Food Chemistry 129 (2011) 1211–1216
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The function of lactic acid bacteria and brine solutions on biogenic amine formation by foodborne pathogens in trout fillets Esmeray Kuley a,⇑, Fatih Özogul a, Yesim Özogul a, Ismail Akyol b a b
Department of Fish Processing Technology, Faculty of Fisheries, Cukurova University, 01330 Balcali, Adana, Turkey Department of Animal Science, Faculty of Agriculture, Sutcu Imam University, Kahramanmaras, Turkey
a r t i c l e
i n f o
Article history: Received 22 October 2010 Received in revised form 11 April 2011 Accepted 23 May 2011 Available online 27 May 2011 Keywords: Food-borne pathogen Lactic acid bacteria Rainbow trout Biogenic amine Ammonia
a b s t r a c t The influences of lactic acid bacteria and brine solutions on the biogenic amine formation by Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterococus faecalis, Pseudomonas aeruginosa, Listeria monocytogenes, Aeromonas hydrophila and Salmonella paratyphi A in fermented trout fillets were investigated. Fish fillets were divided into four groups, group 1 without any lactic acid bacteria inoculation, group 2 and group 3 with different salt concentration inoculated with lactic acid bacteria and food-borne pathogens, and group 4 inoculated with lactic acid bacteria and food-borne pathogens without a salt solution. The histamine content in trout fillets in group 4 was found to be more than 10 mg/100 g, while the other groups contained less than 7.5 mg/100 g. The highest tyramine production was found for group 1 and group 3, ranging from 3 to 18 mg/100 g. Lactic acid bacteria did not seem to play an important role on biogenic amine production by food borne pathogens, while adding brine solution on fillets has inhibitory effects on some of the biogenic amines. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Lactic acid bacteria have a significant role in food fermentations due to their impact on flavour changes and as a preservative, and thus they help to provide food safety by inhibiting pathogen growth (Devlieghere, Vermeiren, & Debevere, 2004). The primary role of lactic acid bacteria strains is to utilise carbohydrate substrates available in the fermentation system (Kopermsub & Yunchalard, 2010) and produce substances such as hydrogen peroxide, weak organic acids, reuterin, diacetyl, bacteriocins and low-molecular-weight metabolites that inhibit pathogenic organisms in fermented foods (Brashears, Amezquita, & Jaroni, 2005). The fermentation process for fish may also provide the conditions required for abundant production of biogenic amines such as availability of free amino acids, the presence of decarboxylase-positive microorganisms and conditions allowing bacterial growth, decarboxylase synthesis and decarboxylase activity (Petaja, Eerola, & Petaja, 2000). Production of biogenic amines was reported in dried and fermented fish (Tsai et al., 2006), vacuum packaged and cold smoked fermented fish products (Petaja et al., 2000; Tome, Pereira, Lopes, Gibbs, & Teixeira, 2008). Some lactic acid bacteria involved in food fermentation, such as Streptococcus thermophilus, Lactococcus lactis and Lactobacillus plantarum, may produce biogenic amines such as tyramine (Bover-Cid & Holzapfel, 1999; Fadda, Vignolo, & Oliver, 2001). ⇑ Corresponding author. Tel.: +90 322 3386084; fax: +90 322 3386439. E-mail address: [email protected] (E. Kuley). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.113
A variety of food-borne pathogens isolated from different types of food have also the ability to produce biogenic amines. Some pathogenic bacteria in the families of Escherichia, Salmonella, Clostridium, and Bacillus have been reported to show histidine decarboxylase activity (Ordonez, Hierro, Bruna, & de la Hoz, 1999). Aeromonas hydrophila produce histamine, cadaverine, and putrescine (Middlebrooks, Toom, Donglas, Harrison, & McDowell, 1988), and Klebsiella pneumoniae and Enterococcus faecalis accumulate cadaverine and tyramine, respectively (Özogul & Özogul, 2007). The interactions between mix bacterial cultures (lactic acid bacteria and food borne pathogens) in terms of biogenic amines production have not been well stated. It has been reported that starter lactic acid bacteria cultures suppressed the accumulation of some biogenic amines in some fermented fish products (Mah & Hwang, 2009; Yongjin, Wenshui, & Xiaoyong, 2007; Zhong-Yi, Zhong-Hai, Miao-Ling, & Xiao-Ping, 2010). Little research has been done on the biogenic amine formation by mixtures of food borne pathogen and lactic acid bacteria (Kuley & Özogul, 2011; Özogul, 2011). Lactic acid bacteria had an important synergetic role in some biogenic amine production by food-borne pathogenic bacteria, although the effect of some lactic acid bacteria strains on biogenic amine production was strain-dependent in vitro conditions (Kuley & Özogul, 2011; Özogul, 2011). However, little information is available on the formation of biogenic amines by mix culture in fish and fish products. Investigating the ability of food-borne pathogenic bacteria and/or lactic acid bacteria to produce biogenic amine in fish and fish products are important due to their potential impacts on human health and food quality. Therefore, the purpose
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of this study was to investigate the effects of lactic acid bacteria on biogenic amine production by food borne pathogens in rainbow trout fillets during fermentation and storage. Moreover, the effect of brine solution on the biogenic amine production by lactic acid bacteria and food borne pathogens was investigated. 2. Materials and methods 2.1. Preparation of fish samples Cultured fresh rainbow trout (Oncorhynchus mykiss) were purchased from a local fish farm in Adana, Turkey. The average weight and length of the samples were 230 ± 1.3 g and 22 ± 2 cm, respectively. Fish were delivered to the laboratory in ice after 4 h of harvesting and they were immediately gutted and filleted without skin removal. After that, the fillets were washed with sterile water and then the trout fillets were divided into four groups. The brine solutions were prepared according to the method of Petaja et al. (2000). The groups of experiment are given in Table 1. For the fermentation process, each group was held in brine in the plastic bags with or without lactic acid bacteria inoculation. After the fish fillets were ripened at chill temperature (4 °C) for one week, they were packed in pouches with polyamide base (Polinas, Manisa, Turkey). The thickness of the pouches was 90 lm and water and oxygen permeability were 8.5 g/m2/24 h and 160 cm3/ m2/24 h, respectively. A Reepack RV50 vacuum packaging machine (Seriate (BG), Italy) was used for vacuum packaging. The fish fillets were stored at 4 °C for 35 days were analysed once a week (on days of 0, 7, 14, 21, 28, 35 and 42). The data were obtained from three fish fillets from each of three bags for each treatment.
incubation, they were initially diluted with nutrient broth to get 1011 cfu/ml bacteria count. After that, absorbance measurements at 550 nm by spectrophotometry (Perkin Elmer, Lambda 25, Shelton, CT, USA) were made for each diluted bacterial suspensions to standardise the number of bacteria. The final bacterial inoculum dose for all treatment was 1011 cfu/g lactic acid bacteria and/or food borne pathogens. One millilitre of each bacterial culture was added to the brine solutions for group 1 (G1), group 2 (G2) and group 3 (G3) or directly to the fish fillets surface (for group 4, G4).
2.3. Microbiological analysis Trout samples were taken to estimate total viable counts from each of the three different fish fillets for each group. Fish muscle (10 g) was mixed with 90 ml of Ringer solution (Merck, 1.15525.0001, Darmstadt, Germany) and then stomachered (IUL instrument, Barcelona, Spain) for 3 min. Further decimal dilutions were made, and then 0.1 ml of each dilution was pipetted onto the surface of Nutrient Agar (NA, Fluka, 70148, Steinheim, Spain) for TVC. MRS (Fluka 69964 Steinheim, Spain), Muller–Hilton Agar, (MHA, Fluka 70191, Steinheim, Spain) and Mac-Conkey MUG Agar (MCA, Fluka 63014, Steinheim, Spain) were used for lactic acid bacteria, P. aeruginosa and E. coli counts, respectively. Listeria Selective Agar (LSA, Fluka 62355, Steinheim, Spain), Salmonella-Shigella Agar (SSA, Oxoid CM99, Hamshire, England) and Baird–Parker Medium (BPA, Oxoid CM0275, Hampshire, England) were used for Salmonella, Listeria and Staphylococcus counts in triplicate, respectively. They were incubated for 48 h at 30 °C except for MRS which incubated at 37 °C for 24 h.
2.2. Bacterial inoculation
2.4. Biogenic amine analysis
The lactic acid bacteria species used were Lactococcus lactic subsp. cremoris MG 1363, Lactococcus lactic subsp. lactic IL 1403, L. plantarum FI8595 and S. thermophilus NCFB 2392 that were obtained from Sutcu Imam University, Kahramanmarasß, Turkey in BGML stock culture. Staphylococcus aureus ATCC29213, Escherichia coli ATCC25922, K. pneumoniae ATCC700603, E. faecalis ATCC29212, Pseudomonas aeruginosa ATCC27853, and Listeria monocytogenes ATCC7677 were purchased from the American Type Culture Collection (Rockville, MD, USA). Aeromonas hydrophila NCIMB 1135 and Salmonella parathypi A NCTC13 were obtained from the National Collections of Industrial Food and Marine Bacteria (Aberdeen, UK) and the National Collection of Type Cultures (London, UK), respectively. Lactic acid bacteria and food borne pathogens were grown separately in nutrient broth (Merck 1.05443.0500, Darmstadt, Germany) for 24 h at 37 °C and 24–48 h at 30 °C, respectively. After
2.4.1. Preparation of standard amine solution All biogenic amine standards were purchased from Sigma– Aldrich (Munich, Germany). The mobile phase for HPLC consisted of acetonitrile and HPLC grade water for amine analyses. Tryptamine hydrochloride (122.8 mg), putrescine dihydrochloride (182.9 mg), 2-phenylethylamine hydrochloride (130.1 mg), cadaverine dihydrochloride (171.4 mg), spermidine trihydrochloride (175.3 mg), spermine tetrahydrochloride (172.0 mg), histamine dihydrochloride (165.7 mg), tyramine hydrochloride (126.7 mg), 5-hydroxytryptamine (serotonin) (133.9 mg), 3-hydroxytyramine hydrochloride (dopamine) (123.8 mg), agmatine sulphate (175. 4 mg), trimethylamine hydrochloride (161.7 mg) and ammonia 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 solution. A standard curve for each of the biogenic amines in the range of 0–5 mg/ml (0, 0.005, 0.05, 0.5 and 5 mg/ml) was prepared.
Table 1 Treatment (groups) of experiment. Groups
G1 G2 G3 G4
Brine solutionsa
Bacterial inoculationsb
NaCl (%)
Ascorbic acid (%)
Glucose (%)
KNO3 (%)
10 10 20 –
0.5 0.5 0.5 –
18 18 18 –
0.62 0.62 0.62 –
LABb (%)
PBSc (%)
+ + +
+ + + +
–: No inoculation of LAB. +: Inoculation of LAB and PBS. : Addition of brine solution content. a The amount of the added brine solution was 5% of the fish fillet weight. b LAB, lactic acid bacteria species, Lactococcus lactic subsp. cremoris, Lactococcus lactic subsp. lactic, Lactobacillus plantarum, Streptococcus thermophilus. c FBS, food borne pathogenic bacteria, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterococus faecalis, Pseudomonas aeruginosa, Listeria monocytogenes, Aeromonas hydrophila, and Salmonella parathypi A. One millilitre of each bacterial culture with 1011 cfu/g final inoculum dose were added to the brine solutions.
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2.4.2. Sample preparation for biogenic amine analysis Ammonia and biogenic amine analysis was performed using a rapid HPLC method (Özogul, Taylor, Quantick, & Özogul, 2002). Fish white muscle without skin (5 g) was taken from each fish fillet and transferred to a 50 ml centrifuge tube. The sample was then homogenised using the Ultra-Turax (T 25 basic IKA-WERKE, Staufen, Germany) at level 2 (9500 1/min) with 20 ml 6% TCA for 3 min, centrifuged using a Hettich 32R centrifuge (Tuttlingen, Germany) at 11,180g (10.000 rpm) for 10 min at 4 °C, and filtered through Whatman No. 1 filter paper (Maidenstone, UK). The aliquot (about 20 ml) was brought to 50 ml with distiled water and was stored in a freezer ( 18 °C) for no longer than 8 weeks until derivatisation. 2.4.3. Derivatisation procedure Benzoyl chloride (%99, Sigma–Aldrich, Steinheim, Germany) was used as a derivatisation reagent. For derivatisation of standard amine solutions, 50 ll were taken (4 ml for extracted bacterial samples) from each free base standard solution (10 mg/ml). One millilitre of 2 M sodium hydroxide was added, followed by 1 ml benzoyl chloride, and mixed on a ZX3 Vortex mixer (Welp Scientifica, Usmate (Milano), Italy) for 1 min. The reaction mixture was left at room temperature (24 °C) for 20 min. The benzoylation was stopped by adding 2 ml of saturated sodium chloride solution and the solution was extracted two times with 2 ml of diethyl ether. The upper organic layer was transferred into a clean tube after mixing and evaporated to dryness in a stream of pure nitrogen (99.9%, Linde Gas, Adana, Turkey). The residue was dissolved in 500 ll of acetonitrile and 5 ll aliquots were injected into the HPLC. 2.4.4. Apparatus and columns 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. For data analysis the LC solution version 1.11 SP1 program (Shimadzu, Kyoto, Japan) was used. The column used was a reverse-phase, Spherisorb 5 Si C18 pH-St, 250 4.6 mm column (Phenomenex, Macclesfield, Cheshire, UK). 2.5. Statistical analysis The mean value and standard deviation were calculated from the data obtained from the three samples for each treatment and for each specific storage time. One way ANOVA was used to determine the significance of the differences at p < 0.05. All statistics were performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. Microbiological changes Table 2 shows the microbiological changes of the trout stored at 4 °C. In fresh trout, the initial total viable counts and lactic acid bacteria counts were 4.76 and 3.92 log cfu/g, respectively. The initial total viable counts in trout were 4–6 log cfu/g (Gonzalez, Lopez, Garcia, Prieto, & Otero, 1999). After bacterial inoculation, the bacterial counts increased rapidly and reached maximum levels at the end of the storage periods. The highest bacterial growth was observed for G4, reaching the value of 11 log cfu/g at the end of the storage period, which indicates that the addition of brine solution has an inhibitory effect on growth of bacteria. Petaja
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et al. (2000) reported that an initial lactic acid bacteria count of 4.9 log cfu/g in fresh trout increased to 8.3 log cfu/g, in fillets inoculated with lactic acid bacteria at doses of 107 log cfu/g, and to 5.9 log cfu/g in control fillets, after 35 days of fermentation. Total viable counts in MHA for G1 and G4 were found to be in the range of 4.63–10.9 log cfu/g during the storage period of fermented trout fillets. Although the Salmonella load in G2 and G3 was below 5.30 log cfu/g, Salmonella counts ranged from 4.45 and 4.11, to 6.60 and 7.85 log cfu/g, for G1 and G4, respectively. The range of Listeria counts in fermented trout fillets were 3.18–6.40 for G1, 3.15–6.38 for G2, 3.00–6.00 for G3 and 3.20–7.2 for G4. Many studies have shown that lactic acid bacteria have an inhibitory effect on Listeria monocytogenes, Salmonella spp. (Fernandez, Boris, & Barbes, 2003), Staphylococcus aureus and E. coli O157:H7 (Tadesse, Ephraim, & Ashenaf, 2005) in different growth media, a fact that is in agreement with this study. Besides lactic acid bacteria inoculation, the brine solution had an important effect on growth of PBS. In this study, the Staphyloccocus counts were between 3.48 and 3.86 log cfu/g at 7 days for all groups and reached the maximum level of 7.15 log cfu/g for G1 at the end of the storage period. Although E. coli counts for all treatments were below 3.5 log cfu/g up to 14 days, towards the end of the storage periods the highest growth of that was observed for G4 and G1, respectively. 3.2. Ammonia and biogenic amine formation Ammonia and biogenic amine production in trout are given in Table 3 for all groups. No significant differences were found in the ammonia concentrations within the treatments until 7 days of storage (p > 0.05), after which the differences were significant within groups (p < 0.05). The initial ammonia content in fresh trout fillets was found to be 3.06 mg/100 g for all groups. G2 had a higher ammonia content than G1 during the storage period. There were similar increases in the ammonia content between G2 and G3. The highest ammonia content was obtained from G4 at the end of the storage, and corresponded to the highest bacterial counts (11 log cfu/g) among the groups. Özogul, Özogul, and Kuley (2008) reported that the initial amount of ammonia was 0.02 mg/100 g and reached 1.76 mg/100 g for white grouper stored at 4 °C at the end of the storage period. Significant changes in the amine contents for each group during the storage period were observed (p < 0.05), with the exception of agmatine. The production of 12 biogenic amines was found in all groups, while 2-phenylethyl amine was not detected in any of the analysed samples. Biogenic amine formation is influenced by brine solution which may suppress some amines accumulation during the fermentation (Gardini et al., 2001). Histamine production was the highest in fermented fish with lactic acid bacteria without any salt, glucose and KNO3 addition (G4). Initial histamine concentration was 2.61 mg/100 g and increased to 6.44 mg/100 g for G4, 4.62 mg/100 g for G3, 7.41 mg/100 g for G2 and 4.11 mg/ 100 g for G1 at the end of storage period. Tsai et al. (2006) found that Bacillus coagulans and Bacillus megaterium in fermented fish products were capable of producing histamine. Moreover, Chang, Kung, Chen, Lin, and Tsai (2008) indicated that Staphylococcus spp., and S. aureus were capable of producing 13–33 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine. In the present study, the highest S. aureus counts were observed for G1 and G4, respectively. Tome et al. (2008) found that lactic acid bacteria strains isolated from vacuum-packaged and cold-smoked salmon were not able to produce histamine, but Enterococcus faecium ET05, Lactobacillus curvatus ET06 and L. curvatus ET30 produced tyramine. In this study, the microbiological results showed that the lactic acid bacteria contents in G1 were lower than in G2, although the histamine content of G1 and G2 were generally
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Table 2 Bacterial counts (log cfu/g) in fresh and fermented trout fillets in different growth medium. Storage days Growth medium
0
7
14
21
28
35
42
Groups
NA
MHA
4.76a ± 0.4b 4.76 ± 0.41 4.76 ± 0.41 4.76 ± 0.41 3.92 ± 0.12 3.92 ± 1.12 3.92 ± 0.12 3.92 ± 0.12 NT
SSA
NT
LSA
NT
MCA
NT
BPA
NT
5.58 ± 0.67 5.45 ± 0.67 5.48 ± 0.69 5.93 ± 0.32 4.10 ± 0.69 4.70 ± 0.58 4.50 ± 0.67 5.25 ± 0.94 4.63 ± 0.07 4.65 ± 0.77 4.90 ± 0.15 5.23 ± 0.94 4.45 ± 0.06 3.62 ± 0.97 3.57 ± 0.35 4.11 ± 0.11 4.34 ± 0.03 3.62 ± 0.77 3.45 ± 0.07 4.00 ± 0.03 3.02 ± 1.32 2.45 ± 1.24 2.98 ± 0.36 2.67 ± 0.27 3.74 ± 0.01 3.86 ± 0.10 3.48 ± 0.49 3.57 ± 1.09
5.56 ± 0.72 5.48 ± 0.42 5.60 ± 0.32 6.00 ± 1.21 5.03 ± 0.76 5.20 ± 1.42 5.21 ± 0.72 6.41 ± 1.32 6.00 ± 0.10 5.00 ± 0.35 5.11 ± 0.50 6.00 ± 1.05 4.86 ± 0.19 3.41 ± 0.49 3.73 ± 0.52 4.32 ± 0.56 3.18 ± 0.07 3.15 ± 0.31 3.23 ± 0.16 3.20 ± 0.17 3.46 ± 0.20 2.80 ± 0.41 3.40 ± 0.43 3.00 ± 0.31 4.34 ± 0.30 3.62 ± 1.02 3.45 ± 0.83 4.00 ± 0.57
7.62 ± 0.65 7.56 ± 0.74 7.26 ± 0.91 8.63 ± 0.88 5.11 ± 0.96 5.57 ± 0.79 5.54 ± 1.71 7.53 ± 0.88 6.57 ± 0.81 6.46 ± 0.55 6.24 ± 0.91 7.89 ± 0.89 5.59 ± 0.98 3.88 ± 0.30 3.18 ± 0.71 7.85 ± 0.42 4.70 ± 0.02 3.40 ± 0.19 3.00 ± 0.29 5.41 ± 0.17 5.30 ± 0.93 4.60 ± 1.01 4.80 ± 0.09 5.30 ± 0.12 5.20 ± 0.07 3.50 ± 0.17 3.70 ± 0.07 4.80 ± 0.64
9.08 ± 0.47 9.48 ± 1.67 9.36 ± 0.45 10.78 ± 0.61 6.34 ± 1.48 7.40 ± 2.20 7.11 ± 0.45 8.15 ± 0.61 7.80 ± 0.55 7.36 ± 0.61 7.18 ± 0.93 9.26 ± 0.99 5.36 ± 0.38 3.61 ± 0.26 3.00 ± 0.55 7.58 ± 0.44 6.00 ± 0.36 5.15 ± 0.62 4.76 ± 0.80 6.48 ± 0.05 6.59 ± 0.81 6.45 ± 0.91 6.18 ± 0.22 7.41 ± 0.16 6.38 ± 0.74 3.60 ± 0.44 5.46 ± 0.68 6.48 ± 0.29
10.52 ± 0.78 9.69 ± 0.76 9.08 ± 0.86 10.18 ± 0.85 5.96 ± 0.79 6.74 ± 0.76 7.32 ± 0.56 7.40 ± 1.15 8.70 ± 0.72 8.28 ± 0.60 7.48 ± 0.32 9.78 ± 1.04 6.23 ± 0.75 3.40 ± 0.78 3.00 ± 0.92 6.18 ± 0.33 6.20 ± 1.10 6.38 ± 0.81 5.81 ± 1.03 6.71 ± 0.22 7.56 ± 0.10 7.20 ± 0.17 6.78 ± 0.14 7.90 ± 0.27 6.73 ± 0.08 5.98 ± 0.09 5.65 ± 0.91 6.18 ± 0.45
9.85 ± 0.97 9.52 ± 0.25 8.94 ± 0.59 11.00 ± 0.51 6.46 ± 0.47 7.00 ± 0.25 7.80 ± 1.54 8.11 ± 0.61 10.90 ± 0.78 9.15 ± 0.63 9.00 ± 0.53 10.85 ± 1.51 6.60 ± 0.29 5.26 ± 0.26 4.48 ± 0.50 6.00 ± 0.63 6.40 ± 0.15 5.90 ± 1.05 6.00 ± 0.46 7.20 ± 0.30 6.32 ± 1.06 6.39 ± 0.22 6.00 ± 0.06 8.32 ± 0.08 7.15 ± 0.92 6.43 ± 1.00 6.48 ± 0.13 6.34 ± 0.39
G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4
MRS
NT, not tested; NA, Nutrient Agar; MRS, de man, Rogosa and Sharpe Agar; MHA, Muller Hilton Agar; SSA, Salmonella Shigella Agar; LSA, Listeria Sellective agar; MCA, Macconkey MUG agar; BPA, Baird Parker Agar. a Mean value. b Standard deviation.
were similar. Significant differences between G1 and G2 were only observed at day 35 and 42, which indicate a minor effect of lactic acid bacteria on histamine formation. Petaja et al. (2000) indicated that histamine and cadaverine production during storage of fermented rainbow trout were higher in the group without any lactic acid bacteria inoculation than the other groups with three different lactic acid bacteria inoculations with staphylococci. Some lactic acid bacteria strains were reported to considerably stimulate histamine production by most of the pathogen in histidine and tyrosine enrichment medium (Özogul, 2011; Kuley & Özogul, 2011). In the current study the presence of lactic acid bacteria strains in fish fillets statistically had no effect on the histamine production by food borne pathogens until 28 days of storage, apart from G4. The histamine content in trout fillets in G4 was found to be more than 10 mg/100 g, while G1, G2 and G3 contained less than 7.5 mg/100 g during storage. This might be due to the fact that G4 did not contain salt, glucose and KNO3. The biogenic amine production was depended on the microbial flora, availability of precursors and physicochemical factors, such as temperature, pH, salt, oxygen and sugar concentration (Zaman, Abdulamir, Bakar, Selamat, & Bakar 2009). Yongjin et al. (2007) reported that the main amines formed were histamine, cadaverine, putrescine and tyramine during a 48 h fermentation of silver carp sausages with 3% NaCl and glucose, and a mixed starter culture. The amine levels were found to be much higher than in the present study. The fish sauces fermented by the lactic acid microflora produced very high amounts of tyramine, cadaverine, putrescine, tryptamine and histamine (Kirschbaum, Rebscher, & Brückner 2000). Cadaverine was the major amine detected in Egyptian saltedfermented bouri fish samples during ripening and storage, followed by putrescine (Rabie, Sarkadi, Siliha, El-seedy, & El Badawy, 2009). In this study, cadaverine was not detected in the fish fillets
at 0 and 7 days, whilst significant increases in the cadaverine content of G4 were observed after 14 days of storage. No significant differences were found in the putrescine concentrations within the treatments during the early stages of the storage period. However, there was a significant difference (p < 0.05) in the putrescine content towards the end of the storage period for all treatments. The spermine and spermidine contents in fish and fish sauce are generally low (Tsai et al., 2006). In this study, the changes in spermidine in G1, G2 and G3 were similar. The initial value of spermidine was 1.06 mg/100 g, whereas G1, G2 and G3 showed slight increases at the end of storage, but not G4. Tryptamine and spermine production among the three groups (G1, G2 and G3) was similar and remained below 2.5 mg/100 g and 1 mg/100 g, respectively. The highest tryptamine and spermine production (2.71 and 3.94 mg/100 g) was observed for G4 at the end of the storage period. Among the analysed biogenic amines, the highest production of biogenic amines during the storage period was found for serotonin and tyramine in G1 and G3, respectively. Serotonin and tyramine were significantly higher in G1, G2 and G3 than G4 at 7 days of storage. These results indicated that adding brine solution had a stimulatory effect on serotonin or tyramine formation. Serotonin production was reported for white grouper (Özogul et al., 2008). Nevertheless, serotonin was not detected in fermented fish sauce (Kirschbaum et al., 2000). Tyramine is quantitatively the most common biogenic amine in fermented meat products (Komprda, Neznalova, Standara, & BoverCid, 2001). The tyramine level of 10–80 mg/100 g in foods has been demonstrated to be toxic (Shalaby, 1997). The initial tyramine concentration was 1.44 mg/100 g in fresh rainbow trout fillets and then reached maximum levels of 18.48 and 16.88 mg/100 g in G4 and G3, respectively, at 14 and 35 days of storage. In our previous work, it was reported that the combination of specific lactic acid
Table 3 The concentration of ammonia and biogenic amines (mg/100 g) in trout fillets. AMN
PUT
CAD
HIS
SPD
TRPT
PHEN
SPM
SER
TYR
TMA
AGM
Groups
0
3.06x ± 0.52y 3.06a ± 0.52 3.06a ± 0.52 3.06a ± 0.52 3.76a ± 0.25 2.81a ± 0.22 3.56a ± 0.34 3.51a ± 0.86 2.81b ± 0.59 4.38a ± 0.78 3.78ab ± 0.85 3.35ab ± 0.30 2.12 b ± 0.36 2.53b ± 0.52 4.60a ± 0.98 2.95b ± 0.09 3.15b ± 0.24 3.38b ± 0.87 3.46b ± 1.25 6.50a ± 0.53 2.54b ± 0.44 7.03a ± 1.16 6.94a ± 0.11 6.38a ± 1.11 5.31b ± 1.27 7.24b ± 0.97 5.87b ± 1.02 13.3a ± 1.45
0.70a ± 0.03 0.70a ± 0.03 0.70a ± 0.03 0.70a ± 0.03 0.50a ± 0.12 0.52a ± 0.00 0.70a ± 0.08 0.84a ± 0.01 0.70a ± 0.20 1.58b ± 0.06 0.58a ± 0.05 1.38b ± 0.91 0.52b ± 0.16 0.55b ± 0.07 0.87b ± 0.02 3.87a ± 0.56 0.61b ± 0.15 0.47b ± 0.06 0.34b ± 0.01 7.70a ± 0.66 0.65c ± 0.29 1.72b ± 0.15 1.10bc ± 0.10 11.8a ± 0.68 1.20b ± 0.15 1.89b ± 0.39 1.07b ± 0.13 20.7a ± 1.50
– – – – – – – – – 1.55b ± 0.34 0.06a ± 0.01 1.32b ± 0.17 – 0.37b ± 0.01 – 3.97a ± 0.79 0.03b ± 0.00 0.03b ± 0.00 0.04b ± 0.01 10.4a ± 0.22 0.33b ± 0.05 0.94b ± 0.07 – 15.0a ± 1.50 0.10c ± 0.01 1.52b ± 0.52 – 30.6a ± 0.65
2.61a ± 0.13 2.61a ± 0.13 2.61a ± 0.13 2.61a ± 0.13 2.47ab ± 0.19 2.12b ± 0.26 2.28ab ± 0.31 2.78b ± 0.15 4.11ab ± 0.79 5.77a ± 2.56 2.55b ± 0.31 5.17a ± 0.81 2.48b ± 0.68 2.41b ± 0.93 3.25b ± 0.36 8.89a ± 0.43 2.42b ± 0.34 2.06b ± 0.18 1.95b ± 0.45 23.0a ± 0.35 2.72c ± 0.78 5.79b ± 0.43 3.45bc ± 0.21 33.3a ± 0.51 4.62c ± 0.21 7.41b ± 0.3 3.73d ± 0.07 6.44a ± 0.24
1.06a ± 0.15 1.06a ± 0.15 1.06a ± 0.15 1.06 a ± 0.15 0.89b ± 0.09 0.85b ± 0.23 0.96b ± 0.05 1.36a ± 0.29 1.21b ± 0.02 0.64a ± 0.13 0.80a ± 0.27 0.56a ± 0.01 0.66ab ± 0.03 0.68ab ± 0.23 1.23a ± 0.14 0.05c ± 0.01 0.88a ± 0.04 0.80ab ± 0.06 0.71b ± 0.08 0.12c ± 0.04 0.70b ± 0.08 1.48a ± 0.14 1.32a ± 0.04 0.12 ± 0.00 1.14a ± 0.15 1.26a ± 0.21 1.28a ± 0.21 –
– – – – 0.93a ± 0.05 – 0.74a ± 0.01 0.16b ± 0.01 0.14a ± 0.04 0.83a ± 0.18 0.57a ± 0.09 0.90a ± 0.05 – 0.47b ± 0.08 0.91a ± 0.07 0.23c ± 0.01 0.59b ± 0.01 0.46b ± 0.01 0.52b ± 0.19 1.14a ± 0.40 0.61b ± 0.00 1.00ab ± 0.15 0.86ab ± 0.04 1.32a ± 0.84 0.91b ± 0.01 0.70b ± 0.03 0.45b ± 0.01 2.71a ± 0.60
– – – – – – – – – – – – – – – – – – – – – – – – – – – –
1.85a ± 0.13 1.85a ± 0.13 1.85a ± 0.13 1.85a ± 0.13 1.51a ± 0.15 1.24a ± 0.26 1.38a ± 0.17 1.65a ± 0.33 1.61a ± 0.54 1.86a ± 0.76 1.35a ± 0.50 1.62a ± 0.68 1.08b ± 0.27 1.06b ± 0.12 1.97a ± 0.25 1.29b ± 0.26 1.53b ± 0.35 1.35b ± 0.01 1.13b ± 0.41 2.79a ± 0.49 1.22c ± 0.64 2.31b ± 0.09 2.42b ± 0.21 3.27a ± 0.56 1.69c ± 0.15 2.07b ± 0.22 1.86bc ± 0.27 3.94a ± 0.08
2.42a ± 0.53 2.42a ± 0.53 2.42a ± 0.53 2.42a ± 0.53 11.6a ± 0.43 11.9a ± 1.82 10.5a ± 1.29 2.69b ± 0.40 18.0a ± 2.51 1.84b ± 0.08 12.8a ± 0.80 1.15b ± 0.06 12.4a ± 1.65 3.20b ± 0.65 11.2a ± 1.20 0.56c ± 0.02 8.05a ± 2.61 4.31b ± 0.57 6.29ab ± 0.68 0.35c ± 0.06 4.97b ± 0.42 6.86b ± 1.61 12.2a ± 2.03 0.33c ± 0.00 12.2b ± 2.08 6.83c ± 1.49 18.8a ± 0.04 2.63d ± 0.43
1.44a ± 0.60 1.44a ± 0.60 1.44a ± 0.60 1.44a ± 0.60 6.32ab ± 0.69 4.36b ± 1.46 8. 84a ± 0.24 0.39c ± 0.32 16.9 a ± 1.33 6.96 ab ± 2.16 8.61a ± 0.64 2.40b ± 1.06 8.57b ± 2.09 6.66b ± 0.92 14.5a ± 1.58 0.45c ± 0.07 4.13a ± 1.65 2.09b ± 0.57 3.70ab ± 0.66 0.79c ± 0.21 5.05b ± 0.20 4.50b ± 0.22 18.5a ± 1.19 1.25c ± 0.19 3.21c ± 0.67 4.49b ± 0.37 12.1a ± 0.37 5.02b ± 0.66
0.61a ± 0.66 0.61a ± 0.66 0.61a ± 0.66 0.6 a ± 0.66 2.65ab ± 0.33 1.89ab ± 0.09 3.71a ± 0.43 0.00c ± 0.00 8.76a ± 1.22 3.11b ± 0.08 3.70b ± 0.10 1.32b ± 0.01 3.64ab ± 1.07 3.13b ± 0.41 6.89a ± 3.05 0.34b ± 0.03 1.63a ± 0.07 1.23a ± 1.06 1.45a ± 0.85 0.40a ± 0.00 2.03b ± 0.86 2.37b ± 0.52 9.72a ± 1.44 1.11c ± 0.20 1.09c ± 0.27 5.80b ± 0.74 4.66b ± 1.07 9.48a ± 0.62
– – – – 3.06a ± 0.14 0.16b ± 0.02 3.06a ± 0.33 – – 10.9a ± 1.88 2.55b ± 0.41 6.23ab ± 0.92 – – – – – – – – 0.73a ± 0.27 – – – 8.39a ± 1.17 0.38c ± 0.01 5.85b ± 0.38 –
G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4
7
14
21
28
35
42
E. Kuley et al. / Food Chemistry 129 (2011) 1211–1216
Storage days
AMN, ammonia; PUT, putrescine; CAD, cadaverine; HIS, histamine; SPD, spermidine; TRP, tryptamine; PHEN, 2-Phenyl–ethylamine; SPN, spermine; SER, serotonin; TYR, tyramine; TMA, trimethylamine; AGM, agmatine. –, not detected. –, Not detected. a Mean value. b Standard deviation (n = 3). Different lowercase letters (a–d) in a row indicate significant differences (p < 0.05) among groups.
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bacteria and foodborne pathogens resulted in significantly higher tyramine accumulation in histidine or tyrosine enriched medium (Kuley & Özogul, 2011; Özogul, 2011). However, in the current study, G4 had significantly lower tyramine formation compared to G1 (in which lactic acid bacteria is not inoculated), during all storage periods, except for 42 days. Moreover, the presence of lactic acid bacteria strains in the brine solution had generally no significant effect on the tyramine production by foodborne pathogens. Agmatine was not detected at 0, 21 and 28 days for all groups. These results suggested that inoculation with lactic acid bacteria and foodborne pathogens did not significantly contribute to the agmatine production of fish fillets. However, major changes in the content of agmatine were observed for G2 and G4 at 14 days, and for G1 and G3 at 7 and 42 days. Özogul and Özogul (2007) demonstrated that bacteria could vary in their relative importance as biogenic amine producers in different decarboxylase broths. They found that K. pneumoniae and E. faecalis did not produce agmatine in arginine decarboxylase broth, while the highest agmatine production by K. pneumoniae and E. faecalis were in lysine decarboxylase broth and in tyrosine decarboxylase broth, respectively. 4. Conclusions Foodborne pathogens in trout fillets had an ability to produce ammonia and biogenic amine during the fermentation and storage of fish. Lactic acid bacteria generally did not seem to play an important role on biogenic amine production by pathogenic bacteria. The result of the study also showed that ammonia and biogenic amine production could be significantly influenced by adding brine solution. Brine solution had an inhibitory effect on ammonia, histamine, putrescine and cadaverine production in trout fillets, but not for serotonin and tyramine production. Acknowledgement The authors would like to thank the Scientific Research Projects Unit in Cukurova University for their financial support (Research Project: SUF 2007 D1). References Bover-Cid, S., & Holzapfel, W. H. (1999). Improved screening procedure for biogenic amine production by lactic acid bacteria. International Journal of Food Microbiology, 53, 33–41. Brashears, M. M., Amezquita, A., & Jaroni, D. (2005). Lactic acid bacteria and their uses in animal feeding to improve food safety. Advances in Food and Nutrition Research, 50, 1–31. Chang, S.-C., Kung, H. F., Chen, H. C., Lin, C. S., & Tsai, Y. H. (2008). Determination of histamine and bacterial isolation in swordfish fillets (Xiphias gladius) implicated in a food borne poisoning. Food Control, 19, 16–21. Devlieghere, F., Vermeiren, L., & Debevere, J. (2004). New preservation technologies: Possibilities and limitations. International Dairy Journal, 14, 273–285. Fadda, S., Vignolo, G., & Oliver, G. (2001). Tyramine degradation and tyramine/ histamine production by lactic acid bacteria and Kocuria strains. Biotechnology Letters, 23, 2015–2019. Fernandez, M. F., Boris, S., & Barbes, C. (2003). Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. Journal of Applied Microbiology, 94, 449–455. Gardini, F., Martuscelli, M., Caruso, M. C., Galgano, F., Crudele, M. A., Favati, F., et al. (2001). Effects of pH, temperature and NaCl concentration on the growth
kinetics, proteolytic activity and biogenic amine production of Enterococcus faecalis. International Journal of Food Microbiology, 64, 105–117. Gonzalez, C. J., Lopez, T. M., Garcia, M. L., Prieto, M., & Otero, A. (1999). Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius), and aquacultured rainbow trout (Oncorhynchus mykiss). Journal of Food Protection, 62, 1270–1277. Kirschbaum, J., Rebscher, K., & Brückner, H. (2000). Liquid chromatographic determination of biogenic amines in fermented foods after derivatization with 3,5-dinitrobenzoyl chloride. Journal of Chromatography A, 881, 517–530. Komprda, T., Neznalova, J., Standara, S., & Bover-Cid, S. (2001). Effect of starter culture and storage temperature on the content of biogenic amines in dry fermented sausage polican. Meat Science, 59, 267–276. Kopermsub, P., & Yunchalard, S. (2010). Identification of lactic acid bacteria associated with the production of plaa-som, a traditional fermented fish product of Thailand. International Journal of Food Microbiology, 138, 200–204. Kuley, E., & Özogul, F. (2011). Synergistic and antagonistic effect of lactic acid bacteria on tyramine production by food-borne pathogenic bacteria in tyrosine decarboxylase broth. Food Chemistry, 127, 1163–1168. Mah, J.-H., & Hwang, H. J. (2009). Inhibition of biogenic amine formation in a salted and fermented anchovy by Staphylococcus xylosus as a protective culture. Food Control, 20, 796–801. Middlebrooks, B. L., Toom, P. M., Donglas, W. L., Harrison, R. E., & McDowell, S. (1988). Effects of storage time and temperature on the microflora and amine development in Spanish Mackerel (Scomberomorus maculatus). Journal of Food Science, 53, 1024–1029. Ordonez, J. A., Hierro, E. M., Bruna, J. M., & de la Hoz, L. (1999). Changes in the components of dry-fermented sausages during ripening. Critical Reviews in Food Science and Nutrition, 39, 329–367. Özogul, F. (2011). Effects of specific lactic acid bacteria species on biogenic amine production by foodborne pathogen. International Journal of Food Science and Technology, 46, 478–484. Özogul, F., & Özogul, Y. (2007). The ability of biogenic amines and ammonia production by single bacterial cultures. European Food Research Technology, 225, 385–394. Özogul, F., Özogul, Y., & Kuley, E. (2008). Nucleotide degradation and biogenic amine formation of wild white grouper (Epinephelus aeneus) stored in ice and at chill temperature (4 °C). Food Chemistry, 108, 933–941. Özogul, F., Taylor, K. D. A., Quantick, P., & Özogul, Y. (2002). Biogenic amines formation in Atlantic herring (Clupea harengus) stored under modified atmosphere packaging using a rapid HPLC method. International Journal of Food Science and Technology, 37, 515–522. Petaja, E., Eerola, S., & Petaja, P. (2000). Biogenic amines in cold-smoked fish fermented with lactic acid bacteria. European Food Research Technology, 210, 280–285. Rabie, M., Sarkadi, L. S., Siliha, H., El-seedy, S., & El Badawy, A. A. (2009). Changes in free amino acids and biogenic amines of Egyptian salted-fermented fish (Feseekh) during ripening and storage. Food Chemistry, 115, 635–638. Shalaby, A. R. (1997). Significance of biogenic amines to food safety and human health. Food Research International, 29, 475–490. Tadesse, G., Ephraim, E., & Ashenaf, M. (2005). Assessment of the antimicrobial activity of lactic acid bacteria isolated from borde and shamita, traditional ethiopian fermented beverages, on some foodborne pathogens and effect of growth medium on the inhibitory activity. International Journal of Food Safety, 5, 13–20. Tome, E., Pereira, V. L., Lopes, C. I., Gibbs, P. A., & Teixeira, P. C. (2008). In vitro tests of suitability of bacteriocin-producing lactic acid bacteria, as potential biopreservation cultures in vacuum-packaged cold-smoked salmon. Food Control, 19, 535–543. Tsai, Y. H., Lin, C. Y., Chien, L. T., Lee, T. M., Wei, C. I., & Hwang, D. F. (2006). Histamine contents of fermented fish products in Taiwan and isolation of histamine-forming bacteria. Food Chemistry, 98, 64–70. Yongjin, H., Wenshui, X., & Xiaoyong, L. (2007). Changes in biogenic amines in fermented silver carp sausages inoculated with mixed starter cultures. Food Chemistry, 104, 188–195. Zaman, M. Z., Abdulamir, A. S., Bakar, F. A., Selamat, J., & Bakar, J. (2009). Microbiological, physicochemical and health impact of high level of biogenic amines in fish sauce. American Journal of Applied Sciences, 6, 1199–1211. Zhong-Yi, L., Zhong-Hai, L., Miao-Ling, Z., & Xiao-Ping, D. (2010). Effect of fermentation with mixed starter cultures on biogenic amines in bighead carp surimi. International Journal of Food Science and Technology, 45, 930–936.
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The function of lactic acid bacteria and brine solutions on biogenic amine formation by foodborne pathogens in trout fillets Esmeray Kuley a,⇑, Fatih Özogul a, Yesim Özogul a, Ismail Akyol b a b
Department of Fish Processing Technology, Faculty of Fisheries, Cukurova University, 01330 Balcali, Adana, Turkey Department of Animal Science, Faculty of Agriculture, Sutcu Imam University, Kahramanmaras, Turkey
a r t i c l e
i n f o
Article history: Received 22 October 2010 Received in revised form 11 April 2011 Accepted 23 May 2011 Available online 27 May 2011 Keywords: Food-borne pathogen Lactic acid bacteria Rainbow trout Biogenic amine Ammonia
a b s t r a c t The influences of lactic acid bacteria and brine solutions on the biogenic amine formation by Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterococus faecalis, Pseudomonas aeruginosa, Listeria monocytogenes, Aeromonas hydrophila and Salmonella paratyphi A in fermented trout fillets were investigated. Fish fillets were divided into four groups, group 1 without any lactic acid bacteria inoculation, group 2 and group 3 with different salt concentration inoculated with lactic acid bacteria and food-borne pathogens, and group 4 inoculated with lactic acid bacteria and food-borne pathogens without a salt solution. The histamine content in trout fillets in group 4 was found to be more than 10 mg/100 g, while the other groups contained less than 7.5 mg/100 g. The highest tyramine production was found for group 1 and group 3, ranging from 3 to 18 mg/100 g. Lactic acid bacteria did not seem to play an important role on biogenic amine production by food borne pathogens, while adding brine solution on fillets has inhibitory effects on some of the biogenic amines. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Lactic acid bacteria have a significant role in food fermentations due to their impact on flavour changes and as a preservative, and thus they help to provide food safety by inhibiting pathogen growth (Devlieghere, Vermeiren, & Debevere, 2004). The primary role of lactic acid bacteria strains is to utilise carbohydrate substrates available in the fermentation system (Kopermsub & Yunchalard, 2010) and produce substances such as hydrogen peroxide, weak organic acids, reuterin, diacetyl, bacteriocins and low-molecular-weight metabolites that inhibit pathogenic organisms in fermented foods (Brashears, Amezquita, & Jaroni, 2005). The fermentation process for fish may also provide the conditions required for abundant production of biogenic amines such as availability of free amino acids, the presence of decarboxylase-positive microorganisms and conditions allowing bacterial growth, decarboxylase synthesis and decarboxylase activity (Petaja, Eerola, & Petaja, 2000). Production of biogenic amines was reported in dried and fermented fish (Tsai et al., 2006), vacuum packaged and cold smoked fermented fish products (Petaja et al., 2000; Tome, Pereira, Lopes, Gibbs, & Teixeira, 2008). Some lactic acid bacteria involved in food fermentation, such as Streptococcus thermophilus, Lactococcus lactis and Lactobacillus plantarum, may produce biogenic amines such as tyramine (Bover-Cid & Holzapfel, 1999; Fadda, Vignolo, & Oliver, 2001). ⇑ Corresponding author. Tel.: +90 322 3386084; fax: +90 322 3386439. E-mail address: [email protected] (E. Kuley). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.113
A variety of food-borne pathogens isolated from different types of food have also the ability to produce biogenic amines. Some pathogenic bacteria in the families of Escherichia, Salmonella, Clostridium, and Bacillus have been reported to show histidine decarboxylase activity (Ordonez, Hierro, Bruna, & de la Hoz, 1999). Aeromonas hydrophila produce histamine, cadaverine, and putrescine (Middlebrooks, Toom, Donglas, Harrison, & McDowell, 1988), and Klebsiella pneumoniae and Enterococcus faecalis accumulate cadaverine and tyramine, respectively (Özogul & Özogul, 2007). The interactions between mix bacterial cultures (lactic acid bacteria and food borne pathogens) in terms of biogenic amines production have not been well stated. It has been reported that starter lactic acid bacteria cultures suppressed the accumulation of some biogenic amines in some fermented fish products (Mah & Hwang, 2009; Yongjin, Wenshui, & Xiaoyong, 2007; Zhong-Yi, Zhong-Hai, Miao-Ling, & Xiao-Ping, 2010). Little research has been done on the biogenic amine formation by mixtures of food borne pathogen and lactic acid bacteria (Kuley & Özogul, 2011; Özogul, 2011). Lactic acid bacteria had an important synergetic role in some biogenic amine production by food-borne pathogenic bacteria, although the effect of some lactic acid bacteria strains on biogenic amine production was strain-dependent in vitro conditions (Kuley & Özogul, 2011; Özogul, 2011). However, little information is available on the formation of biogenic amines by mix culture in fish and fish products. Investigating the ability of food-borne pathogenic bacteria and/or lactic acid bacteria to produce biogenic amine in fish and fish products are important due to their potential impacts on human health and food quality. Therefore, the purpose
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of this study was to investigate the effects of lactic acid bacteria on biogenic amine production by food borne pathogens in rainbow trout fillets during fermentation and storage. Moreover, the effect of brine solution on the biogenic amine production by lactic acid bacteria and food borne pathogens was investigated. 2. Materials and methods 2.1. Preparation of fish samples Cultured fresh rainbow trout (Oncorhynchus mykiss) were purchased from a local fish farm in Adana, Turkey. The average weight and length of the samples were 230 ± 1.3 g and 22 ± 2 cm, respectively. Fish were delivered to the laboratory in ice after 4 h of harvesting and they were immediately gutted and filleted without skin removal. After that, the fillets were washed with sterile water and then the trout fillets were divided into four groups. The brine solutions were prepared according to the method of Petaja et al. (2000). The groups of experiment are given in Table 1. For the fermentation process, each group was held in brine in the plastic bags with or without lactic acid bacteria inoculation. After the fish fillets were ripened at chill temperature (4 °C) for one week, they were packed in pouches with polyamide base (Polinas, Manisa, Turkey). The thickness of the pouches was 90 lm and water and oxygen permeability were 8.5 g/m2/24 h and 160 cm3/ m2/24 h, respectively. A Reepack RV50 vacuum packaging machine (Seriate (BG), Italy) was used for vacuum packaging. The fish fillets were stored at 4 °C for 35 days were analysed once a week (on days of 0, 7, 14, 21, 28, 35 and 42). The data were obtained from three fish fillets from each of three bags for each treatment.
incubation, they were initially diluted with nutrient broth to get 1011 cfu/ml bacteria count. After that, absorbance measurements at 550 nm by spectrophotometry (Perkin Elmer, Lambda 25, Shelton, CT, USA) were made for each diluted bacterial suspensions to standardise the number of bacteria. The final bacterial inoculum dose for all treatment was 1011 cfu/g lactic acid bacteria and/or food borne pathogens. One millilitre of each bacterial culture was added to the brine solutions for group 1 (G1), group 2 (G2) and group 3 (G3) or directly to the fish fillets surface (for group 4, G4).
2.3. Microbiological analysis Trout samples were taken to estimate total viable counts from each of the three different fish fillets for each group. Fish muscle (10 g) was mixed with 90 ml of Ringer solution (Merck, 1.15525.0001, Darmstadt, Germany) and then stomachered (IUL instrument, Barcelona, Spain) for 3 min. Further decimal dilutions were made, and then 0.1 ml of each dilution was pipetted onto the surface of Nutrient Agar (NA, Fluka, 70148, Steinheim, Spain) for TVC. MRS (Fluka 69964 Steinheim, Spain), Muller–Hilton Agar, (MHA, Fluka 70191, Steinheim, Spain) and Mac-Conkey MUG Agar (MCA, Fluka 63014, Steinheim, Spain) were used for lactic acid bacteria, P. aeruginosa and E. coli counts, respectively. Listeria Selective Agar (LSA, Fluka 62355, Steinheim, Spain), Salmonella-Shigella Agar (SSA, Oxoid CM99, Hamshire, England) and Baird–Parker Medium (BPA, Oxoid CM0275, Hampshire, England) were used for Salmonella, Listeria and Staphylococcus counts in triplicate, respectively. They were incubated for 48 h at 30 °C except for MRS which incubated at 37 °C for 24 h.
2.2. Bacterial inoculation
2.4. Biogenic amine analysis
The lactic acid bacteria species used were Lactococcus lactic subsp. cremoris MG 1363, Lactococcus lactic subsp. lactic IL 1403, L. plantarum FI8595 and S. thermophilus NCFB 2392 that were obtained from Sutcu Imam University, Kahramanmarasß, Turkey in BGML stock culture. Staphylococcus aureus ATCC29213, Escherichia coli ATCC25922, K. pneumoniae ATCC700603, E. faecalis ATCC29212, Pseudomonas aeruginosa ATCC27853, and Listeria monocytogenes ATCC7677 were purchased from the American Type Culture Collection (Rockville, MD, USA). Aeromonas hydrophila NCIMB 1135 and Salmonella parathypi A NCTC13 were obtained from the National Collections of Industrial Food and Marine Bacteria (Aberdeen, UK) and the National Collection of Type Cultures (London, UK), respectively. Lactic acid bacteria and food borne pathogens were grown separately in nutrient broth (Merck 1.05443.0500, Darmstadt, Germany) for 24 h at 37 °C and 24–48 h at 30 °C, respectively. After
2.4.1. Preparation of standard amine solution All biogenic amine standards were purchased from Sigma– Aldrich (Munich, Germany). The mobile phase for HPLC consisted of acetonitrile and HPLC grade water for amine analyses. Tryptamine hydrochloride (122.8 mg), putrescine dihydrochloride (182.9 mg), 2-phenylethylamine hydrochloride (130.1 mg), cadaverine dihydrochloride (171.4 mg), spermidine trihydrochloride (175.3 mg), spermine tetrahydrochloride (172.0 mg), histamine dihydrochloride (165.7 mg), tyramine hydrochloride (126.7 mg), 5-hydroxytryptamine (serotonin) (133.9 mg), 3-hydroxytyramine hydrochloride (dopamine) (123.8 mg), agmatine sulphate (175. 4 mg), trimethylamine hydrochloride (161.7 mg) and ammonia 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 solution. A standard curve for each of the biogenic amines in the range of 0–5 mg/ml (0, 0.005, 0.05, 0.5 and 5 mg/ml) was prepared.
Table 1 Treatment (groups) of experiment. Groups
G1 G2 G3 G4
Brine solutionsa
Bacterial inoculationsb
NaCl (%)
Ascorbic acid (%)
Glucose (%)
KNO3 (%)
10 10 20 –
0.5 0.5 0.5 –
18 18 18 –
0.62 0.62 0.62 –
LABb (%)
PBSc (%)
+ + +
+ + + +
–: No inoculation of LAB. +: Inoculation of LAB and PBS. : Addition of brine solution content. a The amount of the added brine solution was 5% of the fish fillet weight. b LAB, lactic acid bacteria species, Lactococcus lactic subsp. cremoris, Lactococcus lactic subsp. lactic, Lactobacillus plantarum, Streptococcus thermophilus. c FBS, food borne pathogenic bacteria, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterococus faecalis, Pseudomonas aeruginosa, Listeria monocytogenes, Aeromonas hydrophila, and Salmonella parathypi A. One millilitre of each bacterial culture with 1011 cfu/g final inoculum dose were added to the brine solutions.
E. Kuley et al. / Food Chemistry 129 (2011) 1211–1216
2.4.2. Sample preparation for biogenic amine analysis Ammonia and biogenic amine analysis was performed using a rapid HPLC method (Özogul, Taylor, Quantick, & Özogul, 2002). Fish white muscle without skin (5 g) was taken from each fish fillet and transferred to a 50 ml centrifuge tube. The sample was then homogenised using the Ultra-Turax (T 25 basic IKA-WERKE, Staufen, Germany) at level 2 (9500 1/min) with 20 ml 6% TCA for 3 min, centrifuged using a Hettich 32R centrifuge (Tuttlingen, Germany) at 11,180g (10.000 rpm) for 10 min at 4 °C, and filtered through Whatman No. 1 filter paper (Maidenstone, UK). The aliquot (about 20 ml) was brought to 50 ml with distiled water and was stored in a freezer ( 18 °C) for no longer than 8 weeks until derivatisation. 2.4.3. Derivatisation procedure Benzoyl chloride (%99, Sigma–Aldrich, Steinheim, Germany) was used as a derivatisation reagent. For derivatisation of standard amine solutions, 50 ll were taken (4 ml for extracted bacterial samples) from each free base standard solution (10 mg/ml). One millilitre of 2 M sodium hydroxide was added, followed by 1 ml benzoyl chloride, and mixed on a ZX3 Vortex mixer (Welp Scientifica, Usmate (Milano), Italy) for 1 min. The reaction mixture was left at room temperature (24 °C) for 20 min. The benzoylation was stopped by adding 2 ml of saturated sodium chloride solution and the solution was extracted two times with 2 ml of diethyl ether. The upper organic layer was transferred into a clean tube after mixing and evaporated to dryness in a stream of pure nitrogen (99.9%, Linde Gas, Adana, Turkey). The residue was dissolved in 500 ll of acetonitrile and 5 ll aliquots were injected into the HPLC. 2.4.4. Apparatus and columns 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. For data analysis the LC solution version 1.11 SP1 program (Shimadzu, Kyoto, Japan) was used. The column used was a reverse-phase, Spherisorb 5 Si C18 pH-St, 250 4.6 mm column (Phenomenex, Macclesfield, Cheshire, UK). 2.5. Statistical analysis The mean value and standard deviation were calculated from the data obtained from the three samples for each treatment and for each specific storage time. One way ANOVA was used to determine the significance of the differences at p < 0.05. All statistics were performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. Microbiological changes Table 2 shows the microbiological changes of the trout stored at 4 °C. In fresh trout, the initial total viable counts and lactic acid bacteria counts were 4.76 and 3.92 log cfu/g, respectively. The initial total viable counts in trout were 4–6 log cfu/g (Gonzalez, Lopez, Garcia, Prieto, & Otero, 1999). After bacterial inoculation, the bacterial counts increased rapidly and reached maximum levels at the end of the storage periods. The highest bacterial growth was observed for G4, reaching the value of 11 log cfu/g at the end of the storage period, which indicates that the addition of brine solution has an inhibitory effect on growth of bacteria. Petaja
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et al. (2000) reported that an initial lactic acid bacteria count of 4.9 log cfu/g in fresh trout increased to 8.3 log cfu/g, in fillets inoculated with lactic acid bacteria at doses of 107 log cfu/g, and to 5.9 log cfu/g in control fillets, after 35 days of fermentation. Total viable counts in MHA for G1 and G4 were found to be in the range of 4.63–10.9 log cfu/g during the storage period of fermented trout fillets. Although the Salmonella load in G2 and G3 was below 5.30 log cfu/g, Salmonella counts ranged from 4.45 and 4.11, to 6.60 and 7.85 log cfu/g, for G1 and G4, respectively. The range of Listeria counts in fermented trout fillets were 3.18–6.40 for G1, 3.15–6.38 for G2, 3.00–6.00 for G3 and 3.20–7.2 for G4. Many studies have shown that lactic acid bacteria have an inhibitory effect on Listeria monocytogenes, Salmonella spp. (Fernandez, Boris, & Barbes, 2003), Staphylococcus aureus and E. coli O157:H7 (Tadesse, Ephraim, & Ashenaf, 2005) in different growth media, a fact that is in agreement with this study. Besides lactic acid bacteria inoculation, the brine solution had an important effect on growth of PBS. In this study, the Staphyloccocus counts were between 3.48 and 3.86 log cfu/g at 7 days for all groups and reached the maximum level of 7.15 log cfu/g for G1 at the end of the storage period. Although E. coli counts for all treatments were below 3.5 log cfu/g up to 14 days, towards the end of the storage periods the highest growth of that was observed for G4 and G1, respectively. 3.2. Ammonia and biogenic amine formation Ammonia and biogenic amine production in trout are given in Table 3 for all groups. No significant differences were found in the ammonia concentrations within the treatments until 7 days of storage (p > 0.05), after which the differences were significant within groups (p < 0.05). The initial ammonia content in fresh trout fillets was found to be 3.06 mg/100 g for all groups. G2 had a higher ammonia content than G1 during the storage period. There were similar increases in the ammonia content between G2 and G3. The highest ammonia content was obtained from G4 at the end of the storage, and corresponded to the highest bacterial counts (11 log cfu/g) among the groups. Özogul, Özogul, and Kuley (2008) reported that the initial amount of ammonia was 0.02 mg/100 g and reached 1.76 mg/100 g for white grouper stored at 4 °C at the end of the storage period. Significant changes in the amine contents for each group during the storage period were observed (p < 0.05), with the exception of agmatine. The production of 12 biogenic amines was found in all groups, while 2-phenylethyl amine was not detected in any of the analysed samples. Biogenic amine formation is influenced by brine solution which may suppress some amines accumulation during the fermentation (Gardini et al., 2001). Histamine production was the highest in fermented fish with lactic acid bacteria without any salt, glucose and KNO3 addition (G4). Initial histamine concentration was 2.61 mg/100 g and increased to 6.44 mg/100 g for G4, 4.62 mg/100 g for G3, 7.41 mg/100 g for G2 and 4.11 mg/ 100 g for G1 at the end of storage period. Tsai et al. (2006) found that Bacillus coagulans and Bacillus megaterium in fermented fish products were capable of producing histamine. Moreover, Chang, Kung, Chen, Lin, and Tsai (2008) indicated that Staphylococcus spp., and S. aureus were capable of producing 13–33 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine. In the present study, the highest S. aureus counts were observed for G1 and G4, respectively. Tome et al. (2008) found that lactic acid bacteria strains isolated from vacuum-packaged and cold-smoked salmon were not able to produce histamine, but Enterococcus faecium ET05, Lactobacillus curvatus ET06 and L. curvatus ET30 produced tyramine. In this study, the microbiological results showed that the lactic acid bacteria contents in G1 were lower than in G2, although the histamine content of G1 and G2 were generally
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Table 2 Bacterial counts (log cfu/g) in fresh and fermented trout fillets in different growth medium. Storage days Growth medium
0
7
14
21
28
35
42
Groups
NA
MHA
4.76a ± 0.4b 4.76 ± 0.41 4.76 ± 0.41 4.76 ± 0.41 3.92 ± 0.12 3.92 ± 1.12 3.92 ± 0.12 3.92 ± 0.12 NT
SSA
NT
LSA
NT
MCA
NT
BPA
NT
5.58 ± 0.67 5.45 ± 0.67 5.48 ± 0.69 5.93 ± 0.32 4.10 ± 0.69 4.70 ± 0.58 4.50 ± 0.67 5.25 ± 0.94 4.63 ± 0.07 4.65 ± 0.77 4.90 ± 0.15 5.23 ± 0.94 4.45 ± 0.06 3.62 ± 0.97 3.57 ± 0.35 4.11 ± 0.11 4.34 ± 0.03 3.62 ± 0.77 3.45 ± 0.07 4.00 ± 0.03 3.02 ± 1.32 2.45 ± 1.24 2.98 ± 0.36 2.67 ± 0.27 3.74 ± 0.01 3.86 ± 0.10 3.48 ± 0.49 3.57 ± 1.09
5.56 ± 0.72 5.48 ± 0.42 5.60 ± 0.32 6.00 ± 1.21 5.03 ± 0.76 5.20 ± 1.42 5.21 ± 0.72 6.41 ± 1.32 6.00 ± 0.10 5.00 ± 0.35 5.11 ± 0.50 6.00 ± 1.05 4.86 ± 0.19 3.41 ± 0.49 3.73 ± 0.52 4.32 ± 0.56 3.18 ± 0.07 3.15 ± 0.31 3.23 ± 0.16 3.20 ± 0.17 3.46 ± 0.20 2.80 ± 0.41 3.40 ± 0.43 3.00 ± 0.31 4.34 ± 0.30 3.62 ± 1.02 3.45 ± 0.83 4.00 ± 0.57
7.62 ± 0.65 7.56 ± 0.74 7.26 ± 0.91 8.63 ± 0.88 5.11 ± 0.96 5.57 ± 0.79 5.54 ± 1.71 7.53 ± 0.88 6.57 ± 0.81 6.46 ± 0.55 6.24 ± 0.91 7.89 ± 0.89 5.59 ± 0.98 3.88 ± 0.30 3.18 ± 0.71 7.85 ± 0.42 4.70 ± 0.02 3.40 ± 0.19 3.00 ± 0.29 5.41 ± 0.17 5.30 ± 0.93 4.60 ± 1.01 4.80 ± 0.09 5.30 ± 0.12 5.20 ± 0.07 3.50 ± 0.17 3.70 ± 0.07 4.80 ± 0.64
9.08 ± 0.47 9.48 ± 1.67 9.36 ± 0.45 10.78 ± 0.61 6.34 ± 1.48 7.40 ± 2.20 7.11 ± 0.45 8.15 ± 0.61 7.80 ± 0.55 7.36 ± 0.61 7.18 ± 0.93 9.26 ± 0.99 5.36 ± 0.38 3.61 ± 0.26 3.00 ± 0.55 7.58 ± 0.44 6.00 ± 0.36 5.15 ± 0.62 4.76 ± 0.80 6.48 ± 0.05 6.59 ± 0.81 6.45 ± 0.91 6.18 ± 0.22 7.41 ± 0.16 6.38 ± 0.74 3.60 ± 0.44 5.46 ± 0.68 6.48 ± 0.29
10.52 ± 0.78 9.69 ± 0.76 9.08 ± 0.86 10.18 ± 0.85 5.96 ± 0.79 6.74 ± 0.76 7.32 ± 0.56 7.40 ± 1.15 8.70 ± 0.72 8.28 ± 0.60 7.48 ± 0.32 9.78 ± 1.04 6.23 ± 0.75 3.40 ± 0.78 3.00 ± 0.92 6.18 ± 0.33 6.20 ± 1.10 6.38 ± 0.81 5.81 ± 1.03 6.71 ± 0.22 7.56 ± 0.10 7.20 ± 0.17 6.78 ± 0.14 7.90 ± 0.27 6.73 ± 0.08 5.98 ± 0.09 5.65 ± 0.91 6.18 ± 0.45
9.85 ± 0.97 9.52 ± 0.25 8.94 ± 0.59 11.00 ± 0.51 6.46 ± 0.47 7.00 ± 0.25 7.80 ± 1.54 8.11 ± 0.61 10.90 ± 0.78 9.15 ± 0.63 9.00 ± 0.53 10.85 ± 1.51 6.60 ± 0.29 5.26 ± 0.26 4.48 ± 0.50 6.00 ± 0.63 6.40 ± 0.15 5.90 ± 1.05 6.00 ± 0.46 7.20 ± 0.30 6.32 ± 1.06 6.39 ± 0.22 6.00 ± 0.06 8.32 ± 0.08 7.15 ± 0.92 6.43 ± 1.00 6.48 ± 0.13 6.34 ± 0.39
G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4
MRS
NT, not tested; NA, Nutrient Agar; MRS, de man, Rogosa and Sharpe Agar; MHA, Muller Hilton Agar; SSA, Salmonella Shigella Agar; LSA, Listeria Sellective agar; MCA, Macconkey MUG agar; BPA, Baird Parker Agar. a Mean value. b Standard deviation.
were similar. Significant differences between G1 and G2 were only observed at day 35 and 42, which indicate a minor effect of lactic acid bacteria on histamine formation. Petaja et al. (2000) indicated that histamine and cadaverine production during storage of fermented rainbow trout were higher in the group without any lactic acid bacteria inoculation than the other groups with three different lactic acid bacteria inoculations with staphylococci. Some lactic acid bacteria strains were reported to considerably stimulate histamine production by most of the pathogen in histidine and tyrosine enrichment medium (Özogul, 2011; Kuley & Özogul, 2011). In the current study the presence of lactic acid bacteria strains in fish fillets statistically had no effect on the histamine production by food borne pathogens until 28 days of storage, apart from G4. The histamine content in trout fillets in G4 was found to be more than 10 mg/100 g, while G1, G2 and G3 contained less than 7.5 mg/100 g during storage. This might be due to the fact that G4 did not contain salt, glucose and KNO3. The biogenic amine production was depended on the microbial flora, availability of precursors and physicochemical factors, such as temperature, pH, salt, oxygen and sugar concentration (Zaman, Abdulamir, Bakar, Selamat, & Bakar 2009). Yongjin et al. (2007) reported that the main amines formed were histamine, cadaverine, putrescine and tyramine during a 48 h fermentation of silver carp sausages with 3% NaCl and glucose, and a mixed starter culture. The amine levels were found to be much higher than in the present study. The fish sauces fermented by the lactic acid microflora produced very high amounts of tyramine, cadaverine, putrescine, tryptamine and histamine (Kirschbaum, Rebscher, & Brückner 2000). Cadaverine was the major amine detected in Egyptian saltedfermented bouri fish samples during ripening and storage, followed by putrescine (Rabie, Sarkadi, Siliha, El-seedy, & El Badawy, 2009). In this study, cadaverine was not detected in the fish fillets
at 0 and 7 days, whilst significant increases in the cadaverine content of G4 were observed after 14 days of storage. No significant differences were found in the putrescine concentrations within the treatments during the early stages of the storage period. However, there was a significant difference (p < 0.05) in the putrescine content towards the end of the storage period for all treatments. The spermine and spermidine contents in fish and fish sauce are generally low (Tsai et al., 2006). In this study, the changes in spermidine in G1, G2 and G3 were similar. The initial value of spermidine was 1.06 mg/100 g, whereas G1, G2 and G3 showed slight increases at the end of storage, but not G4. Tryptamine and spermine production among the three groups (G1, G2 and G3) was similar and remained below 2.5 mg/100 g and 1 mg/100 g, respectively. The highest tryptamine and spermine production (2.71 and 3.94 mg/100 g) was observed for G4 at the end of the storage period. Among the analysed biogenic amines, the highest production of biogenic amines during the storage period was found for serotonin and tyramine in G1 and G3, respectively. Serotonin and tyramine were significantly higher in G1, G2 and G3 than G4 at 7 days of storage. These results indicated that adding brine solution had a stimulatory effect on serotonin or tyramine formation. Serotonin production was reported for white grouper (Özogul et al., 2008). Nevertheless, serotonin was not detected in fermented fish sauce (Kirschbaum et al., 2000). Tyramine is quantitatively the most common biogenic amine in fermented meat products (Komprda, Neznalova, Standara, & BoverCid, 2001). The tyramine level of 10–80 mg/100 g in foods has been demonstrated to be toxic (Shalaby, 1997). The initial tyramine concentration was 1.44 mg/100 g in fresh rainbow trout fillets and then reached maximum levels of 18.48 and 16.88 mg/100 g in G4 and G3, respectively, at 14 and 35 days of storage. In our previous work, it was reported that the combination of specific lactic acid
Table 3 The concentration of ammonia and biogenic amines (mg/100 g) in trout fillets. AMN
PUT
CAD
HIS
SPD
TRPT
PHEN
SPM
SER
TYR
TMA
AGM
Groups
0
3.06x ± 0.52y 3.06a ± 0.52 3.06a ± 0.52 3.06a ± 0.52 3.76a ± 0.25 2.81a ± 0.22 3.56a ± 0.34 3.51a ± 0.86 2.81b ± 0.59 4.38a ± 0.78 3.78ab ± 0.85 3.35ab ± 0.30 2.12 b ± 0.36 2.53b ± 0.52 4.60a ± 0.98 2.95b ± 0.09 3.15b ± 0.24 3.38b ± 0.87 3.46b ± 1.25 6.50a ± 0.53 2.54b ± 0.44 7.03a ± 1.16 6.94a ± 0.11 6.38a ± 1.11 5.31b ± 1.27 7.24b ± 0.97 5.87b ± 1.02 13.3a ± 1.45
0.70a ± 0.03 0.70a ± 0.03 0.70a ± 0.03 0.70a ± 0.03 0.50a ± 0.12 0.52a ± 0.00 0.70a ± 0.08 0.84a ± 0.01 0.70a ± 0.20 1.58b ± 0.06 0.58a ± 0.05 1.38b ± 0.91 0.52b ± 0.16 0.55b ± 0.07 0.87b ± 0.02 3.87a ± 0.56 0.61b ± 0.15 0.47b ± 0.06 0.34b ± 0.01 7.70a ± 0.66 0.65c ± 0.29 1.72b ± 0.15 1.10bc ± 0.10 11.8a ± 0.68 1.20b ± 0.15 1.89b ± 0.39 1.07b ± 0.13 20.7a ± 1.50
– – – – – – – – – 1.55b ± 0.34 0.06a ± 0.01 1.32b ± 0.17 – 0.37b ± 0.01 – 3.97a ± 0.79 0.03b ± 0.00 0.03b ± 0.00 0.04b ± 0.01 10.4a ± 0.22 0.33b ± 0.05 0.94b ± 0.07 – 15.0a ± 1.50 0.10c ± 0.01 1.52b ± 0.52 – 30.6a ± 0.65
2.61a ± 0.13 2.61a ± 0.13 2.61a ± 0.13 2.61a ± 0.13 2.47ab ± 0.19 2.12b ± 0.26 2.28ab ± 0.31 2.78b ± 0.15 4.11ab ± 0.79 5.77a ± 2.56 2.55b ± 0.31 5.17a ± 0.81 2.48b ± 0.68 2.41b ± 0.93 3.25b ± 0.36 8.89a ± 0.43 2.42b ± 0.34 2.06b ± 0.18 1.95b ± 0.45 23.0a ± 0.35 2.72c ± 0.78 5.79b ± 0.43 3.45bc ± 0.21 33.3a ± 0.51 4.62c ± 0.21 7.41b ± 0.3 3.73d ± 0.07 6.44a ± 0.24
1.06a ± 0.15 1.06a ± 0.15 1.06a ± 0.15 1.06 a ± 0.15 0.89b ± 0.09 0.85b ± 0.23 0.96b ± 0.05 1.36a ± 0.29 1.21b ± 0.02 0.64a ± 0.13 0.80a ± 0.27 0.56a ± 0.01 0.66ab ± 0.03 0.68ab ± 0.23 1.23a ± 0.14 0.05c ± 0.01 0.88a ± 0.04 0.80ab ± 0.06 0.71b ± 0.08 0.12c ± 0.04 0.70b ± 0.08 1.48a ± 0.14 1.32a ± 0.04 0.12 ± 0.00 1.14a ± 0.15 1.26a ± 0.21 1.28a ± 0.21 –
– – – – 0.93a ± 0.05 – 0.74a ± 0.01 0.16b ± 0.01 0.14a ± 0.04 0.83a ± 0.18 0.57a ± 0.09 0.90a ± 0.05 – 0.47b ± 0.08 0.91a ± 0.07 0.23c ± 0.01 0.59b ± 0.01 0.46b ± 0.01 0.52b ± 0.19 1.14a ± 0.40 0.61b ± 0.00 1.00ab ± 0.15 0.86ab ± 0.04 1.32a ± 0.84 0.91b ± 0.01 0.70b ± 0.03 0.45b ± 0.01 2.71a ± 0.60
– – – – – – – – – – – – – – – – – – – – – – – – – – – –
1.85a ± 0.13 1.85a ± 0.13 1.85a ± 0.13 1.85a ± 0.13 1.51a ± 0.15 1.24a ± 0.26 1.38a ± 0.17 1.65a ± 0.33 1.61a ± 0.54 1.86a ± 0.76 1.35a ± 0.50 1.62a ± 0.68 1.08b ± 0.27 1.06b ± 0.12 1.97a ± 0.25 1.29b ± 0.26 1.53b ± 0.35 1.35b ± 0.01 1.13b ± 0.41 2.79a ± 0.49 1.22c ± 0.64 2.31b ± 0.09 2.42b ± 0.21 3.27a ± 0.56 1.69c ± 0.15 2.07b ± 0.22 1.86bc ± 0.27 3.94a ± 0.08
2.42a ± 0.53 2.42a ± 0.53 2.42a ± 0.53 2.42a ± 0.53 11.6a ± 0.43 11.9a ± 1.82 10.5a ± 1.29 2.69b ± 0.40 18.0a ± 2.51 1.84b ± 0.08 12.8a ± 0.80 1.15b ± 0.06 12.4a ± 1.65 3.20b ± 0.65 11.2a ± 1.20 0.56c ± 0.02 8.05a ± 2.61 4.31b ± 0.57 6.29ab ± 0.68 0.35c ± 0.06 4.97b ± 0.42 6.86b ± 1.61 12.2a ± 2.03 0.33c ± 0.00 12.2b ± 2.08 6.83c ± 1.49 18.8a ± 0.04 2.63d ± 0.43
1.44a ± 0.60 1.44a ± 0.60 1.44a ± 0.60 1.44a ± 0.60 6.32ab ± 0.69 4.36b ± 1.46 8. 84a ± 0.24 0.39c ± 0.32 16.9 a ± 1.33 6.96 ab ± 2.16 8.61a ± 0.64 2.40b ± 1.06 8.57b ± 2.09 6.66b ± 0.92 14.5a ± 1.58 0.45c ± 0.07 4.13a ± 1.65 2.09b ± 0.57 3.70ab ± 0.66 0.79c ± 0.21 5.05b ± 0.20 4.50b ± 0.22 18.5a ± 1.19 1.25c ± 0.19 3.21c ± 0.67 4.49b ± 0.37 12.1a ± 0.37 5.02b ± 0.66
0.61a ± 0.66 0.61a ± 0.66 0.61a ± 0.66 0.6 a ± 0.66 2.65ab ± 0.33 1.89ab ± 0.09 3.71a ± 0.43 0.00c ± 0.00 8.76a ± 1.22 3.11b ± 0.08 3.70b ± 0.10 1.32b ± 0.01 3.64ab ± 1.07 3.13b ± 0.41 6.89a ± 3.05 0.34b ± 0.03 1.63a ± 0.07 1.23a ± 1.06 1.45a ± 0.85 0.40a ± 0.00 2.03b ± 0.86 2.37b ± 0.52 9.72a ± 1.44 1.11c ± 0.20 1.09c ± 0.27 5.80b ± 0.74 4.66b ± 1.07 9.48a ± 0.62
– – – – 3.06a ± 0.14 0.16b ± 0.02 3.06a ± 0.33 – – 10.9a ± 1.88 2.55b ± 0.41 6.23ab ± 0.92 – – – – – – – – 0.73a ± 0.27 – – – 8.39a ± 1.17 0.38c ± 0.01 5.85b ± 0.38 –
G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4
7
14
21
28
35
42
E. Kuley et al. / Food Chemistry 129 (2011) 1211–1216
Storage days
AMN, ammonia; PUT, putrescine; CAD, cadaverine; HIS, histamine; SPD, spermidine; TRP, tryptamine; PHEN, 2-Phenyl–ethylamine; SPN, spermine; SER, serotonin; TYR, tyramine; TMA, trimethylamine; AGM, agmatine. –, not detected. –, Not detected. a Mean value. b Standard deviation (n = 3). Different lowercase letters (a–d) in a row indicate significant differences (p < 0.05) among groups.
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bacteria and foodborne pathogens resulted in significantly higher tyramine accumulation in histidine or tyrosine enriched medium (Kuley & Özogul, 2011; Özogul, 2011). However, in the current study, G4 had significantly lower tyramine formation compared to G1 (in which lactic acid bacteria is not inoculated), during all storage periods, except for 42 days. Moreover, the presence of lactic acid bacteria strains in the brine solution had generally no significant effect on the tyramine production by foodborne pathogens. Agmatine was not detected at 0, 21 and 28 days for all groups. These results suggested that inoculation with lactic acid bacteria and foodborne pathogens did not significantly contribute to the agmatine production of fish fillets. However, major changes in the content of agmatine were observed for G2 and G4 at 14 days, and for G1 and G3 at 7 and 42 days. Özogul and Özogul (2007) demonstrated that bacteria could vary in their relative importance as biogenic amine producers in different decarboxylase broths. They found that K. pneumoniae and E. faecalis did not produce agmatine in arginine decarboxylase broth, while the highest agmatine production by K. pneumoniae and E. faecalis were in lysine decarboxylase broth and in tyrosine decarboxylase broth, respectively. 4. Conclusions Foodborne pathogens in trout fillets had an ability to produce ammonia and biogenic amine during the fermentation and storage of fish. Lactic acid bacteria generally did not seem to play an important role on biogenic amine production by pathogenic bacteria. The result of the study also showed that ammonia and biogenic amine production could be significantly influenced by adding brine solution. Brine solution had an inhibitory effect on ammonia, histamine, putrescine and cadaverine production in trout fillets, but not for serotonin and tyramine production. Acknowledgement The authors would like to thank the Scientific Research Projects Unit in Cukurova University for their financial support (Research Project: SUF 2007 D1). References Bover-Cid, S., & Holzapfel, W. H. (1999). Improved screening procedure for biogenic amine production by lactic acid bacteria. International Journal of Food Microbiology, 53, 33–41. Brashears, M. M., Amezquita, A., & Jaroni, D. (2005). Lactic acid bacteria and their uses in animal feeding to improve food safety. Advances in Food and Nutrition Research, 50, 1–31. Chang, S.-C., Kung, H. F., Chen, H. C., Lin, C. S., & Tsai, Y. H. (2008). Determination of histamine and bacterial isolation in swordfish fillets (Xiphias gladius) implicated in a food borne poisoning. Food Control, 19, 16–21. Devlieghere, F., Vermeiren, L., & Debevere, J. (2004). New preservation technologies: Possibilities and limitations. International Dairy Journal, 14, 273–285. Fadda, S., Vignolo, G., & Oliver, G. (2001). Tyramine degradation and tyramine/ histamine production by lactic acid bacteria and Kocuria strains. Biotechnology Letters, 23, 2015–2019. Fernandez, M. F., Boris, S., & Barbes, C. (2003). Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. Journal of Applied Microbiology, 94, 449–455. Gardini, F., Martuscelli, M., Caruso, M. C., Galgano, F., Crudele, M. A., Favati, F., et al. (2001). Effects of pH, temperature and NaCl concentration on the growth
kinetics, proteolytic activity and biogenic amine production of Enterococcus faecalis. International Journal of Food Microbiology, 64, 105–117. Gonzalez, C. J., Lopez, T. M., Garcia, M. L., Prieto, M., & Otero, A. (1999). Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius), and aquacultured rainbow trout (Oncorhynchus mykiss). Journal of Food Protection, 62, 1270–1277. Kirschbaum, J., Rebscher, K., & Brückner, H. (2000). Liquid chromatographic determination of biogenic amines in fermented foods after derivatization with 3,5-dinitrobenzoyl chloride. Journal of Chromatography A, 881, 517–530. Komprda, T., Neznalova, J., Standara, S., & Bover-Cid, S. (2001). Effect of starter culture and storage temperature on the content of biogenic amines in dry fermented sausage polican. Meat Science, 59, 267–276. Kopermsub, P., & Yunchalard, S. (2010). Identification of lactic acid bacteria associated with the production of plaa-som, a traditional fermented fish product of Thailand. International Journal of Food Microbiology, 138, 200–204. Kuley, E., & Özogul, F. (2011). Synergistic and antagonistic effect of lactic acid bacteria on tyramine production by food-borne pathogenic bacteria in tyrosine decarboxylase broth. Food Chemistry, 127, 1163–1168. Mah, J.-H., & Hwang, H. J. (2009). Inhibition of biogenic amine formation in a salted and fermented anchovy by Staphylococcus xylosus as a protective culture. Food Control, 20, 796–801. Middlebrooks, B. L., Toom, P. M., Donglas, W. L., Harrison, R. E., & McDowell, S. (1988). Effects of storage time and temperature on the microflora and amine development in Spanish Mackerel (Scomberomorus maculatus). Journal of Food Science, 53, 1024–1029. Ordonez, J. A., Hierro, E. M., Bruna, J. M., & de la Hoz, L. (1999). Changes in the components of dry-fermented sausages during ripening. Critical Reviews in Food Science and Nutrition, 39, 329–367. Özogul, F. (2011). Effects of specific lactic acid bacteria species on biogenic amine production by foodborne pathogen. International Journal of Food Science and Technology, 46, 478–484. Özogul, F., & Özogul, Y. (2007). The ability of biogenic amines and ammonia production by single bacterial cultures. European Food Research Technology, 225, 385–394. Özogul, F., Özogul, Y., & Kuley, E. (2008). Nucleotide degradation and biogenic amine formation of wild white grouper (Epinephelus aeneus) stored in ice and at chill temperature (4 °C). Food Chemistry, 108, 933–941. Özogul, F., Taylor, K. D. A., Quantick, P., & Özogul, Y. (2002). Biogenic amines formation in Atlantic herring (Clupea harengus) stored under modified atmosphere packaging using a rapid HPLC method. International Journal of Food Science and Technology, 37, 515–522. Petaja, E., Eerola, S., & Petaja, P. (2000). Biogenic amines in cold-smoked fish fermented with lactic acid bacteria. European Food Research Technology, 210, 280–285. Rabie, M., Sarkadi, L. S., Siliha, H., El-seedy, S., & El Badawy, A. A. (2009). Changes in free amino acids and biogenic amines of Egyptian salted-fermented fish (Feseekh) during ripening and storage. Food Chemistry, 115, 635–638. Shalaby, A. R. (1997). Significance of biogenic amines to food safety and human health. Food Research International, 29, 475–490. Tadesse, G., Ephraim, E., & Ashenaf, M. (2005). Assessment of the antimicrobial activity of lactic acid bacteria isolated from borde and shamita, traditional ethiopian fermented beverages, on some foodborne pathogens and effect of growth medium on the inhibitory activity. International Journal of Food Safety, 5, 13–20. Tome, E., Pereira, V. L., Lopes, C. I., Gibbs, P. A., & Teixeira, P. C. (2008). In vitro tests of suitability of bacteriocin-producing lactic acid bacteria, as potential biopreservation cultures in vacuum-packaged cold-smoked salmon. Food Control, 19, 535–543. Tsai, Y. H., Lin, C. Y., Chien, L. T., Lee, T. M., Wei, C. I., & Hwang, D. F. (2006). Histamine contents of fermented fish products in Taiwan and isolation of histamine-forming bacteria. Food Chemistry, 98, 64–70. Yongjin, H., Wenshui, X., & Xiaoyong, L. (2007). Changes in biogenic amines in fermented silver carp sausages inoculated with mixed starter cultures. Food Chemistry, 104, 188–195. Zaman, M. Z., Abdulamir, A. S., Bakar, F. A., Selamat, J., & Bakar, J. (2009). Microbiological, physicochemical and health impact of high level of biogenic amines in fish sauce. American Journal of Applied Sciences, 6, 1199–1211. Zhong-Yi, L., Zhong-Hai, L., Miao-Ling, Z., & Xiao-Ping, D. (2010). Effect of fermentation with mixed starter cultures on biogenic amines in bighead carp surimi. International Journal of Food Science and Technology, 45, 930–936.
