Abstract: Lactic acid and chitosan were evaluated for their effects on the growth and survival of V. parahaemolyticus on mussel meat. Mussel (Mytilus sp.) samples were collected from the Samsun region on the middle Black Sea coast of Turkey. Each shelled mussel was decontaminated by immersion in 4% formalin for 3 min. The decontaminated mussel was dipped in TSB broth containing between 8.25 and 7.60 log CFU gG¹ of V. parahaemolyticus and left for 30 min at room temperature 25°C to allow attachment. Initial counts of V. parahaemolyticus in mussel meat immediately after dipping in TSB broth were in the range of 5.38-4.03 log CFU gG¹. Each inoculated mussel (25°C) was dipped in 0.5, 1, 1.5 or 2% of lactic acid (v/v) or 0.05, 0.1, 0.25 or 0.5% of chitosan (v/v) for 5, 15, 30 or 60 min. Initial counts of V. parahaemolyticus in mussel meat decreased following treatment with lactic acid for 5 min by 1.90, 2.13, 2.27 and 2.78 log CFU gG¹, respectively and following treatment with chitosan for 5 min by 1.33, 1.41, 1.56 and >2.03 log CFU gG¹, respectively. Growth of V. parahaemolyticus on mussels was completely inhibited after being dipped in 1.5-2% lactic acid for 15 min and 0.5% chitosan for 5 min.

INTRODUCTION

Vibrio parahaemolyticus, a halophilic marine bacterium is a worldwide cause of food-borne gastroenteritis (Janda et al., 1988). Mussels are filter feeding bivalve molluscs that can concentrate bacteria in contaminated water. V. parahaemolyticus can be a risk factor, when these products are eaten raw or slightly cooked. It was first identified as a foodborne pathogen in Japan in 1950 (Fujino et al., 1953) and in 1977, V. parahaemolyticus caused the largest reported outbreak associated with eating raw oysters involving 209 persons in North America (CDC, 1998). V. parahaemolyticus has been successfully isolated by some researchers from fish (Dileep et al., 2003), shrimp (Robert-Pillot et al., 2004), mussels (Di Pinto et al., 2008; Martinez-Urtaza et al., 2008; Terzi et al., 2009) and oysters (Bej et al., 1999; Lee et al., 2008).

Many methods have been developed to prolong the shelf life of fishery products, such as washing, storage at low temperatures, cold shock, freezing, ultraviolet irradiation, salt treatment and decontamination using chitosan, chlorine, organic acids, ozone and chloroform (Chythanya et al., 2002; Manousaridis et al., 2005; Chaiyakosa et al., 2007; Masniyom and Benjama, 2007). Lactic acid is one of the most widely used among the organic acids for decontamination of meat. Lactic acid inhibits the growth of microorganisms such as gram negative species of the families Enterobacteriaceae and Pseudomonadaceae (Anang et al., 2007).

The other decontaminant that is used in seafood to reduce the bacterial count is chitosan. Chitosan is a b-1, 4 linked N-acetyl-d-glucosamine polymer. It is derived by deacetylation of chitin, a major component of the shells of crustacea such as crab, shrimp and crawfish (Arvanitoyannis et al., 1998; No et al., 2002). Chitosan has been shown to have antibacterial activities against the gram-positive bacteria (Listeria monocytogenes, Bacillus megaterium, B. cereus, Staphylococcus aureus, Lactobacillus plantarum, L. brevis, L. Bulgaris) and gram negative bacteria (E. coli, Pseudomonas fluorescens, Salmonella typhymurium, Vibrio parahaemolyticus) (Chen et al., 2002; Liu et al., 2004; No et al., 2002). The antimicrobial activity of chitosan is reported to be dependent on its degree of deacetylation, molecular weight, concentration and viscosity (Mohy et al., 2008; No et al., 2002; Jeon et al., 2001).

The objective of this study was to investigate the effect of chitosan and lactic acid on the numbers of V. parahaemolyticus in mussels.

MATERIALS AND METHODS

Bacterial strains: V. parahaemolyticus ATCC 17802 was grown in Tryptic Soy Broth (TSB, Merck, Darmstadt, Germany) (with 2% NaCl ) at 37°C for 24 h and then adjusted to 1.6H10⁸ CFU mLG¹ with tube dilution methods.

Mussel samples: A total of 200 mussel (Mytilus sp.) samples were collected from the Samsun region on the middle Black Sea coast of Turkey, in September 2009. All samples were transported to the laboratory in containers with ice bags for analysis within a few hours. The samples were washed in sterile distilled water and disinfected with alcohol and then opened aseptically.

Artificial contamination of mussel samples with V. parahaemolyticus: Twenty five grams of shelled mussel were decontaminated by immersion in 4% formalin for 3 min and washed twice with 1% NaCl to remove any remaining formalin. The decontaminated mussel was dipped in 100 mL Tryptic Soy Broth (TSB, Merck, Darmstadt, Germany) containing 1.6H10⁸ CFU mLG¹ of V. parahaemolyticus and left for 30 min at room temperature (25°C) to allow attachment.

Decontamination with lactic acid or chitosan: Each contaminated mussel was dipped in 100 mL of either a 0.5, 1, 1.5 or 2% solution of lactic acid (v/v) (Merck, Darmstadt, Germany) or low molecular weight chitosan (v/v) (Aldrich-44.886-9, Milwaukee, WI, USA) (MW = 150 kDa, 75-85% deacetylation, viscosity 20-200 cps), at a concentration of 0.05, 0.1, 0.25 or 0.5% for 5, 15, 30 or 60 min. Each inoculated mussel was also dipped in 100 mL of sterile distilled water (2% NaCl) as a control. After dipping, the mussel samples were examined for V. parahaemolyticus.

Microbiological analysis: To determine the V. parahae- molyticus count, 25 g mussel samples were transferred aseptically to a stomacher bag containing 225 mL of alkali peptone water (APW; 1% tryptone peptone, 2% NaCl, pH 8.6) and homogenized for 2 min using a stomacher (Interscience-BagMixer 400, St. Nom., France). For microbial enumeration 0.1 mL samples of serial dilutions (1:10, diluent, alkali peptone water with 2% NaCl) of mussel homogenates were spread on the surface of thiosulphate citrate bile salt sucrose agar (TCBS; Merck, Darmstadt, Germany) and the TCBS plates were incubated at 37°C for 24 h. The formation of colonies that are round (2-3 mm diameter) and green on TCBS was considered positive for V. parahaemolyticus and microbial counts were expressed as CFU mLG¹ (ISO 8914, 1990).

Statistical analysis: Statistical analysis was carried out using the Statistical Package for Social Science (SPSS 10.0 for Windows, SPSS Inc., Chicago, IL). All experiments were replicated three times. The results were evaluated by using one-way ANOVA and the level of significance was set at p<0.05.

RESULTS AND DISCUSSION

Table 1 shows the V. parahaemolyticus counts on mussel meat treated with different concentrations of lactic acid and chitosan. The initial count of V. parahaemolyticus on mussel meat after inoculation ranged from 5.38-4.03 logCFU gG¹. V. parahaemolyticus counts on mussels dipped in 0.5-2% lactic acid for 5 and 15 min were significantly lower (p<0.05). Morover, the growth of V. parahaemolyticus on mussels was completely inhibited after dipping in 0.5-1% lactic acid for 30 min and 1.5-2% lactic acid for 15 min.

The most widely used chemical decontaminants in the meat industry are organic acids (Belk, 2001). The antimicrobial activity of lactic acid has been demonstrated in a number of foods including chicken, meat and seafood (Ramirez et al., 2001; Masniyom and Benjama, 2007; Lin et al., 2005; Anang et al., 2007; McMullin and Steele, 2007). Masniyom and Benjamae (2007) found that mesophilic bacterial counts in green mussels declined from 3.63-2.36 logCFU gG¹, when treated with 0.2 M lactic acid. Sorrells et al. (1989) reported that V. parahaemolyticus was more acid tolerant than other food borne pathogens.

Table 1:	V. parahaemolyticus counts on mussel meat dipped in different concentrations of chitosan and lactic acid
Means in the same row followed by different letters are significantly different (p<0.05), a-cShow number of V. parahaemolyticus differences duration of decontamination (p<0.05), Tukey test, ND: Not Detected (detection limit<log2.0)

Table 2:	Log reduction in V. parahaemolyticus inoculated onto mussel meat dipped in different concentrations of chitosan and lactic acid

In the present study, V. parahaemolyticus counts in mussels declined from 5.38 -3.47 logCFU gG¹, when treated with 0.5% lactic acid for 5 min. Moreover, when treated with 2% lactic acid for 5 min V. parahaemolyticus counts declined from 5.38-2.60 logCFU gG¹. The results indicate that the inhibition of V. parahaemolyticus is related to the concentration of lactic acid and a dipping time period. We observed that V. parahaemolyticus counts declined and even it inhibited completely, when increasing the concentration and also extended the dipping time of lactic acid. Table 2 presents the log reduction in numbers of V. parahaemolyticus on mussel meat dipped in different concentrations of chitosan and lactic acid. The maximum decrease in the initial population of V. parahaemolyticus on mussel meat treated with lactic acid ranged between 2.00 and >3.38 logCFU gG¹, from initial counts of 5.38 logCFU gG¹. The antimicrobial effects of the use of lactic acid have been reported in other studies, with a reduction of 1-2 log (Ransom et al., 2003).

Xiong et al. (1998) observed a 2.2 log CFU cmG² decrease in Salmonella typhimurium following the use of thorough spraying of 1-2% lactic acid solution on chicken skin. In another study, Ramirez et al. (2001) reported a 1.6 logCFU cmG² reduction in Escherichia coli after spraying lamb meat with 2% lactic acid for 9 sec. Ransom et al. (2003) reported a 3.3 log CFU cmG² decrease in Escherichia coli 0157:H7 after dipping beef carcasses in 2% lactic acid solution. Anang et al. (2007) found a decrease in the initial population of L. monocytogenes, S. enteritidis and E. coli O157:H7 of 1.97, 1.71 and 2.59 logCFU gG¹, respectively, after dipping chicken breast in 0.5, 1, 1.5 and 2% lactic acid for 10, 20 and 30. The antibacterial effects of chitosan depend on its molecular weight, viscosity and the type of bacterium. It has been reported that chitosan generally shows stronger antibacterial effects against gram positive bacteria than gram negative bacteria (No et al., 2002; Jeon et al., 2001). Liu et al. (2006) reported that the antibacterial effect of low MW chitosan is higher than that of high MW samples. In this study, we used low MW chitosan (150 kDa) with 75-85% deacetylation. The activity of chitosan against Vibrio sp. has been reported by other researchers (Chaiyakosa et al., 2007; Wang and Chen, 2005). In a study conducted by No et al. (2002), different chitosans with a MW in the range of 28-1671 kDa were used to determine the effect of chitosan MW on the inhibition of V. parahaemolyticus. They found that V. parahaemolyticus counts declined from 6.64-3.29 logCFU gG¹, when treated with 470 kDa MW chitosan. They observed a 2.56 logCFU gG¹ reduction in V. parahaemolyticus.

In the present study, V. parahaemolyticus counts declined from 4.03-2.70 logCFU gG¹, when treated with 150 kDa MW chitosan at a concentration of 0.05% for 5 min. The reduction in V. parahaemolyticus was 1.33 logCFU gG¹.

On the other hand V. parahaemolyticus was not detected after treatment with chitosan at a concentration of 0.05% and contact time of 15 min. Chaiyakosa et al. (2007) reported that in artificially inoculated shrimp, a >90% reduction in V. parahaemolyticus was observed, when the contact time was increased to 30 min and the concentration of chitosan was 1000 ppm (0.1%) and 2000 ppm (0.2%). Similary, in the present study, we found a >90% reduction in V. parahaemolyticus, when 0.05-0.5% chitosan was used for 15 min.

CONCLUSION

This study has demonstrated that V. parahaemolyticus counts decline by between 1.91 and >3.38 logCFU gG¹ after dipping in 0.5-2% lactic acid and between 1.33 and >2.03 logCFU gG¹ after dipping in 0.05-0.5% chitosan. The decontamination of mussel meat by lactic acid and chitosan significantly reduced V. parahaemolyticus counts.