides subsp. cremoris DSMZ 20346. Lactobacillus acidophilus ATCC 11975. Pediococcus acidophilus ATCC 25741. Lactobacillus delbrueckii subsp. lactis ATCC 10697 and Streptococcus thermophilus, AMN, ammonia; PUT, putrescine; CAD, cadaverine; HIM, histamine; SPD, spermidine; TRPT, tryptamine; PHEN, 2-Phenyl-ethylamine; SPN, spermine; SER, serotonin; TYM, tyramine; TMA, trimethylamine; DOP; Dopamine, AGM, agmatine.

Mean value.

Standard deviation (n = 4).

Different lowercase letters (a–g) in a row indicate significant differences (p < 0.05) among bacteria.

Table 2

Biogenic amines production (mg/L) by lactic acid bacteria in sardine (Sardinella aurita) infusion decarboxylase broth.

	Lb.delb.subsp. lactis*	Leu.mes. subsp. crem	Lb.acidophilus	Ped.acidophilus	Lc. cremoris	Lc.lactis	Lb.plantarum	Strep.thermophilus
AMN	399.04x ± 16.44yc	437.18 ± 9.92b	335.78 ± 7.60d	410.71 ± 6.32bc	591.72 ± 15.38a	262.90 ± 4.10e	428.42 ± 18.46b	206.28 ± 8.81f
PUT	58.93 ± 2.71b	66.37 ± 5.42b	43.22 ± 2.48c	26.67 ± 2.16d	75.85 ± 6.82a	18.12 ± 1.58e	33.22 ± 2.08d	29.62 ± 2.29d
CAD	92.14 ± 2.58b	120.42 ± 9.86a	125.04 ± 10.28a	75.69 ± 7.40bc	108.89 ± 8.79a	60.20 ± 3.11c	83.29 ± 5.31b	41.08 ± 3.36d
SPD	101.51 ± 9.19c	146.41 ± 11.97a	57.44 ± 3.33d	43.11 ± 2.36e	124.20 ± 4.54b	66.22 ± 3.14d	66.27 ± 5.48d	28.06 ± 1.49f
TRPT	15.33 ± 1.20a	3.09 ± 0.12de	3.78 ± 0.16d	3.02 ± 0.23de	8.12 ± 0.43b	2.42 ± 0.15e	6.17 ± 0.52c	8.86 ± 0.20b
PHEN	0.00 ± 0.00f	32.45 ± 2.05c	38.06 ± 3.19b	10.45 ± 0.63e	81.35 ± 1.72a	0.00 ± 0.00f	15.27 ± 0.38d	0.00 ± 0.00f
SPN	104.02 ± 8.22a	22.15 ± 1.63f	54.67 ± 3.77d	21.38 ± 0.46f	82.34 ± 3.47b	70.42 ± 4.83c	35.33 ± 2.84e	14.48 ± 0.74f
HIM	21.00 ± 0.09e	101.69 ± 6.99a	67.42 ± 4.93b	11.77 ± 0.25f	24.77 ± 2.07e	59.47 ± 3.63c	20.03 ± 1.66e	35.73 ± 1.22d
SER	110.02 ± 6.30ef	160.32 ± 11.42d	95.31 ± 6.01f	133.14 ± 3.34e	219.31 ± 7.33c	419.25 ± 22.31a	117.06 ± 5.92ef	310.71 ± 6.71b
TYM	2943.51 ± 113.54a	374.25 ± 22.89d	658.86 ± 35.00c	1157.25 ± 80.65b	345.74 ± 18.16d	154.39 ± 9.54e	700.41 ± 42.77c	163.78 ± 11.87e
TMA	0.38 ± 0.03d	1.99 ± 0.08c	2.61 ± 0.25b	0.36 ± 0.02d	0.43 ± 0.02d	4.29 ± 0.03a	2.47 ± 0.16b	0.21 ± 0.01d
DOP	324.66 ± 16.95b	145.79 ± 5.31d	201.42 ± 11.17c	166.21 ± 8.61d	276.74 ± 14.14b	349.46 ± 23.14a	91.08 ± 6.16e	88.42 ± 4.36e
AGM	33.35 ± 1.72cd	56.52 ± 3.25a	37.51 ± 2.56bc	28.93 ± 1.19d	39.64 ± 2.08b	31.38 ± 1.32c	22.41 ± 1.42e	21.19 ± 0.72e

Lactococcus lactis subsp. cremoris MG 1363. Lactococcus lactis subsp. lactis IL 1403. Lactobacillus plantarum FI8595. Leuconostoc mesenteroides subsp. cremoris DSMZ 20346. Lactobacillus acidophilus ATCC 11975. Pediococcus acidophilus ATCC 25741. Lactobacillus delbrueckii subsp. lactis ATCC 10697 and Streptococcus thermophilus, AMN, ammonia; PUT, putrescine; CAD, cadaverine; HIM, histamine; SPD, spermidine; TRPT, tryptamine; PHEN, 2-Phenyl-ethylamine; SPN, spermine; SER, serotonin; TYM, tyramine; TMA, trimethylamine; DOP; Dopamine, AGM, agmatine.

Mean value.

Standard deviation (n = 4).

Different lowercase letters (a–g) in a row indicate significant differences (p < 0.05) among bacteria.

There were also significant differences in BA production among the LAB strains (p < 0.05) for all fish IDB. The strains produced all amine in fish IDB (Tables 1 to 5) apart from spermine (SPN) and 2-phenylethylamine (PHEN). Putrescine (PUT) production by LAB strains was ranged from 1.85 (for Strep. thermophilus in white shark IDB) to 139.98 mg/L (for Ped. acidophilus in anchovy IDB).

Significant differences in histamine (HIM) production was observed among the LAB strains (p < 0.05). Lc. lactis subsp. cremoris, Leuc. mesenteroides subsp. cremoris and Lc. produced cadaverine (CAD) more than 100 mg/L in sardine IDB, whereas CAD production by LAB strains in the other mediums was less than 93 mg/L. In mackerel IDB, the lowest spermidine (SPD) production was found for Lactococcus lactis subsp. cremoris (11.54 mg/L). In anchovy IDB, SPD production by Lactobacillus spp. was 55 mg/L. Lb. delbrueckii subsp. lactis, Lc. lactis subsp. lactis and Strep. thermophilus in sardine IDB as well as Lc. lactis subsp. lactis in mackerel IDB had not an ability to produce PHEN. However, Lc. lactis subsp. cremoris was found as good PHEN producer in gilthead seabream IDB (209.52 mg/L). Lactococcus spp. had also good activity to produce PHEN in anchovy IDB (Table 1). In white shark IDB, significant PHEN production was observed for Lb. delbrueckii subsp. lactis and Leu. mesenteroides subsp. cremoris.

Most of LAB strains produced below 9 mg/L of TMA. However, Lb. plantarum and Strep. thermophilus accumulated significant amount of TMA (784.46 and 786.75 mg/L, respectively) in white shark IDB (p < 0.05) (Table 5). LAB strains i.e. Lb. delbrueckii subsp. lactis, Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris also had a high TMA formation in anchovy IDB (Table 1). Lc. lactis subsp. lactis. was one of the LAB strains produced good amount of TMA in mackerel IDB. Tryptamine (TRP) was one of the lowest produced amine by LAB strains. Agmatine (AGM) production by LAB strains was between 14 and 101 mg/L.

Discussion

Lc. lactis subsp. lactis (4991.38 mg/L) and Ped. acidophilus (4348.38 mg/L) were characterized as the highest AMN producer in shark IDB among the tested LAB strains. In anchovy IDB, Ped. acidophilus produced the highest amount of AMN (670.95 mg/L), whereas Lc. lactis subsp. cremoris accumulated the lowest level of that (281.96 mg/L). Lc. lactis subsp. cremoris and Lb. plantarum in gilthead seabream IDB were the highest ability in AMN production (p < 0.05). In mackerel IDB, AMN production was ranged from 298 mg/L (Lb. delbrueckii subsp. lactis) to 507 mg/L (Lc. lactis subsp. lactis).

The highest accumulated amines by LAB strains were generally TYM, DOP, SER and SPD, although other amines produced at significant levels (p < 0.05). Bunkova reported that Lc. lactis subsp. cremoris, Strep. thermophilus and Lb. delbrueckii subsp. bulgaricus produced TYM but did not produce other tested amines. Herring and tuna fish salads inoculated with Lactobacillus curvatus LTH 975 had PUT, CAD, HIM, TYM accumulation (Leuschner and Hammes, 1999). However, in cold-smoked fish fermented with LAB, the LAB used was unable to produce CAD, HIM or TYM (Leuschner and Hammes, 1999). Similar results were found by Thapa who reported none of the strains of LAB including Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis, Lb. plantarum, Leu. mesenteroides and Pediococcus pentosaceus isolated from traditionally processed fish products produced BA.

Özogul and Özogul (2005) defined four categories in order to simplify the discussion of amine production by bacterial strains, which are: prolific amine former (> 1000 mg/L), good amine former (100–1000 mg/L), medium amine former (10–100 mg/L) and poor amine former (< 10 mg/L). According to this category, LAB strains seemed to poor PUT producer in white shark and mackerel IDB (Tables 4 and 5). However, Leuc. mesenteroides subsp. cremoris in white shark IDB, and Lc. lactis subsp. cremoris in sardine and gilthead seabream IDB accumulated high amount of PUT (> 75 mg/L). In mackerel IDB, PUT production was above the 5.7 mg/L and the highest production was found for Lb. plantarum (30.71 mg/L). Lb. plantarum N4 isolated from wine was able to produce putrescine from ornithine (Arena and Manca de Nadra, 2001). PUT was reported as main amine produced by Leu. mesenteroides and Lactobacillus zeae (Morrena-Arribas and Polo, 2008).

Table 4

Biogenic amines production (mg/L) by lactic acid bacteria in mackerel (Scomber scombrus) infusion decarboxylase broth.

	Lb.delb.subsp. lactis*	Leu.mes. subsp. crem	Lb.acidophilus	Ped.acidophilus	Lc. cremoris	Lc.lactis	Lb.plantarum	Strep.thermophilus
AMN	298.15x ± 21.95ye	349.57 ± 24.08de	439.08 ± 16.68bc	423.11 ± 11.79bc	357.18 ± 35.19de	506.60 ± 20.90a	392.05 ± 13.26cd	455.19 ± 43.94ab
PUT	8.17 ± 0.48c	8.57 ± 0.81c	16.24 ± 1.23b	29.10 ± 1.21a	5.77 ± 0.32c	17.70 ± 0.97b	30.71 ± 2.10a	15.30 ± 1.37b
CAD	6.78 ± 0.67c	6.34 ± 0.39c	42.92 ± 2.49a	18.68 ± 0.97b	3.53 ± 0.17d	8.00 ± 0.64c	7.61 ± 0.27c	8.69 ± 0.38c
SPD	59.72 ± 4.83c	40.14 ± 2.37d	122.93 ± 6.28b	148.55 ± 9.47a	11.54 ± 0.53e	61.48 ± 4.23c	45.36 ± 2.39d	36.96 ± 2.95d
TRPT	3.96 ± 0.33e	3.24 ± 0.22ef	8.25 ± 0.56b	10.45 ± 0.48a	0.56 ± 0.05g	7.04 ± 0.36c	5.96 ± 0.31d	2.56 ± 0.16f
PHEN	12.65 ± 0.37c	9.87 ± 0.73cd	8.64 ± 0.63d	29.11 ± 1.57b	70.50 ± 2.61a	0.00 ± 0.00e	11.39 ± 1.10cd	10.42 ± 0.36cd
SPN	15.52 ± 1.16d	21.61 ± 1.48c	51.30 ± 2.99a	28.56 ± 0.79b	8.62 ± 0.54e	2.61 ± 0.22f	26.64 ± 2.40b	15.43 ± 0.79d
HIM	50.72 ± 1.03c	35.00 ± 2.58d	74.89 ± 4.96b	100.84 ± 6.43a	50.30 ± 3.50c	43.43 ± 2.12c	17.48 ± 0.76e	33.34 ± 2.20d
SER	72.90 ± 1.27f	103.53 ± 7.27de	126.78 ± 3.22d	231.32 ± 17.67b	96.01 ± 9.46de	594.13 ± 41.66a	175.72 ± 6.28c	134.03 ± 6.65d
TYM	156.21 ± 8.58c	312.48 ± 24.85a	120.45 ± 8.99d	228.29 ± 17.52b	217.95 ± 13.07b	158.39 ± 12.15c	88.79 ± 7.43d	171.73 ± 11.42c
TMA	2.88 ± 0.09b	1.75 ± 0.14b	5.23 ± 0.49b	4.84 ± 0.24b	2.57 ± 0.10b	132.71 ± 10.63a	1.58 ± 0.12b	2.05 ± 0.11b
DOP	85.14 ± 3.20d	89.53 ± 4.25d	182.73 ± 6.77b	224.74 ± 9.85a	91.63 ± 2.88d	32.96 ± 2.88e	142.31 ± 9.64c	97.85 ± 1.29d
AGM	44.071.79b	47.471.93b	38.61 ± 1.40c	75.12 ± 4.12a	25.10 ± 1.03e	26.72 ± 1.17de	31.17 ± 2.25d	18.34 ± 1.18f

Lactococcus lactis subsp. cremoris MG 1363. Lactococcus lactis subsp. lactis IL 1403. Lactobacillus plantarum FI8595. Leuconostoc mesenteroides subsp. cremoris DSMZ 20346. Lactobacillus acidophilus ATCC 11975. Pediococcus acidophilus ATCC 25741. Lactobacillus delbrueckii subsp. lactis ATCC 10697 and Streptococcus thermophilus, AMN, ammonia; PUT, putrescine; CAD, cadaverine; HIM, histamine; SPD, spermidine; TRPT, tryptamine; PHEN, 2-Phenyl-ethylamine; SPN, spermine; SER, serotonin; TYM, tyramine; TMA, trimethylamine; DOP; Dopamine, AGM, agmatine.

Mean value.

Standard deviation (n = 4).

Different lowercase letters (a–g) in a row indicate significant differences (p < 0.05) among bacteria.

CAD was one of the most abundant amines in fish sauce with maximum reported value of 1429 ppm (Zaman ). In the present study, Lc. lactis subsp. cremoris, Leuc. mesenteroides subsp. cremoris and Lc. acidophilus were good CAD producer in sardine IDB (> 100 mg/L) (Table 2). Similar production was also observed for Ped. acidophilus in anchovy IDB (Table 1). In the other mediums, CAD production by LAB strains was less than 93 mg/L. In gilthead seabream IDB, the highest CAD production was observed for Lc. lactis subsp. cremoris. In the recently study, Özogul (2011) reported that lower CAD production by Lc. lactis subsp. cremoris and Strep. thermophilus in histidine decarboxylase broth was found (< 17 mg/L). In the current study, the lowest CAD production by LAB strains was generally observed for white shark and mackerel IDB. There were also not significant differences between white shark and mackerel IDB in terms of CAD production by LAB strains apart from Ped. acidophilus and Lc. acidophilus.

SPD was one of the most accumulated amine by LAB strains. Leuc. mesenteroides subsp. cremoris, Ped. acidophilus and Strep. thermophilus produced more than 100 mg/L of SPD in sardine, mackerel and anchovy IDB, respectively. However, SPD production by Lc. acidophilus was 6-fold higher in white shark, compared with those bacteria. However, SPD production by LAB strains did not differ statistically except for Lb. acidophilus in white shark IDB (p > 0.05). The LAB strains produced medium amount of SPD in gilthead seabream IDB. Zaman reported that SPD, TRP, PHEN and SPN were minor amines in fish sauce. Similarly, TRP was one of the lowest produced amine by LAB strains. The lowest TRP production was observed from Lc. lactis subsp. cremoris (mackerel IDB) and Strep. thermophilus (white shark IDB), whereas Leuc. mesenteroides subsp. cremoris and Lb. plantarum accumulated medium amount of TRP in white shark IDB.

Özogul (2011) reported that none of the LAB strains produced SPN in histidine decarboxylase broth. Similar results were obtained from this study associated with SPN production by Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis, Leu. mesenteroides subsp. cremoris in white shark IDB. Lb. plantarum was one of the highest amounts of SPN producer. Similarly, SPN production by Lc. lactis subsp. cremoris in anchovy IDB was negligible level, whereas Strep. thermophilus produced high amount of SPN (116.71 mg/L). No significant differences in SPN were observed between Lc. lactis and Lb. plantarum in anchovy IDB (p > 0.05). There were also not significant differences (p > 0.05) in SPN production among the Ped. acidophilus, Lc. lactis subsp. lactis, Lb. plantarum and Strep. thermophilus in gilthead seabream IDB (~64 mg/L). In mackerel IDB, Lc. lactis subsp. cremoris produced low amount of SPN (8.62 mg/L), while SPN production by Lb. acidophilus was 51.30 mg/L (p < 0.05).

HIM production varies depending on bacterial strains and IDB. Matamoros reported that HIM and tyramine (TYM) production for Leuconostoc gelidum, Lactococcus piscium, Lactobacillus fuchuensis and Carnobacterium alterfunditum isolated from seafood products were below the 5 mg/L. HIM was the only major BA produced by various strains of lactic acid bacteria from fish sauce (Udomsil ). In the recent study, it was shown that HIM production in histidine decarboxylase broth was negligible by Lc. lactis subsp. cremoris, whereas Lc. lactis subsp. lactis, Strep. thermophilus and Lb. plantarum did not produce HIM (Özogul, 2011). However, in the present study, the all strains had an ability to produce HIM in fish IDB, ranging from 2 to 102 mg/L (Tables 1–5) indicating that fish muscle was more favourable than specific medium on HIM production. The lowest HIM accumulation was found for Lc. lactis subsp. lactis (2.69 mg/L) and Ped. acidophilus (2.69 mg/L) in white shark and anchovy IDB, respectively. In anchovy IDB, the highest HIM production was found for Lb. delbrueckii subsp. lactis (47.10 mg/L), whereas Leu. mesenteroides subsp. cremoris, Lb. plantarum and Strep. thermophilus produced similar amount of HIM (p > 0.05). Lactobacillus buchneri LB14 and Lactobacillus buchneri ST2A produced 344 and 401 mg/L HIM in decarboxylase medium contained histidine, lysine, ornitine and tyrosine (Choudhury ). Although most of LAB strains seemed to be medium HIM producer in fish IDB, Leu. mesenteroides subsp. cremoris and Ped. acidophilus was good HIM producer (~100 mg/L) in sardine and mackerel IDB, respectively.

No significant differences in HIM production was observed among the Lb. delbrueckii subsp. lactis, Lc. lactis subsp. cremoris and Lc. lactis subsp. lactis (~ 50 mg/L) in mackerel IDB. In gilthead seabream IDB, the lowest HIM production was found for Lb. delbrueckii subsp. lactis (5.62 mg/L) though there were no significant differences in HIM production between Lc. lactis subsp. cremoris (73.70 mg/L) and Lc. lactis subsp. lactis (74.37 mg/L) (p > 0.05). Tuna and herring salad inoculated with Lb. buchneri revealed 900 ppm and 670 ppm histamine, respectively (Leuschner and Hammes, 1999).

Fadda found that Lb. plantarum, Lb. casei and Pediococcus acidilactici isolated from fermented sausages did not produce TYM in a medium supplemented with tyrosine at 20 mg/L. Udomsil reported trace amounts of TYM production (1–5 mg/100 mL) by various LAB strains from fish sauce. In the current study, TYM was one of the main amines produced by LAB strains. Among the fish IDB, most of LAB strains showed high activity in producing TYM in sardine IDB (> 300 mg/L) (Table 2). The reported upper TYM limit of 100–800 mg kg−1 to be toxic doses in foods (Brink ; Kim ), which were generally exceed by most of LAB strains. Lb. delbrueckii subsp. lactis (2943.51 mg/L) and Ped. acidophilus (1157.25 mg/L) had the highest ability to produce TYM in sardine IDB. In white shark, most of the LAB strains produced less than 55 mg/L. Marino reported that Strep. thermophilus was a TYM-producer strain. In the present study, Strep. thermophilus (241.57 mg/L), Ped. acidophilus (224.42 mg/L), Lb. plantarum (170.30 mg/L) and Lc. lactis subsp. cremoris (153.64 mg/L) produced the highest amount of TYM in anchovy IDB. There were not significant differences in TYM production between Leu. mesenteroides subsp. cremoris and Lb. acidophilus (~90 mg/L), and also for Lc. lactis subsp. lactis, Lb. plantarum and Strep. thermophilus (~180 mg/L) in gilthead seabream IDB.

The LAB strains were generally good SER producer. Among the fish IDB, the highest SER production was found by Lc. lactis subsp. lactis in mackerel IDB and Lb. delbrueckii subsp. lactis in anchovy IDB, whereas Lb. delbrueckii subsp. lactis was produced the lowest amount of SER in gilthead seabream IDB (p < 0.05). Leu. mesenteroides subsp. cremoris was the main LAB produced highest amount of SER in gilthead seabream IDB. LAB strains such as Lc. lactis subsp. lactis, Lc. lactis subsp. cremoris, Lb. plantarum and Strep. thermophilus showed lower activity of SER and DOP (1 and 5.5 mg/L, respectively) in histidine decarboxylase broth (Özogul, 2011). There were significant differences in DOP production among the LAB strains in fish IDB (p < 0.05). LAB strains produced significant amount of DOP, especially for sardine and white shark IDB. Although Lb. delbrueckii subsp. lactis was poor DOP producer in anchovy IDB, the highest DOP production by Lc. lactis subsp. cremoris was found in this medium (p < 0.05). In mackerel IDB, DOP production was ranged from 32.96 mg/L for Lc. lactis subsp. lactis to 224.74 mg/L for Ped. acidophilus.

AGM is formed from arginine by the enzyme of arginine decarboxylase secreted from lactic acid and nitric acid-reducing bacteria during the fermentation process (Umezu ). The lowest AGM production by LAB strains was in white shark IDB (< 42 mg/L) while the highest AGM accumulation was found for Strep. thermophilus (100.32 mg/L) and Ped. acidophilus (75.12 mg/L) in anchovy and mackerel IDB (p < 0.05), respectively. In sardine IDB, AGM production by LAB strains was below the 57 mg/L.

Conclusion

The study results showed that the tested LAB strains had an ability to produce high amount of BA in fish IDB, which were mainly TYM, HIM, DOP, SER and SPD production. Fish species also appeared to play an important role in BA production by LAB strains. Therefore both criteria have to be taken into consideration in order to prevent the bacterial BA production and food poisoning related to fish consumption. Consequently, the ability of LAB strains to produce biogenic amines in fish products can be considered as criteria for the selection of strains and fish species.

Table 3

Biogenic amines production (mg/L) by lactic acid bacteria in gilthead seabream (Sparus aurata) infusion decarboxylase broth.

	Lb.delb.subsp.lactis*	Leu.mes. subsp.crem	Lb.acidophilus	Ped.acidophilus	Lc. cremoris	Lc.lactis	Lb.plantarum	Strep.thermophilus
AMN	446.71x ± 18.96ye	494.40 ± 6.46d	501.95 ± 32.13d	523.20 ± 22.26d	675.14 ± 25.37a	625.78 ± 14.43bc	659.3 ± 15.84ab	589.46 ± 7.92c
PUT	32.32 ± 1.23d	27.49 ± 1.15e	22.48 ± 0.54f	44.21 ± 1.82c	76.98 ± 1.86a	53.91 ± 1.00b	53.92 ± 1.37b	41.05 ± 2.06c
CAD	33.08 ± 2.29e	36.01 ± 2.79e	44.02 ± 1.92d	63.28 ± 4.09b	73.31 ± 2.36a	52.52 ± 3.48c	54.35 ± 1.11c	42.80 ± 2.11d
SPD	42.81 ± 1.57e	60.45 ± 4.10d	33.10 ± 3.11f	87.08 ± 2.43c	96.30 ± 4.16b	116.68 ± 4.35a	121.33 ± 5.64a	69.14 ± 3.55d
TRPT	3.86 ± 0.24e	2.70 ± 0.15f	0.84 ± 0.02g	8.43 ± 0.62c	15.04 ± 0.30a	10.79 ± 0.45b	11.39 ± 0.32b	5.88 ± 0.20d
PHEN	15.43 ± 1.31e	14.37 ± 0.86e	14.87 ± 0.26e	35.76 ± 0.76d	209.52 ± 3.81a	52.15 ± 1.08c	55.06 ± 2.00c	71.88 ± 0.97b
SPN	19.61 ± 1.58d	28.35 ± 0.13c	28.25 ± 1.09c	63.45 ± 1.56b	85.01 ± 2.96a	67.74 ± 3.24b	65.64 ± 4.01b	62.32 ± 2.81b
HIM	5.62 ± 0.48e	13.54 ± 0.29d	14.18 ± 1.38d	30.74 ± 1.25c	73.70 ± 3.05a	74.37 ± 2.13a	41.06 ± 1.42b	29.09 ± 1.69c
SER	36.67 ± 1.38d	228.07 ± 5.62a	234.28 ± 16.92a	123.09 ± 10.93c	191.91 ± 7.57b	142.99 ± 5.56c	133.97 ± 10.84c	127.97 ± 9.12c
TYM	23.22 ± 1.23e	83.65 ± 3.01d	94.84 ± 2.59d	145.12 ± 2.81c	375.17 ± 20.28a	196.85 ± 16.08b	177.01 ± 6.26b	181.62 ± 6.19b
TMA	0.12 ± 0.01e	3.59 ± 0.21c	4.09 ± 0.09b	5.86 ± 0.45a	6.02 ± 0.09a	0.49 ± 0.01de	0.51 ± 0.04de	0.62 ± 0.05d
DOP	172.37 ± 8.15c	323.49 ± 25.14a	91.33 ± 8.71d	45.48 ± 3.66e	225.01 ± 9.17b	100.26 ± 7.61d	149.84 ± 13.72c	77.23 ± 6.63d
AGM	25.93 ± 1.10e	40.39 ± 3.29bc	51.47 ± 3.08a	42.17 ± 3.57b	49.70 ± 0.05a	31.82 ± 2.17d	35.81 ± 1.49cd	31.78 ± 0.76d

Lactococcus lactis subsp. cremoris MG 1363. Lactococcus lactis subsp. lactis IL 1403. Lactobacillus plantarum FI8595. Leuconostoc mesenteroides subsp. cremoris DSMZ 20346. Lactobacillus acidophilus ATCC 11975. Pediococcus acidophilus ATCC 25741. Lactobacillus delbrueckii subsp. lactis ATCC 10697 and Streptococcus thermophilus, AMN, ammonia; PUT, putrescine; CAD, cadaverine; HIM, histamine; SPD, spermidine; TRPT, tryptamine; PHEN, 2-Phenyl-ethylamine; SPN, spermine; SER, serotonin; TYM, tyramine; TMA, trimethylamine; DOP; Dopamine, AGM, agmatine.

Mean value.

Standard deviation (n = 4).

Different lowercase letters (a–g) in a row indicate significant differences (p < 0.05) among bacteria.